Angiogenesis inhibitor, anti-angiogenic pharmaceutical composition based on thereof and method for treatment of malignant neoplasm

FIELD: chemistry of natural compounds, biotechnology, pharmacology, medicine, oncology, pharmacy.

SUBSTANCE: invention relates to a medicinal formulation of angiogenesis inhibitor comprising active compound as liposome form wherein angiostatin or endostatin are used as an active component, and their truncated peptide fragments also, and an anti-angiogenic pharmaceutical composition used for treatment. Also, invention proposes a method for inhibition of tumor growth and metastasizing of malignant neoplasms associated with disorders in angiogenesis. Method involves administration of nontoxic therapeutically effective dose of pharmaceutical composition separately or in combination with adjuvant therapy with other antitumor preparations. Invention provides reducing rate of elimination of active compound (angiostatin, endostatin or their truncated fragments) from body and enhancing effectiveness of its therapeutic effect. Invention can be used for inhibition of tumor growth and metastasizing of malignant neoplasms.

EFFECT: enhanced and valuable medicinal properties of inhibitor.

7 cl, 3 tbl, 5 ex

 

The present invention relates to the field of chemistry of natural compounds, biotechnology, pharmacology and medicine and can be used for therapy of malignant tumors.

The invention relates to a liposomal form antiangiogenic drug for the treatment of malignant tumors, namely for the treatment of primary tumors and metastases in humans, for the treatment of malignant tumors and other pathologies associated with impaired angiogenesis. The advantage of the invention is to enhance antiangiogenic and antitumor activity of the active component of the drug.

Angiogenesis is the process of formation of new blood vessels from preexisting playing a key role in a wide spectrum of physiological and pathological processes. The progression of tumors and for cancer are closely linked to angiogenesis [L.A. Liotta et al., Cell 64: 327-336, 1991; I.J. Fidler and Ellis, L.M., Cell 79: 185-188, 1994; Folkman J., N.Engl. J.Med. 333: 1757-1763, 1995]. Developing the tumor through the secretion of angiogenic factors attracts new capillaries, emerging from nearby vessels. Further progression of the tumor due to the close integration of tumor cells and endothelium of blood vessels. Bring blood oxygen and nutrients cause rapid tumor progression and initiate the process of metastasis. When using antiangiogenic agents that cause the destruction of tumor blood network, in model experiments on animals with transplanted tumors by a number of authors observed a significant therapeutic effect of these drugs in terms of the suppression of tumor growth and the increase in the average life span of experimental animals [M.S. O'reilly et al., Cell 79: 315-328, 1994; M.S. O'reilly et al., Nat. Med. 2: 689-692, 1996; Holmgren L. et al., Nat. Med. 1: 149-153, 1995; Mauceri H.J. et al., Nature 394: 287-291, 1998]. Thus, the destruction of tumor blood network using antiangiogenic agents is a promising approach to therapy of malignant tumors.

One of the most powerful inhibitors of angiogenesis is angiostatin is a proteolytic fragment of plasminogen mol. weight ˜ 40 kDa [M.S. O'reilly et al., Cell 79: 315-328, 1994]. Acting on the endothelial cells of blood vessels, angiostatin inhibits the growth of both primary tumors and regional metastases.

Currently the tools of genetic engineering obtained biologically active recombinant angiostatin, expressed in different strains-producers (Escherichia coli, Pichia pastoris, and others). Recombinant angiostatin induces apoptotic death of endothelial cells and effectively suppresses tumor growth of lung carcinoma Lewis mice at daily doses of 1-6 mg/kg [WuZ. et al., Biochem. Biophys. Res. Commun. 236: 651-654, 1997]. One of the important features of angiostatin is its ability to suppress tumor growth without toxic effects [M.S. O'reilly et al., Nat. Med. 2: 689-692, 1996; Sim, B.K. et al., Cancer Res. 57: 1329-1334, 1997; Lannutti B.J. et al., Cancer Res. 57: 5277-5280, 1997]. Even with the drug in high doses (˜100 mg/kg of body weight) does not show any toxicity or the development of drug resistance of tumors to the drug [Sim, B.K. et al., Cancer Res. 57: 1329-1334, 1997; Mauceri H.J. et al., Nature 394: 287-291, 1998; Drixler T.A. et al., Cancer Res. 60: 1761-1765, 2000].

