The way epidural injection of therapeutic compounds with a supported rate of release, a way to reduce respiratory depression

 

The invention relates to medicine, namely to a regulated release of therapeutic compounds from the drug-delivery systems. Connection encapsulate in a delivery system of the medicinal product, which has slowed the rate of release. Enter the specified system epidurally in one dose. The delivery system of the medicinal product includes multivesicular liposomes obtained from the group consisting of phosphatidylcholines eggs, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine and their appropriate combinations. To reduce respiratory depression epidurally administered encapsulated analgesic agent in multivesicular liposomal delivery system. The method allows to increase the effectiveness of the treatment. 2 C. and 22 C.p. f-crystals, 1 tab., 6 Il.

The technical field of the invention This invention relates to a controlled release of therapeutic compounds from the drug-delivery systems. More specifically, this invention relates to an epidural introduction of therapeutic soedineniya to the way of placing an epidural catheter in a living vertebrate.

Background of the invention the Elimination of postoperative pain is a major problem for patients and physicians, especially in the recovery room when the patient wakes up from anesthesia. Too high dose opioid system, introduced with the attempt to suppress the pain, can potentially cause life-threatening respiratory depression. On the other hand, are either too small, or too late dose of medication against postoperative pain may cause the patient wakes up with unbearable severe pain. In addition, it was shown that weakly repressed postoperative pain following abdominal or thoracic operations, depresses the respiratory movement of the chest walls, abdomen and diaphragm (P. R. Bromage, in Textbook of Pain, P. D. Wall et al. (Eds), Churchill Livingstone, 1989, pages 744-753), which leads to pulmonary atelectasis.

The existence of opioid receptors in the spinal cord was discovered in the 1970's. Following the first reports of clinical efficacy in 1979 (M. Behar et al., Lancet 1: 527-529, 1979) epidural injection of opioids has become very popular for relieving postoperative pain (T. I. lonescu et al., Act. Anaesth. Belg. 40: 65-77, 1989; C. Jayr et al., Anesthesiology, 78: 666-676, 1993; growth et al., European Journal of Obstetrics & Gynecology and Reproductive Biology 49: 147-1 the Aries without loss of propulsion or vasomotor control or without reduced level of consciousness.

Injectable opioids are widely used epidurally in postoperative and postpartum setting. Postoperative and postpartum pain typically last for a few days, however, injectable opioids have a relatively short duration of action (W. G. Brose et al., Pain 45: 11-15, 1991; R. H. That et al., Arzneim-Forsch/Druq Res., 38: 1632-1634, 1988; G. K. Gourlay et al. , Pain, 31: 297-305, 1987). Thus, in order to maintain adequate pain suppression, requires either continuous infusion or repeated injections (J. W. Kwan, Am. J. Hosp. Pharm. 47(Suppl. 1): S18-23, 1990; J. S. Anulty, International Anesthesiology Clinics 28: 17-24, 1990; R. S. Sinatra, The Vale Journal of Biology and Medicine, 64: 351-374, 1991). Continuous infusion or repeated injections additionally necessitate placement of catheter systems with attached infusion pumps or without them, and they all require expensive time of the doctor and nurses to care and maintenance. In addition, repeated injections of a shock dose of the substance or continuous infusion can lead to respiratory depression.

Late respiratory depression and appicella episodes are side effects, which was the greatest problems in earlier studies (P. R. Bromage, Anesthesia and Analgesia 60: 461-463, 1981; E. M. Camporesi et al. , Anesthesia and. Analgesia 62: 633-640, 1983; T. L. Yaksh, P thoracic, abdominal or orthopedic surgery, it was found that the frequency of respiratory depression after epidural morphine is 0.9% (R. Stenseth et al., Acta Anaesthesiol. Scand. 29: 148-156, 1985). As a comparison, the frequency of "life threatening respiratory depression" 860 patients who were given systemic morphine (p. O., C. C., C. M., p. K.), 0.9% (R. R. Miller et al., Drug Effects in Hospitalized Patients. John Wiley & Sons, New York, 1976). Randomized controlled trials comparing epidural with systemic opioids opioids (B. M. or centuries) in high-risk patients showed that suppression of postoperative pain with epidural opioid use leads to excellent analgesia with a low incidence of post operative complications (N. Rawal et al. , Anesth. Play mode display. , 63: 583-592, 1984; M. R. Yeager's et al., Anesth. 60: 729-736, 1987).

