Compositions and methods of treating or preventing inflammatory diseases

 

The invention relates to compositions and methods for the treatment and prevention of such diseases and conditions as graft rejection, surgical adhesions, inflammatory bowel disease, nasal polyps, and includes delivery to the site of inflammation antimicrotubular agent is paclitaxel, or an analogue or derivative. The invention provides a specified treatment and prevention at the expense of disrupting the function of microtubules by depolymerization or stabilization of their education, which interrupts the pathogenesis of these diseases. 4 N. and 42 C.p. f-crystals, 14 tab., 81 Il.

The technical FIELD

This invention relates in General to compositions and methods for treating or preventing inflammatory diseases.

BACKGROUND of the INVENTION

Inflammatory diseases, chronic or acute nature, pose a significant problem in the health system. In short, chronic inflammation is considered as inflammation of the long-term duration (weeks or months) in which active inflammation, tissue destruction and attempts at reparation occur simultaneously (Robbins Patholog inflammatory attack, it can also begin in the form of a gradually evolving process, which progresses over time, for example, as a result of chronic infection (eg, tuberculosis, syphilis, fungal infection), which causes an allergic reaction of the delayed type, prolonged exposure to endogenous (e.g., a high content of plasma lipids) or exogenous (e.g., silica, asbestos, cigarette tar, surgical suture material) toxins, or autoimmune reactions against their own body tissues (for example, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, psoriasis). Thus, chronic inflammatory diseases include many common medical conditions such as rheumatoid arthritis, restenosis, psoriasis, multiple sclerosis, surgical adhesions, tuberculosis and chronic inflammatory lung diseases (e.g. asthma, pneumoconiosis, chronic obstructive pulmonary disease, nasal polyps and pulmonary fibrosis).

Psoriasis

Psoriasis is a common chronic skin disease characterized by bulging, inflamed, thickened and scaly lesions, which causes the pronounced inverse symptoms which are similar to the changes observed in rheumatoid arthritis. Approximately 2-3% of the U.S. population suffers from psoriasis, and annually diagnosed 250000 new cases.

Currently, the cause of psoriasis is not known, although there is sufficient evidence that he is a polygenic autoimmune disorder. In addition, at the present time there is no cure for psoriasis. Available effects include local treatments such as steroid creams and ointments, coal tar, anthralin, and systemic treatment, such as treatment with steroids, UV, PUVA (oral administration of psoralen and subsequent exposure to long-wave ultraviolet light UV-a), methotrexate and cyclosporine. However, the unsatisfactory rate of remission and/or potentially serious side effects characterize most of the methods of treatment of psoriasis. The total cost of treatment of psoriasis in the United States is estimated between 3 and 5 billion dollars a year, which makes psoriasis is a major public health problem.

Multiple sclerosis

Multiple sclerosis (PC), affecting 350,000 people (the ratio of women:men = 2:1) is Connected to specialnym disease, affecting the nervous system. Generally, the PC is manifested clinically in the form of recurring bouts of adverse neurological disorders, taking place over a period of several years. A rough estimate of the half cases PC progress in the more chronic phase. Although this disease does not lead to early death or impairment of the function of knowledge, it makes the patient disabled as a result of violations of visual acuity, stimulation diplopia, mobility impairments that affect walking and using hands, induce incontinence of bowel and bladder, spasticity and loss of sensitivity (sensitivity to touch, pain sensitivity and temperature sensitivity).

The reason the PC is not known, although there is considerable evidence that it is an autoimmune disease. There is currently no means of cure multiple sclerosis, and existing therapeutic regimens are only partially successful. For example, although chemotherapeutic agents such as methotrexate, cyclosporine and azathioprine, have been tested for the treatment of patients with non-treatable progressive disease, to piticescu means, recently approved include interferon-for use in the case of ambulatory patients with recurrence/remission PC (Paty et al., Neurology 43: 662-667, 1993), in particular, Betaseron (recombinant interferon--1; human interferon beta, substituted in position 17, Cys® Ser; Berlex/Chiron) or Avonex (interferon-1; glycosylated human interferon beta produced in mammalian cells; Biogen). Unfortunately, although Betaseron provides improved quality of life for patients with PC, disease progression, apparently, is not improving significantly. Harmful side effects associated with therapy using Betaseron include: reaction at the site of injection (inflammation, pain, allergies and necrosis) and complex flu-like symptoms (fever, chills, anxiety, and confusion).

Rheumatoid arthritis

Rheumatoid arthritis (RA) is a debilitating, chronic inflammatory disease affecting 1-2% of the world's population. This condition causes pain, swelling and destruction of the. Persons with advanced disease have a higher mortality rate than some forms of cancer, and consequently, the treatment regimen was shifted aggressive early drug therapy intended to reduce the likelihood of irreversible damage to the joints. Recent recommendations by the American College of Rheumatology (Arthritis and Rheumatism 39(5): 713-722, 1996) include early initiation of disease modifying therapy Antirheumatic drug (DMARDs) for any patient with a diagnosis and corresponding symptoms. Anticancer drugs have become the primary therapy for the vast majority of patients, with 60-70% of rheumatologists preferred chemotherapeutic agent, methotrexate. The severity of the disease often justifies unlimited weekly therapy with this drug and, in the case of patients, the disease which progresses despite treatment with methotrexate (more than 50% of patients), often used chemotherapeutic drugs second line, such as cyclosporine and azathioprine (separately or in combination).

Restenosis

Restenosis is a form of vascular damage, privateit in response to treatments plastic vessels, including virtually any manipulation, which is aimed at weakening blockages of blood vessels, and is the main factor limiting the effectiveness of invasive treatments for vascular diseases. Restenosis was the focus of cardiovascular research for the past 15 years. Estimated 1994 (U. S. Heart and Stroke Foundation), over 60 million Americans have one or more forms of cardiovascular disease. These diseases that killed approximately 1 million lives in the same year (41% of all deaths in the United States), are considered as the major cause of death and disabilities in a developed society.

There are currently no technically approved methods of preventing restenosis, effective for the individual. Systemic therapies that have been investigated include agents aimed at reducing the loss of endothelial cells, antiplatelet agents (e.g. aspirin), vasodilator (e.g., calcium channel blockers), antithrombotic agents (e.g., heparin), anti-inflammatory drugs (such as steroids), a means of preventing the proliferation of cells in the vascular smooth muscle (VSMC) (e.g., colchicine) and who were investigated, include local delivery of drugs (e.g. heparin) and beta - and gamma-radiation. They were all disappointing as applied to man, primarily because they, apparently, apply to a limited part of the process of restenosis. Systemic treatments also met with the additional problem of achieving adequate absorption and retention of the drug in the site of the violation to ensure long-term biological effect, without inducing adverse systemic complications and toxicity.

Inflammatory bowel disease

The term inflammatory bowel disease (IBD) refers to chronic disorders (originally called Crohn's disease and ulcerative colitis) that cause inflammation or ulceration in the small and large intestines. Briefly, approximately 2 million people in the United States suffer from IBD, with men and women affected equally. The peak frequency of the disease primarily occurs between the ages of 15 and 30 years, and the second reported peak incidence is between ages 55 and 60 years. Although documented many of the distribution of prevalence, the reason for this tabletreeitem recurrence or sudden flare-up. Approximately 50% of patients are in remission at any time, and most suffer at least one recurrence during the period of 10 years. In addition, there are many systemic complications that accompany the disease, and the most common is arthritis. Symptoms of arthritis have 1/4 of all people with IBD. Inflammation of the joints occurs most often when the process involves the colon, and suddenly worsens when the bowel disease is most active. This form of inflammatory arthritis does not cause permanent defect and is often short-lived. Other complications of this disease include inflammation of the eye (iritis, conjunctivitis and episcleritis), inflammation of the mouth (mucositis), skin inflammation (nodosa erythema and pyoderma pyoderma), musculoskeletal disorders (ankylosing spondylitis), renal complications (renal stones and fistulas of the urethra), gall stones, and other liver disease (e.g. hepatitis and biliary system (sclerosing cholangitis). Unfortunately, in many cases, long-term (>10 years) disease can lead to more serious complications such as cancer of the colon and extraintestinal karzi on combating the symptoms of this disease through suppressio inflammation, associated with this disease. The main drugs used to treat IBD, are aminosalicylates and corticosteroids, and for those individuals who do not respond well to these agents, can also be used antibiotics and immunosuppressive drugs. Although drug treatment is effective in 70-80% of patients, for individuals with more active disease, often require surgery. Chronic symptoms and complications associated with active disease, such as bowel obstruction, perforation, abscess, or bleeding, can be loosened and adjusted invasive surgery. Although surgery does not cure this disease permanently and the recurrence rate is high, it really weakens the active symptoms.

Surgical adhesions

Surgical spiloptera, a complex process in which the tissues of the body that normally are separate, grow together, occurs most often as a result of surgical trauma. These postoperative adhesions occur in 60-90% of patients undergoing gynecological surgery, and are one of the most common causes of blockage of blood vessels in Industria radnai cause of bowel obstruction and infertility. Other spikes complications include chronic pain in the pelvic region, blockage of the urethra, and dysfunction of the evacuation (removal of feces or urine output). Now for the inhibition of adhesions use of preventive therapy 4-5 days after surgery. Tested various methods of prevention of adhesions, including (1) preventing the deposition of fibrin, (2) reduction of local inflammation, and (3) removal of deposits of fibrin. The deposition of fibrin is prevented through the use of physical barriers that are either mechanical or consisting of viscous solutions. Although many researchers use the barriers preventing adhesions, there are a number of technical difficulties. Inflammation is reduced by the introduction of drugs such as corticosteroids and non-steroidal anti-inflammatory drugs. However, the results of the use of these drugs in animal models have not been encouraging due to the extent of the inflammatory response and dose limitation due to systemic side effects. Finally, the removal of fibrin accumulation was investigated using proteoliticheskaja the possibility of excessive bleeding.

Inflammatory lung disease

Chronic inflammatory lung diseases, including, for example, asthma, pneumoconiosis, chronic obstructive pulmonary disease, nasal polyps and pulmonary fibrosis, affect many people around the world. Typically, these invasive diseases are characterized by inflammation and thickening of the affected tissues.

For example, nasal polyps are characterized by thickened tissue ensheathing. Polyps can occur in respiratory diseases such as asthma, mukoviszidose, primary ciliary dyskinesia and immunodeficiencies. Believe that nasal polyps develop as a manifestation of chronic inflammatory processes involving the upper respiratory tract. They were found in 36% of patients with intolerance against aspirin, 7% of patients with asthma, 0.1% of children and approximately 20% of patients with cystic fibrosis. Other conditions associated with naselenie polyps are syndrome Churg-Strauss, allergic fungal sinusitis and syndrome ciliary dyskinesia syndrome and young. Approximately 40% of patients with surgical polypectomy have relapses (Settipane, Allergy Asthma Proc. 17(5): 231-236, 1996).

The main symptoms of nasal polyposis are neenie nasal polyps and rhinitis symptoms, (2) the restoration of breathing through the nose and smell, and (3) prevention of relapse. The blockage of the nasal passage smaller number of larger polyps can be treated with a simple polypectomy, to help the patient to breathe through the nose. The aim of surgery is to restore the physiological properties of the nose by releasing this airway from polyps, to the extent possible, and allow drainage of infected sinuses. However, recurrent nasal polyposis is one of the most common unresolved issues of clinical rhinology. Complementary medicinal treatment of polyposis is always necessary, as the surgery cannot cure the inflammatory component of this disease mucosa. Local corticosteroids are the most commonly used drugs to reduce the size of polyps and to prevent recurrence after surgery. Steroids reduce rhinitis, improve breathing through the nose, reduce the size of polyps and reduce the frequency of relapses, but they have little effect on the sense of smell and any abnormality of the sinuses. However, the use of steroids when polyposis is associated with infectious complications, which require used the 1-receptor (for example, azelastine-Hcl) and antidiuretic (diuretics) tools (e.g., furosemide). These treatments are not always effective and the recurrence rate is still very high. Modern medical treatment of nasal polyposis uses corticosteroids to alleviate the symptoms of this disease, but it is not effective against the underlying pathology of this disease. In addition, patients with naselenie polyps observed relapse or resistance to steroid therapy.

Graft rejection

Graft rejection is a complex process by which transplantirovannam fabric is recognized as foreign by the immune system of the host. On the basis of morphology and the basic mechanism of the reaction can be divided into three categories: surgestrip, acute and chronic. With the elimination of the risk of infection and early (acute) rejection using immunosuppressive therapy of chronic rejection has become an increasingly important cause of graft dysfunction and ultimate failure after transplantation. Currently, chronic vascular rejection is the main cause of death or failure transplantation in recipients transplanted hearts about what eupresidency inflammatory diseases. These compositions and methods aimed at solving the problems associated with existing procedures, provide significant advantages when compared with existing procedures and provide other related advantages.

The INVENTION

Briefly, this invention provides methods of treating or preventing inflammatory diseases, including delivery to the site of inflammation antimicrotubule agent, acting on microtubules). Typical examples of such agents include taxanes (e.g. paclitaxel and docetaxel), computacin, eleutherobin, sarcodictyin, epothilone and discodermolide, deuterium oxide (D2O), hexyleneglycol (2-methyl-2,4-pentanediol), tubercidin (7-deazaadenosine), LY290181 (2-amino-4-(3-pyridyl)-4H-naphthol(1,2-b)Piran-3-carbonitrile), aluminum fluoride, bis(Succinimidyl)of ethylene glycol, ethyl ester of glycine, monoclonal antiidiotypic antibodies, a protein that stimulates microtubule Assembly (taxonomically protein, TALP), cellular swelling induced by hypotonic (190 mosmol/l) conditions, insulin (100 nmol/l) or glutamine (10 mmol/l), the binding of dynein, gibberelin, HSNO (kinesiophobia b is sin), buffers with glycerol buffer with Triton X-100, stabilizing microtubules associated with microtubule proteins (for example, MAR, MAR, Tau, big Tau, ensconsed, elongation factor 1-(EF-1) and E-MAP-115), cellular particles (e.g., histone H1, myelin basic protein and kinetochore), endogenous microtubule-based structures (e.g., axoneme structure, macromolecular complexes of enzymes that form pores in the cell membrane and GTP-caps), stable Trubeckoy the only polypeptide (e.g., STOP145 and STOP220) and tension from mitotic forces, as well as any analogues and derivatives of any of the above agents. In other embodiments, antimicrotubule agent is prepared in such a way that it additionally contains a polymer.

Typical examples of inflammatory diseases that can be treated include multiple sclerosis, psoriasis, arthritis, stenosis, graft rejection, surgical adhesions, inflammatory bowel disease and inflammatory disease of the lungs.

In some embodiments of the present invention antimicrotubule agents can be prepared together with other compounds or comp the composition can function as a carrier, which may be polymeric or polimernym. Typical examples of polymeric carriers include poly(ethylene vinyl acetate), copolymers of lactic acid and glycolic acid, poly(caprolactone), poly( lactic acid), copolymers of poly(lactic acid) and poly(caprolactone), gelatin, hyaluronic acid, collagen matrices, and albumin. Typical examples of other suitable carriers include, but are not limited to, ethanol; ethanol and glycols (e.g. ethylene glycol or propylene glycol); a mixture of ethanol and isopropylmyristate or ethanol, isopropylmyristate and water (e.g., 55:5:40); a mixture of ethanol and areola or D-limonene (with or without water; glycols (e.g. ethylene glycol or propylene glycol) and mixtures of glycols, such as propylene glycol and water, phosphatidylglycerol, dioleoylphosphatidylserine, Transcutol®, or terpinolene; a mixture of isopropylmyristate and 1-hexyl-2-pyrrolidone, N-dodecyl-2-piperidine or 1-hexyl-2-pyrrolidone.

In other aspects, antimicrotubule agent can be prepared in such a way that it is contained in a surgical or medical device or implant, or adapted to release such a device or implant, such subramania will become apparent by reference to the following detailed description and the accompanying drawings. In addition, below are various links that describe in more detail certain procedures, device, or composition and, therefore, incorporated by reference in its entirety.

BRIEF DESCRIPTION of DRAWINGS

Fig.1A is a graph showing the reaction chemiluminescence of neutrophils (5×106cells/ml) on opsonization plasma CPPD crystals (50 mg/ml). Effect of paclitaxel (also known as "Taxol") at (o) no paclitaxel, () of 4.5 μm, () 14 μm, () 28 μm,46 µm; n=3.

Fig.1B is a graph showing the concentration dependence of the time course of inhibition of paclitaxel-induced opsonization plasma CPPD crystals chemiluminescence.

Fig.1C is a graph showing the effect of fluoride on aluminum induced opsonization simhasanam activation of neutrophils, as measured by chemiluminescence.

Fig.1D is a chart showing the action of the ethyl ester of glycine on induced opsonization simhasanam activation of neutrophils, as measured by chemiluminescence.

Fig.1E is a graph showing the effect LY290181 on induced opsonization is of an unforgettable lysozyme from neutrophils (5×106cells/ml) in response to opsonization plasma CPPD crystals (50 mg/ml). Effect of paclitaxel at (o) no paclitaxel, () 28 μm, () control (cells alone), () control (cells and paclitaxel at 28 μm); n=3.

Fig.3A is a graph showing the formation of superoxide anion by neutrophils (5×106cells/ml) in response to opsonization plasma CPPD crystals (50 mg/ml). Effect of paclitaxel at (o) no paclitaxel, () 28 μm, () control (cells alone); n=3.

Fig.3B is a graph showing the concentration dependence of the time course of inhibition by paclitaxel induced opsonization plasma crystals CPPD formation of superoxide anion by neutrophils; n=3.

Fig.3C is a graph depicting the effect LY290181 on induced CPPD crystals the formation of superoxide anion by neutrophils.

Fig.4A is a graph showing the reaction chemiluminescence of neutrophils (5×106cells/ml) on opsonizing plasma, zymosan (1 mg/ml). Effect of paclitaxel at (o) no paclitaxel, () 28 µm; n=3.

Fiona. Effect of paclitaxel at (o) no paclitaxel, () 28 μm, () control (cells alone); n=3.

Fig.5A is a graph showing the release of myeloperoxidase from neutrophils (5×106cells/ml) in response to opsonization plasma CPPD crystals (50 mg/ml). Effect of paclitaxel at (o) no paclitaxel, () 28 μm, () control (cells alone), () control (cells with paclitaxel at 28 μm); n=3.

Fig.5B is a graph showing the concentration dependence of inhibition by paclitaxel release of myeloperoxidase from neutrophils in response to opsonization plasma CPPD crystals; n=3.

Fig.5C and 5D graphs showing that LY290181 reduces release as lysozyme and myeloperoxidase in induced CPPD crystals neutrophils.

Fig.6 is a graph showing the proliferation of synoviocytes at various concentrations of paclitaxel.

Fig.7 is a chart showing steps of paclitaxel on keratinocytes in vitro.

Fig.8A and 8B show the effect of paclitaxel on the morphology of astrocytes. Electron microscopic images revealed a thick, good animals, treated with paclitaxel, were morphologically altered astrocytes. Paclitaxel induced rounding of astrocyte, atonal cellular processes and reduced cytoplasmic filaments in comparison with untreated animals.

Fig.9 is a graph showing cell viability EOMA treated with concentrations of paclitaxel, greater than 10-8M

Fig.10 is a chart depicting the percentage of apoptotic cells EOMA in the culture treated with increasing concentrations of paclitaxel.

Fig.11A-11TH - graphs depicting the effect of various antithrombogenic agents on synoviocyte after a period of 24 hours.

Fig.12A-N - blots showing the effect of various antimicrotubule agents in the inhibition of expression of collagenase.

Fig.13A-13H - blots showing the effect of various antimicrotubule agents on the expression of proteoglycan.

Fig.14A and 14B are two pictures of HIMSELF with tumor treated control (unloaded) thermal grease. Briefly, in Fig.14A Central white mass tissue is a tumor. Note the abundance of blood vessels, forming tumor of HIMSELF in all directions. The tumor induces the ingrowth of vascular network of the host by the Ute. Fig.14C is a bottom view ITSELF, as shown in Fig.15A. In short, this view shows a radial view of the blood vessels, which are tumor like the spokes of a wheel. Note that the density of blood vessels more near the tumor than in the surrounding normal tissue ITSELF.

Fig.14C and 14D - two photos of HIMSELF with tumor treated is loaded with 20% paclitaxel by thermal grease. Briefly, in Fig.14C Central white mass tissue is a tumor. Note the paucity of blood vessels near the tumor tissue. Slow release antimicrotubular agent capable of overcoming the angiogenic stimulus produced by the tumor. The tumor is poorly vascularity and progressive decreases in size.

Fig.14D is a bottom view ITSELF, as shown in Fig.14C, and demonstrates the disruption of blood flow in the tumor compared to the control tumor tissue. Note that the density of blood vessels is reduced near the tumor and is more scarce than the density of the normal surrounding tissue ITSELF.

Fig.15A is a photograph showing the culture of eggs without the shell on the 6th day.

Fig.15V - discretione computer image, what's fills, showing capillary network chorioallantoic membrane (CAM), which provided a larger, underlying vessels (arrows; 1300×).

Fig.15D is a photograph depicting a plastic slice thickness of 0.5 mm, perpendicular to ITSELF and registered at the level of the light microscope. This photograph shows the structure ITSELF, including the outer two-layer ectoderm (EE), the mesoderm (M), containing capillaries (arrows) and scattered adventitia cells and a single layer of the endodermis (En) (400×).

Fig.15TH - photography-level electron microscope (3500×), which presents a typical capillary structure, showing thin-walled endothelial cells (heads of arrows) and associated pericic (adventitia cell).

Fig.16A, 16B, 16C and 16D series of sampled images of four different unpainted HIMSELF received after 48 hours of exposure to 10 μg of paclitaxel in 10 ml methylcellulose. Transparent disc methylcellulose ( * ) containing paclitaxel is present on each HIMSELF and is located on a separate avascular zone (A) with the surrounding blood Islands (Is). These devoid of vessels zones extend beyond the disk and usually have a diameter of approximately 6 mm

Fig.16D illustrates proci from the periphery of the avascular zone.

Fig.17A is a photograph (=400×), showing that the capillaries (head arrows), directly relatively peripheral avascular zone, reveal numerous endothelial cells with delayed mitosis. The ectoderm (EC), the mesoderm (m), endodermis (En).

Fig.17V (=400×) shows that in the avascular zone of a typical capillary structure has been eliminated and there are numerous transstilbene blood cells (heads of arrows). Fig.17C (=400×) shows that in the Central part of the avascular zone of the red blood cells dispersed in the mesoderm.

Fig.18A (=2200×) shows a small capillary, lying next to the ectodermal layer (EU) with three of endothelial cells arrested in mitosis (*). Several other types of cells in the ectoderm, and the mesoderm also arrested in mitosis.

Fig.18V (=2800×) displays devoid early vascular phase, which contains transstilbene blood cells near the ectoderm; these cells are mixed with a presumptive endothelial cells (*) and their processes. Shown degraded cell vacuoles (head arrows). Fig.18C (=h) shows that in response to paclitaxel surface section of the ectoderm-mesoderm becomes populated cell is IG.19A schematically depicts the transcriptional regulation of matrix metalloproteinases.

Fig.19C blot demonstrating that IL-1 stimulates the transcriptional activity of AP-1.

Fig.19 (C) is a diagram showing that induced by IL-1 binding activity was decreased in lysates from chondrocytes that were predobrabotki by paclitaxel.

Fig.20 - blot, showing that the induction of IL-1 increases the levels of RNA collagenase and stromelysin in chondrocytes and that this induction is inhibited by pretreatment with paclitaxel.

Fig.21 is a graph showing the effect of paclitaxel on the viability of normal chondrocytes in vitro.

Fig.22 is a graph showing the observed kinetic degradation of pseudobersama order of paclitaxel (20 mg per ml-1in solutions of 10% HPCD and 10% HPCD at 37°C and a pH of 3.7 and 4.9, respectively.

Fig.23 is a graph showing the phase solubility of cyclodextrins and paclitaxel in water at 37°C.

Fig.24 is a graph showing the graphics of the second order complex formation of paclitaxel andCD HPCD and HPCD at 37°C.

Fig.25 is a table showing the melting temperature, enthalpy, molecular weight, polydispersity icalneu scanning calorimetry) PDLLA-PEG-PDLLA and PEG. The heating rate was 10°C/min. Cm. Fig.30 in relation to the melting temperatures and enthalpies.

Fig.27 is a graph showing the cumulative release of paclitaxel-loaded with 20% paclitaxel cylinder PDLLA-PEG-PDLLA in SFR-albumen buffer at 37°C. the error Bars indicate the standard deviation of the 4 samples. Cylinders of 40% PEG ceased to be investigated in 4 days due to the destruction.

Fig.28A, 28C and 28C is graphs showing changes in size, length (A) diameter (b) and wet weight (C) loaded with 20% paclitaxel cylinder PDLLA-PEG-PDLLA at the time of the release of paclitaxel in vitro at 37°C.

Fig.29 is a table showing the mass loss and the change in the polymer composition of the cylinder PDLLA-PEG-PDLLA (loaded with 20% paclitaxel) at the time of the release in SFR-albumen buffer at 37°C.

Fig.30 is a graph showing the gel filtration chromatogram of the cylinder PDLLA-PEG-PDLLA (20% PEG, 1 mm in diameter) loaded with 20% paclitaxel, at the time of the release in SFR-albumen buffer at 37°C.

Fig.31A, B, C and 31D are photographs raster (scanning electronic microscopy (SEM) dried cylinders PDLLA-PEG-PDLLA (loaded with 20% paclitaxel, 1 mm in diameter) before the release of paclitaxel and during release palatinae the release of paclitaxel-loaded with 20% paclitaxel mixtures PDLLA-PCL and PCL in SFR-albumen buffer at 37°C. The error bars represent standard deviation of 4 samples.

Fig.33 is a graph showing the time course of the release of paclitaxel from PCL pastes in SFR at 37°C. PCL pastes contain microparticles of paclitaxel and various additives, prepared with the use of mesh No. 140. The error bars represent standard deviation of 3 samples.

Fig.34 is a graph showing the time course of the release of paclitaxel from pastes paclitaxel-gelatin-PCL SFR at 37°C. This graph shows the effect of the concentration of gelatin (mesh No. 140) and the size of the paclitaxel-gelatin (1:1) microparticles prepared with the use of mesh No. 140 or mesh No. 60. The error bars represent standard deviation of 3 samples.

Fig.35A and 35V - graphs showing the effect of additives (17A; mesh No. 140) and the size of the microparticles (17V; mesh No. 140 or # 60) and the proportion of the additive (mesh No. 140) on the swelling properties of the PCL-pastes containing 20% paclitaxel, after suspension in distilled water at 37°C. Measurements for pasta, cooked with microparticles 270 μm in paclitaxel-gelatin, and a paste containing 30% gelatine was stopped after 4 hours due to the breakdown of the matrix. The error bars represent standard deviation of 3 samples.

Fig.36A, 36V, 36C and 36D are representative skanirovanno water at 37°C for 6 hours. Micrograph 36C and 36D are higher magnifications 36V, showing the delicate relationship of paclitaxel (in the form of sticks) and gelatin matrix.

Fig.37A and 37V constitute a representative micrograph HIMSELF, processed pastes gelatin-PCL (37A) and paclitaxel-gelatin-PCL (20:20:60; 37V), showing the zone, devoid of vessels treated with paclitaxel ITSELF.

Fig.38 is a graph showing the phase solubility of cyclodextrins and paclitaxel in water at 37°C.

Fig.39 is a graph showing the graphics of the second order complex formation of paclitaxel andCD HPCD or HPCD at 37°C.

Fig.40 is a graph showing the phase solubility for paclitaxel at 37°C and hydroxypropyl--cyclodextrin in solutions of 50:50 water:ethanol.

Fig.41 is a graph showing the profiles of dissolution rates of 0.5, 10, or 20% solutions HPCD at 37°C.

Fig.42 is a graph showing the observed kinetic degradation of pseudo-first order of paclitaxel (20 mg/ml) in solutions of 10% HPCD and 10% HPCD at 37°C and a pH of 3.7 and 4.9 their paclitaxel from EVA films and the percentage of paclitaxel, remaining in the same films over time.

Fig.And 43C is a graph showing the swelling of the films EVA/F127 without paclitaxel in time.

Fig.43D - Rafiq, showing the swelling of the films EVA/Span 80 without paclitaxel in time.

Fig.43TH is a graph showing the curve of the dependence of stress on strain (figure voltage) for various blends of EVA/F127.

Fig.44 is a graph showing the effect of opsonization by plasma polymer microspheres on the reaction chemiluminescence of neutrophils (20 mg/ml microspheres in 0.5 ml of cells (conc. 5×106cells/ml)] at PCL microspheres.

Fig.45 is a graph showing the effect of pre-coating plasma +/-2% pluronic F127 on the chemiluminescence reaction of neutrophils (5×106cells/ml) on the PCL microspheres.

Fig.46 is a graph showing the effect of pre-coating plasma +/-2% pluronic F127 on the chemiluminescence reaction of neutrophils (5×106cells/ml) on microspheres emission spectra obtained for pure.

Fig.47 is a graph showing the effect of pre-coating plasma +/-2% pluronic F127 on the chemiluminescence reaction of neutrophils (5×106cells/ml) in PLA microspheres.

Fig.48 is a graph showing the effect of pre-coating plasma +/-2% pluronic F127 on the chemiluminescence reaction n is th coating IgG (2 mg/ml) or 2% pluronic F127 and then IgG (2 mg/ml) to the reaction chemiluminescence of neutrophils on the PCL microspheres.

Fig.50 is a graph showing the effect of pre-coating of IgG (2 mg/ml) or 2% pluronic F127 and then IgG (2 mg/ml) to the reaction chemiluminescence of neutrophils on microspheres emission spectra obtained for pure.

Fig.51 is a graph showing the effect of pre-coating of IgG (2 mg/ml) or 2% pluronic F127 and then IgG (2 mg/ml) to the reaction chemiluminescence of neutrophils on PVA microspheres.

Fig.52 is a graph showing the effect of pre-coating of IgG (2 mg/ml) or 2% pluronic F127 and then IgG (2 mg/ml) to the reaction chemiluminescence of neutrophils on microspheres EVA:PLA.

Fig.53A is a graph showing the profiles of the velocity of the release polycaprolactone microspheres containing 1, 2, 5 or 10% paclitaxel in buffered phosphate solution at 37°C.

Fig.V is a photograph showing HIMSELF treated with control microspheres.

Fig.S is a photograph showing HIMSELF, processed, loaded with 5% paclitaxel microspheres.

Fig.54 is a graph showing the particle size range for the control microspheres (PLLA:GA-85:15).

Fig.55 is a chart showing the range of sizes of particles to be loaded with 20% paclitaxel microspheres (PLLA:GA-5:15).

Fig.56 is a graph showing the particle size range for the control microspheres (PLLA:GA - 85:15).

Fig.57 - ha, 58B and S - graphs showing the dependence of the velocity profiles of the release of paclitaxel from different ranges of size of the microspheres and various ratios of PLLA and GA.

Fig.59A and B - graphs showing the profiles of the velocity of the release of paclitaxel from microspheres with different ratios of PLLA and GA.

Fig.60A and 60B - graphs showing the profiles of the velocity of the release of paclitaxel from microspheres with different ratios of PLLA and GA.

Fig.61A, B and 61C - graphs showing the profiles of the velocity of the release of paclitaxel from microspheres of different sizes and with different ratios of PLLA and GA.

Fig.62 is a graph showing the release of paclitaxel from paclitaxel-nylon microcapsules.

Fig.63A and B - pictures-coated fibronectin PLLA-microspheres in the tissue of the bladder (63A) and poly(L-lysine) microspheres in the tissue of the bladder.

Fig.64 is a graph showing that micellar paclitaxel improves daily average scores of arthritis in a rat model of collagen-induced arthritis.

Fig.65A-65D - the number of radiographs that show the effect of micellar paclitaxel in the rat model of collagen-induced arthritis.

Fig.66A-C - scanning electron micrograph gazirovannaja collagen arthritis.

Fig.68A and B enlarged views of the synovial vasculature in a rat model of collagen-induced arthritis.

Fig.69 is a graph showing the induction of contact allergic reactions in mouse ears oxazolone. Processing 1% gel paclitaxel or media during antigenic stimulation and then once a day. Skin inflammation was assessed quantitatively by measuring the swelling of the ears in comparison with the thickness of the ears before antigenic stimulation. Results represent mean values +/- SD (standard deviation) (n=5). **p<0,01; ***p<0,001.

Fig.70 is a graph showing the induction of contact allergic reactions in mouse ears oxazolone. Initial processing 1% gel paclitaxel or carrier for 24 hours after antigenic stimulation and then once a day. Skin inflammation was quantified by measuring the swelling of the ears in comparison with the thickness of the ears before antigenic stimulation. Results represent mean values +/- SD (standard deviation) (n=5). *p<0,05; **p<0,01.

Fig.71 is a graph showing the induction of skin inflammation in mouse ears by local application of PMA. Initial processing 1% gel paclitaxel or media at 1 hour after the application of the ranking, with thick ears before antigenic stimulation. Results represent mean values +/- SD (standard deviation) (n=5). *p<0,05; **p<0,01.

Fig.72 is a graph showing the induction of skin inflammation in mouse ears by local application of PMA. Initial processing 1% gel paclitaxel or carrier for 24 hours after application of PMA and then once a day. Skin inflammation was quantified by measuring the swelling of the ears in comparison with the thickness of the ears before antigenic stimulation. Results represent mean values +/- SD (standard deviation) (n=5). **p<0,01; ***p<0,001.

Fig.73 illustrates the induction of skin inflammation in mouse ears by local application of PMA. Pretreatment with 1% gel paclitaxel (right ear) or vehicle (left ear). Images were obtained at 48 hours after the application of the RMA. Note the redness and dilated blood vessels handled by the media of the left ear, in comparison with the processed paclitaxel right ears. The same results were obtained in General by 5 mice.

Fig.74 is a graph showing the effect of paclitaxel on the body weight of transgenic mice DM20. Transgenic mice were treated with media or paclitaxel (2.0 mg/kg) three times per week for 24 days and then were killed on day 27. Resultlabel animals showed minimal weight loss, whereas control animals showed a 30% decrease in body weight, with 29 g to 22 g

Fig.75 is a graph showing the effect of therapy with high doses of paclitaxel provided at intervals on the progression of clinical symptoms in transgenic mice. Transgenic mice were treated with 20 mg/kg of paclitaxel once a week for 4 weeks (week 0, 1, 2 and 3) and were followed for 10 weeks, every two days, with points defined for each symptom. The results represent the average score (on a cumulative basis for all symptoms) treated with paclitaxel transgenic mice (n=5) and control mice (n=3). Treatment with paclitaxel reduced the deterioration caused by overexpression of DM20 in transgenic mice, whereas control mice were found very rapid deterioration, and 2 of 3 mice did not survive to the end of the experimental Protocol (as shown).

Fig.76A and B show paste of paclitaxel caused perivascular (adventitious membrane of blood vessels) in a rat model of carotid artery. Adventitious surface of the left common carotid artery was treated with 2.5 mg or control paste (76A), or loaded with 20% paclitaxel pasta (V). The control artery was found to increase the, processed loaded with paclitaxel paste, found no evidence of intimal thickening.

Fig.77A and B depict the influence of the proximity perivascular paste of paclitaxel in rat model of carotid artery. Loaded with paclitaxel paste, applied directly adjacent to the perivascular area of the vessel to prevent restenosis; however, when the paste was not directly next to the vessel wall, was apparent hyperplasia of the newly-forming intima.

Fig.78A, B and C show the effect of paclitaxel on GFAP-staining of astrocytes. The slices of the brain of normal animals and transgenic animals (who developed a neurological disease similar to multiple sclerosis (MS) treated with media or paclitaxel, were stained with GFAP (a marker for activated astrocytes) and examined histologically. In the control transgenic mice had increased numbers of astrocytes and General levels of GFAP in comparison with sections of normal brain. However, the morphology of these cells was the same. The brain slices treated with paclitaxel transgenic mice showed a reduced number of astrocytes and GFAP levels compared to untreated transgenic animals. Histologically observed rounding to the villages inhibits the stimulation of T cells in response to peptide myelin basic protein (GP68-88) and Kona. 48-hour culture, the proliferation of T-cells RT-1 were GP68-88 (A) or Kona () as stimulates. Paclitaxel and its media (micelles) was added at different concentrations at the beginning of antigenic stimulation or after 24 hours. Paclitaxel inhibited the proliferation of T-cells in such low concentrations as 0.02 microns, regardless of stimulate.

Fig.80A, 80V, 80C and 80D - graphs showing that tubercidin and paclitaxel inhibit both as IL-1 and TNF-induced activity of NF-KB.

Fig.A and W - graphs showing the effect of increasing concentrations of paclitaxel or camptothecin on the growth of cancer cells of the prostate man (LNCaP) (2×103cells/ml) measured by crystal violet staining dye (5%) and quantitative determination by absorbance at 492 nm. Percent growth is expressed as % relative to controls, and given an average of 8 results.

DETAILED description of the INVENTION

Before describing the invention may be useful for understanding its present definitions of some terms that will be used next.

The term "inflammatory disease", in the application here, refers to any of a number of diseases that the nature of the capabilities); infiltration of tissues by mononuclear cells; tissue destruction by inflammatory cells, connective tissue cells and their cellular products; and attempted repair by replacement of connective tissue (e.g., chronic inflammatory reactions). Typical examples of such diseases include many common medical conditions such as arthritis, atherosclerosis, psoriasis, inflammatory bowel disease, multiple sclerosis, surgical adhesions, restenosis, tuberculosis, graft rejection, and chronic inflammatory respiratory disease (e.g. asthma, pneumoconiosis, chronic obstructive pulmonary disease, nasal polyps and pulmonary fibrosis).