Despite the obvious benefits and prospects for therapeutic application of angiostatin, a significant disadvantage of the drug is its rapid elimination from the body [Drixler T.A. et al., Cancer Res. 60: 1761-1765, 2000]. In addition, to achieve the desired anticancer effect requires prolonged use of the drug. Thus, the drug is administered to the patient at least twice a day in doses of from 20 to 100 mg/kg of body weight. It is obvious that therapeutic application of angiostatin will not only cause inconvenience to the patients due to the need for regular injections, but will require a huge amount of preparation. In order to avoid this, it is necessary to reduce the rate of excretion of angiostatin and increase the efficiency of its therapeutic action.

Other promising antiangiogenic what Reparata, influencing the survival of endothelial cells, is endostatin - 20 kDa fragment of collagen XVIII, is a component of basement membranes and extracellular matrix [Y. Muragaki et al., Proc. Natl. Acad. Sci. USA, 92: 8763-8767, 1995]. Endostatin specifically inhibits proliferation and migration of capillary endothelial cells and can induce apoptosis in proliferating endothelial cells. Direct effect on tumor cells of various lines in vitro, endostatin does not [M.S. O'reilly et al., Cell: 88, 277-285, 1997]. In vivo, endostatin has a strong inhibitory effect against the growth of various primary tumors and their metastases. Prolonged therapy with high doses of endostatin causes almost complete blockade of tumor angiogenesis and leads to regression of the tumor to microscopic dimensions [Boehm .et al., Nature: 390, 404-407, 1997]. Further micropohone fall into "dormant" state, in which the index of proliferation of tumor cells is balanced by their index of apoptosis. Even after prolonged therapy is not observed the emergence of resistance to the drug or any toxic reactions [Sim, B.K. et al., Cancer Metastasis Rev.: 19, 181-190, 2000]. Endostatin can inhibit induced angiogenic growth factors cascade of intracellular signaling, as well as to block the activation and catalytic activity of the matrix metalloproteinases.

Closest to the technical nature are angiogenesis inhibitors angiostatin, endostatin and shorter fragments for the treatment of malignant tumors /Pat. Of the Russian Federation No. 2240328/. The disadvantages of these inhibitors described above. In addition, the authors of the present invention offer the use of the above inhibitors of angiogenesis in the form homodimeric proteins, in which the molecule inhibitor linked by a polypeptide of the bridge with the Fc region of immunoglobulin gamma, which may further reduce the biological activity of the target an inhibitor of angiogenesis. The use of such fused proteins also does not solve the problem of high rate of excretion of drugs from the body of the patient.

The purpose of the invention is the decrease in the rate of excretion of the active compound (angiostatin and endostatin) from the body and increase the efficiency of its therapeutic action.

The goal is solved by the creation of an inhibitor of angiogenesis, comprising the active compound in the form of liposomes. The active compounds are used angiostatin or endostatin, as well as their shorter peptide fragments. Also available antiangiogenic pharmaceutical composition for the treatment of malignant tumors and other pathologies associated with impaired angiogenesis containing the active compound (Academy of Sciences of histatin, endostatin or shortened fragments) and a pharmaceutical carrier, where the active compound is contained in a liposomal form in an effective amount, and as a pharmaceutical carrier are acceptable solutions for injection.

In addition, a method for treating malignant tumors and other pathologies associated with impaired angiogenesis, which consists in the introduction of a non-toxic therapeutically effective amount of the pharmaceutical composition, either alone or in combination with adjuvant therapy with other anticancer drugs, immunomodulators or antiangiogenic agents. As an anticancer drug for adjuvant therapy using a drug selected from the group of: doxorubicin, karminomitsin, mutamycin, Novantrone, rubomycin, bleocin, cisplatin, Taxol, etoposide, vinblastine, vincristine, Gemzar, fluorouracil, methotrexate, xeloda, Zoladex, dacarbazine, cisplatin, carboplatin, tamoxifen. As an immunomodulator for adjuvant therapy using a drug selected from the group of interferon-αinterferon-γ, interleukin-2, interleukin-4, interleukin-6, interleukin-12. As an antiangiogenic agent for adjuvant therapy using a drug selected from the group of thalidomide, vitaxin, pentosan, suramin, fumagillin, squal the min, combretastatin, prinomastat, marimastat, neovastat.