Prolonged release of various therapeutic agents after the introduction of liposomes, such as multivesicular liposomes, has been well-documented for both systems in vitro and in animals, intrathecal, subcutaneous and intraperitoneal routes of administration, as well as for patients-people for intrathecal route of administration (S. Kim, et al., J. Clin. Oncol., 11: 2186-2193, 1993; V. Russack et al. , Ann. Neurol., 34: 108-112, 1993, and M. C. Chamberlain et al., Arch. Neurol., 50: 261-264, 1993). But still at the level of technology was not yovich and the best routes of administration of opioids and other therapeutic compounds epidurally as a single dose with the in order to achieve the supported speed of release at therapeutically effective levels. The present invention solves the problem of the limitations of the known prior, what is proposed dosage form with a slow release of a therapeutic agent such as an opioid, which leads to maximum analgesia immediately after a single epidural dose and provides a gradually decreasing analgesia in the next few days.

Description of the drawings Fig. 1 is a series of four charts with record analgesic effect in rats over time after a single epidural dose encapsulated in liposomes of martinslife (DTC401) (light circles) or free martinslife (dark circles) for dosages (from top to bottom) 10, 50, 175, or 250 mcg. The intensity of analgesia expressed in percentage of the maximum possible analgesia (% MVA)". Each data point represents the mean and standard error (CO) 5 or 6 animals.

Fig. 2 is a graph showing curves of the dose-response for maximum analgesia measured in rats after a single epidural dose DTC401 (light circles), free marginality % MVAWas received for 5 or 6 animals.

Fig. 3 is a graph which compares the total analgesic effect in rats [measured by the area under the curves analgesia-time (ACC)] for single doses of epidural DTC401 (light circles) or free martinslife (dark circles). Each data point represents the mean and standard error (CO) 5 or 6 animals.

Fig. 4 is a series of five graphs for a comparison of the percent saturation of hemoglobin with oxygen (SpO2) in rats over time after a single epidural dose (from top to bottom) 10, 50, 175, 1000 or 2000 mg epidural DTC401 (light circles) or free martinslife (dark circles). Each data point represents the mean and standard error (CO) 5 animals except group with a dose of 50 mcg, where n=3.

Fig. 5 is a graph which shows the curve of the dose-response for maximum respiratory depression in rats after a single epidural dose DTC401 (light circles) or free martinslife (dark circles). The lowest achieved value SpO2set aside as a function of epidural doses of morphine. Each data point represents the mean and the standard is de compared the pharmacokinetics in cerebrospinal fluid (upper part) and serum (bottom) rats after epidural injection of 250 micrograms DTC401 (light circles) or free martinslife (dark circles). Each data point represents the mean and standard error (CO) 3 or 4 animals.

Summary of the invention an Epidural introduction of therapeutic compounds in the delivery system of medicines has provided a surprisingly long release and a longer duration of therapeutic effect than the use of free therapeutic compounds.

Therefore, one aspect of the invention proposes a method for the prolonged release of therapeutic compounds through the use of the delivery system for drugs that epidurally administered vertebrate in need of such therapy.

Vertebral preferably is a mammal, such as man. In various preferred embodiments, the delivery system of the medicinal product based on lipids, particularly when it is realized in the form multivesicular liposomes.

Characteristic of the invention is the possibility of prolonged delivery of various therapeutic compounds, which in the preferred variants include opioids or opiate antagonists to prevent modulation of analgesia. Alternatives allows the public forms with prolonged release in accordance with the method of the invention facilitates epidural analgesia and reduces total costs by eliminating the need for continuous infusion, multiple injections of a shock dose of the substance or installation of catheters, and reduces the likelihood of infection. Reduced frequency of injection has the advantage even in the presence of an epidural catheter.

Detailed description of the invention the present invention presents a system of lipid based delivery of long-released medicines intended for epidural delivery of therapeutic compounds with epidural efficiency, such as an opioid. With the help of an epidural injection compounds are released into the Central nervous system and cerebrospinal fluid without puncture of the Dura and the supported speed of release.