The term "antimicrotubule agents" includes any protein, peptide, chemical, or other molecule that disrupts the function of microtubules, for example, through the prevention or the stabilization of the polymerization. A great variety of methods can be used to determine antimicrotubule activity of specific compounds, including, for example, the tests described by Smith et al., (Cancer Lett 79(2): 213-219) and Mooberry et al., (Cancer Lett. 96(2): 261-266, (1995).

As noted above, this invention PR is asplenia antimicrotubule agent. Briefly, a wide variety of agents can be delivered to the site of inflammation (or potential site of inflammation), or with the carrier, or without a carrier (e.g., polymer or ointment) for the treatment or prevention of inflammatory diseases. Typical examples of such agents include taxanes (e.g. paclitaxel (discussed in more detail below) and docetaxel) (Schiff et al., Nature 277: 665-667, 1979; Long and Fair-child, Cancer Research 54: 4355-4361, 1994; Ringel and Horwitz, J. Natl. Cancer Inst. 83(4): 288-291, 1991; Pazdur et al., Cancer Treat. Rev. 19(4): 351-386, 1993), computacin, eleutherobin (for example, U. S. Patent No. 5473057), sarcodictyin (including sarcodictyin A), epothilone a and b (Bollag et al., Cancer Research 55: 2325-2333, 1995), discodermolide (ter Haar et al., Biochemistry 35: 243-250, 1996), deuterium oxide (D2O) (James and Lefebvre, Genetics 130(2): 305-314, 1992; Sollott et al., J. Clin. Invest. 95: 1869-1876, 1995), hexyleneglycol (2-methyl-2,4-pentanediol) (Oka et al., Cell Struct. Funct. 16(2): 125-134, 1991), tubercidin (7-deazaadenosine) (Mooberry et al., Cancer Lett. 96(2): 261-266, 1995), LY290181 (2-amino-4-(3-pyridyl)-4H-oil(1,2-b)Piran-3-carbonitrile) (Panda et al., J. Biol. Chem. 272(12): 7681-7687, 1997; Wood et al., Mol. Pharmacol. 52(3): 437-444, 1997), aluminum fluoride (Song et al., J. Cell. Sci. Suppl. 14: 147-150, 1991), bis(Succinimidyl) of ethylene glycol (Caplow and Shanks, J. Biol. Chem. 265(15): 8935-8941, 1990), ethyl ester of glycine (Mejillano et al., Biochembstry 31(13): 3478-3483, 1992), monoclonal antiadipogenic protein, TALP) (Hwang et al., Biochem. Biophys. Res. Commun. 208(3): 1174-1180, 1995), cell swelling induced by hypotonic (190 mosmol/l) conditions, insulin (100 nmol/l) or glutamine (10 mmol/l) (Haussinger et al., Biochem. Cell. Biol. 72(1-2): 12-19, 1994), the binding of dynein (Ohba et al., Biochem. Biophys. Acta 1158(3): 323-332, 1993), gibberellin (Mita and Shibaoka, Protoplasma 119(1/2): 100-109, 1984), XCHO1 (kinesiology protein) (Yonetani et al., Mol. Biol. Cell 7(suppl): 211A, 1996), lysophosphatidic acid (Cook et al., Mol. Biol. Cell 6(suppl): 260A, 1995, lithium ion (Bhattacharyya and Wolff, Biochem. Biophys. Res. Commun. 73(2): 383-390, 1976), components of the cell wall of plants (e.g., poly-L-lysine and extenion) (Akashi et al., Planta 182(3): 363-369, 1990), glycerol buffers (Schilstra et al., Biochem. J. 277(Pt.3): 839-847, 1991; Farrell and Keates, Biochem. Cell. Biol. 68(11): 1256-1261, 1990; Lopez et al., J. Cell. Biochem. 43(3): 281-291, 1990), buffer with Triton X-100, stabilizing microtubules (Brown et al., J. Cell Sci. 104(Pt.2): 339-352, 1993; Safiejko-Mroczka and Bell, J. Histochem. Cytochem. 44(6): 641-656, 1996), associated with microtubule proteins (for example, MAR, MAR, Tau, big Tau, ensconsed, elongation factor 1-alpha (EF-1) and E-MAP-115), (Burgess et al., Cell Motil. Cytoskeleton 20(4): 289-300, 1991; Saoudi et al., J. Cell. Sci. 108 (Pt.1): 357-367, 1995; Bulinski and Bossier, J. Cell. Sci. 107(Pt.10): 2839-2849, 1994; Ookata et al., J. Cell Biol. 128(5): 849-862, 1995; Boyne et al., J. List. Neurol. 358(2): 279-293, 1995; Ferreira and Caceres, J. Neurosci. 11(2): 392-400, 1991; Thurston et al., Chromosoma 105(1): 20-30, 1996; Wang et al. Brain Res. Mol. Brain Res. 38(2): 200-208, 1996; Moore'an who ate protein and kinetochore) (Saoudi et al., J. Cell. Sci. 108(Pt.1): 357-367, 1995; Simerly et al., J. Cell Biol. 111(4): 1491-1504, 1990), endogenous microtubule-based structures (e.g., axoneme structure, macromolecular complexes of enzymes that form pores in the cell membrane and GTP-caps) (Dye et al., Cell Motil. Cytoskeleton 21(3): 171-186, 1992; Azhar and Murphy, Cell Motil. Cytoskeleton 15(3): 156-161, 1990; Walker et al., J. Cell Biol. 114(1): 73-81, 1991; Drechsel and Kirschner, Curr. Biol. 4(12); 1053-1061, 1994), stable Trubeckoy the only polypeptide (e.g., STOP145 and STOP220) (Pirollet et al., Biochem. Biophys. Acta 1160(1): 113-119, 1992; Pirollet et al. Biochemistry 31(37): 8849-8855, 1992; Bosc et al. Proc. Natl. Acad. Sci. USA 93(5): 2125-2130, 1996; Margolis et al., EMBO J. 9(12): 4095-4102, 1990) and tension from mitotic forces (Nicklas and Ward, J. Cell Biol. 126(5): 1241-1253, 1994), as well as any analogues and derivatives of any of the above agents. Such compounds can act or by depolymerization of microtubules (e.g., colchicine and vinblastine) or by stabilizing the formation of microtubules (e.g., paclitaxel).

In one preferred embodiment of this invention therapeutic agent is paclitaxel, a compound that disrupts the formation of microtubules by binding to tubulin with the formation of abnormal mitotic spindles. Briefly, paclitaxel is vysokomineralizovannye diterpenoid (Wani et al., J. Am.ae and endophytic fungus (Endophytic Fungus) Pacific yew (Stierle et al., Science 60: 214-216, 1993). "Paclitaxel" (which should be clear, includes prodrugs, analogues and derivatives such as, for example, TAXOL®, TAXOTERE®, docetaxel, 10-desacetyl-analogues of paclitaxel and 3'N-desbenzoyl-3'N-t-butoxycarbonyl-analogues of paclitaxel) may be readily obtained by using methods known to experts in this field (see, e.g., Schiff et al., Nature 277: 665-667, 1979; Long and Fairchild, Cancer Research 54: 4355-4361, 1994; Ringel and Horwitz, J. Natl. Cancer Inst. 83(4): 288-291, 1991; Pazdur et al., Cancer Treat. Rev. 19(4): 351-386, 1993; WO 94/07882, WO 94/07881; WO 94/07880; WO 94/07876; WO 93/23555; WO 93/10076; WO 94/00156; WO 93/24476; EP 590267; WO 94/20089; U. S. Patent Nos. 5294637; 5283253; 5279949; 5274137; 5202448; 5200534; 5229529; 5254580; 5412092; 5395850; 5380751; 5350866; 4857653; 5272171; 5411984; 5248796; 5248796; 5422364; 5300638; 5294637; 5362831; 5440056; 4814470; 5278324; 5352805; 5411984; 5059699; 4942184; Tetrahedron Letters 35(52): 9709-9712, 1994; J. Med. Chem. 35: 4230-4237, 1992; J. Med. Chem. 34: 992-998, 1991; J. Natural Prod. 57(10): 1404-1410, 1994; J. Natural Prod. 57(11): 1580-1583, 1994; J. Am. Chem. Soc. 110: 6558-6560, 1998) or obtained from various commercial sources, including, for example, (Sigma Chemical Co., St. Louis, Missouri (T7402 from Taxus brevifolia).

Typical examples of such derivatives or analogs of paclitaxel include 7-deoxycortisol, 7,8-cyclopropylamine, N-substituted 2-azetidine, 6,7-epoxyacrylate, 6,7-modified paclitaxel, 10-desiredaccuracy, 10-deacetyltaxol (from 10-deuterostomia 10-deacetoxy-11,12-dihydroxy-10,12(18)-diene, 10-desiredaccuracy, Protocol (2’-and/or 7-O-ester derivatives), (2’-and/or 7-O-carbonate derivatives), asymmetric synthesis of the side chain of Taxol, verticaly, 9-doxetaxel, (13-acetyl-9-deoxyactein III, 9-doxetaxel, 7-deoxy-9-doxetaxel, 10-deacetoxy-7-deoxy-9-doxetaxel, derivatives containing hydrogen or acetyl group and a hydroxy and tert-butoxycarbonylamino, derivatives of sulfonated 2’-acryloyloxy and sulfonated 2’-O-alltexas, succinicacid, 2’-X-aminoethanesulfonic, 2’-acetyloxy, 7-acetyloxy, 7-gitinternational, 2’-OH-7-PEG(5000)-carbomethoxy, 2’-benzoyl and 2’,7-dibenzoylperoxide Taxol, other prodrugs (2’-acetyloxy; 2’,7-deacetyltaxol; 2’-succinicacid; 2’-(beta-alanyl)-Taxol); formate, 2’-gamma-aminobutyrate; containing ethylene glycol derivatives of 2’-succinylcholine; 2’-glutaredoxin; 2’-(N,N-dimethylglycine)Taxol; 2’-(2-(N,N-dimethylamino)propionyl)Taxol; 2’-orthocarboxylic; containing 2'-aliphatic carboxylic acid derivatives of Taxol, prodrugs {2’-(N,N-diethylaminopropyl)Taxol, 2’-(N,N-dimethylglycine)Taxol, 7-(N,N-dimethylglycine)Taxol, 2’,7-di(N,N-dimethylglycine)Taxol, 7-(N,N-diethylaminopropyl)Taxol, 2’, the Sol, 7-(L-alanyl)Taxol, 2’,7-di(L-alanyl)Taxol, 2’-(L-leucyl)Taxol, 7-(L-leucyl)Taxol, 2’,7-di(L-leucyl)Taxol, 2’-(L-isoleucyl)Taxol, 7-(L-isoleucyl)Taxol, 2’,7-di(L-isoleucyl)Taxol, 2’-(L-felled)Taxol, 7-(L-felled)Taxol, 2’,7-di(L-felled)Taxol, 2’-(L-i.e. phenylalanyl)Taxol, 7-(L-i.e. phenylalanyl)Taxol, 2’,7-di(L-i.e. phenylalanyl)Taxol, 2’-(L-prolyl)Taxol, 7-(L-prolyl)Taxol, 2’,7-di(L-prolyl)Taxol, 2’-(L-lysyl)Taxol, 7-(L-lysyl)Taxol, 2’,7-di-(L-lysyl)Taxol, 2’-(L-glutamyl)Taxol, 7-(L-glutamyl)-Taxol, 2’,7-di(L-glutamyl)Taxol, 2’-(L-arginyl)Taxol, 7-(L-arginyl)Taxol, 2’,7-di(L-arginyl)Taxol}, Taxol analogues with modified side chains phenylazomethine, Taxotere, (N-desbenzoyl-N-tert-(butoxycarbonyl)-10-deacetyltaxol, and taxanes (e.g., baccatin III, cephalomannine, 10-deazetil-baccatin III, brevifolia, unintersting and taxesin).

Typical examples of agents depolymerization of microtubules (or destabilization or destruction) include nocodazole (Ding et al., J. Exp. Med. 171(3): 715-727, 1990; Dotti et al., J. Cell Sci. Suppl. 15:75-84, 1991; Oka et al., Cell Struct. Funct. 16(2): 125-134, 1991; Wiemer et al., J. Cell. Biol. 136(1): 71-80, 1997); cytochalasin In (Illinger et al., Biol. Cell 73 (2-3): 131-138, 1991); vinblastine (Ding et al., J. Exp. Med. 171(3): 715-727, 1990; Dirk et al., Neurochem. Res.15 (11): 1135-1139, 1990; Illinger et al., Biol. Cell 73(2-3): 131-138, 1991; Wiemer et al., J. Cell. Biol. 136(1): 71-80, 1997); Vince is, . Exp. Med. 171(3): 715-727, 1990; Gonsales et al., Exp. Cell. Res.192(1): 10-15, 1991; Stargell et al., Mol. Cell. Biol. 12(4): 1443-1450, 1992); CI 980 (similar to colchicine) (Garcia et al., Anticancer Drugs 6(4): 533-544, 1995); colcemid (Barlow et al., Cell. Motil. Cytoskeleton 19(1): 9-17, 1991; Meschini et al., J. Microsc. 176(Pt.3): 204-210, 1994; Oka et al., Cell Struct. Funct. 16(2): 125-134, 1991); podofillotoksin (Ding et al., J. Exp. Med. 171(3): 715-727, 1990); benomyl (Hardwick et al., J. Cell. Biol. 131 (3): 709-720, 1995; Shero et al., Genes Dev. 5(4): 549-560, 1991); oryzalin (Stargell et al., Mol. Cell. Biol. 12(4): 1443-1450, 1992); musculated (Moore, J. Ind. Environ. 16(2): 134-143, 1996); demecolcine (Van Dolah and Ramsdell, J. Cell. Physiol. 166(1): 49-56, 1996; Wiemer et al., J. Cell. Biol. 136(1): 71-80, 1997); and methyl-2-benzo-imidazolecarboxamide (MVS) (Brown et al., J. Cell. Biol. 123(2): 387-403, 1993).

Ready preparative form.

As noted above, therapeutic antimicrotubule agents described herein, can be prepared in various ways and, therefore, can further comprise a carrier. In this respect, there may be selected a wide variety of media or polymer, or polimernogo origin.

For example, in one embodiment of the present invention can be used in a variety of polymeric carriers, which may include and/or be delivered to one or more discussed above therapeutic agents, including, for example, as biodegradable and non-biodegradation, hyaluronic acid, starch, cellulose (methylcellulose, hydroxypropylcellulose, hypromellose, carboxymethyl cellulose, acetate-phthalate cellulose acetate-cellulose succinate, phthalate of hydroxypropylmethylcellulose), casein, dextrans, polysaccharides, fibrinogen, poly(D,L-lactide), copolymers of poly(D,L-lactide-glycolide), poly(glycolide), poly(hydroxybutyrate), poly(alkalicarbonate) and poly(ortho-esters), polyesters, poly(hydroxyvaleric acid), polydioxanone, poly(ethylene-terephthalate), poly(malic acid), poly(Castronovo acid), polyanhydride, polyphosphazene, poly(amino acid) and their copolymers (see in General. Illum, L., Davids, S. S. (eds) "Polymers in Controlled Drug Delivery" Wright, Bristol, 1987; Arshady, J. Controlled Release 17: 1-22, 1991; Pitt, Int. J. Phar. 59: 173-196, 1990; Holland et al., J. Controlled Release 4: 155-0180, 1986). Typical examples degradiruem polymers include copolymers of poly(ethylene vinyl acetate) (EVA), silicone rubber, acrylic polymers (polyacrylic acid, polymethylacrylate acid, polymethylmethacrylate, polyalkylacrylate), polyethylene, polypropylene, polyamides (nylon 6,6), polyurethanes, poly(ester-urethanes), poly(simple ether-urethanes), poly(ether-urea), poly-ethers, (poly(ethylene oxide), poly(propylene oxide), pluronic and the floor is), poly(vinyl acetate-phthalate). Can be obtained polymers, which are either anionic (e.g., alginate, carrageenin, carboxymethyl cellulose and poly(acrylic acid), or cationic (e.g., chitosan, poly-L-lysine, polyethylenimine and poly(allylamine)) (see General Dunn et al., J. Applied Polymer Sci. 50: 353-365, 1993; Cascone et al., J. Materials Sci.: Materials in Medicine 5: 770-774, 1994; Shiraishi et al., Biol. Pharm. Bull. 16(11): 1164-1168, 1993; Thacharodi and Rao, Int’1 J. Pharm. 120: 115-118, 1995; Miyazaki et al., Int’1 J. Pharm. 118: 257-263, 1995). Particularly preferred polymeric carriers include poly(ethylene vinyl acetate), oligomers and polymers, poly(D,L-lactic acid) oligomers and polymers, poly (L-lactic acid), poly(glycolic acid), copolymers of lactic acid and glycolic acid, poly(caprolactone), poly(valerolactone), polyanhydrides, copolymers of poly(caprolactone) or poly(lactic acid) with polyethylene glycol (such as PEG Me) and mixtures thereof.

Polymeric carriers can be prepared in various forms, with desired release characteristics and/or with specific desirable properties. For example, polymeric carriers may be prepared in such a way that they release therapeutic agent upon exposure to the specific trigger event; ang et al., J. Applied Polymer Sci. 48: 343-354, 1993; Dong et al., J. Controlled Release 19: 171-178, 1992; Dong and Hoffman, J. Controlled Release 15: 141-152, 1991; Kirm et al., J. Controlled Release, 28: 143-152, 1994; Cornejo-Bravo et al., J. Controlled Release 33: 223-229, 1995; Wu and Lee, Pharm. Res. 10(10): 1544-1547, 1993; Serres et al., Pharm Res. 13(2): 196-201, 1996; Peppas, "Fundamentals of pH - and Temperature-Sensitive Delivery Systems", in Gurny et al. (eds.), Pulsatile Drug Delivery, Wissenschaftliche Verlagsgesell-all mbH, Stuttgart, 1993, pp.41-55; Doelker, "Cellulose Derivatives", 1993, in Peppas and Langer (eds.), Biopolymers I, Springer-Verlag, Berlin). Typical examples of pH-sensitive polymers include poly(acrylic acid) and its derivatives (including, for example, homopolymers such as poly(aminocarbonyl acid); poly(acrylic acid); poly(methylacrylate acid), copolymers of these homopolymers and copolymers of poly(acrylic acid) and acelmonolo, such as discussed above. Other pH-sensitive polymers include polysaccharides such as acetate-phthalate cellulose; phthalate of hydroxypropylmethylcellulose; acetate-succinate of hydroxypropylmethylcellulose; acetate-trimellitate cellulose; and chitosan. Other pH-sensitive polymers include any mixture of pH-sensitive polymer and a water-soluble polymer.

Likewise can be made of polymeric carriers, which are temperature sensitive (see, for example Mater. 22: 167-168, Controlled Release Society, Inc., 1995; Okano, "Molecular Design of Stimuli-Responsive Hydrogels for Temporal Controlled Drug Delivery," in Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 22: 111-112, Controlled Release Society, Inc., 1995; Johnston et al., Pharm. Res. 9(3): 425-433, 1992; Tung, Int'l J. Pharm. 107: 85-90, 1994; Harsh and Gehrke, J. Controlled Release 17: 175-186, 1991; Bae et al., Pharm. Res.8(4): 531-537, 1991; Dinarvand and D Emanuele, J. Controlled Release 36: 221-227, 1995; Yu and Grainger, "Novel Thermosensitive Amphiphilic Gels: Poly N-isopropylacrylamid-co-sodium acrylate-co-n-N-alkylacrylamide Network Synthesis and Physicochemical Characterization", Dept of Chemical & Biological Sci., Oregon Graduate Institute of Science & Technology, Beaverton, OR, pp.820-821; Zhou and Smid, "Physical Hydrogels of Associative Star Polymers", Polymer Research Institute, Dept. of Chemistry, College of Environmental Science and Forestry, State Univ. of New York, Syracuse, NY, pp.822-823; Hoffman et al., "Characterizing Pore Sizes and Water ‘Structure’ in Stimuli-Responsive Hydrogels", Center for Bioengineering, Univ. of Washington, Seattle, WA, p.828; Yu and Grainger, "Thermosensitive Swelling Behavior in Crosslinked N-isopropylacrylamide Networks: Cationic, Anionic and Ampholytic Hydrogels", Dept. of Chemical & Biological Sci., Oregon Graduate Institute of Science & Technology, Beaverton, OR, pp.829-830; Kim et al., Pharm. Res.9(3): 283-290, 1992; Bae et al., Pharm. Res. 8(5): 624-628, 1991; Kono et al., J. Controlled Release 30: 69-75, 1994; Yoshida et al., J. Controlled Release 32: 97-102, 1994; Okano et al., J. Controlled Release 36:125-133, 1995; Chun and Kim, J. Controlled Release 38: 39-47, 1996; D Emanuele and Dinarvand, Int'l J. Pharm. 118: 237-242, 1995; Katono et al., J. Controlled Release 16: 215-228, 1991; Hoffman, "Thermally Reversible Hydrogels Containing Biologically Active Species", in Migliaresi et al.(eds). Polymers in Medicine III, Elsevier Science Publishers B. V., Amsterdam, 1988, pp.161-167; Hoffman, "Applications of Thermally Reversible Polymers and Hydrogels in Therapeutics and DiagnostiGehrke, J. Controlled Release 18: 1-12, 1992; Paavola et al., Pharm. Res. 12(12): 1997-2002, 1995).

Typical examples thermogenisis polymers and their gelation temperatures (LCST (°C)) include homopolymers such as poly(N-methyl-N-n-propylacetamide), 19,8; poly(N-n-propylacetamide), 21,5; poly(N-methyl-N-izopropilakrilamid), 22,3; poly(N-n-propylbetaine), 28,0; poly(N-izopropilakrilamid), 30,9; poly(N,N-n-diethylacrylamide), 32,0; poly(N-isopropylacrylamide), 44,0; poly(N-cyclopropylamine), 45,5; poly(N-athletically), 50,0; poly(N-methyl-N-ethylacrylate), 56,0; poly(N-cyclopropylmethyl), 59,0; poly(N-ethylacrylate), 72,0. In addition, thermogenisis polymers can be obtained by preparing copolymers between (among) the monomers described above, or Association of such homopolymers with other water-soluble polymers, such as agrimonetary (for example, acrylic acid and its derivatives, such as methylacrylate acid, acrylate and its derivatives, such as butylmethacrylate, acrylamide and N-n-butylacrylamide).

Other typical examples thermogenisis polymers include which contains a simple ester derivatives of cellulose, such as hydroxypropylcellulose, 41°C; methyl cellulose, 55°C; hydroxypropylmethyl-61, 24°C.

A great variety of forms can be prepared using polymeric carriers of the present invention, including the device in the form of sticks, balls, plates or capsules (see, e.g., Goodell et al., Am. J. Hosp. Pharm. 43: 1454-1461, 1986; Langer et al., "Controlled release of macromolecules from polymers", in Biomedical Polymers, Polymeric Materials and Pharmaceuticals for Biomedical Use, Goldberg, E. P., Nakagim, A. (eds.) Academic Press, pp.113-137, 1980; Rhine et al., J. Pharm. Sci. 69: 265-270, 1980; Brown et al., J. Pharm. Sci. 72: 1181-1185, 1983; Bawa et al., J. Controlled Release 1: 259-267, 1985). Therapeutic agents may be linked by occlusion in the matrices of the polymer, bound by covalent linkages, or encapsulated in microcapsules. In some preferred embodiments of the present invention, a therapeutic composition is provided in the unencapsulated public songs, such as microspheres (ranging in size from nanometers to micrometers), pastes, threads of various size, films and sprays.

Preferably, therapeutic compositions of this invention are prepared in a way that is suitable for the proposed use. In some aspects of the present invention, therapeutic composition should be biocompatible, and release one or more therapeutic agents over a period of several days to months. Napra 10, 20 or 25% (m/V) therapeutic agent (e.g., paclitaxel) over a period of 7-10 days. Such compositions "quick release" must be, in some embodiments, is capable of releasing the chemotherapeutic levels (where applicable) of the desired agent. In other embodiments, provided compositions "slow release", which release less than 1% (m/V) of therapeutic agent over a period of 7-10 days. In addition, therapeutic compositions of this invention should preferably be stable for a few months and let their preparation and storage under sterile conditions.

In some aspects of the present invention, therapeutic composition can be manufactured in any size ranging from 50 nm to 500 μm, depending on the particular application. Alternatively, such compositions can also be easily applied as a "spray", which cures in the form of a film or coating. Such sprays can be prepared from microspheres of a wide range of sizes, including, for example, from 0.1 μm to 3 μm, from 10 μm to 30 μm and 30 μm to 100 μm.

Therapeutic compositions of the present invention can also be prepared in a variety of forms of pastes which are liquid at one temperature (for example, at temperatures greater than 37°C, such as 40, 45, 50, 55°s or 60°C) and solid or semi-solid at another temperature (e.g., ambient body temperature, or any temperature lower than 37°C). Such "thermal paste" can be easily made on the basis of this description.

In yet another embodiment, therapeutic compositions of this invention can be manufactured in the form of a film. Preferably, such films have a thickness of less than 5, 4, 3, 2 or 1 mm, more preferably less than 0.75 mm or 0.5 mm, and most preferably less than 500 μm-100 μm. Such films are preferably elastic with good tensile strength (e.g., greater than 50, preferably greater than 100, and more preferably greater than 150 or 200 N/cm2), have good adhesive properties (i.e., easily attached to wet or damp surfaces) and have adjustable permeability.

In further aspects of this invention can be manufactured in therapeutic compositions for topical application. Typical examples include: ethanol; ethanol and glycols (e.g. ethylene glycol or propylene glycol); a mixture of ethanol and isopropylmyristate or ethanol, isopropylmyristate and the Col or propylene glycol) and mixtures of glycols, such as propylene glycol, and water, phosphatidylglycerol, dioleoylphosphatidylserine, Transcutol® or terpinolene; mixtures of isopropylmyristate and 1-hexyl-2-pyrrolidone, N-dodecyl-2-piperidine or 1-hexyl-2-pyrrolidone. Other fillers can be added to the above, including, for example, acids, such as oleic acid and linoleic acid, and Soaps, such as sodium lauryl sulfate. For a more detailed description of the above, see Hoelgaard et al., J. Contr. Rel. 2:111, 1985; Liu et al., Pharm. Res. 8: 938, 1991; Roy et al., J. Pharm. Sci. 83: 126, 1991; Ogiso et al., J. Pharm. Sci. 84: 482, 1995; Sasaki et al., J. Pharm. Sci. 80: 533, 1991; Okabe et al., J. Contr. Rel. 32: 243, 1994; Yokomizo et al., J. Contr. Rel. 38: 267, 1996; Yokomizo et al., J. Contr. Rel. 42: 37, 1996; Mond. et al., J. Contr. Rel. 33: 72, 1994; Michniak et al., J. Contr. Rel. 32: 147, 1994; Sasaki et al., J. Pharm. Sci. 80: 533, 1991; Baker & Hadgraft, Pharm. Res. 12: 993, 1995; Jasti et al., AAPS Proceedings, 1996; Lee et al., AAPS Proceedings, 1996; Ritschel et al., Skin Pharmacol. 4: 235, 1991; and McDaid & Deasy, Int. J. Pharm. 133: 71, 1996.

In some embodiments of the present invention, therapeutic compositions may also contain additional ingredients such as surfactants (e.g., pluronic, such as F-127, L-122, L-92, L-81, and L-61).

In further aspects of the present invention provided with polymeric carriers which are adapted to contain and visualaid, what Elcom or polypeptide. In some embodiments, this polymer medium contains or includes land, on or granules of one or more hydrophobic compounds. For example, in one embodiment of the invention the hydrophobic compounds may be incorporated in the matrix, which contains this hydrophobic compound, with subsequent incorporation of the matrix within the polymeric carrier. A variety of matrices can be used in this regard, including, for example, carbohydrates and polysaccharides, such as starch, cellulose, dextran, methyl cellulose and hyaluronic acid, proteins or polypeptides such as albumin, collagen and gelatin. In alternative embodiments, the hydrophobic compound may be contained in the hydrophobic Central part (the cortex), and this Central part is contained in the hydrophilic shell.

Other carriers described herein, which can also be used for the inclusion and delivery of therapeutic agents include hydroxypropyl--cyclodextrin (Cserhati and Hollo, Int. J. Pharm. 108:69-75, 1994), liposomes (see, for example, Sharma et al., Cancer Res. 53: 5877-5881, 1993; Sharma and Straubinger, Pharm. Res. 11(60): 889-896, 1994; WO 93/18751; U. S. Patent No. 5242073), liposome/gel (WO 94/26254), nanocapsules (by Bartoli et al., J. Mi-croencapsulat, ancer Res. 54: 2201-2212, 1994), nanoparticles (Violante and Lanzafame PAACR), the modified nanoparticles (U. S. Patent No. 5145684), nanoparticles (surface modified) (U. S. Patent No. 5399363), emulsion/solution of Taxol (U. S. Patent No. 5407683), the micelle (surfactant) (U. S. Patent No. 5403858), a synthetic phospholipid compounds (U. S. Patent No. 4534899), carrier gas dispersion (U. S. Patent No. 5301664), liquid, emulsion, foam, spray, gel, lotion, cream, ointment dispersed vesicles, particles or droplets of solid or liquid aerosols, micro-emulsions (U. S. Patent No. 5330756), a polymer membrane (nano - and microcapsule) (U. S. Patent No. 5439686), compositions based on taxoid in surface-active agent (U. S. Patent No. 5438072), emulsion (Tarr et al., Pharm. Res. 4: 62-165, 1987), nanospheres (Hagan et al., Proc. Intern. Symp. Control Rel. Bioact. Mater. 22, 1995); Kwon et al., Pharm. Res. 12(2): 192-195 (in Russian); Kwon et al., Pharm. Res. 10(7): 970-974; Yokoyama et al., J. Contr. Rel. 32: 269-277, 1994; Gref et al., Science 263: 1600-1603, 1994; Bazile et al., J. Pharm. Sci. 84: 493-498, 1994) and implants (U. S. Patent No. 4882168).

As discussed in more detail below, therapeutic agents of the present invention, which are not necessarily included in one of these carriers, can be prepared and applied for treating or preventing a wide variety of diseases.

Treatment or prevention of inflammatory diseases

As oceanically diseases, includes the stage of introduction of the patient antimicrotubule agent. Typical examples of inflammatory diseases that can be treated include, for example, atrophic gastritis, inflammatory hemolytic anemia, graft rejection, inflammatory neutropenia, bullous pemphigoid, coeliac disease, demyelinating neuropathies, dermatomyositis, inflammatory bowel disease (ulcerative colitis and Crohn's disease), multiple sclerosis, myocarditis, myositis, nasal polyps, chronic sinusitis, hand, foot vulgar, primary glomerulonephritis, psoriasis, surgical adhesions, stenosis or restenosis, scleritis, scleroderma, eczema (including atopic dermatitis, irritant dermatitis, allergic dermatitis) and diabetes type I.

Other examples of inflammatory diseases include vasculitis (e.g., giant cell arteritis diagnostics (temporal arteritis diagnostics, Takayasu's arteritis), Nowotny polyarteritis, allergic anghit and Wegener (disease Churg-Strauss), the syndrome of overlapping polyangiitis, allergic vasculitis (purple's disease-Seleina), serum sickness, induced by drugs vasculitis, infectious vasculitis, neopentecostalism complement system, Wegener's granulomatosis, Kawasaki disease, vasculitis of the Central nervous system, Buerger's disease and systemic sclerosis); diseases of the gastrointestinal tract (e.g., pancreatitis, Crohn's disease, ulcerative colitis, ulcerative proctitis, primary sclerosing cholangitis, benign stricture of any origin, including idiopathic (e.g., strictures of the bile ducts, the esophagus, the duodenum, the small intestine or colon); respiratory disease (e.g. asthma, allergic pneumonitis, asbestosis, silicosis and other forms of pneumoconiosis, chronic bronchitis and chronic obstructive disease of the Airways); diseases of the nasolacrimal duct (e.g., stricture of any origin, including idiopathic); and diseases of the Eustachian tubes (e.g., stricture of any origin, including idiopathic).

For further understanding of such diseases characterized by inflammatory diseases are discussed in more detail below.

1. Inflammatory skin diseases (e.g. psoriasis and eczema)

Using the claimed agents, compositions and methods can easily be treated or prevented a large variety of inflammatory skin what does eczema, can be treated or prevented by delivery to the site of inflammation (or potential site of inflammation) agent that inhibits the function of microtubules.

Briefly, skin cells are genetically programmed to perform two possible programs - normal growth or wound healing. In the case of normal growth patterns of skin cells are created in the basal cell layer and are then moved through the epidermis to the skin surface. Dead cells are flushed with a healthy skin at the same speed with which the formation of new cells. Turn-around time (i.e. the time from birth to death of the cells) for normal skin cells is approximately 28 days. During wound healing run faster growth and repair, leading to rapid turnover of skin cells to replace damaged cells and repair wounds), increased blood supply (to meet increased metabolic needs associated with growth and localized inflammation.

In many ways psoriasis such preuvelicheniem the healing process. Skin cells called keratinocytes) are created and pushed out to the surface of the skin in just 2-4 days. The surface of the skin cannot lose the dead skin cells distrust supported new blood vessels in the dermis (supporting tissue under the epidermis), developing to provide nutrients necessary to maintain hyperproliferating keratinocytes. At the same time, lymphocytes, neutrophils and macrophages embedded in the fabric, creating inflammation, swelling and soreness and potentially induce growth factors that enhance the rapid proliferation of keratinocytes. All these cells (keratinocytes, endothelial cells of blood vessels and leukocytes) produce destructive tissue enzymes or proteases that contribute to the progression of the disease and destruction of the surrounding tissue.

Using secured above compositions can be easy to treat inflammatory skin damage. In particular, antimicrotubule agent injected directly into the site of inflammation (or potential site of inflammation) for the treatment or prevention of disease. Suitable antimicrotubule agents are discussed in detail above and include, for example, taxanes (e.g. paclitaxel and docetaxel), computacin, eleutherobin, sarcodictyin, epothilone and discodermolide, deuterium oxide (D2O), hexyleneglycol (2-methyl-2,4-pentanediol), tubercidin (7-deazaadenosine), LY290181 (2-amino-4-(3-pyridyl)-4H-oil(1,2-b)Piran-3-carbonitrile), Florica antibodies protein that stimulates microtubule Assembly (Taxol-like protein, TALP), cellular swelling induced by hypotonic (190 mosmol/l) conditions, insulin (100 nmol/l) or glutamine (10 mmol/l), the binding of dynein, gibberelin, HSNO (kinesin-like protein), lysophosphatidic acid, lithium ion, components of the cell wall of plants (e.g., poly-L-lysine and extensin), buffer with glycerol buffer with Triton X-100, stabilizing microtubules associated with microtubule proteins (for example, MAR, MAR, Tau, big Tau, ensconsed, elongation factor 1-, (EF-1) and E-MAP-115), cellular particles (e.g., histone H1, myelin basic protein and kinetochore), endogenous microtubule-based structures (e.g., axoneme structure, macromolecular complexes of enzymes that form pores in the cell membrane and GTP-caps), stable Trubeckoy the only polypeptide (e.g., STOP145 and STOP220) and tension from mitotic forces, as well as any analogues and derivatives of any of the above agents. In some embodiments antimicrotubule agent is not paclitaxel, Campomarino or epothilones. Such agents, in some embodiments, can be delivered in the form of the center, above and below. In preferred embodiments of the invention, these agents or compositions can be delivered either locally or subcutaneous injection.

Effective antimicrotubular therapy for psoriasis will give at least one of the following results: reducing the number and severity of skin lesions, reducing the frequency of occurrence or duration of active acute illness, increased time spent in remission (i.e., periods when the patient has no symptoms) and/or reducing the severity or duration of concomitant disease symptoms (such as pain and swelling of the joints, pain in the axial skeleton, gastrointestinal symptoms).

Clinically, this treatment will lead to a reduction in the size or number of lesions, reduction of skin symptoms (pain, burning sensation and bleeding of the affected skin) and/or mitigate its accompanying symptoms (such as redness of the joints, fever, swelling, diarrhea, pain in the gut). Pathologically antimicrotubule agent will produce at least one of the following results: inhibition of proliferation of keratinocytes, reducing skin inflammation (e.g., factors of attraversare and increase the inhibition of dermal angiogenesis.

Antimicrotubule agent may be introduced in any way to achieve the above outcomes, but the preferred methods include local and systemic injection. Patients with localized disease can enter the agent in the form containing paclitaxel creams, ointments or softener for local application, applied directly to the psoriatic lesion. For example, a cream for topical administration, containing 0.01 to 10% paclitaxel by weight, may be imposed depending on the severity of the disease and response of the patient to that treatment. In a preferred embodiment, the preparation for topical application containing paclitaxel at a concentration of 0.1-1% by weight, should be entered in psoriatic lesions. Alternative for treatment of various injuries you can use direct intradermal injection of paclitaxel in a suitable pharmaceutical carrier.

In patients with widespread disease or incogniti symptoms (e.g., psoriatic arthritis, Reiter syndrome, concomitant spondylitis associated with inflammatory bowel disease) paclitaxel can be administered systemically. For example, can be carried out intermit the who response and tolerance of the patient; equivalent oral preparation would also be suitable for this indication. Other antimicrotubule agents could be entered in the "equivalent to paclitaxel dosing adjusted relative to the activity and tolerability of a particular agent.

Other conditions may also benefit from antimicrotubule agents, including: eczematous disease (atopic dermatitis, contact dermatitis, eczema), immunobullous disease, precancerous epithelial tumors, basal cell carcinoma (basal cell carcinoma), squamous cell carcinoma, keratoacanthoma, malignant melanoma and viral warts. Creams, ointments and softeners for topical application, containing 0.01 to 10% by weight of paclitaxel, may be suitable for treatment of these conditions.