Angiostatin or endostatin in liposomes can be presented in various forms, differing in length of the peptide chain as whole proteins, and their shorter peptide fragments that differ from the sequence of the whole protein at one or more terminal amino acid. Angiostatin or endostatin for liposomal compositions can be obtained both from natural sources and with the help of genetic engineering technology, and shorter fragments of these proteins are also using genetic engineering methods or by the method of solid-phase peptide synthesis.

Created liposomal form of the inhibitor of angiogenesis is characterized by simplicity and adaptability of receipt shows high biological activity and can effectively suppress the growth of tumor blood vessels and tumors.

The basis of the invention is an inhibitor of angiogenesis, which is in the form of liposomes, which in experiments both in vitro and in vivo is higher antitumor activity than free active component. Inhibitor in the form of liposomes can be easily obtained using standard biotechnological and chemical methods, offers stability and adaptability of receipt. Liposomal form antiangiogenic inhibitor can be successfully used is implemented to treat a number of pathologies, associated with impaired angiogenesis. Thus, the invention fully meets the criterion of "inventive step".

The invention is illustrated by the following examples.

Example 1. Obtaining liposomes loaded with angiostatin or endostatin, or shorter fragments.

1 ml of liposomal dispersion in a round bottom flask is placed 20.5 mg of egg phosphatidylcholine (AFH) and 4.5 mg of cholesterol (AFH/cholesterol 7/3 mole.) in chloroform solutions. A mixture of solutions of lipids evaporated on a rotary evaporator to constant weight at 38°C. the Residues of the solvent is removed in vacuum oil pump within 2 hours of the Obtained lipid film was dissolved in 3 ml of ether and added dropwise into the flask 1 ml of protein solution (angiostatin, endostatin or their fragments) (6 mg/ml) in phosphate-buffered saline (pH 7.4). The flask was rinsed with argon and speak the dispersion in an ultrasonic bath for 3 min at 10°C. Carefully evaporated the ether on a rotary evaporator at room temperature (200 rpm). The obtained lipid dispersion push 19 times through nuclear polycarbonate filter with a pore size of 100 nm. Get 0.9 ml of a dispersion of liposomes of 100 nm (25 mg/ml lipid, 6 mg/ml protein). The effectiveness of the inclusion of protein is 14-26%. Liposomal inhibitor of angiogenesis lyophilizer and stored at 4°C.

Example 2. Getting formcomponents and on the basis of liposomal inhibitor of angiogenesis.

Pharmaceutical composition for injection is obtained by dissolving an effective amount of lyophilized inhibitor of angiogenesis, obtained as described in example 1 in physiological saline or phosphate-saline solution, prepared with water for injection. the medium pH is about 7.4. The concentration of the active substance (a protein) in a solution of 5-10%.

Example 3. Determination of antitumor activity of liposomal inhibitors of angiogenesis, including angiostatin, endostatin or shorter fragments in the individual therapy of mice with transplanted solid tumors melanoma B16.

Melanoma cells lines B16 maintained by weekly intraperitoneal inoculation of mice C57B1/6. For the induction of solid tumors the mice were injected subcutaneously cell suspension (2·105cells in 0.1 ml of physiological solution). Drugs were injected into experimental animals intravenously. A single dose of liposomal forms of the inhibitor of angiogenesis contained: 90 mg/kg angiostatin, endostatin or their fragments. The drugs were injected intravenously once a week, just three injections, starting from the 10th day after the inoculation of the tumor. Each experimental group consisted of six animals, control of ten. The size of the solid tumors were measured once in 2-3 days. Tumor volume was calculated by the formula where a - short; b - the longest diameter of the tumor. The relative size of the tumors (ORO) was determined by the formula:where Ropthe average size of tumors in the experimental group, Ptothe average size of tumors in control animals. The increase in life span of treated animals compared to the control was determined by the formula:where T is the average life expectancy (ALE) treated animals days From - ALE control animals, days.