The term "long release" means that therapeutic connection with the introduction in the form of dose impact encapsulated drug in the form of lipid based, is released over a longer period of time compared with epidural introduction of the same drug in free form in the form of injection loading dose of the substance. This does not necessarily mean that the concentration of therapeutic compound remains constant over an extended period is the patient's need analgesia decreases over time. Using the method of epidural delivery of a medicinal product according to this invention, in the cerebrospinal fluid and/or serum can maintain a therapeutically effective concentration of therapeutic compound for several days, preferably from about 2 to about 7 days.

Used herein, the term "therapeutic compound" means a chemical compound which is suitable for modulation of biological processes in order to achieve the desired effect by modulation or treatment of undesirable conditions in a living organism. The term "therapeutic compound" encompasses non-protein chemical medicines, such as antibiotics and analgesics, as well as protein drugs, such as cytokines, interferons, growth factors, etc.,

System drug delivery are well known in the prior art. The present invention relates to any finished dosage forms with prolonged release, such as synthetic or natural polymers in the form of macromolecular complexes, nanocapsules, microspheres or granules, as well as systems on a lipid basis, including emulsion of the type oil-in-water, micelles, mixed micelles, Severnye system are two-phase systems, in which one phase is distributed in the second phase in the form of particles or droplets. Typically, forming a system of particles have a diameter of 20 nm - 50 μm. The size of the particles allows them to suspendibility pharmaceutical solution to be introduced into the epidural space using the needle or catheter and xprize.

The materials used in the preparation of disperse systems, are usually non-toxic and biodegradable. Thus, it is possible to use, for example, collagen, albumin, ethylcellulose, casein, gelatin, lecithin, phospholipids and soybean oil. Polymer dispersed system can be obtained in a manner similar to koatservatsii or microencapsulation. If required, the density of the dispersed system can be modified by changing the relative weight to make the variance giperborei or Giovanni. For example, the material dispersion can be made more hyperboreum by adding iohexol, iodixanol, metrizamide, sucrose, trehalose, glucose, or other biocompatible molecules with high specific gravity.

One of the types of disperse systems, which can be used in accordance with the invention, consists of a dispersion of a therapeutic agent in a polymer matrix. Therapeutic creodonta, flushed out of the body. For this purpose, investigated several classes of synthetic polymers, including polyesters (Pitt et al., Controlled Release of Bioactive Materials, R. Baker, Ed., Academic Press, New York, 1980); polyamides (Sidman et al., Journal of Membrane Science, 7:277, 1979); polyurethanes (Master et al. , Journal of Polymer Science, Polymer Symposium, 66: 259, 1979); prioritaire (Heller, et al., Polymer Engineering Science, 21: 72 7, 1981) and polyanhydride (Leong. et al., Biomaterials, 7: 364, 1986). Significant research was conducted on complex polyesters PMC and PMC/ISC. Undoubtedly, this is due to considerations of convenience and security. These polymers are easily accessible, as they are used as biodegradable sutures and they decompose into non-toxic lactic and glycolic acid (see U.S. patent 4,578,384, U.S. patent 4,785,973 included as references).

Solid polymer dispersed systems can be synthesized using these methods of polymerization, as the polymerization in bulk, interfacial polymerization, polymerization in solution and polymerization with ring opening (Odian, G. , Principles of Polymerization, 2nd ed., John Viley & Sons, New York, 1981). Using any of these methods receive a variety of different synthetic polymers having a wide range of mechanical, chemical and biodegradation properties; differences in properties, types of solvent and reaction time. If necessary, the solid polymer dispersed system can initially be obtained in the form of greater mass, which is then milled or otherwise processed into particles small enough to maintain the dispersion in a suitable physiological buffer (see , for example, U.S. patents 4,452,025; 4,389,330 and 4,696,258 included as references).