2. Multiple sclerosis

In other aspects of the invention antimicrotubule agents can be used to treat or prevent multiple sclerosis. Briefly, multiple sclerosis (PC) is a debilitating demyelinizing disease of the Central nervous system. Although its etiology and pathogenesis is not known, believe that it is playing the role of genetic, immunological factors and environmental factors. During this the although the exact mechanisms involved in the loss of myelin, is not clear, there is increased proliferation and accumulation of astrocytes in areas of destruction of myelin. In these places there is macrophage-like activity and increased proteasa activity, which is at least partially responsible for the destruction of the myelin sheath.

Antimicrotubule agent may be introduced into the site of inflammation (or potential site of inflammation) for the treatment or prevention of this disease. Suitable antimicrotubule agents are discussed in detail above and include, for example, taxanes (e.g. paclitaxel and docetaxel), computacin, eleutherobin, sarcodictyin, epothilone and discodermolide, deuterium oxide (D2O), hexyleneglycol (2-methyl-2,4-pentanediol), tubercidin (7-deazaadenosine), LY290181 (2-amino-4-(3-pyridyl)-4H-oil(1,2-b)Piran-3-carbonitrile), aluminum fluoride, bis(Succinimidyl) of ethylene glycol, ethyl ester of glycine, monoclonal antiidiotypic antibodies, a protein that stimulates microtubule Assembly (Taxol-like protein, TALP), cellular swelling induced by hypotonic (190 mosmol/l) conditions, insulin (100 nmol/l) or glutamine (10 mmol/l), the binding of dynein, gibberelin, HSNO (kinesin-under the extensin), buffers with glycerol buffer with Triton X-100, stabilizing microtubules associated with microtubule proteins (for example, MAR, MAR, Tau, big Tau, ensconsed, elongation factor 1-(EF-1) and E-MAP-115), cellular particles (e.g., histone H1, myelin basic protein and kinetochore), endogenous microtubule-based structures (e.g., axoneme structure, macromolecular complexes of enzymes that form pores in the cell membrane and GTP-caps), stable Trubeckoy the only polypeptide (e.g., STOP145 and STOP220) and tension from mitotic forces, as well as any analogues and derivatives of any of the above agents. Such agents, in some embodiments, can be delivered in the form of a composition together with a polymer carrier or liposomal composition, as discussed in more detail above and below. In some embodiments of the invention, these agents or compositions can be administered orally, intravenously or by direct introduction (preferably using ultrasound, CT, fluoroscopic, MRI or endoscopic control areas) in the location of pathology.

Effective antimicrotubular therapy for multiple sclerosis will give, at mealeasy; increase the frequency and duration of periods of remission/symptom-free periods; the prevention of persistent deterioration and loss of earning capacity; and/or the prevention/attenuation of chronic progression of the disease. Clinically, this should lead to an improvement in visual symptoms (visual loss, diplopia (double vision in the eyes)), gait disturbance (weakness, axial instability, loss of sensation, spasticity, hyperreflexia, loss of "right-handedness"), dysfunction of the upper extremities (weakness, spasticity, loss of sensitivity), bladder dysfunction (loss of feeling the need to urinate, incontinence, urinary retention, incomplete emptying), depression, emotional lability and disorders cognitive abilities. Pathologically this treatment reduces one of the following events: loss of myelin destruction of the blood-brain barrier, perivascular infiltration of mononuclear cells, immunological disorders, education goticheskij scars and proliferation of astrocytes, education metalloproteinases and worsened the conduction velocity of nerve.

Antimicrotubule agent may be introduced in any way to achieve Visayas is a great introduction or subcutaneous, intramuscular or vnutriobolochechnoe injection. Antimicrotubule agent may be injected in the form of long-term therapy with low doses for prevention of disease progression, prolongation of the remission or reduction of symptoms in the case of active disease. Alternatively, therapeutic agent can be administered in higher doses in the form of "pulse" therapy for induction of remission in acute active disease. The minimum dose capable of provide these results can be used, and it may vary in accordance with the specific patient, severity of disease, the composition of the input agent and by injection. For example, paclitaxel, systemic long-term therapy with low doses can be entered continuously 10-50 mg/m2paclitaxel every 1-4 weeks, depending on therapeutic response: if the system "pulse" therapy with high doses you can enter 50-250 mg/m2every 1-21 days during cycles 1-6. Other antimicrotubule agents can be administered in equivalent doses adjusted relative to the activity and tolerability of a particular agent.

3. Arthritis

Inflammatory arthritis is a serious problem. the for example, one form of inflammatory arthritis, rheumatoid arthritis (RA) is a Multisystem, chronic, relapsing inflammatory disease of unknown origin. Although it can be affected by many authorities, RA is basically a severe form of chronic synovitis, which sometimes leads to destruction and ankylosis of affected joints (Robbins Pathological Basis of Disease, by R. S. Cotran, V. Kumar and S. L. Robbins, W. B. Saunders Co., 1989). Pathologically, this disease is characterized by a marked thickening of the synovial membrane, which forms the villous projections extending into the space of the joint, the formation of multiple layers synoviocytes pavements (proliferation of synoviocytes), infiltration of the synovial membrane cells (macrophages, lymphocytes, plasma cells and lymphoid follicles: the so-called "inflammatory synovitis") and deposition of fibrin with cellular necrosis in Zinovii. The tissue formed as a result of this process, called pannus, and gradually this pannus grows with the filling of the articular space. Pannus develops an extensive network of new blood vessels through angiogenesis, which is essential for the development of synovitis. Vysvobozhdeno inflammatory process (for example, hydrogen peroxide, superoxide, lysosomal enzymes and products of arachidonic acid metabolism) from the cells of the pannus tissue leads to progressive destruction of cartilage. Pannus is embedded in the articular cartilage, leading to erosion and fragmentation of the cartilage. Over time, the erosion under the cartilage of the bone with fibrous ankylosis and, eventually, bone ankylosis of the joint.

Generally considered, but this is not proven conclusively that RA is an autoimmune disease and that the latter many different stimuli activate the immune response in immunogenetic susceptible host. As exogenous infectious agents (virus Epstein-Barr virus, rubella, cytomegalovirus, herpes virus, T-cell lymphotropic virus human Mycoplasma and others), and endogenous proteins (collagen, proteoglycans, the modified immunoglobulins) are causal agent that triggers an inappropriate immune response of the host. Regardless of the exciting agent autoimmunity plays a role in the progression of this disease. In particular, the relevant antigen is absorbed by antigen-presenting cells (macrophages or dendritic cells in the synovial miniroot proliferation and differentiation of b-lymphocytes into plasma cells. The end result is the generation of excessive inappropriate immune response directed against the tissues of the host (for example, antibodies directed against collagen type II antibodies directed against the Fc portion of autologous IgG (called "rheumatoid factor")). This amplificare further immune response and accelerates the degradation of cartilage. Once this cascade is initiated, numerous mediators destruction of cartilage are responsible for the progression of rheumatoid arthritis.

Thus, in one aspect of the present invention are provided methods of treating or preventing inflammatory arthritis (e.g. rheumatoid arthritis), including the stage of the introduction to the patient a therapeutically effective amount antimicrotubule agent. Inflammatory arthritis includes a variety of conditions, including (but not limited to, rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis (scleroderma), mixed disease of connective tissue, Sjogren syndrome, ankylosing spondylitis, syndrome behceta, sarcoidosis, and osteoarthritis, all of which are characterized by inflamed, painful joints as pronounced symptom. In p the intra-articular injection, in the form of a surgical paste or other means, for example, systemically or orally.

Suitable antimicrotubule agents are discussed in detail above and include, for example, taxanes (e.g. paclitaxel and docetaxel), computacin, eleutherobin, sarcodictyin, epothilone and discodermolide, deuterium oxide (D2O), hexyleneglycol (2-methyl-2,4-pentanediol), tubercidin (7-deazaadenosine), LY290181 (2-amino-4-(3-pyridyl)-4H-oil-(1,2-b)Piran-3-carbonitrile), aluminum fluoride, bis(Succinimidyl) of ethylene glycol, ethyl ester of glycine, monoclonal antiidiotypic antibodies, a protein that stimulates microtubule Assembly (Taxol-like protein, TALP), cellular swelling induced by hypotonic (190 mosmol/l) conditions, insulin (100 nmol/l) or glutamine (10 mmol/l), the binding of dynein, gibberelin, HSNO (kinesin-like protein), lysophosphatidic acid, lithium ion, components of the cell wall of plants (e.g., poly-L-lysine and extensin), buffer with glycerol buffer with Triton X-100, stabilizing microtubules associated with microtubule proteins (for example, MAR, MAR, Tau, big Tau, ensconsed, elongation factor 1-(EF-1) and E-MAP-115), cellular aprimer, axoneme structure, macromolecular complexes of enzymes that form pores in the cell membrane and GTP-caps), stable Trubeckoy the only polypeptide (e.g., STOP145 and STOP220) and tension from mitotic forces, as well as any analogues and derivatives of any of the above agents. Such agents, in some embodiments, can be delivered in the form of a composition together with a polymer carrier or liposomal composition, as discussed in more detail above and below. In some embodiments of the invention, this antimicrotubule agent is another agent that is different from paclitaxel, impotecia or epothilone.

Effective antimicrotubular therapy for inflammatory arthritis will give one or more of the following results: (i) reducing the severity of symptoms (pain, swelling and tenderness of affected joints; morning stiffness of the joints, weakness, fatigue, anorexia, weight loss); (ii) decrease the severity of clinical signs of the disease (thickening of the joint capsule, synovial hypertrophy, joint effusion, soft tissue contractures, decreased range of motion, ankylosis and resistant strain of joints); (iii) the reduction of extra-articular manifestations of this sabol is otitis media, iritis, syndrome still's, osteoporosis); (iv) an increase in the frequency and duration of periods of remission/symptom-free periods; (v) prevention of persistent disturbances and loss of earning capacity; and/or (vi) prevention/attenuation of chronic progression of the disease. Pathologically effective antimicrotubular therapy for inflammatory arthritis will produce at least one of the following: (i) reduce the inflammatory response, (ii) to disrupt the activity of inflammatory cytokines (such as IL-1, TNF, FGF, VEGF), (iii) to inhibit the proliferation of synoviocytes, (iv) to block the activity of matrix metalloproteinases and/or (v) to inhibit angiogenesis. Antimicrotubule agent must be administered systemically (oral, intravenous or intramuscular or subcutaneous injection) in the minimum dose to obtain the above results. For patients with only a small number of affected joints or disease, expressed more significantly in a limited number of joints, antimicrotubule agent can inetservices directly (intra-articular injection into the affected joint.

4. Implants and surgical or medical devices, in comity any of the stated antimicrotubule agents, or constructed any other way to contain and/or release antimicrotubule agents. Typical examples include cardiovascular devices (e.g. implantable venous catheters, venous ports, tunneled venous catheters, lines, or ports for continuous infusion, including catheters for infusion hepatic artery, the wire of pacemaker, implantable defibrillator); neurologic/neurosurgical devices (e.g., peritoneal shunts, zheludochno-atrial shunts, devices to stimulate nerves, dural patches and implants to prevent epidural fibrosis after laminectomy, devices for continuous subarachnoid infusion); gastrointestinal devices (e.g., long acting permanent catheters, nutritious tube, portosystemic shunts, shunts for ascites, peritoneal implants for drug delivery, catheters for peritoneal dialysis, implantable mesh for hernia, suspensions or solid implants to prevent surgical adhesions, including grid); genitourinary device (e.g., uterine implants, including intrauterine contraceptive side reversible sterilization devices stents for fallopian tubes, artificial sphincters and okolopochecnuu implants for incontinence, ureteric stents, permanent catheters prolonged, increasing the bladder or wrappers or bus to vasovasostomy); ocular implants (for example, multicompetence and other implants for neovascular glaucoma, drug eluting contact lenses for pterygia, tires failed opening lacrimal SAC, drug eluting contact lenses for revascularization of the cornea, implants for diabetic retinopathy, drug eluting contact lenses for corneal grafts at high risk); otolaryngology devices (e.g., ossicular implants (ear bones), bus or stents of the Eustachian tube for exudative otitis media or chronic otitis as an alternative transtemporal drainage); implants for plastic surgery (e.g., prevention of contracture of muscle fibers in response to containing gel or saline breast implants in subpectorally or abiltity approaches or after mastectomy, or chin implants and orthopedic imple detail above and include, for example, taxanes (e.g. paclitaxel and docetaxel), computacin, eleutherobin, sarcodictyin, epothilone and discodermolide, deuterium oxide (D2O), hexyleneglycol (2-methyl-2,4-pentanediol), tubercidin (7-deazaadenosine), LY290181 (2-amino-4-(3-pyridyl)-4H-oil-(1,2-b)Piran-3-carbonitrile), aluminum fluoride, bis(Succinimidyl) of ethylene glycol, ethyl ester of glycine, monoclonal antiidiotypic antibodies, a protein that stimulates microtubule Assembly (Taxol-like protein, TALP), cellular swelling induced by hypotonic (190 mosmol/l) conditions, insulin (100 nmol/l) or glutamine (10 mmol/l), the binding of dynein, gibberelin, HSNO (kinesin-like protein), lysophosphatidic acid, lithium ion, components of the cell wall of plants (e.g., poly-L-lysine and extensin), buffer with glycerol buffer with Triton X-100, stabilizing microtubules associated with microtubule proteins (for example, MAR, MD.P4, Tau, big Tau, ensconsed, elongation factor 1-(EF-1) and E-MAP-115), cellular particles (e.g., histone H1, myelin basic protein and kinetochore), endogenous microtubule-based structures (e.g., axoneme structure, macromolecular complex is on (for example, STOP145 and STOP220) and tension from mitotic forces, as well as any analogues and derivatives of any of the above agents. Such agents, in some embodiments, can be delivered in the form of a composition together with a polymer carrier or liposomal composition, as discussed in more detail above and below. In some embodiments of the invention (for example, in the case of stents), antimicrotubule agent is an agent that is different from paclitaxel, impotecia or epothilone.

Implants and other surgical or medical devices may be coated with (or otherwise adapted to release) antimicrotubule compositions or antimicrotubule factors of the present invention in a variety of ways, including, for example: (a) direct attachment to the implant or device antimicrotubular agent or composition (e.g., a coating on the implant or device of a film of the polymer/drug or by dipping the implant or device into the polymer solution/drug or other covalent or noncovalent means); (b) coating the implant or device substance such as a hydrogel, which, in turn, will absorb antimicrotubule composition of the thread (or the polymer, formed into a thread) into the implant or device; (d) inserting the implant or device into a sleeve or mesh consisting of antimicrotubule composition or coated with such composition; (e) the design of the implant or device with antimicrotubule by the agent or the composition; or (f) a contrivance, implant or device in any way to release antimicrotubule agent. In preferred embodiments of the invention compositions must be firmly attached to the implant or device during storage and during insertion. Antimicrotubule the agent or the composition preferably should not deteriorate during storage, prior to inserting or when heated to body temperature after insertion into the body (if required). In addition, they should preferably cover the implant or device smoothly and evenly, with a uniform distribution antimicrotubule agent, without changing the contour of the stent. In a preferred embodiment of the invention antimicrotubule the agent or the composition should provide a uniform, predictable, prolonged release antimicrotubular factor in the tissue surrounding the implant or device, as toent thrombogenic (cause the formation of blood clots), or cause significant turbulence in blood flow (more than would be expected with the introduction of not having the coating of the stent).

In the case of stents can be designed in a great variety of stents for content and/or release antimicrotubule agents, including esophageal stents, gastrointestinal stents, vascular stents, biliary stents, stents for colon, stents for pancreatic cancer, ureteric and urethral stents, lacrimal stents, the stents of the Eustachian tubes, stents for fallopian tubes and tracheal/bronchial stents. Stents can be easily obtained from commercial sources or constructed in accordance with well known methods. Typical examples of stents include stents, described in U. S. Patent No. 4768523, entitled "Hydrogel Adhesive"; U. S. Patent No. 4776337, entitled "Expandable Intraluminal Graft, and Method and Apparatus for Implanting and Expandable Intraluminal Graft"; U. S. Patent No. 5041126, entitled "Endovascular Stent and Delivery System"; U. S. Patent No. 5052998 entitled "Indwelling Stent and Method of Use"; U. S. Patent No. 5064435, entitled "Self-Expanding Prosthesis Having Stable Axial Length"; U. S. Patent No. 5089606, entitled "Water-insoluble Polysaccharide Hydrogel Foam for Medical Applications"; U. S. Patent No. 5147370 entitled "Nitinol Stent for Hollow Body Conduits"; U. S. Patent No. 5176626 entitled "Indwelling Stent"; U. S. Patent No. 5213580, entitled "Biodegradable Polymeric Endoluminal Sealing Process"; and U. S. Patent No. 5328471, entitled "Method and Apparatus for Treatment of Focal Disease in Hollow the passage of the body, comprising inserting a stent into the passageway, the stent has a generally tubular structure and the surface of this structure is covered with (or adapted in some other way to release) antimicrotubule composition (or only antimicrotubule agent), so that the duct has expanded. The following describes the different ways in which the lumen of the passage of the body extends to resolve biliary, gastrointestinal, esophageal, tracheal/bronchial, ureter or vascular obstruction.

Typically, the stent is inserted in the same way regardless of where or disease to be treated. Briefly, prior to inserting usually first survey conducted, usually by means of the procedure of diagnostic imaging, endoscopy, or visually during surgery to determine an appropriate location for insertion of the stent. Then after the damage or the proposed location of the insertion promote leader (guide wire) and it passes the catheter of the delivery, allowing you to insert a stent in his spasams. Typically, the stent can be compressed, so that they can be inserted through a thin cavity through small catheters, and then expanded to a larger diameter once they beriwal them open. As such they can be inserted through a small hole and still able to keep open cavity or passage of large diameter. The stent may be self-expanding (e.g., Wallstent and Gianturco stents), extensible cylinders (for example, the Palmaz stent and stent Strecker) or be implanted with the aid of temperature change (e.g., Nitinol stent).

Stents are usually carried out by maneuvering into place when radiological or direct visual control, paying particular attention to the location of the stent accurately across a constriction in a subject to treatment of the body. Then delivering the catheter is removed, leaving the stent to stand on their own as "forests". Examination after insertion, usually with the help of x-ray is often used to confirm the correct premises.

In a preferred embodiment of the invention are provided methods of eliminating biliary obstructi providing for the insertion of biliary stent in the bile duct, and the stent has a generally tubular structure and the surface of this structure is coated with (or otherwise adapted to release) an agent or composition, described above, so that biliary obstruction is eliminated. Briefly, tumor overgrowth of normal bile protonema, which drains bile from the liver into the duodenum, the most frequently blocked (1) a tumor composed of cells of the bile duct (cholangiocarcinoma), (2) a tumor that is embedded in the bile duct (e.g., pancreatic cancer) or (3) tumor that exerts outward pressure and squeezes the bile duct (e.g., enlarged lymph nodes).

As primary biliary tumors, and other tumors that cause compression of the biliary tree (all ducts) can be treated with the use of these stents. One example of primary biliary tumors are adenocarcinomas (which are also called Klatskin tumors when they are in the place of bifurcation of common hepatic duct). These tumors are also called biliary carcinomas, choledochoenterostomy or adenocarcinomas of the biliary system. Benign tumors that affect the bile duct (e.g., adenoma of the biliary system), and, in rare cases, squamous cell carcinoma, bile duct and adenocarcinoma of the gallbladder can also cause compression of the biliary tree and, consequently, lead to biliary obstruction.

Compression of the biliary tree enableproxy. Most tumors of the pancreas arise from cells of the pancreatic ducts. This vysokodetalnye form of cancer (5% of all cancer deaths; 26000 new occurrence of one year in the U.S.) with an average survival of 6 months and survival for 1 year only in 10% of cases. When these tumors are located at the head of the pancreas, they often cause biliary obstruction, and this significantly reduces the patient's quality of life. Although all types of pancreatic tumors, usually referred to as "pancreatic cancer", there are histological subtypes: adenocarcinoma, adrenoblokatory carcinoma, cystadenocarcinoma and Cinemateca carcinoma. Hepatic tumors, as discussed above, can also cause compression of the biliary tree and, consequently, can cause obstruction of the bile ducts.

In one embodiment of the invention biliary stent is first inserted into the biliary duct in one of several ways: from the top end of the inserting needle through the abdominal wall and through the liver (percutaneous perhepatic cholangiogram or "RTS"); from the lower end of kanalirovaniem bile duct through an endoscope inserted through the mouth, stomach and duodenum (endoscopy is usually first of survey conducted, usually using a procedure PTC, ERCP or visually during surgery to determine an appropriate location for insertion of the stent. Then through the damage and promote the delivery catheter, allowing you to insert a stent in his spasams. If the diagnostic study is the RTS, the sender and the delivery catheter is inserted through the abdominal wall, whereas, if the survey is done using ERCP, stent put in place through the mouth. Then the stent is injected at the site under the radiological, endoscopic or direct visual control, paying particular attention to put it accurately through the narrowing in the bile duct. Delivering the catheter is then removed, leaving the stent standing in the form of "scaffolding" that keep the bile duct open. Subsequent cholangiogram can be obtained to document that the stent is placed properly.

In another embodiment of the invention provided methods to eliminate esophageal obstructi providing for the insertion of the esophageal stent in the esophagus, and the stent has a generally tubular structure and the surface of this structure is covered with (or adapted in some other way to release) antimicrotubule agentase a hollow tube, which carries food and liquids from the mouth to the stomach. Cancer of the esophagus or invasion of cancer that occur in adjacent organs (e.g. stomach cancer, or lung), leading to inability to swallow food or saliva. In this embodiment, should be performed prior to inserting the examination, usually swallowing barium swallow or endoscopy to determine an appropriate position for insertion of the stent. Then the catheter or endoscope can be placed through the mouth and the sender of the wire advancing through the point of blockage. A catheter for delivering the stent passes through the sender under radiological or endoscopic control, and the stent is placed exactly over the narrowing in the esophagus. Examination after insertion, usually x-ray swallowed barium, can be used to confirm the appropriate areas of the stent.

In another embodiment of the invention provided methods to eliminate obstructi colon, providing for the insertion of the stent into the large intestine, and the stent has a generally tubular structure and the surface of this structure is covered with (or adapted in some other way to release) antimicrotubule agent or composition, as described above, so as to eliminate the obstruction tol and waste digestion from the small intestine to the anus. Cancer of the rectum and/or colon or invasion of cancer arising in the adjacent organs (e.g., cancer of the uterus, ovary, bladder), leads to the inability of the excretion of faeces from the bowel. In this embodiment, should normally be conducted inspection before loading, usually with an enema with barium or a colonoscopy to determine an appropriate location for insertion of the stent. Then the catheter or endoscope may be inserted through the anus and guidewire promote through the place of obstruction. The catheter delivery of the stent passes through the sender under radiological or endoscopic control and the stent is placed exactly over the narrowing in the colon or rectum. Examination after insertion, typically by x-ray barium enema may be used to confirm proper placement of the stent.

In other embodiments of the invention are provided methods to resolve tracheal/bronchial obstructi providing for the insertion of tracheal/bronchial stent into the trachea or bronchi, the stent has a generally tubular structure and the surface of this structure is covered with (or adapted in some other way to release) antimicrotubule agent or composition, is the quiet carry the air from the mouth and nose to the lungs. Obstruction of the trachea cancer, invasion of cancer arising in the adjacent organs (e.g. lung cancer) or collapse of the trachea or bronchi caused by chondromalacia (softening of the cartilage rings), leading to inability to breathe. In this embodiment of the invention, should normally be conducted inspection before loading, usually endoscopy, to determine an appropriate location for insertion of the stent. Then the catheter or endoscope is placed through the mouth and promote the sender through the point of blockage. Then the catheter delivery of the stent passes through the guiding equipment for inserting spaceghost stent. The stent is placed under radiological or endoscopic control to put it accurately through the constriction. Then, the delivery catheter can be removed, leaving the stent, standing alone in the form of "forests". Examination after insertion, usually bronchoscopy may be used to confirm the appropriate areas of the stent.

In another embodiment of the invention provided methods for eliminating urethral obstructions, providing for the insertion of urethral stent into the urethra, and the stent has a generally tubular structure and the surface of this structure is covered with (or adapted in any other way is appcatalog channel. Briefly, the urethra is a tube that empties the bladder through the penis. External constriction of the urethra as it passes through the prostate gland, caused by hypertrophy of the prostate gland, found in virtually every male over the age of 60 years and causes progressive difficulty urinating. In this embodiment, must first be examined before loading, usually endoscopy or urethrogram to determine the correct location to insert the stent, which is above the external sphincter of the urethra at its lower end and close to the junction to the bladder neck at the upper end. Then the endoscope or catheter is placed through the hole of the penis and the leader of the advance into the bladder. Then the catheter delivery of the stent passes through the sender to insert the stent. Then the delivery catheter is removed and the stent is expanded into place. Examination after insertion, usually endoscopy or retrograde urethrogram, can be used to confirm proper position of the stent.

In another embodiment of the invention provided methods to eliminate the HT has a generally tubular structure and the surface of this structure is covered with (or adapted in some other way to release) antimicrotubule agent or composition, so to blockage of the vessel. Briefly, the stents may be placed in a large series of blood vessels, both arteries and veins, to prevent recurrent stenosis in failed angioplasty for treatment of stenoses, which, in all probability, will not be able to resolve angioplasty, and for treatment of stenosis after surgery (e.g., stenosis dialysis transplant). Typical examples of suitable locations include iliac artery gland, renal and coronary arteries, upper Vena cava and in dialysis grafts. In one embodiment, the first conduct angiography to determine where to place the stent. This is usually performed by injection radioprotecao contrast through a catheter inserted into an artery or vein, when receiving x-rays. Then the catheter can be inserted percutaneous or surgically into the femoral artery, brachial artery, femoral vein, or brachial vein and advanced into a suitable blood vessel direction it through the vasculature by using fluroscopy. Then the stent can be placed across the narrowed vessels. Angiogram after insertion can be used to confirm the correct areas of the stent.

Usually used by the left artery is released from the endothelium by intraluminal passage of a balloon catheter, introduced through the external carotid artery (Clowes et al., Lab. Invest. 49(2) 208-215, 1983). At 2 weeks carotid artery is significantly narrowed due to compression of smooth muscle cells, but between 2 and 12 weeks intima is doubled in thickness, which leads to the reduction luminale size.

5. Inflammatory bowel disease

With the application described here, the agent, the compositions and methods can be achieved in the treatment or prevention of a large variety of inflammatory bowel disease. Inflammatory bowel disease (IBD) is a term for a group of chronic inflammatory disorders of unknown etiology that involves the gastrointestinal tract. Chronic IBD can be subdivided into 2 groups: ulcerative colitis and Crohn's disease. In Western Europe and in the United States ulcerative colitis has a prevalence of 6-8 cases per 100,000.

Although the cause of this disease remains unknown, as causal factors were assumed to be genetic, infectious, immunologic, and psychological factors. In the case of ulcerative colitis is an inflammatory reaction, covering the mucous membrane of the colon, leading to ulceration of the surface. Normal is infilt and long-term ulcerative colitis epithelium may become displeasurement and in the end, malignant. Crohn's disease is characterized by chronic inflammation extending through all layers of the intestinal wall. With progression of the disease, the intestine becomes thickened and stenosis of the lumen. Ulceration of the mucous membrane takes place, and these ulcers can penetrate into the submucosal membrane and the muscular layer with the formation of fistulas and fissures.

Antimicrotubule agents can be used to treat inflammatory bowel disease in several ways. In particular, antimicrotubule agent may be introduced into the site of inflammation (or potential site of inflammation) for the treatment of this disease. Suitable antimicrotubule agents are discussed in detail above and include, for example, taxanes (e.g. paclitaxel and docetaxel), computacin, eleutherobin, sarcodictyin, epothilone and discodermolide, deuterium oxide (D2O), hexyleneglycol (2-methyl-2,4-pentanediol), tubercidin (7-deazaadenosine), LY290181 (2-amino-4-(3-pyridyl)-4H-oil(1,2-b)Piran-3-carbonitrile), aluminum fluoride, bis(Succinimidyl) of ethylene glycol, ethyl ester of glycine, monoclonal antiidiotypic antibodies, a protein that stimulates the Assembly Mikami, insulin (100 nmol/l) or glutamine (10 mmol/l), the binding of dynein, gibberelin, HSNO (kinesin-like protein), lysophosphatidic acid, lithium ion, components of the cell wall of plants (e.g., poly-L-lysine and extensin), buffer with glycerol buffer with Triton X-100, stabilizing microtubules associated with microtubule proteins (for example, MAR, MAR, Tau, big Tau, ensconsed, elongation factor 1-(EF-1) and E-MAP-115), cellular particles (e.g., histone H1, myelin basic protein and kinetochore), endogenous microtubule-based structures (e.g., axoneme structure, macromolecular complexes of enzymes that form pores in the cell membrane and GTP-caps), stable Trubeckoy the only polypeptide (e.g., STOP145 and STOP220) and tension from mitotic forces, as well as any analogues and derivatives of any of the above agents. Such agents, in some embodiments, can be delivered in the form of a composition together with a polymer carrier or liposomal composition, as discussed in more detail above and below.

An ideal model for studies of IBD should be naturally occurring or induced by disease of the animal, which sushestwah primates, intestinal inflammation, which was not detected causal organism is the causative agent. The first model, Tamarin cotton-top tamarin), has a high incidence of spontaneous colitis is not associated with identifiable pathogens and, as in the case of humans, the activity of this disease process spontaneously ebb and flow (Madara et al., Gastroenterology 88 : 13-19, 1985). Another spontaneous chronic colitis also occurs in juvenile rhesus monkeys (Adier et al., Gastroenterology 98 : A436, 1990). There are many animal models with experimentally induced colitis. In mice, rats, Guinea pigs and rabbits colitis can be induced by oral administration of sulfated polysaccharides (amylopectin sulfate carrageenan, doctranslate) (Marcus and Watt, Lancet 2: 489-490, 1969), rectal introduction of chemical stimuli (diluted acetic acid) (MacPherson and Pfeiffer. Digestion 17 : 135-150, 1978) and allergic reaction of the delayed type on dinitrochlorobenzene (Glick and Falchuk., Gut 22 : 120-125, 1981) or trinitrobenzenesulfonic acid (Rabin and Rogers, Gastroenterology 75 : 29-33, 1978).

Since there is no pathogenomics (typical for this disease) symptoms or specific diagnostic tests for IBD, efficiency antimicrotubular agent in the treatment of atomania least in the form of the following results: reduction in seizure frequency, increase the amount of time spent in remission (i.e., periods when the patient has no symptoms) and/or reducing the severity or duration related manifestations (the formation of abscesses, fistula formation, colon cancer, bowel perforation, bowel obstruction, toxic megacolon, peripheral arthritis, ankylosing spondylitis, cholecystitis, sclerosing cholangitis, cirrhosis, nedaznai erythema, iritis, uveitis, episcleritis, venous thrombosis). Specific symptoms such as bloody diarrhea, pain in the abdomen, pyrexia, weight loss, rectal bleeding, tenesmus and abdominal swelling will be reduced or weakened.

Antimicrotubule agent may be introduced in any way to achieve the above outcomes, but the preferred methods include oral, rectal or peritubular introduction (preferably with ultrasound, CT (computed tomography), fluoroscopically, MRI or endoscopic control directions; the administration can also be performed by direct introduction during surgery in the abdominal cavity). For some patients in the treatment of this disease can also be used CNAME symptoms fit systemic treatment (e.g., oral administration, intravenous, subcutaneous, intramuscular injection). In a preferred embodiment, paclitaxel may be administered orally in a dose of 10-75 mg/m2every 1-4 weeks depending on therapeutic response and tolerance of the patient. For the treatment of severe acute exacerbations higher doses of 50-250 mg/m2paclitaxel provided oral (or intravenous) can be defined as "pulse" therapy. In patients with localized rectal disease (rectum is affected in 95% of patients with ulcerative colitis), paclitaxel can be administered locally in the form of rectal cream or suppository. For example, a cream for topical application, containing 0.01 to 10% paclitaxel by weight, may be imposed depending on the severity of the disease and the patient's response to this introduction. In a preferred embodiment, the preparation for local administration, containing 0.1-1% paclitaxel by weight, could be introduced into the rectum daily if necessary. Peritubular paclitaxel (i.e., the introduction of drugs to the outside or the mesenteric surface of the intestine) may be injected into areas with active bowel disease. In a preferred embodiment, 0.5 to 20% paclitaxel by ve the professionals", "film" or "wrapper", which releases the drug over a certain period of time. In all variants other antimicrotubule agents could be administered in equivalent doses adjusted relative to the activity and tolerability of a particular agent.

6. Surgical procedures

As noted above, antimicrotubule agents and compositions can be used in a variety of surgical procedures. For example, in one aspect of the present invention antimicrotubule agent or composition (e.g., in the form of a spray or film) may be used for coating or spraying to the area before removal of the tumor, in order to isolate the surrounding tissues from malignant tissue, and/or to prevent the disease from spreading to the surrounding tissue. In other aspects of this invention, antimicrotubule agents or compositions (e.g., in the form of a spray) may be delivered via endoscopic procedures to cover a tumor or inhibiting the disease in a desirable location. Still other aspects of this invention, a surgical mesh, which were covered antimicrotubule agents or compositions or propulsively in any procedure, which can be used surgical mesh. For example, in one embodiment of the invention, a surgical mesh, loaded antimicrotubule composition, can be used during isbecause cancer surgery of the abdominal cavity (for example, after resection of the colon) to provide support for this structure and for the release of a number antimicrotubule agent.

In further aspects of the present invention are provided methods for the treatment places excision (excision) of tumors, providing for the introduction antimicrotubule agent or composition, as described above, in the margins of resection of the tumor after excision, so that the local recurrence of cancer in this location is inhibited.

In one embodiment of the invention antimicrotubule composition (compositions) (or only antimicrotubule factor (factors)) injected directly into the site of excision of a tumor (for example, coating by means of a swab or brush the edges (boundaries) tumor resection antimicrotubule compositions or factors)). Alternatively, antimicrotubule composition or factors can be included in a well-known surgical pastes before the introduction. In particularly preferred embodiments, izobretali neurosurgical operations.

In one aspect of the present invention, antimicrotubule agent or composition can be introduced into the border of the resection of a large variety of tumors, including for example, breast cancers, head and neck, tumors of the colon, brain and liver. For example, in one embodiment of the invention, antimicrotubule agents or compositions can be entered in place of neurological tumor after excision, so that inhibited the recurrence of this tumor. Briefly, the brain is vysokodotatsionnym functional; i.e., each specific anatomical region is specialized for carrying out specific functions. Therefore, it is often more important is the location of brain pathology than its type. Relatively little damage in the critical area can be much more destructive than much more damage in less important areas. In this way damage to the surface of the brain can be easy for surgical excision, whereas the same tumor located deep in the brain, may not be easy for excision (will need to cut too many vital structures to access it). Also, even benign tumors can be dangerous for several reasons: Alena surgical excision, this may be impossible; and, finally, if left uncontrolled, they can increase intracranial pressure. The skull is a closed space, incapable of expansion. Therefore, if something is growing in the same location, something else has to be compressed in a different location - the result is increased pressure in the skull, or increased intracranial pressure. If this condition is left untreated, vital structures can be sdavlennym, leading to death. The incidence of malignant diseases of the Central nervous system (CNS) is 8-16 per 100,000. The prognosis of primary malignant brain tumors is dark, with an average survival of less than one year, even after surgical removal. These tumors, especially gliomas represent a local disease that recurs in 2 cm from the source center of the disease after surgical removal.

Typical examples of brain tumors that can be treated using the described here agents, compositions and methods include glial tumors such as anaplastic astrocytoma, polymorphic glioblastoma, hair astrocytoma, oligodendroglioma, is (for example, neuroblastoma, ganglioneuroblastoma, ganglionevroma and medulloblastoma); tumors of the pineal body (for example, pineoblastoma, pineocytoma); meningeal tumors (e.g., meningioma, meningeal hemangiopericytoma, meningeal sarcoma); and tumors cells perineurial (for example, Sanoma (neurilemoma) and neurofibroma); lymphomas (e.g., Hodgkin's disease and lymphoma non-Hodgkin's lymphoma (including numerous subtypes, both primary and secondary); congenital tumors (e.g., craniopharyngioma, epidermoid cyst, dermoid cysts and colloid cysts); and metastatic tumors (which can be formed essentially from any tumor, most commonly from lung tumors, breast cancer, melanoma, kidney, and gastrointestinal tract).

Suitable antimicrotubule agents are discussed in detail above and include, for example, taxanes (e.g. paclitaxel and docetaxel), computacin, eleutherobin, sarcodictyin, epothilone and discodermolide, deuterium oxide (D2O), hexyleneglycol (2-methyl-2,4-pentanediol), tubercidin (7-deazaadenosine), LY290181 (2-amino-4-(3-pyridyl)-4H-oil-(1,2-b)Piran-3-carbonitrile), aluminum fluoride, bis(Succinimidyl) of ethylene glycol, ethyl ester of glycine, mono-clone is the hoots cells, induced by hypotonic (190 mosmol/l) conditions, insulin (100 nmol/l) or glutamine (10 mmol/l), the binding of dynein, gibberelin, HSNO (kinesin-like protein), lysophosphatidic acid, lithium ion, components of the cell wall of plants (e.g., poly-L-lysine and extensin), buffer with glycerol buffer with Triton X-100, stabilizing microtubules associated with microtubule proteins (for example, MAR, MAR, Tau, big Tau, ensconsed, the elongation factor 1-, (EF-1) and E-MAP-115), cellular particles (e.g., histone H1, myelin basic protein and kinetochore), endogenous microtubule-based structures (e.g., axoneme structure, macromolecular complexes of enzymes that form pores in the cell membrane and GTP-caps), stable Trubeckoy the only polypeptide (e.g., STOP145 and STOP220) and tension from mitotic forces, as well as any analogues and derivatives of any of the above agents. Such agents, in some embodiments, can be delivered in the form of a composition together with a polymer carrier or liposomal composition, as discussed in more detail above and below. In some embodiments of the invention, this antimicrotubule agent is other the x aspects of the invention are provided methods of treating and/or preventing surgical adhesions introduction patient antimicrotubule agent. Briefly, the formation of surgical adhesions is a complex process in which the tissues of the body that normally are separate, grow together. These postoperative adhesions occur in 60-90% of patients undergoing major gynecological surgery. Surgical trauma, as a result of drying of tissues, ischemia, thermal damage, infection or the presence of a foreign body, long recognized as the stimulus for the formation of tissue adhesions. These adhesions are a major cause of failed surgical therapy and are the leading cause of bowel obstruction and infertility. Other adhesive complications include chronic pain in the pelvic region, blockage of the urethra and emptying dysfunction (removal of feces and urine output).