The results of the experiment are given in table 1. Compared to the mild effect of the application liposonix forms of angiostatin, endostatin and its truncated fragments therapy liposomal forms has led to a significant inhibition of tumor growth both during treatment and after its abolition and the increase in the average life span of the treated animals.

100
Table 1

The effectiveness of antitumor activity of liposomal inhibitor of angiogenesis in relation to melanoma In 16 mice in comparison with liposome forms.
The group of animalsORO, %USPS, %
24 day35 day
control100-
Liposome forms of medicinesangiostatin64,882,420,8
angiostatin shortened58,374,027,1
endostatin59,071,324,6
endostatin shortened53,668,529,8
Liposomal inhibitorsangiostatin36,148,941,8
angiostatin shortened32,643,8to 49.9
endostatinof 31.442,847,3
endostatin shortened28,737,152,1

Example 4. Antimetastatic activity of liposomal inhibitors of angiogenesis.

To evaluate the antimetastatic activity of liposomal antiangiogenic inhibitors, we conducted an experiment on mice with subcutaneously transplanted tumor Lewis lung carcinoma (3LL) in the background of the removal of the primary tumor site.

The primary tumor was removed on day 8 after inoculation. As the anesthesia used geksenal. Tumor site was deleted along with adjacent areas of the skin, for p is eupresidency the possibility of recurrence. The defect of the skin was closed by the overlapping seam, smeared with a 5% alcohol solution of iodine. The first introduction of the studied drugs was performed for 12 hours prior to surgery. The drugs were injected intravenously in doses of 90 mg/kg 1 time in 4 days, just 5 injections. Each experimental group consisted of 8 animals.

Antimetastatic effect was evaluated at the beginning of the loss of control animals. The intensity of metastasis was evaluated by comparing the average mass of lung metastases in the control and experimental groups of animals. At the same time used conditional intensity indicators of metastasis - the number and size of metastatic nodes in the lung tissue, the frequency of metastasis of the tumor. Statistical processing of the obtained results was performed by the t-test.

The growth inhibition of metastases was calculated by the formula:where Lto- average weight of the lung with metastases in the control group, Labout- average weight of the lung with metastases in the group treated mice.

The results of the experiment are presented in table 2. Compared with the control animals, in which the lung metastases detected in 100% of cases in the groups treated animals was observed in the absence of metastases and reduced the intensity of metastasis. Thus the m the application of the proposed drug for the prevention of metastasis is very promising.

Table 2

Influence of liposomal inhibitors of angiogenesis on the intensity of metastasis subcutaneously inoculated tumor Lewis lung carcinoma (3LL) in the background of the removal of the primary tumor site.
The group of animalsTRM, %The frequency of metastasisThe intensity of metastasis (the average number of metastases/mouse)
Control-8/8[5,8±3,1]
liposomal form of angiostatin42,75/8[2,7±1,3]
liposomal form of angiostatin (abridged)38,24/8[2,1±1,9]
liposomal form of endostatin40,65/8[2,2±0,8]
liposomal form of endostatin (abridged)34,65/8[1,9±0,6]
Notes:

1. The data are given at the time of death.

2. In the column "Frequency of metastasis" is number of animals with metastases/number of animals in the group.

3. In gra the e Intensity metastasis" (in square brackets) - the average number of metastatic colonies in the lungs of the animal.

Example 5. The effectiveness of antiangiogenic pharmaceutical compositions, including angiostatin, endostatin or shorter fragments, in relation to melanoma mice in their application in the mode of combination therapy with anticancer drugs, immunomodulators and other antiangiogenic agents.