Optionally, therapeutic compound can be introduced in non-dispersible structure, which epidurally implanted using surgical or mechanical means. Non-dispersible structure is a structure having a certain overall shape, such as a plate, cylinder or sphere. The allocation mechanism a therapeutic agent from a biodegradable plates, cylinders or spheres described Hopfenberg (Conrolled Release Polymeric Formulations, pages 26-32, Paul, D. R. and Harris, F. W., Eds., American Chemical Society, Washington, D. C., 1976). A simple expression describing the additive release of these devices, where the release is mainly controlled by the decomposition of the matrix as follows: Mt/M= 1-[1-k0t/C0]nwhere n= 3 for a sphere, n= 2 for the cylinder and n=1 for the plate. The symbolrepresent the mass of drug released at time t and infinity, respectively.

In the practice of the invention can use any system drug delivery lipid based. For example, you can use multivesicular liposomes (ials), mnogoplatnye liposomes (also known as mnogoplatnye vesicles or "SRM"), a single-disk liposomes, including small single-disk liposomes (also known as a single-disk vesicles or "MOU") and large single-disk liposomes (also known as large single-disk vesicles or "scale"), provided that can be installed supported the rate of release of the encapsulated therapeutic compounds. However, in the preferred embodiment of the invention, the delivery system of a drug in the lipid base is a system multivesicular liposomes. A method of manufacturing systems drug delivery controlled release based on multivesicular liposomes are described fully in patent applications U.S. 08/352,352, filed December 7, 1994, and 08/393,724, filed February 23, 1995, and PCT-eawtah US94/12957 and US94/04490, which are all on the s, usually in combination with steroids, especially cholesterol. You can also use other phospholipids or other lipids.

Examples of lipids suitable when receiving vesicles with a synthetic membrane, are phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine and dioleoylphosphatidylserine.

Upon receipt of the vesicles containing therapeutic agent should take into account such variables as the efficiency of encapsulation of drugs, the lability of the medicinal product, homogeneity and size of the resulting population of vesicles, the ratio of drug/lipid permeability, instability of the preparation, and pharmaceutical acceptability of the dosage form (Szoka et al., Annual Reviews of Biophysics and Bioengineering, 9: 467, 1980; Deamer, et al., Liposomes, Marcel Dekker, New York, 1983, 27: Hope, et al., Chem. Phys. Lipids, 40: 89, 1986).

The use of dosage forms of opioids on lipid basis were studied by other authors with limited success and no Odie is poidev and their activity in vitro (F. Reig., et al./ J. Microencapsulation 6: 277-283, 1989) without any epidural study in vivo. In addition, researched antinociception and side effects Alfentanil encapsulated in liposomes dosage forms and entered using spinal delivery to rats (M. S. Wallace et al., Anestch. Play mode display. 79: 778-786, 1994; C. M. Bernards et al., Anesthesiology 77: 529-535, 1992). However, neither the pharmacokinetics or pharmacodynamics of these compounds did not differ significantly from those for standard opioids to guarantee their application in clinical practice. In these studies has not been studied dosage forms with prolonged release of opioids, administered epidurally.

The delivery system of a drug in the lipid base, including therapeutic connection, you can enter as a single dose, for example, through an epidural catheter. However, in the preferred embodiment, the delivery system of a drug in the lipid base is injected as a single dose into the epidural space around the spinal cord using a small calibrated needle, so avoiding the location of the catheter. Preferably use a needle 18-25 caliber.

Representative list of therapeutic compounds suitable for epidermization, buprenorphine, nalbuphine, butorphanol, pentazocine, methadone, fentanyl, Sufentanil and Alfentanil. In addition, epidurally, you can enter opiate antagonists such as naloxone and naltrexone, using the method according to the invention, to pay or to anlagenservice opiate effect.

I believe that opioids are peptides and peptidomimetics that are associated with one or more neuroreceptors, such as Delta-opioid, mu-opioid, Kappa-opioid and Epsilon-opioid receptors, and they can be administered for therapeutic action in accordance with the method according to the invention. These compounds include enkephalins, endorphins, casomorphin, kyotorphin and their biologically active fragments. The term "biologically active fragment" means any part of therapeutic compound, which retains the biological activity of the whole therapeutic molecules. The person skilled in the art will know or can easily determine whether basically a fragment of the biological activity of the whole molecule.