Usually spiloptera is an inflammatory reaction which can release factors that increase vascular permeability and leading to fibrinogen influx and deposition of fibrin. This deposition forms a matrix, which connects by bridge adjacent tissue. Fibroblasts accumulate, join this matrix, lay collagen and induce angiogenesis. If this cascade of events mo is to aromatise.

Thus, as noted above, this invention provides methods of treating and/or preventing the formation of surgical adhesions. A great variety of animal models can be used to assess a particular therapeutic composition or scheme of treatment. Briefly, peritoneal adhesions occur in animals as a result suffered serious damage, which usually covers two adjacent surfaces. Damage can be mechanical, caused by ischemia, or be a result of the introduction of alien material. Mechanical damage includes punches in the gut (Choate et al., Arch. Surg. 88 : 249-254, 1964) and skinning or scraping of the outer layers of the gut wall (Gustavsson et al., Acta Chir. Scand. 109 : 327-333, 1955). The separation of the major vessels on the loop of the intestine induces ischemia (James et al., J. Path. Bact. 90 : 279-287, 1965). The foreign material, which may be entered in this field includes talc (Green et al., Proc. Soc. Exp. Biol. Med. 133: 544-550, 1970), gauze swabs (Lehman and Boys, Ann. Surg. 111: 427-435, 1940), toxic chemicals (Chancy, Arch. Surg. 60 : 1151-1153, 1950), bacteria (Moin et al., Am. J. Sci. 250 : 675-679, 1965) and faeces (Jackson, Surgery 44 : 507-518, 1958).

Currently, a typical model of preventing formation of adhesions include the AOC is -20, 1987), modified by devascularization rabbit model of uterine horns, which is characterized by erosion and disruption of the blood supply of the uterus (Wiseman et al., J. Invest. Surg. 7: 527-532, 1994) and rabbit model leparisien side wall including cutting the flap parietal peritoneum and the erosion of the caecum (Wiseman and Johns, Fertil. Steril. Suppi : 25S, 1993).

Typical antimicrotubule agents for the treatment of adhesions are discussed in detail above and include taxanes (e.g. paclitaxel and docetaxel), computacin, eleutherobin, sarcodictyin, epothilone and discodermolide, deuterium oxide (D2O), hexyleneglycol (2-methyl-2,4-pentanediol), tubercidin (7-deazaadenosine), LY290181 (2-amino-4-(3-pyridyl)-4H-oil(1,2-b)Piran-3-carbonitrile), aluminum fluoride, bis(Succinimidyl)of ethylene glycol, ethyl ester of glycine, monoclonal antiidiotypic antibodies, a protein that stimulates microtubule Assembly (Taxol-like protein, TALP), cellular swelling induced by hypotonic (190 mosmol/l) conditions, insulin (100 nmol/l) or glutamine (10 mmol/l), the binding of dynein, gibberelin, HSNO (kinesin-like protein), lysophosphatidic acid, lithium ion, components of the cell wall of plants (e.g., poly-L-lysine and extensin), buffet is emer, MAR, MAR, Tau, big Tau, ensconsed, elongation factor 1-(EF-1) and E-MAP-115), cellular particles (e.g., histone H1, myelin basic protein and kinetochore), endogenous microtubule-based structures (e.g., axoneme structure, macromolecular complexes of enzymes that form pores in the cell membrane and GTP-caps), stable Trubeckoy the only polypeptide (e.g., STOP145 and STOP220) and tension from mitotic forces, as well as any analogues and derivatives of any of the above agents. Such agents, in some embodiments, can be delivered in the form of a composition together with a polymer carrier or liposomal composition, as discussed in more detail above and below. In some embodiments of the invention, this antimicrotubule agent is another agent that is different from paclitaxel, impotecia or epothilone.

Using the bottom here of the agents, compositions and methods can be treated or prevented a large variety of surgical spikebravo and complications. Spiloptera or undesirable accumulation/encapsulation of scar tissue complicates many surgical procedures. As described above, hereroland. Encapsulating surgical implants is also a complication of reconstructive (plastic) surgery, joint replacement surgery, reconstructive surgery of hernias, surgery, artificial vascular grafts and neurosurgery. In each case, the implant becomes encapsulated by a capsule of fibrous connective tissue, which impairs or disrupts the function of the surgical implant (e.g., breast implant, artificial joint replacement, surgical meshes, vascular grafts, dural patch). Chronic inflammation and scarring occurs during surgery regarding correction of chronic sinusitis or remove other sites of chronic inflammation (for example, foreign bodies, infections (fungal, mycobacterial)).

Antimicrotubule agent may be introduced in any way to achieve the above outcomes, but the preferred methods include peritubular introduction (either direct application to the site of surgery, or endoscopic, ultrasound, CT, MRI or fluoroscopically direction control introduction); "floor" of the surgical implant; and placing eluting ate by weight loading in the polymeric carrier (as described in the following examples) and put on peritubular (mesenteric) surface in the form of a "paste", "film" or "wrapper", which releases the drug over a certain period of time, so that the incidence of surgical adhesions is reduced. During endoscopic procedures, the preparation of paclitaxel-polymer applied as a "spray", through the ports of delivery in the endoscope, the mesentery of the abdominal and pelvic cavity, which is manipulated during surgery. In a particularly preferred embodiment, peritubular composition comprises 1-5% paclitaxel by weight. In another preferred embodiment, the polymer coating containing 0.1-20% paclitaxel, is applied to the surface of the surgical implant (e.g., breast implant, artificial joint replacement, vascular graft) to prevent encapsulation/unsuitable scarring near the implant. In another preferred embodiment, the polymer implant containing 0.1-20% paclitaxel by weight, is applied directly to the surgery (for example, directly into the sinus cavity, the cavity of the chest, abdominal cavity or into the surgical site during neurosurgery), so that the recurrence of inflammation, spiloptera or scarring are reduced. In all these cases, other antimicro is inosinate specific agent.

8. Chronic inflammatory disease of the Airways

In other aspects of the invention antimicrotubule agents (and composition) can be used to treat or prevent such diseases as chronic inflammatory disease of the respiratory tract. In particular, antimicrotubule agent may be injected to the site of inflammation (or potential site of inflammation) for the treatment of this disease. Suitable antimicrotubule agents for the treatment of adhesions are discussed in detail above and include taxanes (e.g. paclitaxel and docetaxel), computacin, eleutherobin, sarcodictyin, epothilone D and, discodermolide, deuterium oxide (D2O), hexyleneglycol (2-methyl-2,4-pentanediol), tubercidin (7-deazaadenosine), LY290181 (2-amino-4-(3-pyridyl)-4H-oil(1,2-b)Piran-3-carbonitrile), aluminum fluoride, bis(Succinimidyl)of ethylene glycol, ethyl ester of glycine, monoclonal antiidiotypic antibodies, a protein that stimulates microtubule Assembly (Taxol-like protein, TALP), cellular swelling induced by hypotonic (190 mosmol/l) conditions, insulin (100 nmol/l) or glutamine (10 mmol/l), the binding of dynein, gibberelin, HSNO (kinesin-like protein), lysophosphatidyl KIS the buffer with Triton X-100, stabilizing microtubules associated with microtubule proteins (for example, MAR, MAR, Tau, big Tau, ensconsed, elongation factor 1-(EF-1) and E-MAP-115), cellular particles (e.g., histone H1, myelin basic protein and kinetochore), endogenous microtubule-based structures (e.g., axoneme structure, macromolecular complexes of enzymes that form pores in the cell membrane and GTP-caps), stable Trubeckoy the only polypeptide (e.g., STOP145 and STOP220) and tension from mitotic forces, as well as any analogues and derivatives of any of the above agents. Such agents, in some embodiments, can be delivered in the form of a composition together with a polymer carrier or liposomal composition, as discussed in more detail above and below. In preferred embodiments of the invention, the agents or compositions can be administered intranasally, systemic, inhalation, local administration (for example, in the case of polyps in the nose) or oral sinuses.

Asthma

In some aspects of the invention, antimicrotubule agents can be used for treating or preventing asthma. In summary, asthma is a condition, characterizes the treatment. Although the exact etiology of asthma is not known, this condition is a condition of excessive narrowing of the lumen of the bronchi and inflammatory responses to stimuli, which affects 5% of the population. Effective antimicrotubular therapy for asthma would change one or more pathological characteristics of this condition, such as the reduction of infiltration and activity of inflammatory cells (T cells, mastocytes, eosinophils), reduced proliferation and thickening of the epithelium of the respiratory tract, inhibition of cell proliferation of smooth muscle and hypertrophy in the wall of the respiratory tract, reducing secretion of mucus in the lumen of the respiratory tract, blocking the activity of inflammatory cytokines (IL-3, IL-4, IL-5, GMSF), which induce and perpetuate inflammation and inhibit the hyperplasia and hypertrophy of the secreting glands of the respiratory tract.

Clinically effective antimicrotubular therapy asthma could produce one of the following: decrease the severity of symptoms, decrease the duration of exacerbations, increase the frequency and duration of periods of remission, to prevent persistent deterioration and disability and to prevent chronic progression of dyspnea, cough and stridor to resist the flow of air and hypocapnic/respiratory alkalosis and improve the mismatch V:Q (ventilation:perfusion).

Antimicrotubule agent may be injected in any way to achieve the above outcomes. Preferred routes of administration include inhalation therapy (e.g., using an inhaler with a metered-dose spray, through the endotracheal tube, inhalation of microparticles) or systemic therapy (intravenous, subcutaneous or intramuscular injection or introduction of the oral drug). System dose should be administered to patients with severe exacerbations or for patients unsuitable for which inhalation therapy. Must use the minimum dose capable of producing clinical or pathological improvement.

For example, paclitaxel prolonged therapy with low doses may include the introduction of 10-50 mg/m2every 1-4 weeks depending on response; "pulse" therapy with high doses may include the introduction of 50-250 mg/m2for a patient with an acute illness. For inhalation therapy of 0.01-1% paclitaxel can be entered directly by inhalation using the above delivering media/songs. This would lead to the delivery of 1-50 mg/m2paclitaxel directly into the respiratory tract. Friend the spine and tolerability of a particular agent.

Chronic obstructive pulmonary disease (COPD)

COPD includes various conditions (chronic bronchitis, asthmatic bronchitis, chronic obstructive bronchitis and emphysema), which lead to chronic obstruction of the respiratory tract. These conditions can cause serious disability and are the fourth leading cause of death in the United States. Clinically, they are characterized by shortness of breath, cough, stridor and recurrent respiratory tract infections. The signs of this disease include reduced FEV (forced expiratory volume in one second), increased residual volume, the mismatch V:Q and hypoxemia. Pathologically, there is increased mucus formation, hyperplasia of mucous glands, increased activity of proteases (mainly elastase), inflammation of the Airways and destruction of the alveolar walls. Despite the wide range of etiologies (most common is Smoking), the improvement of any of the above symptoms, signs or pathological processes would have a beneficial effect on this condition; therefore, antimicrotubule therapy for COPD modifies at least one of the above symptomology paclitaxel, provided at 1-50 mg/m2if necessary, in the case of systemic therapy 10-50 mg/m2provided every 1-4 weeks during prolonged introduction, or 50-250 mg/m2provided in the form of "pulse" patients with acute disease. Other antimicrotubule agents could be administered in a clinically equivalent doses.

9. Stenosis, neoplastic disease and obstruction

As noted above, this invention provides methods of treating or preventing a wide variety of diseases, concomitant obstruction of passageways of the body, including, for example, vascular diseases, neoplastic obstructi, inflammatory diseases and infectious diseases.

For example, in one aspect of this invention a wide variety described here antimicrotubule agents and compositions may be used for the treatment of vascular diseases that cause obstruction of the vascular system. Typical examples of such diseases include atherosclerosis of blood vessels (about any artery, vein or graft, including (but not only): coronary arteries, aorta, iliac arteries, carotid artery, common femoral artery, superficial Beynost); restenosis (blockage of a vessel in place of the previous intervention, such as balloon angioplasty, insertion of the stent and the graft is inserted); inflammatory and autoimmune conditions (e.g., temporal arteritis diagnostics, vasculitis).

Briefly, vascular diseases such as atherosclerosis, leukocytes, particularly monocytes and T-lymphocytes attach to endothelial cells, especially in places of branching arteries. After attaching to the endothelium leukocytes migrate through the lining of endothelial cells in response to hemostatically incentives and accumulate in an intim of the arterial wall with smooth muscle cells. This initial damage to the developing atherosclerosis is known as the "fatty streak". Monocytes in this fat strip differentiate into macrophages, and macrophages and smooth muscle cells progressing way absorb lipids and lipoprotein, becoming kantamneni (foamy) cells.

The accumulation of macrophages lying below the endothelium becomes mechanically destroyed and chemically modified oxidized lipids produced from oxygen free radicals and proteases secreted by macrophages. Foamy cells break through the surface is liannah tissues such as collagen and other proteins) with the components of blood flow leads to the attachment of platelets to the sites of damaged endothelium. The attachment of platelets and other events trigger the release and secretion of growth factors in the environment, including PDGF, platelet activating factor (PAF), IL-1 and IL-6. It is believed that these paracrine factors that stimulate migration and proliferation of cells in the vascular smooth muscle (VSMC).

In normal (non-pathological) wall of the blood vessel VSMC possess contractile phenotype and low mitotic index. However, under the influence of cytokines and growth factors released by platelets, macrophages and endothelial cells, VSMC undergo a phenotypic change from a Mature contractile cells to immature secretory cells. Converted VSMC proliferate in the environment of a wall of a blood vessel, migrate into the intima, continue to proliferate in an intim and generate large amounts of extracellular matrix. This makes developing damage in fibrous (fibrotic) the plaque. Extracellular matrix released secretory VSMC, includes collagen, elastin, glycoprotein, and a glycosaminoglycan, and collagen is the main component of the extracellular matrix of atherosclerotic plaques. Elastin and glycosaminoglycans bind lipoproteins and also contribute to growing the ins, containing smooth muscle cells and the underlying macrophages, T-cells and extracellular material.

In addition to PDGF, IL-1 and IL-6, cells are produced by other mitogenic factors which infiltrate the vessel wall, including: TGFFGF, thrombospondin, serotonin, thromboxane a2, norepinephrine, and angiotensin II. This leads to the recruitment of additional cells, the release of additional extracellular matrix and accumulation of additional lipid. This progressive increases atherosclerotic damage until it intrudes into the lumen of the vessel. First, obstructed blood flow through the tube of the vessel causes ischemia, distal relative to the atherosclerotic plaque, only if necessary, increase blood flow, and later, as further blocked artery damage, ischemia develops in the remaining part.

The increased macrophages in the atherosclerotic plaque release of oxidized lipids, free radicals, elastase and collagenase, which cause cell damage and necrosis of adjacent tissue. Damage develops necrotic Central part and turns into a complex plaque. Complex plaques are unstable rupture of blood vessel blood vessels (vasa vasorum), supplying this plaque, which leads to the blockage of the lumen due to the rapid spread of damage); or pitting and the formation of fissures (this exposes thrombogenic necrotic center part to blood flow, producing local thrombosis or distal embolization). Even if none of these effects has not occurred, the attached thrombus can be organized and included in this plaque, accelerating its growth. In addition, by increasing the local concentrations of fibrinogen and thrombin cell proliferation of vascular smooth muscles in this environment and in an intim stimulated; the process that leads eventually to the additional narrowing of the vessel.

Intima and media (middle part of the membrane of the blood vessels) normal arteries are oxygendemanding and supplied with nutrients from the lumen Eretria or from vessels vessels (vasa vasorum in the adventitia shell. With the development of atherosclerotic plaque microvessels arising from the adventitia of the vessel vessels, extend in the thickness intima and media (the middle part of the shell). This vascular network becomes more extensive as the deterioration of plaques and decreases with regression of plaques.

Bleeding from these is ulceration or thrombosis. It was also reported that the leakage of plasma proteins from these microvessels may atregional inflammatory infiltrates in this area and these inflammatory cells may contribute to the rapid growth of atherosclerotic plaques and related complications (through local swelling and inflammation).

For the treatment of vascular diseases, such as described here, antimicrotubule agent (with the carrier or without carrier) can be delivered to the outer end of the passage of the body or cells of smooth muscles through adventitious membrane of the passage of the body. Especially preferred in this regard antimicrotubule agents include, for example, taxanes (e.g. paclitaxel and docetaxel), computacin, eleutherobin, sarcodictyin, epothilone and discodermolide, deuterium oxide (D2O), hexyleneglycol (2-methyl-2,4-pentandiol), tubercidin (7-deazaadenosine), LY290181 (2-amino-4-(3-pyridyl)-4H-oil(1,2-b)Piran-3-carbonitrile), aluminum fluoride, bis(Succinimidyl) of ethylene glycol, ethyl ester of glycine, monoclonal antiidiotypic antibodies, a protein that stimulates microtubule Assembly (Taxol-like protein, TALP), cellular swelling induced by hypotonic (190 mosmol/l) conditions, insulin (100 nmol/l of the lot, lithium ion, components of the cell wall of plants (e.g., poly-L-lysine and extensin), buffer with glycerol buffer with Triton X-100, stabilizing microtubules associated with microtubule proteins (for example, MAR, MAR, Tau, big Tau, ensconsed, elongation factor 1-(EF-1) and E-MAP-115), cellular particles (e.g., histone H1, myelin basic protein and kinetochore), endogenous microtubule-based structures (e.g., axoneme structure, macromolecular complexes of enzymes that form pores in the cell membrane and GTP-caps), stable Trubeckoy the only polypeptide (e.g., STOP145 and STOP220) and tension from mitotic forces, as well as any analogues and derivatives of any of the above agents. In some embodiments, antimicrotubule agent may be an agent that is different from paclitaxel, impotecia or epothilone. Such agents, in some embodiments, can be delivered in the form of a composition together with a polymer carrier or liposomal composition, as discussed in more detail above and below. In preferred embodiments of the invention, the agents or compositions can be administered by means of a balloon catheter, oral, perivascular, using the icii can be used to treat neoplastic obstructi. Briefly, in the application here, the term "neoplastic obstruction" should be understood as neoplastic (benign or malignant) obstruction of the tubular passage body regardless of the location of the tube or histological type present malignancy. Typical examples include gastrointestinal disease (e.g., oral-farinella carcinoma, adenocarcinoma, esophageal cancer (squamous cell carcinoma, adenocarcinoma, lymphoma, melanoma), stomach cancer (adenocarcinoma, diffuse cancer of the stomach (linitis plastica), lymphoma, leiomyosarcoma), small intestine tumor (adenoma, leiomyoma, lipoma, adenocarcinoma, lipoma, carcinoid tumors), cancer of the colon (adenocarcinoma) and rectal cancer), diseases of the biliary tract (for example, neoplasma, leading to biliary obstruction, such as pancreatic cancer (running adenocarcinoma, tumor islet cells, cystadenocarcinoma), cholangiocarcinoma and hepatocellular carcinoma); lung diseases (e.g., lung cancer and/or tracheal-bronchial passages (small cell lung cancer, non-small cell lung cancer); diseases of the female reproductive organs (e.g., malignant tumors fallopieve, cancer of the epididymis, tumor semyavynosyaschego duct, tumors of the prostate gland, benign prostatic hypertrophy); and diseases of the urinary tract (e.g., renal cell carcinoma, tumors of the renal pelvis, tumors of the system for urine collection, such as transitional cell cancer, bladder and ureteral obstruction caused by benign strictures or tumors).

As an example, benign prostatic hyperplasia (national Department of standardization) is an enlargement of the prostate, in particular the Central part of the prostate that surrounds the urethra, which occurs in response to prolonged androgenic stimulation. It affects more than 80% of men aged over 50 years. This increase can lead to compression of the part of the urethra passing through the prostate gland, which leads to the blockage of the path of the outflow of the bladder, i.e., required pathologically high pressure bladder for generating the expiration of urine. In 1980 the United States was made 367000 transurethral resections of the prostate as a treatment national Department of standardization. Other treatments include retransferring pyrexia, transurethral ultrasonic method, transrectal microwave method, transrectal hyperthermia, transrectal ultrasound method and surgical removal. They all have disadvantages, including the violation of the sphincter mechanism, leading to incontinence and the formation of strictures.

For the treatment of neoplastic diseases, such as described above, a variety of therapeutic agents (without polymer carrier or polymer carrier) can be delivered to the outer end of the passage of the body or cells of smooth muscles through adventitious membrane this passage body. For example, in one preferred embodiment, the needle or catheter is directed into the prostate gland near the urethra by transrectal (or, alternatively, by transperitoneal) controlled injection with ultrasound and through this introduction deliver a therapeutic agent, preferably in several quadrants of the gland, especially around the urethra. The needle or catheter can also be placed in direct palpation or endoscopic, CT or MRI control direction and be entered at intervals. Alternatively, can be performed n the AI with the location of the stent into the urethra of the prostate. Avoid the urethral instrumentation or damage to the urethra sphincter mechanism may remain intact, thus avoiding incontinence, and the formation of strictures is unlikely.

In other aspects of the invention are provided methods of preventing or treating inflammatory diseases that affect the passages of the body or cause the obstruction of the passages of the body. Inflammatory diseases include both acute and chronic inflammation, leading to obstruction of the various tubular passageways of the body. Typical examples include vasculitis (e.g., giant cell arteritis diagnostics (temporal arteritis diagnostics, Takayasu's arteritis), Nowotny polyarteritis, allergic anghit and Wegener (disease Churg-Strauss), the syndrome of overlapping polyangiitis, allergic vasculitis (purple's disease-Seleina), serum sickness, induced by drugs vasculitis, infectious vasculitis, neoplastic vasculitis, vasculitis associated with connective-tissue disorders, vasculitis associated with congenital nedostatocnosti complement system), Wegener's granulomatosis, Kawasaki disease, vasculitis of the Central nervous system, the disease Berge is it ulcerative proctitis, primary sclerosing cholangitis, benign stricture of any origin, including idiopathic (e.g., stricture of the biliary tract, esophagus, duodenum, small intestine or colon)); respiratory disease (e.g. asthma, allergic pneumonitis, asbestosis, silicosis and other forms of pneumoconiosis, chronic bronchitis and chronic obstructive airway disease); diseases of the nasolacrimal passages (e.g., stricture of any origin, including idiopathic); and diseases of the Eustachian tubes (e.g., stricture of any origin, including idiopathic).

For the treatment of inflammatory diseases, such as described above, antimicrotubule agent (with the carrier or without carrier) can be delivered to the outer end of the passage of the body or cells of smooth muscles through adventitial shell of this passage of the body.

In other embodiments of the present invention are provided methods of treating or preventing infectious diseases that are associated with obstruction of the passage of the body or caused by obstruction of the passage of the body. Briefly, infectious diseases include some acute or chronic infsci genital tract (e.g., stricture caused by urethritis, epididymitis, prostatitis); obstruction of the female genital tract (e.g., vaginitis, cervicitis, pelvic inflammatory disease (e.g. tuberculosis, gonococcus, hamidin, Enterococcus and syphilis); obstruction of the urinary tract (eg, cystitis, urethritis); airway obstruction (eg, chronic bronchitis, tuberculosis, other mycobacterial infections (MAI and so on), anaerobic infections, fungal infections and parasitic infections); and cardio-vascular obstruction (for example, mycotic aneurysm, and endocarditis).

For the treatment of infectious diseases, such as discussed above, a wide variety of therapeutic agents (with the carrier or without carrier) can be delivered to the outer end of the passage of the body or cells of smooth muscles through adventitious membrane this passage body. Particularly preferred agents in this regard include discussed above antimicrotubule agents.

10. Graft rejection

The above antimicrotubule agents and compositions may also be used for treating or preventing transplant rejection. Briefly, the two main histological manifestations of chronic was otorcycles in surviving renal allografts (Hume et al., J. Clin. Invest. 34 : 327, 1955; Busch et al., Human Pathol. 2 : 253, 1971), and heart transplant (Johnson et al., J. Heart Transplantation 8 : 349, 1989), liver (Demetris et al., Am. J. Pathol. 118 : 151, 1985) and lung (Burke et al., Lancet I : 517 : 1986). Heart transplants are extremely sensitive to luminale narrowing due to the dependence of infarction from coronary blood flow.

Many animal models used to study chronic rejection of heart transplants. Model of heart transplantation in rats Lewis-F344 gives cardiac allografts with chronic rejection, characterized by the formation arteriosclerotic damage. This model is applicable, since more than 80% of the recipients survive for more than 3 weeks, and 90% of them find damage to the intima of the coronary vessels (Adams et al., Transplantation 53 : 1115-1119, 1992). In addition to the detection of high frequency and severity of damage, the inflammatory stage of damage can be easily detected, since this system does not require immunosuppression. Although the degree of infiltration of mononuclear cells and necrosis are more severe arterial injury in this model is very similar to clinical arteriosclerosis grafts.

Effective antimicrotubular therapy for transplant rejection m) reduced side effects, concomitant immunosuppressive therapy and, (iii) the reduction of the accelerated atherosclerosis associated with transplants.

Suitable antimicrotubule agents for the treatment of transplant rejection include taxanes (e.g. paclitaxel and docetaxel), computacin, eleutherobin, sarcodictyin, epothilone and discodermolide, deuterium oxide (D2O), hexyleneglycol (2-methyl-2,4-pentanediol), tubercidin (7-deazaadenosine), LY290181 (2-amino-4-(3-pyridyl)-4H-oil-(1,2-b)Piran-3-carbonitrile), aluminum fluoride, bis(Succinimidyl) of ethylene glycol, ethyl ester of glycine, monoclonal antiidiotypic antibodies, a protein that stimulates microtubule Assembly (Taxol-like protein, TALP), cellular swelling induced by hypotonic (190 mosmol/l) conditions, insulin (100 nmol/l) or glutamine (10 mmol/l), the binding of dynein, gibberelin, HSNO (kinesin-like protein), lysophosphatidic acid, lithium ion, components of the cell wall of plants (e.g., poly-L-lysine and extensin), buffer with glycerol buffer with Triton X-100, stabilizing microtubules associated with microtubule proteins (for example, MAR, MAR, Tau, big Tau, ensconsed, elongation factor 1-(EF-1Antimicrotubule agent may be injected with grafts in any way to achieve the above outcomes. However, preferred methods include oral administration or intravenous, subcutaneous or intramuscular injection. Antimicrotubule agent may be injected in the form of long-term therapy with low doses to prevent transplant rejection or in high doses to prevent acute transplant rejection. For example, paclitaxel in the event of a system of long-term therapy with low doses may be given at 10-50 mg/m2every 1-4 weeks depending on therapeutic response; when "pulse" therapy high doses, it can be entered at 50-250 mg/m2ka is equivalent doses adjusted relative to the activity and tolerability of a particular agent.

11. Systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a disease of unknown etiology characterized by inflammation in many different organ systems associated with the formation of antibodies reactive with nuclear, cytoplasmic antigens and antigens of the cell membranes. SLE is a common disease with an incidence of 1 in 2500 in some populations (Michet et al., Mayo Clini. Proc. 60 : 105, 1985). SLE is a disease mostly women, with incidence of 1 out of 700 women between the ages of 10 and 64 years and the ratio of women:men is 9:1. Overall annual incidence of SLE is equal to approximately 6 to 35 new cases per population of 100,000 people per year, depending on the risk of the population.

SLE represents, apparently, the complex disorder of multifactorial origin, arising from the interactions among genetic, hormonal factors and environmental factors acting simultaneously, for activation of T - and b-cells, which leads to the secretion of several types of antibodies. SLE is often classified as an autoimmune disorder characterized by uveal - AHA) and phospholipids. Antiphospholipid antibodies are present in 20-40% of patients with lupus and how it was discovered, react with a number of anionic phospholipids.

Morphological changes in SLE are extremely diverse, reflecting the variability of clinical manifestations and course of disease in individual patients. The most typical damage arise from deposition of immune complexes and are found in blood vessels, kidneys, connective tissue and skin. Acute necrotising vasculitis, covering the small arteries and arterioles, may be present in any tissue, although it most often affects the skin and muscles. In organs affected by vasculitis of small vessels, the first damage is usually characterized by infiltration of granulocytes and colourability edema. Fibrinoid deposits in the walls of blood vessels also characterize the Takayasu. In chronic cases the vessels are exposed to fibrous thickening with luminally narrowing. In the spleen, these vascular lesions cover the Central artery and characterized by a noticeable oxaloacetate fibrosis, forming a so-called "onion" (layered) damage.

Suitable antimicrotubule agents for the treatment of SLE include mold, deuterium oxide (D2O), hexyleneglycol (2-methyl-2,4-pentanediol), tubercidin (7-deazaadenosine), LY290181 (2-amino-4-(3-pyridyl)-4H-oil(1,2-b)Piran-3-carbonitrile), aluminum fluoride, bis(Succinimidyl) of ethylene glycol, ethyl ester of glycine, monoclonal antiidiotypic antibodies, a protein that stimulates microtubule Assembly (Taxol-like protein, TALP), cellular swelling induced by hypotonic (190 mosmol/l) conditions, insulin (100 nmol/l) or glutamine (10 mmol/l), the binding of dynein, gibberelin, HSNO (kinesin-like protein), lysophosphatidic acid, lithium ion, components of the cell wall of plants (e.g., poly-L-lysine and extensin), buffer with glycerol buffer with Triton X-100, stabilizing microtubules associated with microtubule proteins (for example, MAR, MAR, Tau, big Tau, ensconsed, elongation factor 1-, (EF-1) and E-MAP-115), cellular particles (e.g., histone H1, myelin basic protein and kinetochore), endogenous microtubule-based structures (e.g., axoneme patterns, makromolekulare enzyme complexes that form pores in the cell membrane and GTP-caps), stable Trubeckoy the only polypeptide (e.g., STOP145 and is such agents, in some embodiments, can be delivered in the form of a composition together with a polymer carrier or liposomal composition, as discussed in more detail above and below. In preferred embodiments of the invention, the agents or compositions can be administered intranasally, systematically, by inhalation, or topically (for example, in the case of polyps in the nose).

PREPARATION of READY PREPARATIVE FORMS AND INTRODUCTION

As noted above, antimicrotubule agents of this invention can be prepared in various forms (for example, in the form of microspheres, pastes, films, sprays, ointments, creams, gels, etc). In addition, the compositions of this invention can be prepared so that they contain more than one antimicrotubule agent, contain many additional connections have certain physical properties (e.g. elasticity, specific melting point or a specific allocation rate). In some embodiments of the invention compositions can be combined to achieve both quick and slow or sustained release of one or more antimicrotubule agents.

Antimicrotubule agents can be administered either alone or in combination with Fermi must be non-toxic in relation to recipients in the applied doses and concentrations. Typically, the preparation of such compositions involves combining therapeutic agent with buffers, antioxidants such as ascorbic acid, low molecular weight (less than 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with serum albumin nonspecific serum, are suitable examples of solvents.

As noted above, the claimed antimicrotubule agents, compositions, or pharmaceutical compositions can be prepared for the introduction of a number of different ways, including, for example, topically at the site of inflammation, oral, rectal, intracranial, vnutriobolochechnoe through the nose, vnutriglazna, intravenously, subcutaneously, intraperitoneally, intramuscularly, sublingual and intravesical. Other preferred routes of administration include direct introduction (preferably with ultrasound, CT, fluoroscopic, MRI or endoscopic control input direction) to the site of disease.

Therapeutic agents, therapeutic cnym material, which provides instructions regarding the use of such materials. Typically, these instructions comprise a description of reagent concentration, as well as, in some embodiments, the relative amounts of ingredients, fillers or diluents (e.g., water, saline or SFR), which may be necessary to recreate antimicrotubule agent, antimicrotubule compositions or pharmaceutical compositions.

The following examples are presented to illustrate but not to limit.

EXAMPLES

As discussed above, chronic inflammation is a process characterized by tissue infiltration by leukocytes (macrophages, lymphocytes, neutrophils and plasma cells), tissue destruction by inflammatory cells and cell products (reactive oxygen molecules that destroy tissue enzymes such as matrix metalloproteinases) and repeated repair attempts by compensation connective tissue (angiogenesis and fibrosis).

To assess antimicrotubule agents on their ability to act on chronic inflammation use the following pathological/biological end resultantly cascade; (2) the inhibition of proliferation of mesenchymal cells (fibroblasts, synoviocytes and so on), which leads to the development of fibrosis and loss of function of an organ; (3) inhibition of the production/activity of matrix metalloproteinases, which causes tissue damage; (4) violation of angiogenesis, which may enhance the inflammatory response and to provide metabolic support the growth and development of fibrous tissue; and (5) all this should be achieved without significant toxicity to normal parenchymal cells or disruption of the synthesis of matrix components (e.g., collagen and proteoglycans).

As described in more detail below, the activity of the agents, stabilizing microtubules, such as, for example, paclitaxel, experienced in multiple tissues and inflammatory pathological conditions. These agents demonstrate the ability to modify the above indicators of the disease.

EXAMPLE 1

ACTION ANTIMICROTUBULE AGENTS

ON the ACTIVITY of NEUTROPHILS

This example describes the effect antimicrotubule agents on the response of neutrophils stimulated opsonization CPPD crystals or opsonization simhasanam. As shown by the experiments described below, antimi is arena on chemiluminescene, the production of superoxide anion and degranulation in response to opsonisation the microcrystals or zymosan.

A. Materials and methods

Throughout this study used a balanced salt solution Hanks (SRH). All chemicals were purchased at Sigma Chemical Co. (St. Louis, MO), unless otherwise noted. All experiments were performed at 37°C, unless otherwise noted.

1. Fabrication AND CHARACTERIZATION of CRYSTALS

Received CPPD crystals (triclinic). The size distribution of these crystals was approximately 33% less than 10 microns, 58% between 10 and 20 microns and 9% more than 20 μm. The crystals obtained under the above described conditions are pyrogen-free, and the crystals obtained in sterile, pyrogen-free conditions, give the same amplitude response of neutrophils, and that the crystals obtained under normal, non-sterile laboratory conditions.

2. OPSONIZATION of CRYSTALS AND ZYMOSAN

All the experiments investigated the response of neutrophils to the crystals or zymosan in the presence of paclitaxel, was performed using opsonizing plasma CPPD or zymosan. The opsonization of crystals or zymosan performed with 50% heparinization plasma at a concentration of 75 mg of CPPD or 12 mg Simeona per ml of 50% plasma. The crystals their NEUTROPHILS

Neutrophils were obtained from freshly processed citrate whole blood person. Briefly, 400 ml of blood was mixed with 80 ml of 4% dextran T500 (Pharmacia LKB, Biotechnology AB, Uppsala, Sweden) in SRH and allowed to settle for 1 hour. Plasma was collected continuously and 5 ml was applied to a 5 ml Ficoll Paque (Pharmacia) in polypropylene tubes 15 ml (Coming, NY). After centrifugation at 500 g for 30 minutes precipitation neutrophils were washed from the red blood cells 20 by 20-second hypotonic shock. Neutrophils resuspendable in SRH, kept on ice and used for experiments within 3 hours. The viability and purity of neutrophils was always over 90%.

4. INCUBATION of NEUTROPHILS WITH ANTIMICROTUBULE AGENTS

(a) Paclitaxel

The original solution of paclitaxel at a concentration of 12 mm in dimethyl sulfoxide (DMSO) were prepared in the form of fresh solution before each experiment. This original solution was diluted in DMSO to obtain solutions of paclitaxel in a concentration range of 1-10 mm. Equal volumes of these diluted solutions of paclitaxel was added to neutrophils at a concentration of 5,000,000 cells per ml with gentle mixing by vortex with obtaining concentrations of 0-50 µm final concentration of DMSO of 0.5%. Tile is and zymosan.

(b) an aluminum Fluoride

The original solution of aluminum fluoride (lF3) at a concentration of 1 M in SRH cooked in a fresh solution. This original solution was diluted in SRH obtaining solutions lF3in the concentration range of 5-100 mm. Equal volumes (50 µl) of these diluted solutions lF3added to neutrophils at 5,000,000 cells per ml and incubated for 15 minutes at 37°C. was Added luminal (1 μm) and then 20 μl opsonizing zymosan (final concentration = 1 mg/ml) for cell activation.

(c) Ethyl ester of glycine

The original solution of the ethyl ester of glycine was prepared at a concentration of 100 mm in SRH in the form of fresh solution. This original solution was diluted in SRH obtaining solutions of ethyl ester of glycine in a concentration range of 0.5-10 mm. Equal volumes (50 µl) of these diluted solutions of ethyl ester of glycine was added to neutrophils at 5,000,000 cells per ml and incubated for 15 minutes at 37°C. was Added luminal (1 μm) and then 20 μl opsonizing zymosan (final concentration = 1 mg/ml) for cell activation.