Antiangiogenic pharmaceutical composition was injected as described in example 3, on the background of adjuvant therapy with other drugs. Thus, animals of group 1 were additionally introduced antitumor antibiotic doxorubicin (2 mg/kg) in the same pattern as liposomal preparations, group 2 - interferon-α daily for 3 weeks, group 3 - angiogenic drug thalidomide. The results of the experiment are given in table 3. As can be seen from the above data, the application of the proposed angiogenic pharmaceutical compositions in the modes of combination therapy with other drugs significantly increases the effectiveness of their anti-tumor activity.

Table 3

The effectiveness of the antitumor activity of antiangiogenic pharmaceutical compositions against melanoma line B16 mice when combined with other therapy and drugs.
The group of animalsORO, % (35 day)USPS, %
control100-
Individual therapyLiposomal form of angiostatin48,941,8
Liposomal form of angiostatin (abridged)43,8to 49.9
Liposomal form of endostatin42,847,3
Liposomal form of endostatin (abridged)37,152,1
DoxorubicinLiposomal form of angiostatin41,649,8
Liposomal form of angiostatin (abridged)37,7of 56.4
Liposomal form of endostatin38,455,2
Liposomal form of endostatin (abridged)32,6of 58.9
Interferon-αLiposomal form of angiostatin46,146,2
Liposomal form of angiostatin (abridged)39,854,9
Liposomal form of endostatin39,751,2
Liposomal form of endostatin (koroch the config) 35,356,0
ThalidomideLiposomal form of angiostatin40,252,9

Similar results were obtained in experiments to study the effectiveness of the claimed angiogenic pharmaceutical compositions in the mode of combination therapy with other anticancer drugs - karminomitsin, mutamycin, Novantrone, rubomycin, bleocin, cisplatin, Taxol, etoposide, vinblastine, vincristine, Gemzar, fluorouracil, methotrexate, xeloda, Zoladex, dacarbazine, cisplatin, carboplatin, tamoxifen; immunomodulators interferon-γ, interleukin-2, interleukin-4, interleukin-6, interleukin-12; antiangiogenic agents - vitaxin, pentosan, suramin, fumagillin, squalamine, combretastatin, prinomastat, marimastat, neovastat.

Proposed by the applicant approach is devoid of the aforementioned drawbacks and combines the simplicity and adaptability of receiving liposomal forms of antiangiogenic drugs with high efficiency antitumor activity due to an increase in the residence time of the drug in the blood and its selective accumulation in the area of tumor blood vessels.

1. Dosage form of the inhibitor of angiogenesis, comprising as active substance angiostatin or endostatin in Linden is som characterized in that the active substance it contains a full-size form or a shortened peptide fragment of the indicated inhibitors, when this substance is enclosed in liposomal membrane based on phosphatidylcholine and cholesterol levels when a molar ratio of 7:3, the obtained liposome containing an active substance not less than 19 wt.%.

2. Antiangiogenic pharmaceutical composition for the treatment of malignant tumors, containing the active ingredient and the pharmaceutical carrier, characterized in that the active substance it contains a dosage form according to claim 1 and saline or phosphate-saline solution, and the concentration of the active substance in the solution is 5-10% for protein.

3. Method of inhibiting tumor growth and metastasis of malignant tumors associated with impaired angiogenesis, characterized in that the patient is given a non-toxic therapeutically effective amount of the pharmaceutical composition according to claim 2.

4. The method of inhibition according to claim 3, characterized in that it further conduct of combined adjuvant therapy with other anticancer drugs, immunomodulators or antiangiogenic agents.

5. The method according to claim 4, characterized in that as an anticancer drug for adjuvant therapy using repeat, selected from the group doxorubicin, karminomitsin, mutamycin, Novantrone, rubomycin, bleocin, cisplatin, Taxol, etoposide, vinblastine, vincristine, Gemzar, fluorouracil, methotrexate, xeloda, Zoladex, dacarbazine, cisplatin, carboplatin, tamoxifen.

6. The method according to claim 4, characterized in that as an immunomodulator for adjuvant therapy using a drug selected from the group of interferon-αinterferon-γ, interleukin-2, interleukin-4, interleukin-6, interleukin-12.