In addition to opioids, in practice the method according to the invention it is also possible to use a series of compounds that are useful therapeutically in the epidural introduction supported / min net is neurotropic factor and nerve growth factors; neurotransmitters and their antagonists, such as dopamine, epinephrine, norepinephrine and gamma-aminobutyric acid; local anesthetics, such as tetracaine, lidocaine, bupivacaine and mepivacaine; substance P and related peptides; and antagonists of alpha-2 receptors such as clonidine, dexmedetomidine. In addition, the effectiveness of epidural opioids may be increased by the joint administration of a local anaesthetic, such as lidocaine, bupivacaine and tetracaine.

In the present invention it is shown that the delivery system of a drug in the lipid base containing opioid, such as martinslife, has minimal possibility of respiratory depression, measured as a decrease in the percent saturation of hemoglobin with oxygen (SpO2) from the maximum saturation of blood oxygen or base value before the introduction of the drug compared with epidural introduction of free medicines. The person skilled in the art will understand that the content of oxygen in the blood can easily be measured using such commercially available devices as pulse oxigemometry.

Also it is shown that a single dose of long-allocated opioid contained in the composition of the mule is Ziya, moreover, the maximum concentration of therapeutic drug in cisternal CSF (cerebrospinal fluid) was observed within 60 minutes after [the introduction of] a single epidural dose, and then gradually decreases over the next few days, for example up to eight days. Although the maximum concentration of CSF (cerebrospinal fluid) was reduced compared to the concentration after epidural injection of free martinslife, General analgesia (as shown, for example, the area under the curve (CPD) Fig.1, 3 and table 1), was increased many times compared with the free martinslife delivered epidurally. For example, rats were observed for 17 - and 3.1-fold decrease in the maximum concentration of morphine in serum and CSF, respectively, however, the CPD for CSF was 2.8 times increased after epidural injection of 250 micrograms of martinslife encapsulated in multivesicular liposomes (DTC401), compared with the same dose decapsulating of martinslife.

Due to the reduction of the maximum concentrations of morphine in serum and CSF in the regulated release of epidurally injected morphine was not depressed respiration; while epidurally entered St is gained. First, the way epidural delivery of a single dose of long-redundant connection provides the advantage that the patient reduces the risk associated with the dose negative effects such as respiratory depression, which is usually associated with epidural injections percussion or infusion of therapeutic compounds. Secondly, through the introduction of therapeutic compounds epidurally, and not directly into the cerebrospinal fluid therapeutic connection does not migrate throughout the brain and spinal cord and therapeutically effective dosage of therapeutic compound is released locally into the epidural space for an extended period of time, for example up to eight days. And finally achieved prolonged analgesia without multiple injections or continuous infusions.

The person skilled in the art will understand that the time period during which in the practice of the invention is supported therapeutic rate of release will vary depending on the disease condition that should be treated, characteristics of therapeutic compound and the delivery system for drugs with a long release, as well as from the General to the m, in which he refers to compositions according to the invention, means that therapeutic compound is released from the delivery system drugs at a concentration sufficient to achieve a particular medical action for which it was intended therapeutic agent. For example, if therapeutic compound is an opioid, then the required medical action is analgesia without respiratory depression. The exact dosage will vary depending on such factors as the particular therapeutic compound and the desired medical effect, as well as factors such as age, sex, General condition of the patient etc., Experts in this field can easily take into account these factors and use them to establish effective therapeutic concentrations, without resorting to undue experimentation.

For example, the interval of dosages that are suitable for epidural injection martinslife people is 1 to 60 mg. For more effective compounds may be required even these low dosages as 0.01 mg, and less effective, you may need 5000 mg Although you can give a dose outside of the above range of doses, this interval covers the width primenenie published methods epidural space rats include drilling a hole through the bone of the lumbar vertebrae and the introduction of the catheter 1 cm up into the epidural space. The present invention allows to place the catheter at the top (i.e. from the neck) without injury due to a surgical procedure. In addition, the tip of the catheter can be placed anywhere along the spine, but not limited to the lumbar region, as described in the prior art. This way of placing the catheter from above is also applicable to animals other than the rats, such as rabbits, dogs and humans.

The following examples illustrate how the invention may be embodied in practice. However, it is clear that the examples are for the purpose of illustration, and should not assume that the invention is limited to any specific materials or conditions listed here.

EXAMPLE 1 A. Getting multivesicular liposomes encapsulating martinslife (DTC401) in the presence of hydrochloride.