(d) LY290181

The original solution LY290181 was prepared at a concentration of 100 μm in SRH in the form of fresh solution. This original solution was diluted in SRH with getting the Lyali to neutrophils at 5,000,000 cells per ml and incubated for 15 minutes at 37°C. Added luminal (1 μm) and then 20 μl opsonizing zymosan (final concentration = 1 mg/ml) for cell activation.

5. CHEMILUMINESCENT TEST

All studies chemiluminescence was performed at a concentration of 5,000,000 cells/ml in SRH with CPPD (50 mg/ml). In all experiments, 0.5 ml of cells were added to 25 mg of CPPD or 0.5 mg zymosan in Eppendorf tubes with lids 1.5 ml 10 ál lyuminola dissolved in 25% DMSO in SRH, was added to a final concentration of 1 μm and the samples were mixed to initiate the activation of neutrophils crystals or simhasanam. The chemiluminescence was observed using lumenera LKB Luminometer (Model 1250) at 37°C for 20 minutes with shaking immediately before the measurements to resuspendable crystals or zymosan. Control tubes contained cells, medicines and luminal (the crystals were absent).

6. The GENERATION of SUPEROXIDE ANION

The concentration of superoxide anion was measured using the test inhibiting superoxide dismutase recovery of cytochrome C. Briefly, 25 mg of crystals or 0.5 mg zymosan was placed in a test tube of Eppendorf with lid for 1.5 ml and heated to 37°C. was Added 0.5 ml of cells at 37°With together with ferricytochrome (ConECs the s time the tubes were centrifuged at 10000 g for 10 seconds and the supernatant was collected for measurement of the absorption at 550 nm. Control tubes were kept under the same conditions with the inclusion of superoxide dismutase at 600 units per ml.

7. TEST DEGRANULATION of NEUTROPHILS

Tubes Eppendorf-half milliliter containing either 25 mg of CPPD or 1 mg zymosan, pre-heated to 37°C. 0.5 ml of cells at 37°C was added, followed by strong shaking to initiate reactions. At suitable points in time the tubes were centrifuged at 10000 g for 10 seconds and 0.4 ml of the supernatant was stored at -20°C for subsequent analysis.

Secrete lysozyme was measured by the decrease in absorption at 450 nm suspension of Micrococcus lysodeikticus. Briefly, Micrococcus lysodeikticus suspended at 0.1 mg/ml in 65 mm potassium-phosphate buffer, pH of 6.2, and the absorption at 450 nm was brought up to 0.7 units dilution. The crystal (or zymosan) and cell supernatant (100 ál) was added to 2.5 ml of a suspension of Micrococcus and observed a decrease in absorption. Prepared standards lysozyme (protein chicken eggs) in the range 0-2000 units/ml and receive a calibration curve of the concentration of the lysozyme, depending on the speed of decrease of the absorption at 450 nm.

The activity of myeloperoxidase (MPO) was measured by the increase of absorption at 450 nm, which is accompanied by the oxidation of dianisidine. 7.8 mg of dianisidine Rustaveli 0,89 ml of dianisidine followed by adding 50 μl of 1% Triton X-100, 10 μl of 0.05% solution of hydrogen peroxide in water and 50 μl of the supernatant of the cells with crystal. The activity of MPO was determined on the basis of changes in absorbance (450 nm) per minute, Delta450, using the following equation:

Oxidation of dianisidine (nmol/min) = 50 × Delta450

8. The VIABILITY of NEUTROPHILS

To determine the effect of antimicrotubule agents on the viability of neutrophils was measured by the release of marker enzymes in the cytoplasm, lactate dehydrogenase (LDH). Control tubes containing cells with a drug (without crystals) from experiments on the degranulation also analyzed for LDH.

C. Results

In all experiments, statistical significance was determined using t-test, t-test, and significance was at p<0,05. In those cases, when shown the error bars, they describe the standard deviation around the average value for a specified number n.

1. The VIABILITY of NEUTROPHILS

(a) Paclitaxel

Neutrophils treated with paclitaxel at 46 μm for one hour at 37°C, did not find any increased level of LDH release (always less than 5% of the total the Oia

Neutrophils treated with aluminum fluoride in a concentration range of 5-100 mm for 1 hour at 37°C, did not find any increased level of LDH release above controls, which indicates that the aluminum fluoride did not cause cell death.

(c) Ethyl ester of glycine

Neutrophils treated with ethyl ester of glycine in a concentration range of 0.5-20 mm for 1 hour at 37°C, did not find any increased level of LDH release above control that indicates that the ethyl ester of glycine did not cause cell death.

2. CHEMILUMINESCENCE

(a) Paclitaxel

Paclitaxel at 28 μm was produced strong inhibition induced opsonization plasma CPPD and induced opsonization plasma simhasanam chemiluminescence of neutrophils, as shown in figures 1A, 1B and 2A, respectively. Inhibition of the maximum response chemiluminescence was 52% (+/-12%) and 45% (+/-11%) for CPPD and zymosan respectively. Inhibition by paclitaxel at 28 μm as induced opsonization plasma CPPD and induced opsonization plasma simhasanam chemiluminescence of neutrophils was significant at all time points from 3 to 16 minutes (figures 1 and 4A). Figure 1m plasma CPPD chemiluminescence of neutrophils. In all experiments, a control sample never gave values of chemiluminescence more than 5 mV and the addition of paclitaxel at all concentrations used in this study had no effect on values of chemiluminescence controls.

(b) an aluminum Fluoride

The aluminum fluoride at concentrations of 5-100 mm produced a strong inhibition induced opsonization plasma simhasanam chemiluminescence of neutrophils, as shown in figure 1C. This figure shows the concentration dependence of inhibition lF3induced opsonization plasma simhasanam chemiluminescence of neutrophils. Adding AlF3at all concentrations used in this study had no effect on values of chemiluminescence controls.

(c) Ethyl ester of glycine

Ethyl ester of glycine at concentrations of 0.5-20 mm produced a strong inhibition induced opsonization plasma simhasanam chemiluminescence of neutrophils, as shown in figure 1D. This figure shows the concentration dependence of inhibition of the ethyl ester of glycine induced opsonization plasma simhasanam chemiluminescence of neutrophils. Add ethyl ester of glycine in all co

LY290181 in concentrations of 0.5-50 μm produced a strong inhibition induced opsonization plasma simhasanam chemiluminescence of neutrophils, as shown in figure 1E. This figure shows the concentration dependence of inhibition LY290181 induced opsonization plasma simhasanam chemiluminescence of neutrophils. Adding LY290181 at all concentrations used in this study had no effect on values of chemiluminescence controls.

3. The GENERATION of SUPEROXIDE

The time course of induced opsonization CPPD crystal production of superoxide anion, measured inhibiting superoxide dismutase (SOD) recovery of cytochrome C, are shown in figure 3. Treatment of cells with paclitaxel at 28 μm produced a decrease in the amount of superoxide generated in all time points. This reduction was significant at all time points, are shown in figure 3A. The concentration dependence of this inhibition are shown in figure 3B. Stimulation of the production of superoxide anion opsonization simhasanam (figure 4B) showed the same time course with CPPD-induced activation. The inhibition induced simhasanam producyrovaniem all time points, shown in figure 4B.

Processing-induced CPPD crystal neutrophils LY290181 at 17 µm also produced a decrease in the number of generated superoxide (figure 3C).

4. DEGRANULATION of NEUTROPHILS

The degranulation of neutrophils was observed in induced panierowany plasma CPPD crystals release of myeloperoxidase and lysozyme or induced opsonization plasma simhasanam the release of myeloperoxidase. It was shown that sufficient amounts of these two enzymes are released in the extracellular environment, when covered with plasma CPPD crystals are used for stimulation of neutrophils, without having to add cytochalasin In to the cells. Figures 5 and 2 show the time course of release of MPO and lysozyme respectively from neutrophils stimulated with coated plasma CPPD crystals. Figure 5A shows that paclitaxel inhibits the release of myeloperoxidase from activated opsonization CPPD neutrophils in the first 9 minutes of incubation crystal cells. Paclitaxel significantly inhibited CPPD-induced release of myeloperoxidase in all time points, as shown in figure 5A. Figure 5B shows the concentration dependence and Igal the release of lysozyme and this inhibition of degranulation was significant at all time points, as shown in figure 2.

Only a small number of MPO and lysozyme released when neutrophils were simplerules opsonization simhasanam. Despite these low levels, it was possible to observe a 50% inhibition of release of LRO after 9 minutes of incubation in the presence of paclitaxel at a concentration of 28 mm, which was statistically significant (p<0,05) (data not shown). Processing induced by CPPD crystals of neutrophils LY290181 at a concentration of 17 μm reduced the release as lysozyme and myeloperoxidase from these cells (figures 5C and 5D).

C. Discussion

These experiments show that paclitaxel and other antimicrotubule agents are strong inhibitors induced crystals activation of neutrophils. In addition, due to the detection of the same levels of inhibition reactions of neutrophils in another form consisting of particles of activator opsonizing zymosan, it is evident that the inhibitory activity of paclitaxel and other antimicrotubule agents is not limited to the reactions of neutrophils in the crystals. It has been shown that paclitaxel, aluminum fluoride, ethyl ester of glycine and LY290181 are strong inhibitors induced simhasanam activation of neutrons and the degranulation induced by CPPD crystals of neutrophils.

EXAMPLE 2

T-CELL RESPONSE TO ANTIGENIC STIMULUS

To determine whether paclitaxel on the activation of T cells in response to stimulate, T-cell clones TR1 stimulated or peptide of the myelin basic protein, GP68-88 or by the lectins CONA, within 48 hours in the absence or in the presence of increasing concentrations of paclitaxel in the micelle composition. Paclitaxel was added at the beginning of the experiment or 24 hours after stimulation of cells with peptide or Kona. The inclusion titiraupenga thymidine was determined as a measure proliferation of T cells in response to stimulation by the peptide or Kona.

The results showed that stimulation of T cells was increased in response to peptide GP68-88 and Kona. In the presence of a control micellar polymer molecules stimulation of T cells in response to both agonists was not changed. However, processing micelles for paclitaxel or at the beginning of the experiment, or 24 hours after stimulation was reduced T-cell response is dependent on concentration. In both conditions, the proliferation of T-cells completely inhibited 0,02 µm paclitaxel (figure 79).

These results indicate that paclitaxel is a strong inhibitor of proliferation of T cells in response to induced the Antiga is iment conducted to evaluate the effect of different concentrations of paclitaxel for inclusion titiraupenga thymidine (measurement of DNA synthesis of synoviocytes) and cell proliferation in vitro.

A. Materials and methods

1. The INCLUSION of3H-THYMIDINE IN SYNOVIOCYTE

Synoviocyte incubated with various concentrations of paclitaxel (10-5M, 10-6M, 10-7M and 10-8M) continuously for 6 or 24 hours in vitro. These time points were added to 1×10-6pulse/min3H-thymidine to the cell culture and incubated for 2 hours at 37°C. the Cells were collected using a harvester cells were washed through the filter, the filters cut and determined the amount of radiation contained in the pieces of the filter. After determining the amount of thymidine incorporated into the cells, this number is used to determine the speed of cell proliferation. The experiment was repeated three times and the results were compared.

2. The PROLIFERATION of SYNOVIOCYTES

Bovine synovial fibroblasts were grown in the presence and absence of various concentrations (10-5M, 10-6M, 10-7M and 10-8M) of paclitaxel for 24 hours. At the end of this period of time was determined visually the total number of viable cells of synoviocytes by calculating the displacement of the dye using coloring Trifanova blue. This experiment was performed 4 times and the results were compared.

C. Sharp concentrations inhibits the inclusion of3H-thymidine (and in a broader sense DNA synthesis) in synoviocyte in such low concentrations as 10-8M At six hours was not significant difference between the degree of inhibition produced higher concentrations compared to lower concentrations of paclitaxel (figure 8). However, at 24 hours, some portion of the effect was lost at lower concentrations of drug (10-8M), but the inclusion was still lower than that observed in control animals.

2. The PROLIFERATION of SYNOVIOCYTES

This study showed that paclitaxel was cytotoxic against proliferation of synovial fibroblasts and its cytotoxicity was dependent on concentration. Paclitaxel in such low concentrations as 10-7M, is able to inhibit the proliferation of synoviocytes (figure 9). At higher concentrations of paclitaxel (10-6M and 10-5M) this drug was toxic in respect of synovial fibroblasts in vitro.

C. Discussion

The above study shows that paclitaxel is able to inhibit the proliferation of fibroblasts derived from synovia at relatively low concentrations in vitro. Thus, in the inflammatory diseases of blocking cellular proliferation will favorably influence the outcome of the disease in vivo.

EXAMPLE 4

The CHARACTERISTIC ACTIVITY of PACLITAXEL ON EPIDERMAL

The HUMAN KERATINOCYTES in vitro

Investigated time-dependent and dose effect of paclitaxel on actively proliferating keratinocytes of the healthy person and the keratinocytes Nasat (spontaneously immortalized epidermal keratinocytes).

A. Materials and methods

Effect of paclitaxel on keratinocytes was assessed by determining the number of cells and including3H-thymidine these cells. To enable thymidine keratinocytes, seeded at low density in DMEM, supplemented with 10% FCS, glutamine, antibiotics), were treated with concentrations of paclitaxel from 0 to 10-4M for 6 hours during the logarithmic phase of growth. To the cells was added3H-thymidine and incubated them for another 6 hours. The cells were collected and radioactivity was determined. To determine the total number of cells keratinocytes were sown as described above, and incubated in the presence and in the absence of paclitaxel for 4 days. After incubation the cells were collected and counted using a test wytestone Trypanosoma blue.

C. Results

To determine the number of viable cells in percent of the untreated kontrola, while 10-8M viability was less than 87% (figure 7). Had a significant drop in cell viability at concentrations of paclitaxel 10-7M or higher concentration.

C. Discussion

Paclitaxel was extremely cytotoxic against human keratinocytes in such low concentrations as 10-7M In the case of psoriasis keratinocytes are atypical proliferating cells and as paclitaxel stabilizes microtubules, you can see his influence in this mitotically active system. In other studies it was found that paclitaxel is a cytotoxic against proliferating synoviocytes, but does not affect or neproliferirute chondrocytes. Thus, paclitaxel can act on hyperproliferative cells in lesions of the prostate, although it is non-toxic with respect to the normal epidermal cells.

EXAMPLE 5

EFFECT of PACLITAXEL ON PROLIFERATION of ASTROCYTES

It was determined that the damage in PC (multiple sclerosis) is an increase in fibrous) astrocytes, which are believed to participate in the destruction of myelin by producing cytokines and metalloproteinases m and glial fibrillar acidic protein (GFAP), which serves as a biochemical marker for proliferation of fibrous astrocytes. The ability of micelles for paclitaxel to inhibit the proliferation of astrocytes was evaluated in the model demyelinizing disease transgenic mice (Mastronardi et al., J. Neurosci. Res. 36: 315-324, 1993).

A. Materials and methods

Continuous therapy with subcutaneous injection of paclitaxel (2 mg/kg; 3× a week, a total of 10 injections) started at clinical appearance of the disease (at the age of approximately 4 months). Five animals received micellar paclitaxel, two mice were used as controls; one mouse was unhandled standard mouse and one was raw odnomomentnoe transgenic mouse. Only one transgenic mouse was used as control, as the course of the disease was well established in this laboratory. Four animals were injected with micellar paclitaxel after it was obvious the initial signs of neurological pathology PC.

Three days after the tenth injection of this experimental study was completed and the brain tissue was processed for histological analysis. For light microscopy, tissues were fixed in formalin and poured (patch) in paraffin. Giovannini with NRR. The sections were stained for NRR and secondarily stained with hematoxylin. For electron microscopy, tissues were fixed in 2,5% glutaraldehyde the aldehyde and phosphate buffered saline (pH of 7.2) and then fixed with 1% osmium tetroxide. Cooked sliced and viewed them in a transmission electron microscope EAT JEOL 1200 EX II.

C. Results

With progression of neurological pathology GFAP levels increased in the brain of transgenic mice; this, according to the authors, reflects the increase in the number of attendees fibrous astrocytes. In contrast, transgenic mice treated with paclitaxel, have near normal levels of GFAP (table 1). These results suggest that paclitaxel may inhibit the proliferation of astrocytes in vivo, which may contribute to the prevention of demyelination PC.

Additionally GFAP in brain tissue was evaluated histologically. Figure 78 illustrates the slices of the brain of normal mice, transgenic control mice not treated with paclitaxel, and transgenic mice treated with paclitaxel.

Although the control transgenic mice have higher numbers of fibrous astrocytes, the morphology of these astrocytes Odinak However, in transgenic mice, treated with paclitaxel, the number of fibrous astrocytes was significantly reduced. In addition, watched two morphological changes: the cell body of fibrous astrocytes seems noticeably rounding (which, as we have seen, leads to apoptosis in culture) and cellular processes become very thin around the cell body.

Additional analysis of the ultrastructure using electron microscope showed that astrocytes of transgenic mice are characterized by densely painted processes of astrocytes extending from the cell body. These broad processes contain a well-organized series of filaments (fibers), which proves viable, the activated cell. However, the morphology of astrocytes in transgenic mice treated with paclitaxel, characterized by rounding of cells, thin fibrous processes and intracellular depletion and disruption of protein filaments.

C. Conclusions

These results indicate that paclitaxel induces changes in fibrous astrocytes in vivo, most of the proliferating cell type in damage to the PC. It is possible that paclitaxel inhibits the function of the processes of astrocytes and, therefore, may alter the cellular events involved in d definitions inhibits whether paclitaxel proliferation of endothelial cells, EOMA (lines endothelial cells) were sown at low density and incubated in the absence and in the presence of increasing concentrations of paclitaxel for 48 hours. After incubation to determine the number of viable cells using a test displacement Trypanosoma blue. The results (presented in figure 9) show that paclitaxel at concentrations of 10-8M inhibited the proliferation of endothelial cells by more than 50%, and a concentration of 10-7M or more fully inhibited cell proliferation. All tests of cellular toxicity was performed three times and each measurement was performed in triplicate.

To determine the effect of paclitaxel on turnover and apoptosis of endothelial cells cells EOMA incubated in the absence and in the presence of increasing concentrations of paclitaxel for 24 hours. The cells were fixed with 3.7% formaldehyde in phosphate buffered saline for 20 minutes, stained with DAPI (4’-6-diamidino-2-phenyl-indole), 1 μg/ml, and were studied by means of lens 40× under epifluorescent optics. Apoptotic cells was evaluated by counting cells on fraglie, than 10-8M, induce apoptosis of endothelial cells (figure 10).

EXAMPLE 7

The TEST PROTOCOL PROLIFERATION (MTT)

On the first day of 5-10×104synoviocytes were sown per well (96-well plate). Column No. 1 was kept free from cells (blind experiment). On the second day Wednesday shook clicks and added 200 μl of medium containing various concentrations of the drug. Cells were exposed for 6 hours, 24 hours or 4 days. The drug was not added in columns # 1 and # 2 (the blind experience and untreated control, respectively). The medium containing the drug was thrown out and was added 200 μl of fresh complete medium. Then the cells were allowed to grow for 3-4 days. On the fifth day was added 20 μl bromide salt dimethylterephthalate (MTT) (5 mg/ml SPR) and allowed to incubated for 4 hours at 37°C. Environment decantation and was added 200 μl of DMSO. The tablet was shaken for 30 minutes and the absorption was recorded at 562 nm.

Results

The results were expressed as % survival, which was obtained by dividing the number of cells remaining after treatment, the number of cells in untreated control column No. 2 (this is the number of cells was obtained from a standard prigotovleie to be interpolated from figures 11A-E. In the case of a 24-hour exposure was found that the combination of LY290181 is the most powerful antimicrotubule agent in respect of the reduction and inhibition of cell proliferation with IC50less than 5 nm (figure C). Paclitaxel, epothilone In and tubercidin were somewhat less strong, with IC50about 30 nm (figure A), 45 nm (figure F) and 45 nm (figure), respectively. Finally, the IC50for aluminum fluoride (lF3and hexyleneglycol were significantly higher, with values of about 32 μm (figure E) and 64 mm (figure D), respectively.

EXAMPLE 8

EFFECT of PACLITAXEL AND OTHER ANTIMICROTUBULE AGENTS ON the PRODUCTION of MATRIX METALLOPROTEINASES

A. Materials and methods

1. STIMULATED IL-1 TRANSCRIPTIONAL ACTIVITY of AP-1 is INHIBITED by PACLITAXEL

The chondrocytes were transfusional structures containing run AP-1 reporter gene CAT (chlorophenylacetyl-transferase), and stimulated with IL-1, was added to IL-1 (50 ng/ml) and incubated for 24 hours in the absence and in the presence of paclitaxel at various concentrations. Treatment with paclitaxel was reduced CAT activity dependent on the concentration follows (mean ± SD (standard deviation)). Results marked with an asterisk (litty represent three independent experiments.

2. EFFECT of PACLITAXEL ON INDUCED IL-1 DNA-BINDING ACTIVITY of AP-1 DNA AP-1

Binding activity was determined with a radioactively labeled probe sequences AR-1, and with the help of test bias mobility in the gel. Extracts from chondrocytes, not treated or treated with different amounts of paclitaxel (10-7-10-5M), followed by the addition of IL-1(20 ng/ml) were incubated with an excess of probe on ice for 30 minutes followed sedentarism gel electrophoresis. The track "com" contains an excess of unlabeled oligonucleotide AR-1. The results represent three independent experiments.

3. EFFECT of PACLITAXEL ON INDUCED IL-1 mRNA EXPRESSION of MMP-1 And MMP-3

Cells were treated with paclitaxel at various concentrations (10-7-10-5M) for 24 hours. Then they were treated with IL-1(20 ng/ml) for 18 hours in the presence of paclitaxel. Total RNA was isolated and mRNA levels of MMP-1 was determined by Northern blot analysis. Then the blots was stripped and re-probed32R-radioactively labeled cDNA of rat GAPDH, which was used as a gene "household" (bond is init the mRNA expression of collagenase-1 and stromelysin. The levels of expression of MMP-1 and MMP-3 were normalized with GAPDH.

4. The EFFECTS of OTHER ANTIMICROTUBULE AGENTS ON the EXPRESSION of COLLAGENASE

Primary cultures of chondrocytes were isolated from cartilage of calf in the form of fresh chondrocytes. Cells were sown at a 2.5×106cells per ml in culture tablets 100×20 mm and incubated in medium Ham F12 containing 5% FCS, over night at 37°C. the Cells were starved in serum-free medium overnight and then treated them antimicrotubule agents at various concentrations for 6 hours. Then for each tablet was added to IL-1 (20 mg/ml) and the plates were incubated for another 18 hours. Total RNA was isolated according to the method acidified with guanidinosuccinic and subjected to electrophoresis on a denaturing gel. Denatured samples RNA (15 µg) were analyzed by gel-electrophoresis in 1% denaturing gel, transferred to nylon membrane and hybridized with32P-labeled cDNA probe collagenase.32P-labeled cDNA of glyceraldehydes (GAPDH) was used as internal standard to ensure approximately the same load. Exposed films were scanned and quantitatively analyzed using ImageQuant.

C. Results

1. The PROMOTION is held transcriptional elements AP-1 and REA-3, except Gelatinase Century it Was well established that the expression of matrix metalloproteinases, such as collagenase and stromelysin depends on the activation of transcription factors AP-1. Thus, inhibitors of AP-1 to inhibit the expression of matrix metalloproteinases.

2. EFFECT of PACLITAXEL AT the TRANSCRIPTIONAL ACTIVITY of AP-1

As shown in figure 19C, IL-1 stimulated the transcriptional activity of AP-1 in 5 times. Preprocessing temporarily transfected chondrocytes by paclitaxel reduced induced IL-1 activity of the reporter gene CAT AP-1. Thus, induced IL-1 activity of AP-1 was reduced in chondrocytes by paclitaxel dependent on the concentration of way (10-7-10-5M). These results showed that paclitaxel is a strong inhibitor of the activity of AP-1 in chondrocytes.

3. EFFECT of PACLITAXEL ON DNA-BINDING ACTIVITY of AP-1

To confirm that inhibition by paclitaxel activity of AP-1 is not due to a nonspecific effect, studied the effect of paclitaxel on induced IL-1 binding to AP-1 oligonucleotides with nuclear lysates of chondrocytes. As shown in figure 19 (C), induced IL-1 binding activity is up>-5M within 24 hours. Inhibition by paclitaxel transcriptional activity of AP-1 was strongly correlated with a decrease in the binding of AP-1 to DNA.

4. EFFECT of PACLITAXEL ON the EXPRESSION of COLLAGENASE AND STROMELYSIN

Because paclitaxel was a strong inhibitor of the activity of AP-1, studied the effect of paclitaxel on induced IL-1 expression of collagenase and stromelysin. Briefly, as shown in figure 20, IL-1 increases the mRNA levels of collagenase and stromelysin in chondrocytes. Pretreatment of chondrocytes with paclitaxel for 24 hours significantly reduced the mRNA levels of collagenase and stromelysin. At 10-5M paclitaxel, there was complete inhibition. These results indicate that paclitaxel completely inhibited the expression of two matrix metalloproteinases in concentrations similar to the concentrations, any abscopal activity of AP-1.

5. The EFFECTS of OTHER ANTITOBACCO AGENTS ON the EXPRESSION of COLLAGENASE

Figures 12A-N show that antimicrotubule agents inhibited the expression of collagenase. Expression of collagenase was stimulated by the addition of IL-1, which is a proinflammatory cytokine. Preincubate chondrocytes with various antimicrotubule agents in the belly and bis-(succinimidylester) of ethylene glycol, prevented induced IL-1 expression of collagenase in such low concentrations as 1×10-7M

C. Discussion

Paclitaxel was able to inhibit the expression of collagenase and stromelysin in vitro at concentrations of 10-6M. Because this inhibition may be due to inhibition of the activity of AP-1, a necessary stage in the induction of all matrix metalloproteinases except gelatinase, it is expected that paclitaxel should inhibit and other matrix metalloproteinases, which are AP-1-dependent. The levels of these matrix metalloproteinases are elevated in all inflammatory diseases and play an important role in the decomposition matrix, cell migration and proliferation and angiogenesis. Thus, inhibition by paclitaxel expression of matrix metalloproteinases, such as collagenase and stromelysin, will have a beneficial effect in inflammatory diseases.

EXAMPLE 9

ACTION ANTIMICROTUBULE AGENTS ON the EXPRESSION of PROTEOGLYCANS

Primary cultures of chondrocytes were isolated from cartilage of calf in the form of fresh chondrocytes. Cells were sown at a 2.5×106cells per ml in culture tablets 100×20 mm and incubated in medium Ham is x processed antimicrotubule agents at various concentrations (10-7M, 10-6M, 10-5M and 10-4M) for 6 hours. Then for each tablet was added to IL-1 (20 mg/ml) and the plates were incubated for another 18 hours. Total RNA was isolated according to the method acidified with guanidinosuccinic and subjected to electrophoresis on a denaturing gel. Denatured samples RNA (15 µg) were analyzed by gel-electrophoresis in 1% denaturing gel, transferred to nylon membrane and hybridized with32P-labeled cDNA probe of proteoglycan (aggrecan).32P-labeled cDNA of glyceraldehydes (GAPDH) was used as internal standard to ensure approximately the same load. Exposed films were scanned and quantitatively analyzed using ImageQuant.

Results

Figures 13A-H show that antimicrotubule agents who had inhibitory effect on the expression of collagenase (example 8), in particular LY290181, hexyleneglycol, deuterium oxide, ethyl ester of glycine, lF3tubercidin, epothilone and bis-(Succinimidyl) ethylene glycol had no effect on the expression of aggrecan, a major component of cartilage matrix, at all tested concentrations.

EXAMPLE 10

TEST the activity of NF-KB (CELL-BASED)

IL-1 and TNF transcriptional factor called NF-KB, is also involved in inflammatory processes. The inflammatory effect of IL-1 and TNF can therefore be estimated indirectly using the test reporter gene (NF-KB) that respond to stimulation by IL-1 and TNF.

On the first day, 5×104NIH-3T3 (mouse fibroblast), stably transfected with the reporter construct NF-kV (Luciferase, Promega Corp.), were sown per well (24 well plate). After reaching confluentes (3-4 day) cells was starving when replacing full environment 1 ml serum-free medium. After a 24-hour starvation cells were treated with various concentrations antimicrotubule agents within 6 hours before the addition of IL-1 (20 ng/ml) and TNF (20 ng/ml). Cells were exposed to IL-1 and TNF for 1 hour and 16 hours and the activity of NF-KB was measured after 24 hours. On the fifth day, the medium was discarded and cells were washed once SFR. Then the cells were extracted for 15 minutes with 250 ál lisanova buffer (Promega Corp., Wisconsin). The transcriptional activity of NF-KB was measured by adding 25 μl of luciferase substrate in a test tube containing 2.5 μl of cell extract. The tube was immediately inserted into the luminometer (Turner Designs) and light emission was measured for 10 seconds. Then the results of the luciferase test normalized in otnoshenii induction of NF-KB (induction into the specified number of times). As shown in figure 80A, 80V, 80C and 80D, tubercidin and paclitaxel inhibited the activity of NF-KB induced by both IL-1 and TNF. Inhibitory effect of tubercidin and paclitaxel with 6-hour and 24-hour treatments were equal to 10 μm and 2 μm, respectively.

EXAMPLE 11

INHIBITION of TUMOR ANGIOGENESIS by PACLITAXEL

Fertilized embryos homemade chicken were incubated for 4 days before removing their shells. Egg contents were isolated by removal of the sheath located around the airspace, and output the contents of the eggs cautious slip from a blunt end. The contents were collected in sterilized round bottom glass tanks were covered with lids of Petri dishes and incubated at a relative humidity of 90% and 3% carbon dioxide.

Cells MDAY-D2 (lymphoid tumor mice) were injected with in of mice and gave them grow into tumors with a weight of 0.5-1.0, Mice were killed, tumors rubbed with alcohol, cut out, placed in a sterile environment for the culture of tissue and cut into cubes 1 mm in a laminar box. Before placing the dissected tumors in 9-day-old chick embryos surface ITSELF was carefully viscerale needle 30 gauge (G) to ensure the implantation of tumors. Then what of the four days to establish vascular supply. Using this method were obtained four of the embryo, and every embryo had 3 tumors. On the 12th day of each of the three tumors on the embryos received either loaded with 20% paclitaxel thermal grease, unloaded thermal grease, or received no treatment (control). These treatments were continued for two days before registration results.

Explantion tumor MDAY-D2 secrete angiogenic factors that induce the ingrowth of capillaries (originating from ITSELF) in tumor weight and make it grow in size. Since all tumor vessels originate from HIMSELF, whereas all the tumor cells originate from the Explant, it is possible to evaluate the effect of therapeutic interventions independently on these two processes. This test is used to determine the effectiveness of actions loaded with paclitaxel thermal paste on: (a) inhibition of tumor vascularization and (b) inhibition of growth of tumor cells themselves.

Direct stereomicroscopic evaluation of in vivo and histological test of fixed tissues from this study showed the following. In tumors treated loaded with 20% paclitaxel thermal grease was a reduction in the number of blood vessels supplying the tumor (figures 14C and 14D), Umana, which is usually the most vascularized solid tumors) compared with the control tumors (figures 14A and 14B). The tumor began to shrink in size and weight during the two days while conducting this study. In addition, it was possible to see numerous endothelial cells stopped cell division, indicating that there was activity on the proliferation of endothelial cells. Observed also often tumor cells with delayed mitosis. All 4 of the embryo found consistent picture with the suppression of tumor vascularization loaded with 20% paclitaxel thermal compound, while not loaded with paclitaxel thermal grease not provided such action.

As a comparison, in ITSELF, processed unloaded thermal grease tumors were well vascularity with the increasing number and density of blood vessels in comparison with normal vascularization of the surrounding tissue and dramatically large number of vessels compared with tumors treated loaded with paclitaxel pasta. The newly formed vessels were included in the tumor from all angles, appearing in the form of spokes attached to the center of the wheel (figures 14A and 14B). Control tumors continued to grow in size and weight in the course of the history of the tumor and only a few endothelial cells were in a state of cell division. Tumor tissue was well vascularity and viable throughout the experiment.

As an example, in two tumors of the same size (the original, at the time of explantation), placed on the same HIMSELF, the following results were obtained. The tumor treated is loaded with 20% paclitaxel thermal grease had a size of 330 mm × 597 mm; in the immediate periphery of the tumor has 14 of the blood vessels, while the tumor mass has only 3-4 small capillary. The tumor treated with unloaded thermal grease had a size of 623 mm × 678 mm; the immediate periphery of this tumor has 54 blood vessel, while the tumor mass has 12-14 small blood vessels. In addition, surrounding HIMSELF had more blood vessels in comparison with the area surrounding treated with paclitaxel tumor.

This study demonstrates that the compound releases a sufficient amount of paclitaxel to inhibit pathological angiogenesis that accompanies the growth and development of tumors. Under these conditions, angiogenesis is maximally stimulated by tumor cells that produce angiogenic factors able to induce the ingrowth of capillaries from ogranichivat the ability of tumor tissue to maintain an adequate blood supply. This results in reduction in tumor mass by means of the cytotoxic effect of drugs on tumor cells themselves, or by deprivation of this tissue nutrients needed for growth and stretching.

EXAMPLE 12

INHIBITION of ANGIOGENESIS by PACLITAXEL

A. Tests with chicken chorioallantoic membrane ("SAM")

Fertilized embryos homemade chicken were incubated for 3 days before cultivation without the shell. In this process the contents of the eggs were isolated by removal of the sheath located around the airspace. Then the inner shell membrane was damaged and the opposite end of the shell was performable, giving the contents of the eggs gently to leave a blunt end. The contents of the eggs were placed in a round bottom sterilized glass containers and covered with lids of Petri dishes. Then they were placed in thermostat at a relative humidity of 90% and 3% CO2and incubated for 3 days.

Paclitaxel (Sigma, St. Louis, MI) was mixed at concentrations of 0,25, 0,5, 1, 5, 10, 30 µg aliquot of 10 μl of 0.5% aqueous methylcellulose. Because paclitaxel is insoluble in water, to obtain fine particles used glass beads. Aliquots of 10 µl of this rasurada paclitaxel, carefully placed at the growing edge of each HIMSELF on the 6th day of incubation. Controls received location not containing paclitaxel methylcellulose disks on HIMSELF during the same time course. After 2-day exposure (8-day incubation) vascular network was examined using a stereomicroscope. Liposyn II, white opaque solution, were injected with ITSELF to increase the visibility of parts of vessels. Vascular network unpainted, live embryos were observed on the images of the stereomicroscope Zeiss, which was paired with a video camera (Dage-MTI Inc., Michigan City, IN). These video signals are then displayed with increasing h and information gathered using system image analysis (Vidas, Kontron; Etching, Germany). Then produced negative images on the graphic chart recorder (Model 3000; Matrix Instruments, Orangeburg, NY).

Membrane 8 days without shell germ filled in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer; an additional amount of the retainer were injected with under HIMSELF. After 10 minutes of in situ CAM was removed and placed in fresh fixative for 2 hours at room temperature. Then the fabric was washed overnight in cacodylate buffer containing 6% sucrose. Interest areas post the x alcohols, the solvent was replaced by propylene oxide and embedded in Spurr resin. Did thin slices diamond microtome knife, placed on copper grids, stained and examined in the electron microscope Joel 1200EX. In this way did the slices of 0.5 mm and were stained with toluene blue to a light microscope.

On the 11th day of the development of chicken embryos used for corrosion pouring. Mercox Resin (Ted Pella, Inc., Redding, CA) were injected with in the vascular network ITSELF using 30 G needle for subcutaneous injection. Casting material consisted of 2.5 grams of polymer Mercox CL-2B and 0.05 grams of catalyst (55% benzoyl peroxide) having a curing time of 5 minutes. After injection, the plastic was allowed to remain in situ for one hour at room temperature and then overnight in an incubator at 65°C. Then placed in 50% sodium hydroxide solution to break down all organic components. Plastic fill washed copiously with distilled water, air-dried, coated with gold/palladium and viewed using a scanning electron microscope Philips 501B.

The results of the above experiments are shown in figures 15-18. Briefly, the General features of the normal culture of chicken, without the shell, embryo screening and blood vessels; ITSELF develops near the Bud. These growing vessels lie near the surface and are easily visible, which makes this system ideal model for the study of angiogenesis. Live unpainted capillary network ITSELF can be rendered non-invasive by using a stereomicroscope. Figure 15 illustrates this vascular area in which the cellular components of blood in the capillaries are registered using the video computer interface. Three-dimensional architectonics such capillary networks ITSELF is shown as a method of corrosion casting and observed in a scanning electron microscope (figure 15C). These fill identified below vessels, which act in the direction of the surface ITSELF, where they form a single layer anastomotic capillaries.

Cross sections through ITSELF show the outer ectoderm, consisting of a double cell layer, wider mesoderm layer containing the capillaries, which lie adjacent to the ectoderm, adventitia cells and inner endodermal layer of cells (figure 15D). At the level of the electron microscope shows a typical structural details of the capillary ITSELF. Typically, these vessels are in close proximity to the inner cell layer of the ectoderm (Figo and in terms of its viability using the stereomicroscope, equipped with a computerized video interface for evaluating the effect on angiogenesis. This gives the image setting used to increase 160× that allowed the direct visualization of blood cells in the capillaries; as a result the blood flow to interest areas can be easily evaluated and registered. For this study, the inhibition of angiogenesis is defined as the area ITSELF (with 2-6 mm in diameter) without capillary network and vascular blood flow. In all experiments, avascular zone was evaluated on a 4-point avascular gradient (table 2). This scale represents the degree of General inhibition with maximum inhibition, are presented as number 3 scale avascular gradient. Paclitaxel was very persistent and induced maximum avascular zone (6 mm in diameter or 3 according to the scale avascular gradient) within 48 hours depending on its concentration.