7. The method according to claim 4, characterized in that as an antiangiogenic agent for adjuvant therapy using a drug selected from the group thalidomide, vitaxin, pentosan, suramin, fumagillin, squalamine, combretastatin, prinomastat, marimastat, neovastat.



 

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SUBSTANCE: invention relates to compositions comprising a substance possessing regulating activity, for example, inhibition of proliferation of cells expressing AILIM, for suppression of onset of intestine inflammatory diseases, in particular Crohn's disease, and colitis. Invention provides enhancing the therapeutic effect.

EFFECT: valuable medicinal properties of agents.

10 cl, 12 dwg, 2 ex

FIELD: medicine, oncology.

SUBSTANCE: method involves ligature of lung radix during the operation followed by taking off 400 ml of blood from pulmonary artery into capacity with glugicire, centrifugation at 1500 rev/min for 10 min and taking off plasma into another flask, and antibiotics are added into flask with remaining cellular suspension in the dose corresponding to the maximal daily dose, and neupogen is added to flask with plasma in the dose 60 mcg/kg of body mass. Both flasks are incubated at temperature 37°C for 30 min, and administrated in a patient by drops during the continuing operation. Method provides alignment of reduced resistance of body to infectious complications due to stimulating secretion of functional neutrophiles by bone marrow, elongation of time antibiotic residence in the body associated with formed blood elements and with absence of transient depression. Invention can be used in prophylaxis of suppurative-septic complications during the post-operative period in patients with the central lung cancer subjected for pneumoectomy operation.

EFFECT: improved method for prophylaxis.

1 ex

FIELD: experimental medicine, oncology.

SUBSTANCE: the present innovation deals with increasing body resistance, at carrying out anti-tumor chemotherapy, as well. For this purpose one should sample about 0.7-1.0 ml venous blood, phylgrastim at the dosage of 10 mg/kg body weight should be incubated with this blood during 45 min at 37° C followed by reinfusion. The innovation provides total increase of body resistance due to stimulating the development of not neutrophilic leukocytes and monocytes only, but erythrocytes, as well, and decreasing the number of mast cells and reticular cells of stroma, and, moreover, it leads to decreased toxicity of anti-tumor chemopreparations.

EFFECT: higher efficiency.

1 ex, 1 tbl

FIELD: medicine, dermatology, in particular treatment of trophic ulcer (TU) and long-term open septic wound (LTOSW).

SUBSTANCE: claimed method includes application of wound-healthing composition onto TU and LTOSW in the first step of wound process. Said composition contains (mass %): methyluracil 0.9-1.1; pepsin 3.4-4.4; bentaine hydrochloride 3.6-4.4; sodium chloride 9.0-11.0; and balance up to 100,0: distilled water. In the second and third phases fine cut collagen preparations in form of 3-4 mm thickness layer are applied on purified TU and LTOSW surface. Then cotton carrier freely moistened with wound-healthing composition diluted with water in ratio of 1:2 is layered onto collagen layer. Multihole microirrigator and further sterile cotton carrier are sequentially applied over abovementioned cotton carrier.

EFFECT: effective treatment due to accelerated abruption of necrotic tissues, inhibition of microbial growth, decreased risk of infective, toxic and allergic affects, and improved tissue regeneration.

2 ex, 3 cl

FIELD: medicine.

SUBSTANCE: method involves introducing to a mammal therapeutic dose of drug having at least one subunit or at least one oligomer of mannan-binding lectin comprising one mannan-binding lectin subunit.

EFFECT: reduced risk of infection development; enhanced effectiveness in acting upon agents resistant to usual therapy.

43 cl, 2 dwg

FIELD: medicine, pediatrics.

SUBSTANCE: the present innovation deals with treating motor-autonomic disorders in children associated with affected function of central nervous system. For this purpose one should puncture perineural areas in the region of the main nervous trunks with alfetin dissolved in cerebrolysine. Additionally, one should puncture in projection area of cervical and lumbar spinal thickenings and areas that correspond to segmentary innervation of organs with affected function and, also, in scalp areas depending upon the character of patient's disorders. The method suggested provides improved autonomic-trophic impact of nervous system.

EFFECT: higher efficiency of therapy.

2 ex

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