Stage 1) In a clean odnotrahniki glass bubble (inner diameter 1.3 cmheight 4.5 cm) were placed 1 ml of a solution in chloroform (Spectrum Corp., Gardena, CA), containing 9.3 µmol of dialogicity (Avanti Polar Lipids, Alabaster, AL), 2.1 μmol of dipalmitoylphosphatidylcholine (Avanti Polar Lipids), 15 μmol of cholesterol (Avanti Polar Lipids) and 1.8 mmol of triolein (Sigma). This solution is called the lipid component.

Study obavljale in the above odnotrahniki bubble, containing the lipid component.

Stage 3) For the preparation of emulsions of the type water-in-oil" glass bubble containing a mixture of "stage 2", have sealed, attached horizontally to the head of the device for shaking and mixing (# catalog S8223-1, American Scientific Products, McGaw Park, IL) and were shaken at maximum speed for 6 minutes.

Stage 4) To obtain Sterol chloroform, suspended in water, obtained in stage 3" emulsion-type water-in-oil" was divided into equal volumes and quickly squeezed through a Pasteur pipette with a narrow tip in each of the two odnotrahniki glass bubbles (inner diameter 1.3 cmheight 4.5 cm), each of which contained 2.5 ml of water, glucose (32 mg/ml) and lysine in the form of the free base (40 mm) (Sigma). Each vial was then sealed and attached them to the head of the same device for shaking and mixing, which is used to "stage 3", and was shaken for 3 seconds at maximum speed for education Sterol chloroform.

Stage 5) in order to get multivesicular liposomes suspension Sterol chloroform, obtained in two bubbles on the "stage 4", then poured into the bottom of the Erlenmeyer flask of 250 machinemy water bath, after the flask was passed a stream of nitrogen gas with a flow rate of 7 l/min to slowly evaporate chloroform over a period of 10-15 minutes. After that liposomes were isolated by centrifugation at 600g for 5 minutes; then washed three times with 0.9% NaCl solution.

B. Getting ready medicinal forms Before epidural injection of drugs DTC401 and control decapsulating ("free") martinslife brought to such a state that in 50 µl contained a dose of 10, 50, 175, 250, or 1000 micrograms. In addition, 75 ál volume for injection was dosage form ials, containing a dose of martinslife 2000 mcg for use in the study of respiratory depression. The concentration of morphine in different liposomal formulations was determined by dissolving 50 µl of each drug in 1 ml isopropylalcohol alcohol with further dilution in water. Morphine concentrations were determined by HPLC using published methods (Joel S. P. et al., Journal of Chromatography, 430: 394-399, 1988). For control in the form of placebo was prepared blank composition multivesicular liposomes by replacing martinslife glucose.

EXAMPLE 2
A. Preparation of animals
Six-to 8-month-old male rats Sprague-Dolny light and darkness for 12 hours and gave them unlimited access to food and water. Before each study, animals were habituated to the environment. Each animal is examined only once. All animals were kept in accordance with the guidelines of the Committee on the use of laboratory animals and their care Institute resources laboratory animals. The national research Council.

B. Epidural catheterization
Caudal epidural catheterization in rats was carried out as follows: were anesthetized with halothane gas and the animals were placed in a stereotactic device for the horizontal position height 7 see Head straight, paying attention to the fact that the animals maintained normal breathing. The needle 19 caliber short cut was introduced at an angle of about 170oin the spine Caudalie to the occipital protrusion on the Central line, and the cut of the needle facing down. The needle pushed Caudalie towards the C1 vertebra up until the end of the needle does not touch the vertebral bone or posterior lamina of C1. The tip of the needle is gently pushed into the ventral edge of the posterior lamina. At this point I felt a slight weakening of the needle and pushed for another 1-2 mm. Tried not to allow the needle to penetrate the Dura. Accidental violation of the Dura can opno catheter. In the posterior epidural space through a needle injected polyethylene catheter (PE-10; 12 cm length; int. diameter: 0.28 mm; volume: 7,4 ml (Becton Dickinson, Sparks, MD). The catheter is slowly pushed through the needle and stopped at the level L of about 1.8 cm from C1. The outer part of the catheter subcutaneously tunnelirovanie under the scalp and secured silk purse string suture 3-0. Finally, the catheter was rinsed 10 µl of normal saline and was clogging the stainless steel wire. The procedure from the beginning of anesthesia until suturing lasted approximately 10-15 minutes. Animals were allowed to return to normal and watched them for 60 of ancient Nations. In subsequent studies have used only those animals that are fully recovered from the procedure.