Dependent on the dose of the experimental data of the effects of various therapeutic agents at different concentrations are shown in table 3.

Also shown is a typical treated with paclitaxel ITSELF with a transparent disk methylcellulose, is what Aceh avascular zones is clearly distinguishable (figure 16C); surrounding functional vessels were often directed away from the source of paclitaxel (figures 16C and 16D). Such changes are at an angle to the direction of flow has never been observed under normal conditions. Another symptom of action of paclitaxel was the formation of blood Islands in the avascular zone, representing the aggregation of blood cells.

Related morphological changes of treated with paclitaxel ITSELF is clearly visible as on the level of SS, and at the level of electron microscope. For ease of presentation, shows three different phases of a major transition from the normal to the avascular state. Near the periphery of the avascular zone ITSELF is notable for the abundance of mitotic cells from all three germ layers (figures 17A and 18A). This increased mitotic division also firmness was observed for capillary endothelial cells. With further destruction ITSELF is disintegration and dissolution of capillaries (figures 17B and 18B). Putative endothelial cells, usually delayed in mitosis, still maintain a close spatial relationship with the blood cells and lie near the ectoderm; however, these cells are not connected by the joint. The most Central part of avascular Soldini, cell junction remained intact and layers retained their structural characteristics. In the mesoderm were abundant scattered cells with delayed mitosis; these cells were not detected polarized endothelial cells observed in the first phase. Also, in this avascular area of degenerating cells were normal, as can be seen by electron-dense vacuoles and cellular debris (figure 18C).

In General, this invention showed that after 48 hours after application of paclitaxel on the angiogenesis inhibited. Inhibition of blood vessels formed in the avascular zone was represented by three transitional phases of action of paclitaxel. The Central most area affected avascular zone contained ruined capillaries with transstilbene erythrocytes; this indicates that the intercellular junction between the endothelial cells are absent. Cell endoderm and ectoderm kept their intercellular junction and, therefore, these germ layers remained intact; however, they were slightly thickened. As it approaches the normal vascular zone of the blood vessels kept them socistudies complexes and, consequently, also remained in the Xia, what was evident from the typical changes of direction or effect "elbow" bend blood vessels (figure 16D).

Treated with paclitaxel avascular zone also discovered the abundance of cells arrested in mitosis, in all three germ layers ITSELF; it was unique to paclitaxel, as no previous research has not demonstrated such an event. Arrested in mitosis of endothelial cells could not be their normal metabolic functions involved in angiogenesis. As a comparison, avascular zone, formed by the. and cortisone acetate, not mitotically produces detained cells in HIMSELF; they only prevent further growth of blood vessels in the treated area. Thus, even though these agents are antiangiogenic, there are many points in which the process of angiogenesis can be affected.

Also observed the effect of paclitaxel for 48 hours. During this period of observation, it was noted that the inhibition of angiogenesis occurs as early as 9 hours after application. Histological sections revealed a similar morphology with the morphology observed in the first transition phase avascular zone at 48 hours, the sludge is earlier. It was found that avascular zone formed by heparin and angiostatic steroids became revascularizing after 60 hours after application. In one study treated with paclitaxel avascular zones were not revascularizable for at least 7 days after application, suggesting a stronger long-term action.

EXAMPLE 13

EFFECT of PACLITAXEL AND CAMPOMARINO ON the PROLIFERATION

CELLS LNCaP

Materials and methods

The LNCaP cells were sown at concentrations of 2×1031×103cells/well, respectively, in 96-well plates. After 48 hours in each culture well was added with different concentrations of paclitaxel or camptothecin (25 μl) and the plates were incubated at 37°C for 5 days. After incubation the cells were fixed with 1% solution of glutaraldehyde and stained for 5 minutes with 0.5% crystal violet dye. The dye was consistently suirable 100 μl of buffer solution and the absorption was recorded on a microplate reader Titertek Multiskan at a wavelength of 492 nm. Cell growth was expressed relative to control wells containing no test compound (taken as 100%).

Results

the cells in the wells after treatment with paclitaxel using test DNA fragmentation. Observed strong apoptosis, indicating that paclitaxel was cytotoxic in accordance with the mechanism of apoptosis.

Computacin was very strong in its cytotoxic activity against LNCaP cells. Such low concentrations as 0.001 nm, were toxic to more than 60% of the cells. Thus, EC50for this drug against LNCaP cells must lie in the range femtomolar concentrations (table.4, 5).

EXAMPLE 14

ANTIANGIOGENIC ACTIVITY of OTHER ANTIMICROTUBULE AGENTS

In addition to paclitaxel, other antimicrotubule agents can also be incorporated into polymeric carriers. Typical examples are described below, include competitin and Vinca alkaloids such as vinblastine and vincristine, and stabilizing microtubule agents such as tubercidin, aluminum fluoride and LY290181.

A. the Inclusion of agents in PCL

Agents rubbed pestle in a mortar to reduce the particle size to less than 5 microns. Then this powder was mixed as a dry powder with polycaprolactone (PCL) (molecular weight 18000 Birmingham Polymers, AL USA). The mixture was heated to 65°C for 5 minutes and the mixture of Rapla the syringe 1 ml was dispensed with obtaining granules 3 mg These granules were then placed HIMSELF on day 6 of gestation to assess their antiangiogenic properties.

C. Action loaded Campomarino paste PCL on HIMSELF

Loaded Campomarino the compound was effective in the inhibition of angiogenesis in comparison with the control PCL pellets. At 5% load medicines 4/5 proven HIMSELF revealed a strong inhibition of angiogenesis. In addition, when the load of 1% and 0.25% 2/3 and 3/4 HIMSELF, accordingly, found the inhibition of angiogenesis. Thus, from these data it is obvious that computacin sufficiently released from thermal paste PCL and that he has antiangiogenic therapeutic efficacy.

C. Action loaded with vinblastine and vincristine paste

PCL on HIMSELF

When testing these songs to MYSELF it was evident that these agents were released from granules PCL in sufficient quantities for the induction of biological effects. As vinblastine and vincristine induced angiogenic effects in the test in comparison with a control pellet thermal paste PCL.

At concentrations of 0.5% and 0.1% load drugs vincristine induced inhibition of angiogenesis in all tested HIMSELF. At test concentrations of previsor>Vinblastine was also effective in the inhibition of angiogenesis by HIMSELF at concentrations of 0.25%, 0.5% and 1%. However, at concentrations greater than 2%, vinblastine was toxic in respect of the embryo.

D. Action-loaded tubercidin paste PCL on HIMSELF

Loaded tubercidin the compound was effective in the inhibition of angiogenesis in comparison with the control PCL pellets. At a load of 1% tubercidin induced inhibition of angiogenesis in 1/3 proven HIMSELF. However, at higher concentrations of the medicinal product 5% load tubercidin strongly inhibited angiogenesis in 2/3 HIMSELF. Thus, from these results it was evident that tubercidin sufficiently released from the paste PCL and that he has a strong antiangiogenic activity.

That is, the Action-loaded aluminum fluoride toothpaste PCL ITSELF Loaded with aluminum fluoride (lF3) paste PCL were effective in the inhibition of angiogenesis at 20% load of the drug in comparison with control pellets. At 20% load medicines 2/4 HIMSELF discovered the inhibition of angiogenesis, as is evident from the formation of an avascular zone with a diameter of 2-6 mm, But at lower load drugs, 1% and 5 is effektivnym in the induction of inhibition of angiogenesis only at higher concentrations of the drug.

F. Action loaded LY290181 paste PCL on HIMSELF

Assessment paste PCL loaded with 5% LY290181, HIMSELF revealed that LY290181 induced inhibition of angiogenesis in 1/3 proven HIMSELF. However, when the load of the medicinal product 1% LY290181 not induced angiogenic response (n=2).

EXAMPLE 15

EFFECT of PACLITAXEL ON the VIABILITY of NONPROLIFERATIVE CELLS

While it is important that disease modifying agent is able to inhibit various inappropriate cellular activity (proliferation, inflammation and the production of proteolytic enzymes) that are in abundance during the development of chronic inflammation, it should not be toxic to normal tissues. Especially critical is the fact that normal cells should not be damaged, as this would lead to the progression of the disease. In this example investigated the effect of paclitaxel on the viability of normal non-dividing cells in vitro using cultured chondrocytes grown to confluently.

Briefly, chondrocytes were incubated in the presence (10-5M, 10-7M and 10-9M) or in the absence (control) of paclitaxel for 72 hours. At the end of this period of time total number of giant conducted 4 times and the results were compared.

The results of this experiment are shown in figure 21. In short, as can be seen from figure 21, paclitaxel does not affect the viability of normal nonproliferative cells in vitro even at high (10-5M) concentrations of paclitaxel. More specifically, even when the concentrations of the medicinal product, sufficient to block pathological processes described in the preceding examples, no cytotoxicity against normal chondrocytes.

EXAMPLE 16

The CHOICE of AMPLIFIER PENETRATION FOR SONGS

PACLITAXEL FOR LOCAL APPLICATION

A. the solubility of paclitaxel in various amplifiers

Had the following penetration enhancers: Transcutol®, ethanol, propylene glycol, isopropylmyristate, oleic acid and the mixture Transcutol:isopropylmyristate (9:1 vol./vol.). One milliliter of each amplifier in glass vials pre-heated to 37°C and was added an excess of paclitaxel. A sample of 0.5 ml of liquid from each vessel was centrifuged at 37°C and 13000 rpm for 2 minutes. Aliquots (0.1 ml) of the supernatant from the centrifuge tubes was transferred into a volumetric flask and diluted with methanol. The content of paclitaxel was evaluated using liquid chromatography Wysocki in the volume of the amplifier, heated to 37°C. Aliquots (1 ml) of this solution was added to 1 ml of octanol in a glass vial of 4 ml was Then added to phosphate buffered saline solution (1 ml) (pH 7.4) and the vessels were shaken by vortex with obtaining emulsion. The vials were placed in a thermostat at 37°C for 16 hours, and then from each vial was taking 0.1 ml of octanol and was diluted with 9.9 ml of methanol. For aqueous phase samples of 0.5 ml were taken from the vials with oleic acid and isopropylmyristate and samples of 0.5 ml were taken from the vials with propylene glycol and diluted with 0.5 ml of methanol. From vials Transcutol took samples of 0.1 ml and was diluted with 9.9 ml of a mixture of 50:50 Transcutol:SFR and vials with ethanol took samples of 0.1 ml and was diluted with a mixture of 50:50 ethanol:SFR. The content of paclitaxel was determined using VGH. Each determination was performed in triplicate.

C. Results

The solubility of paclitaxel in each amplifier at 37°C is shown in table 6.

The distribution coefficients octanol-water, Ko/shown in table 7.

For effective action paclitaxel must penetrate the skin to a lower layer of the viable epidermis. It was found that drug is close to 100 (Hadgraft J. H. and K. Walters, Drug absorption enhancements, A. G. de Boers Ed., Harwood Publishers, 1994). Based on the results in tables 1 and 2, it is seen that the propylene glycol and Transcutol find the best combination of solubilization of paclitaxel and strengthening its distribution of the oil phase in the aqueous phase.

However,o/produced by Transcutol and propylene glycol, may be somewhat low, so they were combined with isopropylmyristate, which has infiniteo/in an attempt to increase the solubility of paclitaxel in the phase of octanol. Isopropylmyristate and Transcutol were mixed in the volume ratio 1:9. Isopropylmyristate was easily dissolved at room temperature in Transcutol. For the formation of a homogeneous phase propylene glycol and isopropylmyristate was mixed with ethanol in a ratio of 4:3,5:0,5 propylene glycol:ethanol:isopropylmyristate. Receivedo/shown in table 8.

Add isopropylmyristate to Transcutol led to the significant increase in the distribution coefficient. However, a solution of propylene glycol:ethanol:isopropylmyristate not led to the significant improvement of the distribution coefficient in comparison with one propylene glycol. This last result is the combination of amplifiers from further consideration. In addition, adding isopropylmyristate actually increased the solubility of paclitaxel relative to its solubility in one Transcutol. The solubility of paclitaxel in Transcutol was 346,9 mg/ml, whereas in combination Transcutol:isopropylmyristate solubility was increased to 353,9 mg/ml Thus, this combination of amplifiers was selected studies of skin diseases.

EXAMPLE 17

PREPARATION AND ANALYSIS of COMPOSITIONS of PACLITAXEL

FOR LOCAL USE

A. Preparation of ointments And paclitaxel

Transcutol (3.2 g), isopropylmyristate (0.3 g), Lubrizol (2.5 g), paclitaxel (0.01 g) and 0.5 MCI/ml3H-paclitaxel (0.3 ml) were combined in a scintillation vial of 20 ml In a separate scintillation vial United labrafil (2.5 g), arlacel 165 (1.2 g) and Capitol (0.3 g) and was heated to 70°C to full fusion. The contents of the first scintillation vial was added to the melt, were shaken in a vortex to homogeneity and allowed to cool.

C. Preparation of ointments In the paclitaxel

Transcutol (2.5 g), isopropylmyristate (1.0 g), Lubrizol (2.5 g), paclitaxel (0.01 g) and 0.5 MCI/ml3H-paclitaxel (0.3 ml) were combined in a scintillation vial of 20 ml In a separate scintillation placodermi first scintillation vial was added to the melt, were shaken in a vortex to homogeneity and allowed to cool.

C. Preparation of the skin and the study penetration

Frozen skin mini-pigs Yucatan miniature pigs for medical experiments) were stored at -70°C to use. The skin samples were prepared with the use of cork Sverd No. 10 to knock out discs from the frozen skin. The samples were washed with a solution of streptomycin-penicillin and placed in bags for the freezer and kept at -70°C.

Sections of skin were placed in the diffusion cell Franz up the cornified layer (stratum corneum). The lower receptor solution was 0.05% solution of amoxicillin in R. O. water. The donor cell was attached to each surface of the skin. Ointment of paclitaxel was heated to melting (40-50°C) and fill the syringe. Still in molten form 0.1 ml was dispensed on each surface of the skin. Donor cells were covered with a glass disk and the whole ensemble was left for 24 hours.

After 24 hours the cells were disassembled, the excess ointment was removed and stored in scintillation vial. Skin surface is easily washed with 3 ml of dichloromethane (DHM) and dried. Wash DHM kept in the same vial, which was the excess ointment. Sections of the skin and receptor solutions were placed in a separate scintillation the s slices skin was dissolved by adding 0.5 ml of tissue solubilizer in each bottle. The samples were left overnight to dissolve at room temperature. The next day in the vials was added 3 ml of scintillation mixture. In the case of wash DHM solutions 100 µl was transferred into 1 ml of acetonitrile and then added 3 ml of scintillation mixture. The radioactivity of all solutions was measured using a beta counter.

The skin samples were placed in a diffusion cell Franz and were divided into three groups. Each sample was processed accordingly (without processing or ointment with or without paclitaxel paclitaxel). After 24 hours the samples were removed and processed using standard histological methods.

D. Results

Based on the study of histological sections, it was found that the raw skin has a thickness of 50-120 μm, whereas the viable epidermis has a thickness of 400-700 microns. In the case of an ointment containing 3% m/m isopropylmyristate (ointment), the concentration of paclitaxel in the skin was essentially constant at 1 μg/ml (1,2×10-6M) in stratum cogeim and across the viable epidermis. In the case of an ointment containing 10% m/m isopropylmyristate (ointment), the concentration of paclitaxel was standing in the stratum corneum and viable epidermis, but higher in the stratum corneum (6 µg/ml vs. 2 mcg/ml). When Israel not completely passed through the cut skin.

Did not observe large differences in the application of ointments containing paclitaxel.

EXAMPLE 18

The DEVELOPMENT of SYSTEM COMPOSITIONS of PACLITAXEL FOR the TREATMENT of PSORIASIS

In severe cases of psoriasis are considered to be acceptable more aggressive treatment and, therefore, may be acceptable toxicity associated with systemic treatment with paclitaxel.

System composition for paclitaxel consists of amphiphilic diblock copolymers, which in aqueous solutions to form micelles consisting of a hydrophobic core and hydrophilic shell in the water. Diblock copolymers of poly(DL-lactide)-block-methoxypolyethyleneglycol (PDLLA-MePEG), polycaprolactone-block-methoxypolyethyleneglycol (PCL-MePEG) and poly (DL-lactide-co-caprolactone)-block-methoxypolyethyleneglycol (PDLLACL-MePEG) can be synthesized using the procedure of polymerization of the melt in the mass or similar methods. In short, a certain number of monomers DL-lactide, caprolactone and methoxypolyethyleneglycol with different molecular masses were heated (130°C) to melt while bubbling nitrogen and stirred. To the molten monomer was added to the catalyst octoate tin (0.2% m/m). The polymerization was carried out for 4 hours. Molecular is, is luorescence and testing solubilization, respectively (figure 22). Received high load-bearing capacity for paclitaxel. The ability to solubilize paclitaxel depends on the compositions and concentrations of these copolymers (figures 22 and 23). PDLLA-MePEG gave the most stable the solubilized paclitaxel (figures 23 and 24).

Strong coupling in the inner Central part (core) of polymeric micelles provides an environment high capacity for carrying hydrophobic drugs such as paclitaxel. Drugs can be covalently linked block copolymers with formation of micellar structures or they may be physically included in the hydrophobic core of these micelles. Mechanisms of release of drug from micelles include diffusion from the core and the exchange between the individual chains of the polymers and micelles. The small size of the micelles (usually less than 100 nm) will resolve the difficulties associated with injecting larger particles.

EXAMPLE 19

The PROCEDURE for preparation of thermal paste

Weighed five grams of polycaprolactone mol. weight of 10,000 to 20,000 (Polysciences, Warrington, Penn. USA) in a glass scintillation vial of 20 ml, which was placed in chemical beaker 600 ml containing 50 ml of water. Hline polymer. A known sample of paclitaxel or other inhibitor of angiogenesis thoroughly mixed in the molten polymer at 65°C. the Molten polymer was poured into a preheated (thermostat at 60°C) the form and allowed to cool to solidification of the polymer. The polymer is cut into small pieces (approximately 2 mm by 2 mm) and placed in 1 ml-new glass syringe.

A glass syringe was then placed vertically (closed lid with the tip down) in glass chemical glass for 500 ml) containing distilled water at 65°C (hot plate Corning), until complete melting of the polymer. Then put the piston in the syringe to compress the molten polymer in the viscous mass at the tip of the syringe cylinder. The syringe was closed with a lid and gave it to cool at room temperature.

To use this syringe was re-heated to 60°C and injected in liquid form, which overides when cooled to body temperature.

EXAMPLE 20

Modification of the RELEASE of PACLITAXEL FROM thermal paste USING PDLLA-PEG-PDLLA AND low-MOLECULAR

POLY(D,L-LACTIC ACID)

A. Preparation of PDLLA-PEG-PDLLA and low molecular weight PDLLA

DL-lactide was purchased from Aldrich. Polyethylene glycol (PEG) with molecular weight molecular weight of 20,000 was obtained from Birmingham Polymers (Birmingham, AL). Paclitaxel was obtained from Hauser Chemicals (Boulder, CO).

The polystyrene standards with a narrow distribution of molecular masses were obtained from Polysciences (Warrington, PA). Acetonitrile and methylenchlorid had VIH-purity (Fisher Scientific).

Triblock-copolymer of PDLLA-PEG-PDLLA was synthesized by polymerization with ring opening. The monomers of DL-lactide and PEG in different ratios were mixed and added to 0.5 wt.% octoate tin. The polymerization was carried out at 150°C for 3.5 hours. Low molecular weight PDLLA was synthesized by polycondensation DL-lactic acid. The reaction was carried out in a glass flask under conditions of slow blowing nitrogen, mechanical stirring and heating at 180°C for 1.5 hours. Molecular weight PDLLA was about 800, as measured by titration of end carboxyl groups.

C. Preparation of compositions in the form of pastes

Paclitaxel at loads of 20% or 30% was thoroughly mixed with copolymers of PDLLA-PEG-PDLLA or mixtures PDLLA-PCL 90:10, 80:20 and 70:30, molten at about 60°C. Pasta, loaded with paclitaxel, weighed in syringes of 1 ml and stored at 4°C.

C. Characteristics of PDLLA-PEG-PDLLA and pasty mixtures

Molecular weight and distribution of the copolymers of PDLLA-PEG-PDLLA was determined at ambient temperature inanaga with gel column 104-Hewlett Packard PI. Mobile phase was chloroform with a flow rate of 1 ml/min Injection volume of sample was 20 μl at a concentration of polymer of 0.2% (m/V). Molecular weights of polymers were determined relative to polystyrene standards. The characteristic viscosity of PDLLA-PEG-PDLLA in l3at 25°With measured using a viscometer Cannon-Fenske.

Thermal analysis of the copolymers was performed differential scanning calorimetry (DSC) using THE controller Instruments 2000, DuPont DSC 910S (Newcastle, Delaware). The heating rate was 10°C/min, and the sample matrix of the copolymer matrix paclitaxel/copolymer weighed (3-5 mg) in curved open aluminum trays for samples.

1H-nuclear magnetic resonance (NMR) was used to determine the chemical composition of the polymer. Spectra1H-NMR loaded with paclitaxel PDLLA-PEG-PDLLA were obtained in CDCl3using NMR instrument (Bruker AC-200E) at 200 MHz. The concentration of polymer was 1-2%.

The morphology of the paste paclitaxel/PDLLA-PEG-PDLLA was investigated using scanning electron microscopy (SEM) (Hitachi F-2300). The sample was covered 60% Au and 40% Pd (thickness of 10-15 nm) using a hammer device (Technics, USA).

D. Release of paclitaxel in vitro

The sky is eat molten paste through a syringe loaded with 20% paclitaxel paste PDLLA-PEG-PDLLA were placed in a closed lids glass vials 14 ml, containing 10 ml of phosphate buffered saline (pH 7.4) with 0.4 g/l albumin. The tube was incubated at 37°C with slow rotational mixing. Supernatant were taken periodically for analysis of paclitaxel and replaced with fresh buffer SFR/albumin. The supernatant (10 ml) was extracted with 1 ml of methylene chloride. The aqueous phase decantation and the methylene chloride phase was dried under a stream of nitrogen at 60°C. the Dried residue was recreated in a mixture of 40:60 water:acetonitrile and centrifuged at 10,000 g for about 1 minute. Then the amount of paclitaxel in the supernatant was analyzed using VGH. Analysis WICH were performed using pump 110A and columns Ultrastar S-8 (Beckman) and UV-detector setup SPD-6A at 232 nm, autoinjector SIL-9A and integrator C-R3A (Shimadzu). Injection volume of 20 µl and the flow rate 1 ml/min Mobile phase consisted of 58% acetonitrile, 5% methanol and 37% of distilled water.

E. Results and discussion

The molecular weight and distribution of molecular masses of PDLLA-PEG-PDLLA relative to polystyrene standards was measured using GPC (figure 30). The characteristic viscosity of the copolymer in l3at 25°C was determined using a viscometer Canon-Fenske. Molecular weight and characteristic of VASO copolymer with 70% PEG had a narrow molecular weight distribution with a polydispersity of 1.21. This may be due to high content of PEG, which reduces the chance of adverse reactions such as transesterification, which leads to a wide distribution of molecular masses of the polymer. Alternatively, the spiral structure of the hydrophobic-hydrophilic block copolymers could lead to an artificially low value of polydispersity.

The DSC scans of pure PEG and copolymers PDLLA-PEG-PDLLA presented in figures 25 and 26. PEG and PDLLA-PEG-PDLLA content PEG 70% and 40% showed endothermic peaks with decreasing enthalpy and temperature with decreasing content of PEG copolymer. Endothermic peaks in the copolymer with 40% and 70% PEG, probably due to the melting of land PEG, which indicates the occurrence of phase separation. While PEG had sharp peak melting, copolymers with 70% and 40% of PEG was found broad peaks with a prominent shoulder in the case of 70% PEG. Broad melting peaks could be the result of interference of PDLLA with the crystallization of PEG. This shoulder in the case of 70% PEG may represent a transition from highly elastic to plastic state in glassy plot PDLLA. Not observed thermal changes in the copolymers with PEG content of 10%, 20% and 30% within the temperature 10-250°C, which indicates that not happened z0:10) without paclitaxel or with 20% paclitaxel discovered endothermic peak at about 60°C arising from the melting of the PCL.

Due to the amorphous nature of PDLLA and its low molecular weight (800) melting and transitions in glassy state PDLLA was not observed. Not observed thermal changes due to recrystallization or melting of paclitaxel.

The copolymers of PDLLA-PEG-PDLLA content PEG 20% and 30% were chosen as the optimal composite materials for pasta for the following reasons: PDLLA-PEG-PDLLA with 10% PEG could not melt at a temperature of about 60°C; copolymers of 40% and 70% PEG easily melted at 60°C and a copolymer of 20% and 30% PEG became viscous fluid between 50°s and 60°C; and swelling of 40% and 70% copolymers of PEG in water was very high, leading to a rapid dispersion pastes in water.

The release profiles of paclitaxel in vitro of the cylinders PDLLA-PEG-PDLLA shown in figure 27. The experiment measuring the release of 40% PEG cylinders stopped because these cylinders had a very high degree of swelling (approximately 200% water absorption within one day) and was destroyed in a few days. The released fraction of paclitaxel from the cylinder 30% PEG gradually increased for 70 days. The released fraction of paclitaxel from the cylinders 20% PEG was slowly increased to 30 days and undertaking to the extent which individual cylinder (PEG content 20%) was detected abrupt change in the release of paclitaxel. Facing a sharp increase in the released fraction of paclitaxel was lower for polymers with lower content of PEG in the same cylinder diameter (1 mm). 40% and 30% PEG cylinders found a much higher speed of release of paclitaxel than the cylinders 20% PEG. For example, the cylinder 30% PEG released 17% paclitaxel 30 days, compared with 2% release of cylinder 20% PEG. Cylinders with smaller diameters led to higher rates of release (e.g., within 30 days of the cylinders 30% PEG diameter of 0.65 mm and 1 mm were freed 26% and 17% of paclitaxel, respectively (figure 27)).

The above observations can be explained by the mechanisms of the release of paclitaxel from the cylinder. Paclitaxel dispersed in the polymer in the form of crystals, as observed using optical microscopy. These crystals begin to dissolve in the matrix copolymer in 170°C and completely dissolved at 180°C, as observed in thermograms microscopy DSC with heated table loaded with 20% paclitaxel paste PDLLA-PEG-PDLLA (30% PEG), which revealed a small ectothermy recrystallization (16 j/g, 190°xela from the melt of the copolymer after 180°C. In this type of matrix, the drug/polymer paclitaxel could be released by diffusion and/or erosion of the polymer.

In the regulated diffusion case, the drug may be released by molecular diffusion in the polymer and/or through open channels formed bound particle drugs. Therefore, at 20% load, some particles of paclitaxel stood out and paclitaxel could be released by dissolving the copolymer followed by diffusion. Other particles of paclitaxel could form a cluster that connects to the surface and be released through the channel diffusion. In both cases, the cylinders of the small size gave more rapid release of drug due to shorter diffusion path (figure 27).

At the time of release was registered resize and water absorption of the cylinder (figure 28). Changing the length, diameter and fresh weight of the cylinder 30% PEG was increased rapidly to a maximum within 2 days, remained unchanged for about 15 days, then gradually decreased. The original diameter of the cylinder does not affect the characteristic of swelling. For a cylinder of 20% PEG length decreased by 10% in one DSEE the amount of PEG in the copolymer absorbed more water to facilitate diffusion of paclitaxel, observed a more rapid release (figure 27).

The destruction of the molecular weight of the copolymer paste PDLLA-PEG-PDLLA was observed using GPC. For a cylinder of 20% PEG volume elution position of the peak increased with time, indicating that the reduced molecular weight of the polymer in the course of the experiment for release (figure 30). Two-phase molecular weight distribution was observed on the 69th day. The molecular weight of the polymer decreased for cylinders 30% PEG (1 mm and 0.65 mm). However, two-phase distribution was not observed.

The NMR spectra showed peak PEG 3.6 M. D. and peaks PDLLA when 1,65 M. D. and 5.1 m D. the peak Area of the PEG relative to the PDLLA in the copolymer decreased significantly after 69 days (figure 29), indicating the dissolution of PEG after its dissociation from PDLLA. The loss of dry weight of the cylinder was also recorded (figure 29), and it shows a decrease in the decomposition rate in a sequence of 30% PEG-0.65 mm>30% PEG-1 mm>20% PEG-1 mm.

Morphological changes of the dried cylinders before and during the time of the release of paclitaxel was observed using SEM (figure 31). Briefly, solid crystals of paclitaxel and non-porous polymeric matrices was observed before release (figures 31A and B). After 69 days of release were not observed Cristallo>elendra 30% PEG found a strong swelling after only two days in the water (figure 28) and, therefore, the inhibition of diffusion internationally unrecognized breakaway water-soluble PEG block and degraded PDLLA (i.e., oligomers DL-lactic acid) decreased. Because weight loss and destruction of the cylinders 30% PEG were continuous, the contribution of erosion of release increased gradually, resulting in slow release of paclitaxel without any abrupt changes (figure 27). Cylinders 20% PEG swelling was initially low (figure 28), leading to a slow diffusion of the degradation products. Thus, the degradation products on the domestic portion of the original was maintained, whereas the less the degradation products was in the outer area due to short diffusion path. These degradation products have accelerated the rate of degradation, as of the end carboxyl groups of these oligomers catalyzed hydrolytic degradation. This led to heavier shell and low-molecular-weight internal parts, as shown in the two-phase molecular weight distribution of the copolymer (figure 30, the 69th day). Since rupture of the membranes depended on factors such as strength, thickness, and defects of this shell is the second part were very variable. Because rupture of membranes was not persistent and the drug in the polymer was not microscopically homogeneous, the time point for release in the form of emissions and the extent of this release were different for the 4 tested samples (figure 27).

The release of paclitaxel from mixtures of PDLLA and PCL and pure PCL is shown in figure 32. Briefly, the released fraction increased with increasing content of PDLLA in this mixture. For example, within 10 days of released paclitaxel from mixtures of 80:20, 70:30 and 0:100 PDLLA:PCL was 17%, 11% and 6%, respectively. After the initial spike on the first day it was received approximately constant release of paste 80:20 PDLLA:PCL. He observed a significant degree of swelling at the time of the release. For mixtures PDLLA:PCL, because PDLLA has a very low molecular weight of about 800, he hydrolizable quickly to water-soluble products without a long delay in the weight loss. PCL served as the "holding" of the material, preventing the pasta from rapid destruction. Therefore, the rate of release increased with increasing content of PDLLA in the mixture due to enhanced degradation. Continuous erosion PDLLA regulate the release of paclitaxel and led to postie low speed degradation (1-2 years) PCL.

Difficulties were encountered in the study release loaded with 20% paclitaxel paste 90:10 PDLLA:PCL due to the destruction of the ball of paste for 24 hours incubation. Briefly, during the first 12 hours of incubation took samples every hour to ensure conditions of sedimentation for the release of paclitaxel. Released paclitaxel pasta 90:10 25-35% within 10 hours.

Pasta 90:10 PDLLA: PCL containing 30% of paclitaxel was released more paclitaxel than pasta 90:10 PDLLA:PCL containing 20% paclitaxel. Thus, modulating the speed of release of paclitaxel, which is governed by the properties of the polymer and chemotherapeutic agents, as well as by injection, was important in the development of local therapy.

EXAMPLE 21

OBTAINING POLYMER COMPOSITIONS CONTAINING

Water-SOLUBLE ADDITIVES AND PACLITAXEL

A. Preparation of polymer compositions

Received microparticles coprecipitates of paclitaxel/supplements and then added to PCL with getting pastes. Briefly, paclitaxel (100 mg) was dissolved in 0.5 ml of ethanol (95%) and was mixed with the additive (100 mg), previously dissolved or dispersed in 1.0 ml of distilled water. The mixture was ground to obtain a smooth paste. This pasta raspredvalami mortar and pestle and passed through a sieve with mesh No. 140 (106 microns) (Endecotts Test Sieves Ltd, London, England). Then these microparticles (40%) included in the molten PCL (60%) at 65°C, which corresponds to 20% load of paclitaxel. Additives used in this study were gelatin (Type b, 100 bloom. Fisher Scientific), methylcellulose (British Drug Houses), dextran, T500 (Pharmacia, Sweden), albumin (Fisher Scientific) and sodium chloride (Fisher Scientific). Microparticles of paclitaxel and gelatin or albumin was prepared as described above, but they were passed through a sieve with mesh No. 60 (270 μm) (Endecotts Test Sieves Ltd, London, England) to assess the impact of particle size on the release of paclitaxel from the paste. Also cooked pasta containing 10, 20 or 30% gelatin and 20% paclitaxel in PCL to study the effect of the share of additives on the release of the drug. If there are no other instructions, pasta containing 20% paclitaxel dispersed in the PCL was prepared for use as controls for studies of the speed of release.

Century Studies of release of the medicinal product

The pellet approximately 2.5 mg loaded with paclitaxel paste suspended in 50 ml 10 mm SFR (pH 7.4) in a closed screw-capped test tubes. Tubes turned from end to end at 37°C and at certain intervals of 49.5 ml of the supernatant was removed, filtered cerasale maintain conditions of sedimentation throughout the study. To analyze the filtrate was extracted with 3×1 ml dichloromethane (DHM), extracts DHM evaporated to dryness under a stream of nitrogen and re-dissolved in 1 ml of acetonitrile. The analysis was carried out using VIH using as mobile phase a mixture of water:methanol:acetonitrile (37:5:58) at speed of current of 1 ml/min (Beckman Isocratic Pump), reversed-phase C18 column (Beckman) and UV detection (Shimadzu SPD) at 232 nm.

C. Research swelling

Paste paclitaxel/additive/PCL prepared with the use of microparticles of paclitaxel/additive with mesh size No. 140 (and # 60 only for gelatin) was extrudible education cylinders, cut to pieces, weighed and the diameter and the length of each piece was measured using a micrometer (Mitutoyo Digimatic). Pieces suspended in distilled water (10 ml) at 37°C and at predetermined intervals the water was removed and the diameter and length of the cylindrical pieces were measured and the samples were weighed. The morphology of the samples (before and after suspension in water) was investigated using scanning electron microscopy (SEM) (Hitachi F-2300). The samples were covered 60% Au and 40% Pd (thickness of 10-15 nm) using a hammer device (Technics, USA).

D. Studies using chorioallantoic membrane of chicken embryo (CAM)

Fertilized saravali at 90% relative humidity and 3% CO2and on the 6th day of incubation, the slices loaded with paclitaxel paste weighing 1 mg (containing 6% paclitaxel, 24% gelatin and 70% PCL) or control paste (30% gelatin in PCL) were placed directly on the surface ITSELF. After 2-day exposure of the vascular network was examined using a stereomicroscope connected to the camera; then the signals were displayed on a computer and video images were recorded using image analysis.

E. Results and discussion

Microparticles soosazhdennykh of paclitaxel and gelatin or albumin were hard and brittle and is easily included in the PCL, while other additives made of soft particles, which tended to disintegrate during cooking pasta.

Figure 33 shows the time course of the release of paclitaxel from pastes containing 20% paclitaxel in PCL or 20% paclitaxel, 20% additive and 60% PCL. The release of paclitaxel from the PCL with or without additives had a biphasic pattern of release; the first was a faster rate of release followed by a slower release of the drug. The authors suggest that an initial period of faster release of these pas is chestnykh areas paste. Subsequent slower phase of release profiles can be associated with a decrease in effective surface area of the particles of the drug in contact with the buffer, slow entry buffer into the polymer matrix or to an increase in average ways of diffusion of drug through the polymer matrix.

Both phases of the release profiles of paclitaxel from PCL increased in the presence of hydrophilic additives, and gelatin, albumin and methylcellulose produced the greatest increase in the speed of release of the drug (figure 33). Additional increase in the rate of release of paclitaxel from the polymer matrix were in the case when larger particles of paclitaxel-additive (270 μm) used for cooking pasta, in comparison with cases when using smaller particles of paclitaxel-additive (106 µm) (figure 34). Increase in the amount of additive (e.g., gelatin) produced a corresponding increase in the release of drug (figure 34). Figure 35A shows the characteristic swelling pastes containing 20% paclitaxel, 20% additive and 60% PCL. The rate of swelling followed the following order: gelatin > albumin > methylcellosolve polymer was added to the paste (figure 35V). Paste containing gelatin or albumin, swollen rapidly within the first 8-10 hours and then the rate of swelling was reduced when the volume change of the sample was more than 40%. Pasta made with larger (270 μm) particles of paclitaxel-gelatin, swollen at a higher speed than pasta cooked with less (106 microns) particles of paclitaxel-gelatin. All pastes were broken up, when the volume was increased by more than 50%. Studies using SEM showed that the swelling of the pastes was accompanied by cracking of the matrix (figure 36). Higher magnifications (figure 36C and 36D) were visible crystals of paclitaxel in the form of needles or sticks on the surface of the paste and in close connection with gelatin after swelling (figure 36C and 36D).

Osmotic or nabukenya hydrophilic agents, shipped in the form of discrete particles in a hydrophobic polymer, resulted in the release of drugs by a combination of erosion of the matrix, diffusion of drug through the polymer matrix and/or diffusion and/or convection flow through the pores formed in the matrix by dissolving water-soluble additives. Osmotic agents or nabukenya polymers, dispersed in a hydrophobic polymer, will be itity, is AutoRAE could break the wall (polymer layer) between neighboring particles, creating micro-channels and, therefore, facilitating the release of the drug molecules into the environment by diffusion or convection current. Swelling and cracking of the matrix paste (figure 36), probably led to the formation of micro-channels through the interior of the matrix. Various speed and the degree of swelling of the polymers (figure 35) can cause differences in observed speeds release of paclitaxel (figures 33 and 34).