Century Antinociceptive
Baseline nociception after placing an epidural catheter was determined by exposing animals standard test hot plate (52,50,5o(C) as described in M. S. Wallace et al. (Anesth. Play mode display. 79: 778-786, 1994). Delayed response to nociception (in seconds) was measured from the time when the animals were placed on a hot plate until the time they either licked his hind leg, or jumping. Basic (after preprocessing) value Sogo. Thereafter, each animal was doing an epidural injection of 50 ál or DTC401 containing doses of epidural morphine in the range from 10 to 250 micrograms, solution decapsulating of martinslife control idle ials. In addition, it was determined antinociceptive effect introduced subcutaneously of martinslife in doses ranging from 250 μg to 1 mg After epidural injection of the investigated solutions through a catheter inserted as described above, an epidural catheter was rinsed 10 μl of 0.9% sodium chloride.

Then the animals were again tested hot plate to measure antinociceptive effect at given points in time: 0,5, 1, 2, 3, 4, 6, 12 and 24 hours after injection decapsulating of martinslife and 0.5, and 1.6 hours and 1, 2, 3, 4, 5, 6, 7 and 8 days after injection as DTC401 and idle ials. Antinociception was determined for 5 or 6 animals for each dose of each drug. To prevent tissue damage to the pads on the feet, used a time limit of 60 seconds. In accordance with this 100% MVA was determined as antinociceptive lasting60 seconds. The delay interval of 102 to 60 seconds, responsible MBA from 0 to 100%, respectively, was sensitive to what Netanya respiration as functions of time for each of the administered dose. Response to the hot plate was calculated as the percentage of the maximum possible analgesia (% MVA) as described in Wallace and others (see above):

All the area under the curve was calculated by the trapezoid rule to the last data point using computer programs RSTRIP [Micromath, Salt Lake City, UT].

Used univariate analysis of variance (YES) in order to separately determine the dose dependence for different dosage forms and routes of administration; while the two-factor used for comparison dosage forms, with an equal dose. If all YES exercised control on the Newman Coil (Newman-Keuls) in order to determine statistical significance in all tests p<0,05 believed statistically significant. All data cited as averagethe standard error ().

According to the data of Fig.1, an epidural introduction DTC401 resulted in the equivalent of the beginning of analgesia, but the duration of analgesia was significantly increased compared with the entered epidurally free martinslife. When epidural injection idle control MVV antinociceptive effect was not evident (data not shown). Maximize, as shown in Fig.2, and the maximum possible analgesia epidural free martinslife was greater than that for epidural DTC401, which was significantly longer than in the case of subcutaneously introduced free martinslife (p<0.05 for each comparison).

Substantial prolongation analgesic effects in animals, which has entered the epidural DTC401, it is easy to see in Fig.1 and large values of the areas under the curves (CPD) for DTC401 in Fig.3. At the dose of 250 mcg, leading to maximum effect, close to 100% MVA for DTC401 and free martinslife, time reduction up to 50% MVA was 3, 4 days to DTC401 compared to 0.17 per day for martinslife.

, Respiratory Depression
Respiratory depression quantitatively assessed using pulse oxigenate. Animals were removed from their cells, were placed in polystyrene means of mechanical retention in rats (Plas Labs, Lansing, MI) and left to adapt for 5 minutes. Oxygen saturation was determined at baseline and after one epidural loading dose martinslife or DTC401, at given points in time, placing the probe pulse oxigenate on the right hind paw (Ohmeta Medical Systems, model 3740, Madison, WI). Dose DTC401 and St is whether 6 animals for each data point, except doses 50 mcg, where used 3 animals. Values of the saturation of hemoglobin with oxygen in percent (SpO2) according to the pulse oxigenate continuously monitored in real time, the maximum value that can be reached within a 3-minute recording period, defined as oxygen saturation.