Figure 37 shows HIMSELF treated control paste gelatin-PCL (figure 37A) and paste 20% paclitaxel-gelatin-PCL (figure 37V). Paste on the surface ITSELF of the arrows shown in the figures. HIMSELF with the control paste shows normal architecture of the capillary network. HIMSELF, processed pasta paclitaxel-PCL, bravely showed regression of the vascular network and areas that do not have capillary vascular network. The inclusion of additives in the paste significantly increased the diameter of the avascular zone (figure 37).

This study showed that the release in vitro of paclitaxel from PCL could be increased by the inclusion of microparticles consisting of paclitaxel/hydrophilic polymer in the PCL matrix. Issledovania paclitaxel/gelatin/PCL significantly reduced the weight of the tumors. It has been shown that factors such as water-soluble agent, the size of the microparticles and the share of additives affect the release characteristics of the drug.

EXAMPLE 22

The PROCEDURE for preparation of NANOPASTE

Nanopaste is a suspension of microspheres in a hydrophilic gel. In one aspect of this invention, the gel or paste can be spread on the fabric as a way of placing the loaded drug microspheres near the target tissue. With its basis in the water, the paste is quickly becoming dissolved in the fluids of the body, causing a reduction in the stickiness of the paste and the tendency of the microspheres to be on the nearest tissue. Pool encapsulated in the microspheres of the drug in the result is placed near the target tissue.

Reagents and equipment which were used in these experiments include glass chemical glasses, Carbopol 925 (pharmaceutical purity, Goodyear Chemical Co.), distilled water, sodium hydroxide (1 M) in aqueous solution, sodium hydroxide solution (5 M) in aqueous solution, the microspheres in the size range of 0.1-3 LM, suspended in water at 20% m/about (see above).

1. PREPARATION of 5% m/about GEL of CARBOPOL

A sufficient amount of carbopol to the mixture was allowed to stand for about one hour. During this period, the mixture was stirred and after one hour the pH was brought to 7.4 by using 5 M sodium hydroxide until complete dissolution of carbopol. Upon reaching pH 7.4 gel was covered and allowed him to stand for 2-3 hours.

2. The PROCEDURE for preparation of NANOPASTE

A sufficient number of microspheres of 0.1-3 μm) was added to water to obtain a 20% suspension of microspheres. The gel of carbopol (8 ml of 5% m/V) were placed in glass chemical beaker and add 2 ml of 20% suspension of microspheres. The mixture was stirred for thorough dispersion of the microspheres in gel. This mixture was stored at 4°C.

EXAMPLE 23

COMPLEXATION of PACLITAXEL WITH CYCLODEXTRINS

A. Materials

Paclitaxel was obtained from Hauser Chemicals Inc. (Boulder Colorado Drug). Centripetal (Fisher), citric acid (British Drug Houses), hydroxypropyl--cyclodextrin (HPCD)-cyclodextrin (-CD) and hydroxypropyl--cyclodextrin (HPCD) was obtained from American Maize-Products Company (Hammond, Indiana) and used in the form in which they were received.

C. Methods

1. SOLUBILITY STUDIES

Excess 47.gif" border="0">-CD, HP-CD or HP-CD and turned slowly for approximately 24 hours at 37°C. After equilibration aliquot of the suspension was filtered through a membrane filter of 0.45 μm (Millipore), appropriately diluted and analyzed using VGH. The mobile phase consisted of acetonitrile, methanol and water (58:5:37) when the speed of the current of 1.0 ml/min solubility of paclitaxel in a solvent consisting of 50:50 water and ethanol (95%) containing different concentrations, up to 10% HPCD also investigated. In addition, the profiles of the velocity of dissolution of paclitaxel was investigated by adding 2 mg of paclitaxel (in such form in which it was received) to 0, 5, 10 or 20% solutions HP-CD or 2 mg pre-hydrated paclitaxel (suspendirovanie in water for 7 days) to clean water and turning slowly at 37°C. At different time intervals were taking aliquots and analyzed for paclitaxel.

2. STABILITY STUDIES

Solutions containing 20% HPCD or HPCD, had a pH of 3.9 and 5.2, respectively. The stability of paclitaxel in solution �.gif" border="0">-CD or HP-CD, either in water or in a mixture of 50:50 water-ethanol at 37°C or 55°C at different time intervals. In addition, we determined the stability of paclitaxel in solution (1 μg/ml) containing 1%, 2% or 5% HPCD, with 55°C.

C. Results

1. SOLUBILITY STUDIES

The solubility of paclitaxel was increased throughout the investigated range of concentrations of CD; HPCD gave the greatest increase in solubility of paclitaxel (figure 38). The shape of the curves of solubility suggests that the stoichiometry were of a higher order than the complex is 1:1. Paclitaxel formed curves of type apas with HP-CD and HP-CD curves of type AN-CD. The solubility of paclitaxel in a 50% solution HPCD in water was 3.2 mg/ml at 37°C, which represented approximately 2000-fold increase relative to the solubility of paclitaxel in water. Estimated stability constants (figure 39) for complexes of the first order paclitaxel-cyclodextrins were to 3.1, 5.8 and 7.2 M-1forCD HPCD HPCD and HPCD, respectively. The magnitude of the observed stability suggest that the inclusion complexes formed by paclitaxel with cyclodextrins were mainly complexes of the second order.

The solubility of paclitaxel in a mixture of 50:50 water-ethanol increased with increasing concentration of cyclodextrins (figure 40), with the observation of complex formation in pure water. The apparent stability constant of for the complexation of paclitaxel and HP-CD in the presence of 50% ethanol (26,57 M-1) was significantly lower (about 300 times) than the stability constant of in the absence of ethanol. Lower constant resistance can be attributed to changes in the dielectric constant or the polarity of the solvent in the presence of ethanol.

The diffusion profiles of paclitaxel in 0, 5, 10 and 20% solutions-cd (figure 41) illustrate the formation of a metastable solution of paclitaxel in pure water or in solutions of cyclodextrins; the amount of paclitaxel in the solution was gradually increased, the achievement of the seat reservation gidratirovana suspendirovanie in water for 48 hours, did not show the formation of a metastable solution. In addition, DSC analysis of hydrated paclitaxel (dried in a vacuum thermostat at room temperature) showed two broad endothermic peak between 60 and 110°C. These peaks were followed by about 4.5% weight loss (determined by thermogravimetric analysis), which indicates the presence of hydrate (hydrate). Weight loss of approximately 2.1% would suggest the formation of the monohydrate of paclitaxel. Thus, the appearance of peaks DSK between 60°C and 110°C and weight loss of about 4.5% suggest the presence of the dihydrate. For samples of paclitaxel in the form in which it was received, there was an endothermic peak (peaks) between 60°C and 110°C (results DSK) or weight loss (TGA results). Thus, paclitaxel (in the form in which it was received) was waterless and at suspendirovanie dissolved in water with the formation of a supersaturated solution, which was precrystallization in the form of a hydrate with a lower solubility (figure 41).

2. STABILITY STUDIES

Destruction of paclitaxel was dependent on the concentration of cyclodextrin and had the kinetics of degradation of pseudobersama order (for example, figure 42). The decomposition rate of the paclitaxel in raster>-1) than at higher concentrations of cyclodextrin. The rate constants of degradation of 1.78×10-3h-1and 0.96×10-3h-1observed for paclitaxel in 10% HP-CD and HP-CD, respectively. Solutions of paclitaxel (1 mg/ml) containing 2, 4, 6 or 8% HP-CD did not show any significant difference in the decomposition rate in comparison with that obtained with solutions of 10 or 20% HP-CD (20 µg/ml). The presence of ethanol had no harmful effect on the stability of paclitaxel in solutions of cyclodextrins.

D. Conclusion

This study showed that the solubility of paclitaxel can be increased by complexation with cyclodextrins. These cyclodextrine composition based on water can be used in the treatment of various inflammatory diseases.

EXAMPLE 24

POLYMERIC COMPOSITIONS WITH INCREASED CONCENTRATIONS

PACLITAXEL

PDLLA-MePEG and PDLLA-PEG-PDLLA are block copolymers with hydrophobic (PDLLA) and hydrophilic (PEG or PEG Me) areas. When appropriate molecular mass and chemical composition, they can form very small aggregates of hydrophobic core PDL is eaten with increased "solubility".

A. Materials

D, L-lactide was purchased from Aldrich, octoate tin, poly(ethylene glycol) (mol. weight 8000), MePEG (mol. weight of 2000 and 5000) were from Sigma. MePEG (mol. weight 750) was obtained from Union Carbide. The copolymers synthesized by the procedure of polymerization with ring opening using octoate tin as catalyst (Deng et al., J. Polym. Sci., Polym, Lett. 28: 411-416, 1990; Cohn et al., J. Biomed, Mater. Res. 22: 993-1009, 1988).

For the synthesis of PDLLA-MePEG a mixture of DL-lactide/Mered/octoate tin was added in a glass ampoule of 10 ml. The ampoule was connected to the vacuum and sealed in the flame. Polymerization was performed by incubating the vial in an oil bath at 150°C for 3 hours. For the synthesis of PDLLA-PEG-PDLLA mixture of D, L-lactide/ED/octoate tin was transferred to a glass flask was sealed with a rubber stopper and heated for 3 hours in a thermostat at 150°C. the Original compositions of the copolymers are given in tables 1 and 2. In all cases, the number of octoate tin was 0,5-0,7%.

C. Methods

The polymer was dissolved in acetonitrile and centrifuged at 10,000 g for 5 minutes to remove insoluble impurities. Then a solution of paclitaxel in acetonitrile was added to each polymer solution with 10 wt.% solution of paclitaxel (paclitaxel + polymer). Then the solvent is acetonitrile UDI distilled water, 0.9% saline solution NaCl or 5% dextrose in weight, 4 times the weight matrix. Finally, this matrix is "dissolved" by mixing using a vortex and periodic heating at 60°C. In all cases were obtained transparent solution. The particle sizes were below 50 nm, as defined in the classifier sub-micron particles (NICOMP Model 270). The compositions are presented in table 9.

In the case of PDLLA-PEG-PDLLA (table 10), because the copolymers can't dissolve in water, paclitaxel and polymer were co-dissolved in acetone. Water or mixture of water/acetone was gradually added to this solution paclitaxel-polymer to induce the formation of spheres paclitaxel/polymer.

C. Results

Many compositions PDLLA-MePEG form clear solutions in water, 0.9% saline solution or 5% dextrose, which indicates the formation of tiny units in the range of pressure gauges. Paclitaxel has been successfully loaded into micelles PDLLA-MePEG. For example, if % load (it means 10 mg of paclitaxel in 1 ml paclitaxel/PDLLA-MePEG/water system) was obtained a clear solution of 2000-50/50 and 2000-40/60. The particle size was about 60 nm.

EXAMPLE 25

The PROCEDURE FOR PREPARATION of the FILM

the te thin flexible sheet of polymer or polymer disk with a thickness of 2 mm, each of which can be deposited on the surface of the fabric to prevent further scarring or adhesions. This film can be manufactured for use on a Nude fabric so that the encapsulated drug could be released from the polymer over a long period of time in this section of the cloth. Films can be manufactured in a variety of ways, including, for example, by casting or spraying.

In the method of casting the polymer either melted or poured into a form or dissolved in dichloromethane and poured into the form. Then the polymer or utverjdali when cooled, or utverjdali evaporation of the solvent, respectively. In the method of spraying the polymer was dissolved in a solvent and deposited on the glass and upon evaporation of the solvent, the polymer was utverjdala on the glass. Repeat the spraying was possible to obtain a polymer in the form of a film which can be removed from the glass.

Reagents and equipment which were used in these experiments include small chemical glass, mixer with hot plate Corning, forms to fill (for example, cap centrifuge tubes 50 ml) and the device for holding forms, saclandchamningdo ("PCL" - molecular weight 10000-20000; Polysciences), paclitaxel (Sigma 95% purity), ethanol, "washed" (see above) ethylene vinyl acetate ("EVA"), poly(DL)lactic acid ("PLA" - molecular weight 15000-25000; Polysciences), DHM (VGH-purity; Fisher Scientific).

1. The PROCEDURE of PRODUCING FILMS - CASTING MELT

A small glass chemical glass with a known sample PCL put more chemical beaker containing water (acting as a water bath), and was placed on a hot plate at 70°C and kept at it until complete melting of the polymer. To the molten polymer was added to a known sample of the medicinal product and the mixture is thoroughly stirred. The molten polymer was poured into a form and gave it to cool.

2. The PROCEDURE of PRODUCING FILMS - CASTING SOLVENT

A known hinge PCL weighed directly into a glass scintillation vial, 20 ml) was added a sufficient amount of DHM to get 10% m/R solution. The solution was stirred and then adding a sufficient amount of paclitaxel to obtain the desired final concentration of paclitaxel. The solution was mixed by vortex for dissolution of paclitaxel, allowing it to stand for one hour (to reduce the presence of the who.

3. The PROCEDURE for MANUFACTURING FILM - COATED

A sufficient amount of polymer was weighed directly into a glass scintillation vial, 20 ml) was added a sufficient amount of DHM to obtain a 2% m/R solution. The solution was stirred to dissolve the polymer. With the use of automatic pipettes suitable amount (minimum 5 ml) of 2% solution of the polymer was transferred into another glass scintillation vial, 20 ml of the solution was added a sufficient amount of paclitaxel was dissolved by shaking the closed lid of the vial. To prepare the spray cap of the vial was removed and the cylinder TLC sprayer was immersed in the polymer solution.

The tank with nitrogen was connected to the inlet for the gas nozzle and the pressure was gradually increased until, until he started atomization and spraying. Form deposited using 5-second oscillating spraying with a drying time of 15 seconds between deposition. Spraying was continued until the form was not created by a suitable thickness of the polymer.

EXAMPLE 26

LOADED with a THERAPEUTIC AGENT, a POLYMERIC FILM COMPRISING ethylene vinyl acetate AND a SURFACTANT

In this example, examined two t is, loaded with paclitaxel.

Tested surfactants were two hydrophobic surfactant (Span 80 and pluronic L101) and one hydrophilic surfactant (pluronic F127). Surfactant type pluronic are polymers that were attractive because they can be mixed with EVA to optimize the properties of the delivery of various drugs. Span 80 is a smaller molecule, which is dispersed in the polymer matrix and does not form a mixture.

Surfactants applicable in modulating the speed of the release of paclitaxel from films and optimization of some physical parameters of the films. One aspect mixed with the surface-active substance films, indicating that the speed of release of the medicinal product may be regulated, was the ability to adjust the speed and degree with which a connection is swollen in water. Diffusion of water in the matrix polymer, the drug was critical for the release of drug from the carrier. Figures and 43C and 43D shows the degree of swelling of these films, when the level of surfactant in the mixture has changed. Pure EVA-plovernet-active substances, added to EVA, it was possible to increase the degree of swelling of the joints and as a result of increasing hydrophilicity swelling increased.

Results of experiments with these films are shown below in figures 43A-that is, in a nutshell, figure 43A shows the release of paclitaxel (mg) over time of pure EVA films. Figure V shows the percentage of drug remaining in the same films. As can be seen from these two figures, under increasing load of paclitaxel (i.e., increasing the percentage of paclitaxel by weight) speed of release of paclitaxel was increased, showing the expected concentration dependence. When the loading increases, the percentage of paclitaxel paclitaxel remaining in the film, was also increased, indicating that a higher load may be more favorable for compositions for long-term release.

Physical strength and elasticity of the films were evaluated and are presented in figure 43TH. In short, the figure of the 43TH shows curves of voltage from deformation (chart voltage) for films of pure EVA and films from mixtures of EVA/surfactant. This rough estimate voltage showed that the elasticity of the films Wiseman concentration, when you add pluronic F127. Flexibility and strength are important considerations in the design of the film, which should be used for specific clinical applications without causing permanent deformation of the connection.

The above results demonstrate the ability of some additives in the form of surface-active substances to regulate the rate of release of medicaments and to modify the physical characteristics of the media.

EXAMPLE 27

The PROCEDURE for PREPARATION of NANOSPRAY

Nanospray is a suspension of small microspheres in saline solution. If the microspheres are very small (i.e. less than 1 micron in diameter), they form a colloid, so that the suspension will not settle under gravity. As described in more detail below, the suspension of particles of 0.1-1 μm can be made suitable for aerosol premises directly to the tissue during surgery (e.g., the vascular adhesions through laproscopically injection or by manual injection of the aerosol (e.g., for local delivery). The equipment and materials that are used to obtain nanospray include a glass water jacket 200 ml (Kimax or Pyrex), cirkuliuoti stainless steel with 4 blades Fisher brand), glass, 500 ml, mixer with hot plate (Corning brand), 4×50 ml polypropylene centrifuge tubes (Nalgene), glass scintillation vials with plastic inserted covers, table centrifuge (Beckman), high speed centrifuge - floor model (JS 21 Beckman), analytical balance of Mettler (AJ 100, 0.1 mg), digital scale with a top load of Mettler (AE 163, 0.01 mg), automatic pipette (Gilson), sterile pipette tips, aerosol, supplied by a pump (Pfeiffer pharmaceuticals) 20 ml, laminar box, PCL (molecular weight 10000-20000; Polysciences, Warrington, Pennsylvania USA), "washed" (see above) EVA, PLA (molecular weight 15000-25000; Polysciences), polyvinyl alcohol ("PVA" - molecular weight 124000-186000; hydrolyzed 99%; Aldrich Chemical Co., Milwaukee, WI, USA), DHM or "methylenchlorid"; VIH-purity, Fisher Scientific), distilled water, sterile saline solution (Becton and Dickenson or equivalent).

1. PREPARATION of 5% (m/V) POLYMER SOLUTIONS

Depending on the prepared polymer solution was weighed directly into a glass scintillation vial, 20 ml: 1,00 g PCL or PLA or 0.50 g of each of PLA and washed EVA. Using the measuring cylinder was added 20 ml DHM and the bottle firmly closed lid. The vial was allowed to stand at to the PVA

The solution was prepared according to the following procedure or by dilution of 5% (m/R) of the original PVA solution prepared for the manufacture of microspheres (see example 28). Briefly, 17,5 g of PVA was weighed directly into a glass chemical beaker 600 ml and was added to 500 ml of distilled water. The Cup was closed and placed in a glass beaker 2000 ml containing 300 ml of water. PVA was stirred at 300 rpm at 85°C to dissolve.

3. The PROCEDURE for OBTAINING NANOSPRAY

Briefly, 100 ml of a 3.5% solution of PVA was placed in the beaker with the water jacket 200 ml with attached water bath Haacke. The contents of the beaker was stirred at 3000 rpm, and 10 ml of polymer solution (used polymer solution based on the type of prepared nanospray) was immersed in a mixed PVA for 2 minutes using an automatic pipette, 5 ml After 3 minutes, the stirring speed was brought up to 2500 rpm (+/- 200 rpm) and was stirred for 2.5 hours. After 2.5 hours of stirring blades were removed from the drug nanospray and was rinsed with 10 ml of distilled water, giving promiseme the solution to drain into the drug nanospray.

The preparation of the microspheres was poured in chemical beaker 500 ml Bath with the water jacket was washed with 70 ml of distillirovanna delannoy stick and poured equally into four polypropylene centrifuge tubes 50 ml, which was centrifuged at 10000 g (+/- 1000 g) for 10 minutes. The PVA solution was delayed from each sediment microspheres and threw. In each centrifuge tube was added distilled water (5 ml) and was mixed by vortex. Four suspensions of microspheres were combined into one centrifuge tube using 20 ml of distilled water and centrifuged for 10 minutes at 10000 g (+/- 1000 g). The supernatant was delayed precipitation of the microspheres was added 40 ml of distilled water and the preparation of the microspheres was mixed by vortex (this process was repeated 3x). Then the preparation of the microspheres was transferred into pre-weighed glass scintillation vial.

The vial was allowed to stand for 1 hour at room temperature (25°C) to give microspheres with a diameter of 2 μm and 3 μm, deposited under the action of gravity. After 1 hour the top 9 ml of the suspension was delayed, was placed in a sterile centrifuge tube 50 ml with cap and centrifuged at 10000 g (+/- 1000 g) for 10 minutes. The supernatant was discarded and the sediment resuspendable in 20 ml of sterile saline by centrifugation of the suspension at 10,000 g (+/- 1000 g) for 10 minutes. The supernatant was discarded and the precipitate resuspendable in sterile saline. nospray added to the aerosol.

EXAMPLE 28

PREPARATION of MICROSPHERES

The equipment used for the preparation of microspheres, includes: a glass water jacket 200 ml (Kimax or Pyrex), circulating water bath Haacke inserted from above the mixer and controller with a diameter of 2 inches (stirrer propeller type stainless steel with 4 blades, Fisher brand), glass, 500 ml, mixer with hot plate (Corning brand), 4×50 ml polypropylene centrifuge tubes (Nalgene), glass scintillation vials with plastic inserted covers, table centrifuge (Beckman GPR), high-speed centrifuge - floor model (JS 21 Beckman), analytical balance of Mettler (AJ 100, 0.1 mg), digital scale with a top load of Mettler (AE 163, 0.01 mg), automatic pipette (Gilson). The reagents include PCL (molecular weight 10000-20000; Polysciences, Warrington, Pennsylvania USA), "washed" (see method wash below) EVA, PLA (molecular weight 15000-25000; Polysciences), polyvinyl alcohol ("PVA" - molecular weight 124000-186000; hydrolyzed 99%; Aldrich Chemical Co., Milwaukee, WI, USA), DHM or "methylenchlorid"; VIH-purity, Fisher Scientific) and distilled water.

A. Preparation of 5% (m/V) polymer solutions

DCL (1,00 g) or PLA or 0.50 g of each of PLA and washed EVA weighed neposredno the lid and kept at room temperature (25°C) for one hour (can be applied periodic shaking) or until complete dissolution of the entire polymer. The solution can be stored at room temperature for at least two weeks.

C. Preparation of 5% (m/V) initial solution PVA

Twenty-five grams of PVA was weighed directly into a glass chemical beaker 600 ml and was added to 500 ml of distilled water, covered with a Teflon rod for stirring a size of 3 inches. The glass was covered with glass and placed in a glass beaker 2000 ml containing 300 ml of water. PVA was stirred at 300 rpm at 85°C (mixer hot plate Corning) for 2 hours or until complete dissolution. The dissolution of PVA was determined by visual inspection; the solution should be transparent. Then the solution was transferred into a glass storage container with a screw cap and stored at 4°With a maximum of two months. However, the solution should be warmed to room temperature before use or dilution.

C. the Procedure for preparation of microspheres

On the basis of the size of the prepared microspheres (see table 1) 100 ml of PVA solution (concentrations are given in table 1) were placed in a chemical glass with the water jacket 200 ml. of Circulating water bath Haacke was connected to this chemical beaker and contents were given the 1) set the initial speed at the top of the agitator and the upper blade stirrer was placed in the middle of the PVA solution. Then the stirrer was started and 10 ml of polymer solution (used the polymer solution on the basis of the type prepared microspheres) was immersed in a mixed solution of PVA for 2 minutes using an automatic pipette, 5 ml After 3 minutes, the stirring speed was adjusted (see table 1) and the solution was stirred for 2.5 hours. Then mixing blade knife was removed from the preparation of microspheres and washed with 10 ml of distilled water, giving promiseme the solution to drain into the preparation of the microspheres. Then the preparation of the microspheres was poured in chemical beaker of 500 ml and bath with the water jacket was washed with 70 ml of distilled water, giving promiseme the solution to drain into the preparation of the microspheres. Then 180 ml of microspheres was stirred with a glass rod and poured equally into four polypropylene centrifuge tubes 50 ml Then the tubes were centrifuged for 10 minutes (the centrifugal force is given in table 11). Forty-five milliliters of a solution of PVA was delayed from each sediment microspheres.

Then in each centrifuge tube was added five milliliters of distilled water and was mixed by vortex for resuspendable microspheres. Then four suspensions of microspheres were United in one sea power given in table 1). This process was repeated two more times with getting just three washes. Then the microspheres were centrifuged last time and resuspendable in 10 ml of distilled water. After the last wash, the preparation of the microspheres was transferred into pre-weighed glass scintillation vial. The vial was closed with a lid and left overnight at room temperature (25°C) to allow the microspheres to settle under gravity. As microspheres, which are in the size range of 0.1-3 μm, not deposited under the action of gravity, they remained in 10 ml of suspension.

D. Drying the microspheres with a diameter of 10-30 μm or 30-100 microns

After standing microspheres at room temperature overnight, the supernatant was delayed from precipitated microspheres. The microspheres were allowed to dry in an open cap vial with a thrust within the period of one week or until they are fully dry (up to a constant weight of the bottle). More rapid drying can be achieved by leaving an open bottle under a slow stream of nitrogen (current of approximately 10 ml/min) in a fume hood. After complete drying (up to a constant weight of the vial) vial is weighed and close the lid. Indoor signed vial store at room teacher with a diameter of 0.1-3 μm

Microspheres of this size range is not deposited, so that they remain in suspension at 4°With a maximum of four weeks. To determine the concentration of microspheres in 10 ml of suspension sample 200 µl of this suspension is placed in a pipette in a pre-weighed microporous tube 1.5 ml Then the test tube is centrifuged at 10,000 g in a tabletop Eppendorf centrifuge), the supernatant removed and the tube allowed to dry at 50°C for the night. Then the tube is re-weighed to determine the weight of the dried microspheres in vitro.

F. Preparation loaded with paclitaxel microspheres

For preparation containing paclitaxel microspheres suitable number weighted of paclitaxel (based on the percentage of paclitaxel, which should be encapsulated) were placed directly in a glass scintillation vial of 20 ml and Then into the vial containing paclitaxel was added 10 ml of a solution of a suitable polymer and then mixed by vortex to dissolve the paclitaxel.

Then the microspheres containing paclitaxel, can be prepared as described in stage (C)-(E).

EXAMPLE 29

MICROSPHERES COATED with SURFACE-ACTIVE SUBSTANCE

A. Materials and methods

ICRI is unilateral (EVA):PLA essentially as explained above.

Human blood was obtained from healthy volunteers. Neutrophils (leukocytes) was separated from the blood by means of dextran sedimentation and centrifugation in Ficoll Hypaque. Neutrophils were centrifuged at 5 million cells per ml in SRH.

The activation levels of neutrophils was determined by generation of reactive molecules of oxygen determined by chemiluminescence. In particular, the chemiluminescence was determined using an LKB luminometer with 1 μm lyuminola as the amplifier. Preliminary floor (or opsonization) plasma microspheres was performed by suspendirovanie 10 mg of microspheres in 0.5 ml of plasma and turning at 37°C for 30 minutes.

Then the microspheres were washed in 1 ml SRH and centrifuged sediment of the microspheres was added to a suspension of neutrophils at 37°C at time t=0. The surface of the microspheres modified with the use of surfactants pluronic F127 (BASF) suspendirovanie 10 mg of microspheres in 0.5 ml of 2% m/m solution of F127 in SRH for 30 minutes at 37°C. Then the beads were washed twice in 1 ml SSR before adding to neutrophils or plasma for further pre-coating.

C. Results

Figure 44 shows that the raw Trefilov. As a comparison, inflammatory microcrystals can give values close to 1000 mV, soluble chemical activators can give values close to 5000 mV. However, when microspheres pre-coated with plasma, all values chemiluminescence increase to the range of 100-300 mV (figure 44). These levels of response or activation of neutrophils can be seen as srednetemperaturnyi. Emission spectra obtained for pure gave the highest reaction and could be considered as the most inflammatory. PLA and PCL, both became 3-4 times stronger in the activation of neutrophils after preprocessing of the plasma (or opsonization), but there was little difference between the two polymers in this regard. EVA:PLA, apparently, cannot be used in compositions for angiogenesis, as these microspheres are difficult to dry and resuspending in aqueous buffer. This action of the plasma is called opsonization, and it leads to the adsorption of antibodies or molecules of complement on the surface. These adsorbed molecules interact with receptors on leukocytes and cause strong activation of the cells.

Figures 45-48 describe the effect of pre-coating plasma PCL, emission spectra obtained for pure PLA and EVA:PLA, respectively, and also show the effect of coating plural) pre-coating the plasma enhances the reaction. (2) pre-coating pluronic F127 has no effect by itself; (3) the amplified response of neutrophils caused by pre-coating plasma can strongly inhibited by pretreatment of the surface of the microspheres 2% pluronic F127.

The nature of the adsorbed protein molecules from plasma was also investigated by means of electrophoresis. Using this method it was shown that the pretreatment of polymer surfaces by pluronic F127 inhibited the adsorption of antibodies on the polymer surface.

Figures 49-52 also show the effect of pre-coating of PCL microspheres, emission spectra obtained for pure PLA and EVA:PLA (respectively) or IgG (2 mg/ml) or 2% pluronic F127 and then IgG (2 mg/ml). As can be seen from these figures, the amplified response caused by pre-coating the microspheres IgG, could be ingibirovany treatment pluronic F127.

This result shows that by pretreatment of polymer sequences of all four types of microspheres by pluronic F127 inflammatory response of neutrophils to the microspheres can be ingibirovany.

EXAMPLE 30

ENCAPSULATING theRAPEUTIC AGENT IN MICROSPHERES

POLY(-CAPROLACTONE). INHIBITION of ANGIOGENESIS IN Scania in vitro paclitaxel from biodegradable microspheres of poly(-caprolactone) (PCL) and demonstrates in vivo antiangiogenic activity of paclitaxel released from these microspheres, when placed on HIMSELF.

The reagents used in these experiments include: PCL (molecular weight 35000-45000; purchased in Polusciences (Warrington, PA), DHM from Fisher Scientific Co. Canada; polyvinyl alcohol ("PVP" - molecular weight 12000-18000, hydrolyzed 99%) from Aldrich Chemical Co. (Milwaukee, Wis.) and paclitaxel from Sigma Chemical Co. (St. Louis, MO). Unless otherwise noted, all chemicals and reagents used in the form in which they were delivered. In all cases, used distilled water.

A. Preparation of microspheres

Microspheres were prepared essentially as described in example 28, using the method of solvent evaporation. Briefly, loaded with 5% m/m paclitaxel microspheres were prepared by dissolving 10 mg of paclitaxel and 190 mg of PCL in 2 ml DHM with the addition of 100 ml of 1% aqueous solution of PVP and stirring at 1000 rpm, at 25°C for 2 hours. Suspension of microspheres was centrifuged at 1000×g for 10 minutes (Beckman GPR), the supernatant was removed and the microspheres were washed three times with water. The washed microspheres were air-dried overnight and stored at room temperature. Control microspheres (without p the spheres was determined using an optical microscope with a micrometer on the table.

C. the Efficiency of encapsulation

A known sample loaded drug microspheres (about 5 mg) was dissolved in 8 ml of acetonitrile was added 2 ml of distilled water to precipitate the polymer. The mixture was centrifuged at 1000 g for 10 minutes and the number of encapsulated paclitaxel was calculated from the absorbance of the supernatant measured in a UV spectrophotometer (Hewlett-Packard 8452A Diode Array Spectrophotometer) at 232 nm.

C. Studies of release of the medicinal product

Approximately 10 mg loaded with paclitaxel microspheres suspended in 20 ml of 10 mm SFR (pH 7.4) in test tubes with screw caps. Tubes turned from end to end at 37°C and at certain time intervals were removed to 19.5 ml of the supernatant after incubation of the microspheres for deposition on the bottom), filtered through a membrane filter of 0.45 μm and preserved for analysis of paclitaxel. Equal volume SFR was added to each tube to preserve the conditions of sedimentation throughout the study. The filtrate was extracted with 3×1 ml DHM, extracts DHM evaporated to dryness under a stream of nitrogen, pererestorani in 1 ml of acetonitrile and analyzed using VIH using as mobile phase a mixture of water:methanol:ACE the Shimadzu SPD) at 232 nm.

D. Studies using HIMSELF

Fertilized embryos hens were incubated for 4 days before cultivation without the shell. On the 6th day of incubation aliquots of 1 mg loaded with 5% paclitaxel or control (not containing paclitaxel) microspheres were placed directly on the surface ITSELF. After 2-day exposure of the vascular network was examined using a stereomicroscope with an attached video camera; then the signals were displayed on the computer and their information was collected using a system of image analysis.

E. Scanning electron microscopy

Microspheres were placed in holders for samples coated with gold plating, and then placed in SAM Phillips 501B, operating at 15 kV.

F. Results

The range of sizes of samples of the microspheres was between 30 and 100 microns, although all loaded with paclitaxel or control series microspheres some microspheres were outside this range. Efficiency load of PCL microspheres by paclitaxel was always above 95% for all investigated loads medicines. Scanning electron microscopy showed that all microspheres were spherical and many have found rough or covered with dimples marasti microspheres.

The time course of release of paclitaxel loaded from 1%, 2% and 5% paclitaxel microspheres PCL presented in figure 53A. Profiles of rates of release were biphasic. Observed initial rapid release of paclitaxel or "phase emissions at all loads medicines. The phase of the ejection took place for 1-2 days at 1% and 2% loads of paclitaxel and for 3-4 days for loaded with 5% paclitaxel microspheres. For the initial phase of fast release phase followed at a much slower release of the drug. For microspheres containing 1% or 2% paclitaxel, no additional release of drug after 21 days. At 5% loading of paclitaxel microspheres released about 20% of the total content of the medicinal product after 21 days.

Figure V shows HIMSELF treated control PCL microspheres, and figure C shows processing loaded with 5% paclitaxel microspheres. HIMSELF with control microspheres found normal architecture of the capillary network. HIMSELF-treated microspheres paclitaxel-PCL showed marked regression of blood vessels and areas that had no capillary vascular network.

G. Discussion

Two-phase release profile for paclitaxel was the typical pattern of release for many drugs from biodegradable polymeric matrices. Poly(-caprolacton) is an aliphatic polyester, which can be broken by hydrolysis under physiological conditions, and it is non-toxic and donesomething. The degradation of PCL is much slower than the destruction of the well-studied polymers and copolymers of lactic and glycolic acids, and therefore it is suitable for construction of long-term drug-delivery systems. It is believed that the initial rapid phase of release or phase of the release of paclitaxel due to diffusional release of drug from the surface area of the microspheres (close to the surface of the microspheres). The release of paclitaxel in the second (slower) phase of the release profiles were not, apparently, caused by the destruction or erosion of the PCL, as research pok is inogo period. The slower phase of the release of paclitaxel was probably caused by the dissolution of the drug in the fluid-filled pores in the polymer matrix and diffusion through the pores. Faster release at higher loading of paclitaxel was probably a result of more abundant network of pores in the polymer matrix.

It was shown that the microspheres of paclitaxel with a 5% load release a sufficient quantity of a drug to induce a strong inhibition of angiogenesis in the room ITSELF. Inhibition of angiogenesis has led to the formation of an avascular zone, as shown in figure S.

EXAMPLE 31

PREPARATION of PLGA MICROSPHERES

Microspheres were prepared from copolymers of lactic acid - glycolic acid (PLGA).

A. the Way

Microspheres were prepared in the size ranges of 0.5-10 μm, 10-20 μm, and 30-100 microns using standard methods (polymer was dissolved in dichloromethane and was emulsiable in solution of polyvinyl alcohol with stirring, as described earlier in the methods of preparation of PCL microspheres or PDLLA). Different ratios of PLLA to GA was used as polymers with different molecular weights (presented in their original polymers:

Paclitaxel at 10% or 20% loads successfully integrated in all these microspheres. Examples of size distributions for one of the original polymer (85:15, I. V.=0,56) are given in figures 54-57. Experiments on the release of paclitaxel was performed using the microspheres of different sizes and different compositions. Speed of release is shown in figures 58-61.

EXAMPLE 32

ENCAPSULATION of PACLITAXEL IN NYLON

The MICROCAPSULES

Therapeutic agents can be encapsulated in a wide variety of media that can be formed in the form of the selected form or device. For example, as described in more detail below, paclitaxel may be included in nylon microcapsules, which can be prepared in the form of artificial heart valves, vascular grafts, surgical mesh or suture material.

A. Preparation loaded with paclitaxel microcapsules

Paclitaxel was encapsulated in nylon microcapsules by the means of interfacial polymerization (polymerization at the phase boundary). Briefly, 100 mg of paclitaxel and 100 mg pluronic F-127 was dissolved in 1 ml DHM and was added 0.4 ml (approximately 500 mg) editorchoice (ADC). This Organizatsii was added dropwise a solution of 1,6-hexanediamine (HMD) in 5 ml of distilled water. The mixture is homogenized for 10 seconds after adding the solution HMD. The mixture was transferred into a chemical beaker and stirred with a magnetic stirrer for 3 hours. The mixture was centrifuged, collected and resuspendable in 1 ml of distilled water.

C. the encapsulation Efficiency/load paclitaxel

Approximately 0.5 ml of the suspension was filtered and the microspheres were dried. Approximately 2.5 mg of microcapsules were weighed and suspended in 10 ml of acetonitrile for 24 hours. The supernatant was analyzed for paclitaxel and the results were expressed as the percentage of paclitaxel. Preliminary studies have shown that paclitaxel could be encapsulated in nylon microcapsules under high load (up to 60%) and high encapsulation efficiency (>80%).

C. Studies of the release of paclitaxel

Approximately 2.5 mg of microspheres paclitaxel-nylon suspended in 50 ml of water containing 1 M sodium chloride and 1 M urea, and periodically analyzed. The release of paclitaxel from these microcapsules was fast, with 95% of the drug released after 72 hours (figure 62).

EXAMPLE 33

BIOADHESIVE MICROSPHERES

A. Preparation bioadhesive microspheres

Micro is hydroxide sodium obtaining carboxyl groups on the surface by hydrolysis of the polyester. The reaction was characterized in terms of concentration of sodium hydroxide and time of incubation by measuring the surface charge. The reaction was terminated after 45 minutes incubation in 0.1 M sodium hydroxide. After treatment with alkali microspheres covered with dimethylaminopropylamine (DEC), a crosslinking agent, by suspension of the microspheres in ethanol solution DEC and drying the mixture to a dispersible powder. The weight ratio of microspheres to DEC was 9:1. After drying of the microspheres were dispersively under stirring in 2% m/about the solution of poly(acrylic acid) (PAA) and gave DEC to react with PAA with the formation of water-insoluble network crosslinked PAA on the surface of the microspheres. Scanning electron microscopy was used to confirm the presence of PAA on the surface of the microspheres.

Differential scanning calorimetry of these microspheres before and after treatment with alkali was found that there were no changes in the overall thermal properties of (Cu, melting and crystallinity) in the study with SAM.

C. Speed of release of paclitaxel in vitro

Prepared loaded with paclitaxel microspheres (10% and 30% m/m load) with the same range of sizes and diameters were determined medicines with 400 μg of paclitaxel released from 5 mg loaded 30% paclitaxel microspheres for 10 days and 150 μg of paclitaxel released from the loaded 10% paclitaxel microspheres for the same period of time. The encapsulation efficiency was about 80%. Loaded with paclitaxel microspheres were incubated in 0.1 M sodium hydroxide for 45 minutes and measured the Zeta potential before and after incubation in sodium hydroxide. Surface charge loaded with paclitaxel microspheres was lower than microspheres without paclitaxel, both before and after treatment with alkali.

S. Preparation and evaluation in vitro PLLA coated polylysine or fibronectin

Prepared PLLA microspheres containing 1% Sudan black (for staining of the microspheres). These spheres suspended in 2% (m/V) solution polylysine (Sigma Chemicals - Hydrobromell form) or fibronectin (Sigma) for 10 minutes. Microspheres were washed once in buffer and placed on the inner surface of svezhepriobretenny urinary bladders of rats. Bladders were left for 10 minutes and then washed three times in buffer. Residual (related) microspheres were present in the bladder wall after this process, indicating, therefore, the presence of bioadhesive (figures 63A and B) ATTACK of ARTHRITIS by PACLITAXEL

In a RAT MODEL ClA

A. Materials and methods

Syngeneic female rats Louvain weighing 120-150 grams were intradermally injected with 0.5 mg of native chicken collagen II (Genzyme, Boston, MA) dissolved in 0.1 M acetic acid and emulsified in FIA (Difco, Detroit, MI). After about 9 days after immunization the animals found polyarthritis with histological changes in the formation of pannus and bone/cartilage erosions. Used only 45 rats in 4 protocols: control group (n=17), which received only the carrier, and 3 groups of treatment with paclitaxel, consisting of groups with preventive Protocol and 2 groups with suppressive protocols. For evaluating the effect of paclitaxel paclitaxel (the solubilized in a dilution of 1:1 ethanol:rmhr E. L.® (Sigma) and added to a saline solution to a final concentration of 4.8 mg/ml of paclitaxel in 5% m/about ethanol and rmophor E. L.) were injected intraperitoneally (i.p.), starting from day 2 after immunization (preventive Protocol) or if you have any arthritis at day 9 (suppressive Protocol). For preventive Protocol (n=8) of paclitaxel was given at a concentration of 1 mg/kg body weight starting on day 2 with 5 subsequent doses on days 5, 7, 9, 12 and 14. For suppressive Protocol with high dose (n=10) paclitaxel (day 1 to day 21. Control and experimental animals was evaluated relative to the severity of the disease both clinically and rentgenograficheski persons who did not know the setting of the experiment (treated groups).

The severity of inflammation for each limb was assessed daily and evaluated at points on the basis of standardized levels of swelling and periarticular erythema (0 for normal and 4 for heavy). Animals were evaluated rentgenograficheski on the 28th day of the experiment. Radiographs of both hind limbs were placed on the degrees of swelling of soft tissues, narrowing of the joint space, bone destruction and periosteal bone tissue. To quantify each limb used a scale of 0-3 (0 = normal, 1 = soft tissue swelling, 2 = early bone erosion, 3 = severe bone destruction and/or ankylosis). Histological evaluation of the joints was performed at the end of the experiment.

Allergic reaction of the delayed type (DTH) on collagen II (S) was determined by radiometric ear test performed on the 28th day. Radiometric performance ear test1,4 indicate a significant response to S. The presence of IgG antibodies against S determined immunofermentnyi the de average optical density at 490 nm, in four replications. Background levels in the serum of normal rats at this dilution is equal to 0 and readily distinguishable from serum immunized with collagen of rats.

C. Results

In this model, treatment with paclitaxel taken prior to the occurrence of arthritis, completely prevented the development of this disease in all treated rats (even after interruption of treatment with paclitaxel) in comparison with the control group treated with the carrier.

In control animals was a progressive increase in clinical symptoms up until not occurred deformity and loss of function of joints. Animals treated with low doses and high doses of paclitaxel after the onset of arthritis, showed significant clinical improvement. The average clinical scores were equivalent to scores observed at the beginning of treatment, indicating the ability of paclitaxel to prevent the progression of this disease.

Animals treated with paclitaxel, were able to withstand your weight and move and have found toxic effects (or no toxic effects) of treatment. The treated animals were observed wound healing and re-growth of fur in places the Rys preventive Protocol (with the effect of paclitaxel on the prevention of arthritis) did not find any radiographic changes or clinical arthritis. Both groups, with high and low doses of paclitaxel, had significantly less radiographic disease compared with the control group. Histological evaluation revealed that the rats of the control group showed a marked pannus, with bone and cartilage erosions, however, treated with paclitaxel rats had minimum pannus (or no pannus) with preservation of the articular cartilage.

When applying the test, ELISA, IgG antibodies to collagen II were significantly lower in treated with paclitaxel rats compared with the control group; rats in the preventive Protocol had significantly fewer antibodies IgG compared with rats in suppressive protocols with high and low doses of paclitaxel.

C. Discussion

Paclitaxel is a viable means of treatment for arthritis and potentially for other types of autoimmune diseases, as it blocks the process of the disease when introduced after immunization, but prior to the occurrence of arthritis. These results indicate that paclitaxel could completely suppress the occurrence of arthritis at the beginning of his introduction 2 days after immunization S. During treatment with paclitaxel in suppressive Protocol severity of arthritis of Senegalese in both suppressive protocols. However, the early introduction of paclitaxel, apparently, reduces the need for continuous therapy.

EXAMPLE 35

REGRESSION of collagen-INDUCED ARTHRITIS

The PACLITAXEL

Paclitaxel showed disease modifying effects in the CIA model with the systemic administration in the form of micellar composition. To assess the potential disease modifying action of paclitaxel micelle (without Cremophor) paclitaxel was administered intraperitoneally (i.p.) every four days (q.o.d.) at 5 mg/kg (group 1) or 10 mg/kg (group 2) immunized animals in the emergence of clinically detected arthritis (day 9). Paclitaxel was administered during the evaluation period. For comparison with standard therapy, the third group received methotrexate at 0.3 mg/kg i.p. (equivalent to the human dose) on days 0, 5 and 10 after the onset of arthritis. The fourth group received a combinatorial therapy with the provision of methotrexate (0.3 mg/kg) and micellar paclitaxel (10 mg/kg). Control group (group 5) and experimental animals was evaluated in relation to the severity of the disease both clinically and rentgenograficheski by persons not familiar with group processing.

The severity of inflammation for each limb was determined daily and an assessment is 4 for heavy). Animals were evaluated rentgenograficheski on the 28th day of the experiment. Radiographs of both hind limbs were placed on the degrees of swelling of soft tissues, narrowing of the joint space, bone destruction and periosteal new bone formation. To quantify each limb used a scale of 0-3 (0 = normal, 1 = soft tissue swelling, 2 = early bone erosion, 3 = severe bone destruction and/or ankylosis) (Brahn et al. Arthritis Rheum. 37: 839-845, 1994; Oliver et al., Cell. Immunol., 157:291-299, 1994). Histological evaluation of the joints was performed at the end of the experiment.

In this model, processing micellar paclitaxel taken prior to the occurrence of arthritis, completely prevented the development of this disease even after interruption of treatment with paclitaxel. Control animals was a progressive increase in clinical symptoms (figure 64), until there was a deformity and loss of function of joints. Animals receiving treatment with methotrexate, were not statistically improved in comparison with controls (figure 64 and table 12). Animals that received the low dose of micellar paclitaxel (5 mg/kg) after the onset of arthritis, has shown some improvement, but animals that p is tion (figure 64). The average clinical scores were equivalent to scores observed at the beginning of treatment, indicating the ability of micellar paclitaxel to prevent clinical progression of this disease (table 12).

Animals receiving micellar paclitaxel, were able to hold the weight and move and did not show any toxic effects of the treatment. The treated animals were observed wound healing and resumption of hair growth at the site of vaccination. Processed micellar paclitaxel animals gained weight relative to untreated controls. Animals receiving as micellar paclitaxel and methotrexate was well tolerated by this therapy and has shown impressive clinical improvement (pof 0.0001) compared with controls (figure 64). When using enzyme-linked immunosorbent assay (ELISA) levels of IgG antibodies to collagen type II were lower in treated with paclitaxel and the combination of MTX/paclitaxel rats in comparison with controls.

Radiographic studies have also shown significant improvement in the case of therapy with paclitaxel. While the control and treated methotrexate animals discovered the x-ray is ostalnoe the formation of bone tissue, treated with paclitaxel animals had almost normal signs of joints in radiography (figure 65).

In fact, only a small percentage (17-18%) animals treated only micellar paclitaxel (10 mg/kg) or paclitaxel in combination with methotrexate, has developed a cartilage erosion. Cartilage erosion, an important indicator of progression/outcome of the disease, there are 4 times more frequently in control animals (72%) or animals treated only methotrexate than in animals treated with therapy micellar paclitaxel (table 13).

Micrograph of scanning electron microscope illustrate chondro-protective effects of therapy with paclitaxel in vivo. Normal surface of the joint is characterized by intact cartilage matrix, surrounded by a thin synovial lining (figure 66A). In ClA cartilage pierced DFID produced pannus tissue and inflamed synovium (figure V). The surface of the cartilage matrix is split, exposing the chondrocytes or the empty gaps that they once held (insert figure V). In animals with ClA receiving treatment with paclitaxel after the occurrence of clinical arthritis, the surface of the joints remains intact (figure C) and cartilage the Finance tissue pannus and synovial hypertrophy was not observed in treated with paclitaxel groups.

Histologically CIA characterized by marked synovial hypertrophy, infiltration of inflammatory cells in sinovia and destruction of the cartilage (figure 67A). In treated with paclitaxel animal sinovi seems normal, with only 1-2 layers of synoviocytes and no infiltration of inflammatory cells (figure V).

Corrosion casting was evaluated to determine whether paclitaxel to block angiogenesis in Zinovii animals with ClA. The polymer Megson was administered by infusion into the femoral artery dead animals at a pressure of 100 mm RT. Art., allowing it to cure in situ, and then the tissues were digested with obtaining castings of the vascular network of the lower limb. Micrograph of scanning electron microscope castings synovial vasculature in animals with CIA revealed slipscandace capillary processes acting inside the articular space (figure 68A). These vessels seem to be morphologically similar to growing angiogenic vessels described for solid tumors and other angiogenic conditions (box figure 68A). In contrast, synovial vessels treated with paclitaxel animals placed in the form of capillary loops (figure V) without the appearance of neovascular is the recipient of paclitaxel/MTX, like this picture, found in no processed anything the controls. These studies suggest that therapy with the use of micellar paclitaxel and the combination of paclitaxel/methotrexate may lead to a reverse development (regression) neovascularization, inhibit inflammation, and cause the opposite of the steady development of synovitis and prevent joint destruction.

It was demonstrated that systemic injection of paclitaxel is a viable therapy for arthritis. The natural course of the disease suddenly escalates and detects remission, and each of a sudden exacerbation leads to additional damage that eventually leads to joint destruction. There is the potential for short-term therapy, therapy with high doses of systemic therapy, which should be used for the induction of remission of the disease, or long-term therapy with low doses to maintain control of the disease. Alternative methods of delivery of paclitaxel include direct intra-articular injection of the drug in the affected joints of patients with predominant disease 1 or 2 joints.

EXAMPLE 36

EVALUATION of COMPOSITIONS PAK is lsout for research specific to your skin angiogenesis. Immunodeficient SCID mice are used as recipients for surface grafts lines of human keratinocytes, transfected factor vascular endothelial growth (VEGF) in sense and antisense orientation. Keratinocytes transplanted by applying the modified test with silicone camera for transplantation on the skin of a mouse recipient. The keratinocytes give to differentiate and to induce cutaneous angiogenesis. Then injected paclitaxel either systemically or topically (cream, ointment, lotion, gel) and perform morphometric measurements of the number and size of vessels in untreated and treated groups.

Century Murine model for allergic skin reactions of the delayed type.

Murine model for allergic skin reactions of the delayed type were used for studying the action of paclitaxel on induced skin inflammation. Briefly, mice were senzibilizirani oxazolone local application of this compound to the skin. Five days later the mice were provoked by oxazolone by local application to the skin of the ear (left ear: oxazole, right ear: one medium), leading to skin inflammation, allergic reactions of the delayed type". The degree of inflammation was determined by the PMAS, painted Giesma, 1 μm tissue sections were evaluated for the presence of inflammatory cells, the presence of mastocytes in the fabric and their state of activation and the degree of epidermal hyperplasia. Paclitaxel was given systemically or topically for the quantification of dermal inflammatory response in this model in vivo.

C. Results

These studies showed that local injection of 1% of paclitaxel in comparison with one carrier in the treatment of experimentally induced skin inflammation in mice revealed that paclitaxel exerts inhibitory effects on skin inflammation. In experimentally induced allergic reactions of the delayed type was a significant reduction in the swelling of the ears treated topically with 1% paclitaxel, compared with one carrier. Local application of the composition 1% of paclitaxel significantly inhibited the swelling of the ears and skin erythema (redness), induced by local application of PMA (phorbol-12-myristol-13-acetate) (see figures 71 and 72). As shown in figure 73, treated with paclitaxel ear (right ear) was normal in appearance in comparison to the controls (left ear). Similar results were obtained in General for 5 mice.

To assess skin irritation 1% paclitaxel in sraa. After 8 days there was no irritation of the skin after application of one carrier or composition 1% of paclitaxel in normal or inflamed skin of the ears of a mouse.

EXAMPLE 37

ASSESSMENT of CHRONIC TRANSPLANT REJECTION IN a MODEL ANIMAL

Accelerated form of atherosclerosis develops in the majority of recipients of heart transplants and limits long-term survival of the graft. The model of chronic rejection in heterotopic heart transplant rats Lewis-F344 is a valuable experimental model because it produces atherosclerotic damage Paladino, medium and long-term survival of the allografts. The advantages of this model Lewis-F344 are as follows: (i) the occurrence and severity of atherosclerotic lesions in dogovarivaya the grafts is quite high; and (ii) the inflammatory stage of development of damage can be easily detected, since this system does not require immunosuppression.

Adult male Lewis rats served as donors and rats (F-344 as recipients. Twenty heterotopic abdominal cardiac allografts transplanted, making a long abdominal incision along the mid-line of the shot R is internai tissue and small clamps are placed on these vessels. Longitudinal sections (2-3 mm) are produced in each vessel at the site of anastomosis.

Belly shot donor rats open for injection of 300 units of aqueous heparin into the inferior Vena cava. Chest wall opening for exposure of the heart. Hollow veins are ligated, followed by transection of the ascending aorta and the main pulmonary artery, so that the beginning of vessels 2-3 mm long remain attached to the heart. Cava relatively distal ligatures are divided and placed a ligature around the mass of the left atrium and pulmonary veins. Vessels on the pulmonary side of the ligature is separated and the heart removed.

The donor heart is placed into the abdominal cavity of the recipient and the aorta sew together at the site of incision in the recipient vessel. Similarly pulmonary artery connected to the cut location on the inferior Vena cava in the same way. Clamps for vessels exempt (proximal Vena cava, the distal Vena cava and the aorta and proximal aorta) to minimize bleeding from the holes of the needles.

After transplantation paclitaxel (33%) in the paste of polycaprolactone (PCL) (n=10) or only pasta PCL (n=10) injected through the epicardium over the length of the outer surface of the coronary artery in 10 rats, so that the area of the artery, immersed in the myocardium, the CI) at the time of transplantation. Grafts examined daily by palpation and their function evaluated on a scale of 1-4, with 4 points, representing a normal heartbeat, and 0 representing the absence of mechanical activity. Five rats from each group of death at 14 days and the remaining five at 28 days. Rats were observed weight loss and other signs of systemic disease. After 14 or 28 days anaesthetize animals and heart naked, as in the beginning of the experiment. Covered and uncovered coronary artery allocate, fixed in 10% buffered formaldehyde and examined histologically.

The original experiment can be modified to apply the film paclitaxel/EVA or covered stents in the coronary arteries after transplantation. The EVA film is applied on extraluminal surface of the coronary artery just as described above, while the stent is coated is placed intraluminally.

In addition, these studies can be further expanded so that they included the transplants of other organs and tissue grafts (e.g., veins, skin).

EXAMPLE 38

EFFECT of PACLITAXEL IN the MODEL PC (MULTIPLE SCLEROSIS)

ANIMAL

Investigated the ability of micelles for paclitaxel Inga the mouse (Mastronardi et al., J. Neurosci. Res. 36: 315-324, 1993). These transgenic mice contain 70 copies transgenic DM20, proteolipid myelin. Clinically, the animals appear normal until 3 months of age. After 3 months appear and progressive increase in their severity of symptoms of neurological diseases, such as seizures, tremor, motility of the rear limbs, unsteady gait, limp tail, staggering gait, and reducing the degree of activity, while these animals do not die at the age of between 6 and 8 months. Clinical signs correlate histologically with demielinizaciei and increased proliferation of fibrous astrocytes in the brain (Mastronardi et al., J. Neurosci. Res. 36: 315-324, 1993).

A. Materials and methods

Two animal studies were performed using subcutaneous injection or according to the Protocol of continuous therapy with paclitaxel at low doses (2.0 mg/kg; 3× a week, a total of 10 injections), or according to the Protocol "pulse" therapy with paclitaxel in high doses (20 mg/kg; four times, once a week), starting with the clinical appearance of the disease (approximately 4 months of age). For a Protocol with low doses of 5 animals received micellar paclitaxel, two mice were used as controls; one mouse bidno transgenic mouse was used as a control, as the course of the disease was well established in this laboratory. Four animals were injected with micellar paclitaxel after initial signs of a PC has reached a score of 1+ for categories of symptoms described above. Processing was carried out for 24 days (2.0 mg/kg of paclitaxel, 3× week ×10 doses). Body weight and clinical signs were determined on each day of the injection.

C. Results

5 animals treated with paclitaxel, found no significant weight loss. However, untreated transgenic mouse found a 30% reduction of body weight, with 29 g to 22 g (figure 74), as is the norm associated with the progression of this disease.

Clinical indicators PC, such as tremor, motility of the rear limbs, seizures, head tremor, unsteady gait, limp tail and the decrease in activity was observed daily. At the beginning of treatment, the animals had a score of 1+ in the main categories of symptoms. Untreated animals progressed from grade 1 to grade 4+ over the next 27 days on a range of symptoms; indicator 3+ was characterized by balance, one of the main signs of this disease. In treated with paclitaxel group all five animals remained at odusami mice were treated with 20 mg/kg of paclitaxel once a week for 4 weeks to simulate pulse therapy with intervals (once a month for breast cancer and ovarian cancer), used for cancer patients. Animals were monitored for 10 weeks, every two days and the scores were determined for each symptom. Three untreated animals neurological symptoms progressed rapidly and two animals died (at 5 weeks and 9 weeks) during the experiment; the third surviving animal had severe clinical symptoms. 5 transgenic animals receiving treatment with paclitaxel, which began after the first week of observation was the reduction of points PC (multiple sclerosis) relative to the controls after the first week of observation and after that neurological deterioration was observed. In these animals the progression of the disease is not observed, and the animals remained clinically in remission during therapy (weeks 0-3), and after cessation of drug treatment (weeks 4-10) (figure 75).

C. Conclusions

Paclitaxel prevented a rapid progression of neurological symptoms observed in this model PC animal at low doses and at high doses. These results suggest that paclitaxel may be a potential therapeutic agent for demyelinating disease.

EXAMPLE 39

EVALUATION of PACLITAXEL AND OTHER STABILIZING MICROCR the CSOs polyp used to evaluate the effectiveness of the compositions, containing paclitaxel or other agents, in the treatment of nasal polyps. This approach is based on the premise that the epithelial cells release cytokines and contribute to chronic inflammation that can be detected by natalina the polyposis, as well as in rhinitis and asthma, and that treatment with drugs with prolonged action will prevent eosinophilia and to inhibit gene expression of cytokines.

Compositions of paclitaxel, including mortars (using cyclodextrins) or suspension containing paclitaxel encapsulated in mucoadhesive polymers for use in the form of nasal sprays, and/or microencapsulating paclitaxel in mucoadhesive the polymers used in the form of insufflate. These compositions are used in the research, described in detail below.

A. Effect of paclitaxel in vitro

The manipulation of tissues - Normal samples of the nasal mucosa (NM) obtained from patients without clinical signs of rhinitis and negative test skin prick during plastic surgery of the nose. Samples of nasal polyp (NP) obtained from patients with positive and negative test skin prick subjected nasal polypectomy. Nasal samples placed transportyou in the laboratory.

Culture of epithelial cells - nasal epithelial cells of the NM and NP allocate proteases splitting as follows. Tissue samples are washed 2-3 times environment Ham F12 supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 μg/ml amphotericin b, and then incubated in 0.1% protease type XIV in Ham F12 at 4°C for the night. After incubation, add 10% FCS for neutralization by activity and epithelial cells separated by slow shaking. The cell suspension is filtered through a sieve for dissociation of cells with 60 mesh and centrifuged at 500 g for 10 minutes at room temperature. Then the precipitate cells resuspended in hormonally defined culture medium Ham F12 (Ham HD) containing the following reagents: 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 μg/ml of amphotericin b, 150 μg/ml glutamine, 5 μg of transferrin, 5 μg/ml insulin, 25 ng/ml epidermal growth factor, 15 µg/ml growth supplements endothelial cells, 200 PM trijodthyronin and 100 nm hydrocortisone. Then the cell suspension (105cells/well) were seeded on the coated collagen wells in 2 ml US HD and cultured in a humidified atmosphere of 5% CO2at 37°C. the Culture medium replaced during the day and then each subsequent on the epithelial cells of the human environment (NASM) - When cultures of epithelial cells was achieved confluently, environment Ham replace the HD medium RPMI 1640 (Irvin, Scotland), supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 μg/ml of amphotericin b, 150 μg/ml glutamine and 25 mm Hepes-buffer (RPMI 10%). NASM, which is generated after 48 hours incubation with RPMI (10%), collected from cultures, centrifuged at 400 g for 10 minutes at room temperature (RT), sterilized by filtration through 0.22 μm filters and stored at -70°C to use.

Survival of eosinophils and the effect of paclitaxel - Eosinophils isolated from the peripheral blood and the action of NESM of the NM and NP on the survival of eosinophils define two different ways: by analysis of the time course and the analysis of dose-response. In experiments with a time course of eosinophils at concentrations of approximately 250000/ml incubated in sectionone cultures fabric with 50% of NASM or without 50% of NASM (negative control) and the index of survival assessed at days 2, 4, 6 and 8. Other experiments carried out with 1-50% of NESM. In the experiments, which have the effect of drugs (e.g. paclitaxel) induced on NASM survival of eosinophils, a drug (paclitaxel) from 0.1 nm to 10 μm incubated with eosinophils at 37°With during the social environment) and positive control (culture medium with NASM). To study whether these medicines any toxic effect, the viability of eosinophils incubated with drug (various concentrations), compared with eosinophils cultured only with RPMI (10%), over a period of 24 hours.

C. Effect of paclitaxel on gene expression of cytokines and the release of epithelial cells

Epithelial cells derived from nasal polyps and normal mucosa of the nose, to cultivate confluently, generate air-conditioned epithelial cells of the human environment with paclitaxel and without paclitaxel (or other agents) and supernatant measured by ELISA. Gene expression of the cytokines examined using polymerase chain reaction with reverse transcription (RT-PCR), as described Mullol et al., Clinical and Experimental Allergy 25: 607-615, 1995.

The results show that modulates whether paclitaxel gene expression of cytokines as a means of inhibiting the survival of eosinophils. The main disadvantage of the use of primary cell cultures is the fact that it takes 10 days to reach the cells confluently, dissociation of cellular functions from the local environment, as well as systemic effects, which is vitro/ex vivo for studies of growth factors, regulatory function and proliferation of structural cells (e.g. epithelial cells), and thereby clarify some aspects of inflammation of the mucous membrane.

C. Immunological release of chemical modulators of nasal polyps person

Mediating with paclitaxel and other agents - Polyps receive during resection and washed 5 times with buffer Tirade and fragmenting small pair of scissors on the replica with the raw weight of about 200 mg. Replica is suspended in 3 ml of buffer containing various concentrations of paclitaxel, at 37°C and provoke (after 5 minutes) of 0.2 μg/ml of antigen that is, After 15 minutes of incubation with the antigen diffusate removed and the cloth is boiled in fresh buffer for 10 minutes for extraction of residual histamine. The released histamine and SRS-A analyze with the help of VGH.

EXAMPLE 40

OXALOACETATE INTRODUCTION AGENTS THAT VIOLATE

The FUNCTION of MICROTUBULES

Studies have been conducted to evaluate the effectiveness loaded with paclitaxel/camptothecine surgical paste (PCL) and/or film EVA when allocatedata introduction in the treatment of restenosis.

A. Materials and methods

The Wistar rats weighing 250-300 g were anestesiologi intramuscular injection of Innovara is her anesthesia wool in the neck shaved, the skin was fixed and rubbed the swab with Betadine. Did a vertical incision is made over the left carotid artery was exposed external carotid Eretria. Two ligatures were placed around the external carotid artery and conducted cross arteriotomy. Then in the carotid artery was introduced French catheter-balloon Fogarty 2, and missed it in the left common carotid artery and the balloon is filled with saline. Endothelium exposed by holding the filled container up and down carotid artery three times. Then the catheter was removed and the ligature of the left external carotid artery were unleashed.

Rats randomized into groups of 10 to retrieve cases: without treatment, one polymer (EVA film or paste PCL) or polymer plus 20% paclitaxel. The polymer mixture (2.5 mg) were placed peripheral image around the carotid artery. Then the wound was closed. Five rats of each group were killed at 14 days and the remaining five at 28 days. In the span of rats was observed regarding weight loss or other signs of systemic disease. After 14 or 28 days the animals were anestesiology and left carotid artery were isolated, fixed with 10% buffered formaldehyde and examined histologically.

As a preliminary study, two rats were treated diseases.

C. Results

The results of these studies have found that loaded with paclitaxel (20%) polymers completely prevent restenosis, whereas control animals and animals receiving only the polymer, developed 28% and 55% luminale violation at 14 days and 28 days after balloon injury (figure 76A and B).

Saw an absolute inhibition of intimal hyperplasia in those places where paclitaxel was in contact with the vessel wall. However, this effect was very local, as evidenced by the unequal effect of paclitaxel in those places where it was not possible to keep the drug is contiguous with the wall of the vessel (figures 77A and B).

Preliminary results showed that loaded camptothecine the EVA film was effective in preventing the reaction of restenosis in this disease model animal. Camptothecin completely inhibited intimal hyperplasia in two of the tested animals.

EXAMPLE 41

EFFECT of PACLITAXEL IN the MODEL of SURGICAL ADHESIONS ANIMAL

Application loaded with paclitaxel film EVA to reduce adhesions experience in a rabbit model of uterine horns (keratosis).

Female new Zealand white rabbits were anestesiology and vypolaskivat using the blade of a scalpel. This scraping is sufficient to remove the serous membranes, leading to the point of bleeding. Rabbits were randomly assigned to either the control or treated with paclitaxel group and postoperative evaluation periods with a duration of two, four and eight weeks. In the processing of the paclitaxel group each fallopian keratome fully wrap loaded with paclitaxel film EVA after scraping. Musculo-peritoneal layer is closed with sutures, and the skin layer skin staples.

Animals are assessed in respect of adhesions after two, four or eight weeks after surgery. Animals asteniziruth humanely and conduct the autopsy. Uterine keratosis explore in General form and histologically using standard microscopic methods. In General form adhesions have degrees using a standard scoring system, which is based on the fact that injured 5 cm of the uterine horns; therefore, the degree of adhesions determine the length measurement zone containing adhesions. Use the following distribution system in degrees: 0 = no adhesions, 1 = spike by 25% in this zone, 2 = spikes by 50% in this zone, and 3 = full involvement in spike. The severity of adhesions esmerala) and 1 = required sharp dissection. The total degree is additive (total), and the range of scores of adhesions 0-4, which represents both the extent and severity of adhesions.

EXAMPLE 42

MICELLAR PACLITAXEL IN the TREATMENT of INFLAMMATORY

BOWEL DISEASE (IBD)

Inflammatory bowel disease (IBD), namely Crohn's disease and ulcerative colitis, is characterized by periods of relapse and remission. The best available model of IBD get in rats by injection into the colon 2,4,6-trinitrobenzenesulfonic acid (TNB), dissolved in ethanol and saline (Morris et al., Gastroenterology, 96: 795-803, 1989). One introduction initiates acute and chronic inflammation that persists for several weeks. However, it has been shown pharmacologically that the rabbit colon more similar to the colon of a person than the rat colon (Gastroenterology, 99: 13424-13432, 1990).

In all experiments use female new Zealand white rabbits. Animals anaesthetize intravenous (i.v.) pentobarbital. Feeding tube for enteral feeding infants insert rectally, so the tip is 20 cm proximal to the anus, for the introduction of TNB (0.6 ml; 40 mg in 25% ethanol in saline). One week after the introduction of TNB is only micelles (i.v.), or get micellar paclitaxel (i.v.). Repeat this every 4 days, just 4 processing.

In the course of this study rabbits have every week with the help of endoscopy using pediatric bronchoscope under General anesthesia performed as described above. Damage scored by the endoscopist (who knows variants of the experiment) in accordance with the following scale: 0 - no violations; 1 - inflammation, but no pitting; 2 - inflammation and ulceration in the 1st place (<1 cm); 3 - two or more places inflammation and ulceration or one main focus of inflammation and ulceration (>1 cm) along the entire length of the colon.

After the last treatment of rabbits killed by Autonoom at 24 hours and 1, 2, 4 and 6 weeks. All the colon secrete, excised and opened along antimesenteric the border, washed with saline and placed in a balanced salt Hanks solution containing antibodies. The colon examined using a stereomicroscope and scored in accordance with the same criteria as endoscopy. Also select samples of the colon at necropsy, both from clearly inflamed and ulcerated areas, and from normal colon along the entire length o; make the cuts and paint hematoxylin and eosin. Microscopic specimens examined for the presence or absence of histopathology IBD.

The original experiment can be modified for oral administration of paclitaxel after induction of colitis in rabbits introduction TNB through the colon. Animals randomizer into 3 groups: not receiving processing of receiving only the media or prepared for oral administration of paclitaxel.

EXAMPLE 43

EFFECT of PACLITAXEL IN the MODEL SYSTEM

Lupus ERYTHEMATOSUS ANIMAL

The effectiveness of paclitaxel in systemic lupus erythematosus determine the treatment (processing) of female mice NZB/NZW F1(B/W) micellar paclitaxel. This strain of mice develop a disease like SLE (systemic lupus erythematosus) person. At the age of one month, these mice have an increased level of spleen cells spontaneously secreting immunoglobulin, in comparison with normal mice. High levels of antibodies against single-stranded DNA are observed at the age of 2 months. At the age of 5 months immunoglobulin accumulates along the glomerular capillary walls. Develops severe glomerulonephritis and at the age of 9 months, 50% of mice B/W is ISA B/W arbitrarily assigned to the processing group and the control group. The processing group receive either continuous treatment with low doses of micellar paclitaxel (2.0 mg/kg; 3 times per week, a total of 10 injections), or "pulse" treatment with high doses of micellar paclitaxel (20 mg/kg; four times, once a week). The control group received the control micelles.

At given time intervals treated with paclitaxel and untreated control mice (B/W comparable age killed, their spleens removed aseptically and suspension of individual cells is prepared for counting lymphocytes. To identify subpopulations of lymphocytes in the spleen spend fluorescence analysis. The number of cells per million cells of spleen, spontaneously secreting immunoglobulin (IgG, IgM, total immunoglobulin or antibody against single-stranded DNA, determined using ELISA.

From the preceding description it should be clear that, although there is described the characteristic variations of the present invention for purposes of illustration, may be made of various modifications without departing from the idea and scope of the invention. Thus, this invention is not limited by anything except the attached claims.

Claims

1. A method of treating or preventing transplant rejection, comprising the administration to a patient a therapeutically effective amount antimicrotubular agent and specified antimicrotubule agent is paclitaxel, or an analogue or derivative.

2. A method of treating or preventing surgical adhesions, comprising the administration to a patient a therapeutically effective amount antimicrotubular agent and specified antimicrotubule agent is paclitaxel, or an analogue or derivative.

3. A method of treating or preventing inflammatory bowel disease, comprising the administration to a patient a therapeutically effective amount antimicrotubular agent and specified antimicrotubule asalnya polyps, includes introduction to the patient a therapeutically effective amount antimicrotubular agent and specified antimicrotubule agent is paclitaxel, or an analogue or derivative.

5. The method according to p. 1, wherein said paclitaxel, or an analogue or derivative is administered orally, topically, peritubular, subcutaneous, systemic, rectally or intramuscularly.

6. The method according to p. 1, wherein said paclitaxel, or an analogue or derivative applied to the surface of the surgical implant.

7. The method according to p. 2, wherein said paclitaxel, or an analogue or derivative is administered topically or peritubular.

8. The method according to p. 3, wherein said paclitaxel, or an analogue or derivative is administered orally, topically, peritubular, subcutaneous, systemic, rectally or intramuscularly.

9. The method according to p. 8, wherein said paclitaxel, or an analogue or derivative is administered under ultrasound, CT, fluoroscopy, NMR imaging or endoscopy.

10. The method according to p. 3, wherein said paclitaxel, or an analogue or derivative is administered orally in a dose of from 10 to 75 mg/m2every 1-4 weeks.

11. The method according to p. 3, wherein said paclitaxel, or an analogue or derivative centuries is, or its analogue, or derivative is administered rectally in the form of rectal cream or suppository.

13. The method according to p. 12, wherein said cream comprises from 0.01 to 10 wt.% paclitaxel, or its analogue, or derivative.

14. The method according to p. 3, wherein said drug for local injection contains from 0.1 to 1 wt.% paclitaxel, or its analogue, or derivative for daily rectal administration.

15. The method according to p. 3, wherein said paclitaxel, or an analogue or derivative is applied on the mesenteric surface of the intestine in the form of pastes, films or coatings.

16. The method according to p. 3, in which the specified inflammatory bowel disease is ulcerative colitis or Crohn's disease.

17. The method according to p. 3, wherein said paclitaxel, or an analogue or derivative is contained in a surgical or medical device or implant, or adapted for release from surgical or medical device or implant.

18. The method according to p. 4, wherein said paclitaxel, or an analogue or derivative is administered topically.

19. The method according to any of paragraphs.1-18, wherein said paclitaxel, or an analogue or derivative included in a therapeutic composition containing polymem these microspheres have an average size between 0.5 and 200 μm.

22. The method according to p. 19, wherein said polymer comprises poly(caprolactone).

23. The method according to p. 19, wherein said polymer comprises poly(lactic acid).

24. The method according to p. 19, wherein said polymer comprises polyethylene glycol.

25. The method according to p. 19, wherein said polymer includes ethylene vinyl acetate.

26. The method according to p. 19, wherein said polymer is a copolymer of poly(lactic acid) and poly(caprolactone).

27. The method according to p. 19, wherein said polymer comprises urethane.

28. The method according to p. 19, wherein said polymer comprises cellulose.

29. The method according to p. 28, in which the specified cellulose is metilgidroxiatilzelllozu.

30. The method according to p. 19, wherein said polymer is a copolymer of lactic acid and glycolic acid.

31. The method according to p. 19, wherein said polymer is a diblock or tribocorrosion.

32. The method according to any of paragraphs.1-18, wherein said paclitaxel, or an analogue or derivative is included in the formulation together with a carrier.

33. The method according to p. 32, in which the specified carrier is a liposome.

34. The method according to p. 32, in which the specified carrier is a cream, gel, lotion or ointment.

35. The method according to any of paragraphs.1-18, in compiletime permeability.

36. The method according to p. 35, in which the specified amplifier permeability is isopropylmyristate.

37. The method according to p. 35, in which the specified amplifier permeability is ethanol.

38. The method according to p. 35, in which the specified amplifier permeability is glycol.

39. The method according to p. 38, in which the specified glycol is etokxidiglicol.

40. The method according to p. 38, in which the specified glycol is propylene glycol.

41. The method according to p. 38, in which the specified glycol is ethylene glycol.

42. The method according to p. 19, in which the specified therapeutic composition is administered topically in the form of pastes, films, coatings or spray.

43. The method according to p. 19, in which paclitaxel, or an analogue or derivative is from 0.1 to 20 wt.% the specified composition.

44. The method according to p. 19, wherein said paclitaxel, or an analogue or derivative is administered systemically at a dose of from 10 to 75 mg/m2.

45. The method according to any of paragraphs.1-18, in which the specified paclitaxel, or an analogue or derivative is paclitaxel.

46. The method according to p. 19, in which the specified paclitaxel, or an analogue or derivative is paclitaxel.

Priority items:

24.10.1997 on PP.1, 5 and 6;

02.12.1996 on PP.2-4, 7-46.



 

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