In Fig. 4 shows the change with time of saturation of hemoglobin with oxygen in percent (SpO2), the measured pulse oxygenatom at different doses DTC401 and martinslife. Occurred in a dose-dependent increase in respiratory depression at higher doses of martinslife, as shown in Fig.5; while respiratory depression at the same doses DTC401 was minimal. On the other hand, the maximum decrease in SpO2was observed within 1 hour after epidural injection of free martinslife or DTC401 and could not see any delayed respiratory depression in any dosage form. The difference between martinslife and DTC401 maximum respiratory depression was statistically significant (p<0,01).Pharmacokinetic studies were performed by measuring the concentrations of morphine in the peripheral blood and in CSF in due time points after a single epidural dose of DTC401, as described above, and through 0,5, 1, 3, 6, 12, 24 hours after epidural injection of free martinslife. A set of 3 or 4 animals were anestesiologi using halothane gas, and samples of CSF and blood was collected by cisternal puncture and cardiac puncture, respectively. Then the animals were killed by an overdose of halothane gas. Serum was separated from blood by centrifugation and stored along with the CSF samples at -80oTo further study the method of radioimmunoassay (RIA).

The concentration of morphine in serum and CSF was determined using a commercially available kit for RIA, highly specific for morphine [Coat-A-CountTMSerum Morphine, Diagnostic Products Corp., Los Angeles, CA], as suggested by the manufacturer. All measurements were performed twice.

In Fig. 6 shows the concentration of morphine in cisternal CSF and serum of animals injected with 250 µg of free martinslife or DTC401. Table 1 summarizes the pharmacokinetic parameters. The maximum concentration of morphine in CSF and serum after epidural injection DTC401 were respectively 32 and 5.9% from those after the introduction of martinslife. The final half of CSF (for DTC401 was 82 casesa compared with martinslife, however, CPD plasma was very similar. Times the half-life was calculated by fitting a pharmacokinetic curves to biexponential functions. For curve fitting by iterative nonlinear regression used the program RSTRIP.

EXAMPLE 3
Getting DTC401 on a larger scale
Stage 1) Into a clean tube for centrifuge stainless steel with a volume of 50 ml was placed 5 ml solution in chloroform containing 46.5 mmol of dioleoylphosphatidylcholine (Avanti Polar Lipids), 10.5 µmol of dipalmitoylphosphatidylcholine (Avanti Polar Lipids), 75 μmol of cholesterol (Sigma Chemical Co. ), 9.0 mmol triolein (Avanti Polar Lipids). This solution is called the lipid component.

Stage 2) Five ml of an aqueous solution containing 20 mg/ml pentahydrate of martinslife (Mallinckrodt Chemical Inc.) and 0.1 G. hydrochloric acid, was added into the above tube centrifuge stainless steel containing the lipid component.

Stage 3) For the preparation of emulsions of the type water-in-oil mixture from stage 2 were mixed using a stirrer TK (AutoHomoMixer, model M, Tokushu Kika, Osaka, Japan) with a speed of 9,000 revolutions per minute (rpm) for 9 minutes.

Stage 4) To obtain Sterol chloroform, suspended in water, obtained in stage 3 of the emulsion of the type water-in-oil; add the/min for 120 seconds.

Stage 5) to obtain multivesicular liposomes suspension of Sterol of chloroform in a test tube for centrifuge was poured on the bottom of the Erlenmeyer flask 1000 ml, containing 25 ml of 4% glucose and 40 mm lysine in water. The flask was kept at 37oWith to shake the water bath through the flask was passed a stream of nitrogen gas with a flow rate of 7 l/min to slowly evaporate the chloroform for 20 minutes. After that liposomes were isolated by 4-fold dilution of the suspension with normal saline and centrifugation of the suspension at 600g for 5 minutes; the supernatant decantation and the liposomal pellet re-suspended in 50 ml of normal saline. Liposomes were again isolated by centrifugation at 600g for 5 minutes. The supernatant again decantation and the pellet re-suspended in normal saline.

The preceding description of the invention is exemplary for the purpose of illustration and explanation. You should understand what can be carried out in various modifications without deviating from the scope and essence of the invention. Accordingly, it is assumed that the following claims should be interpreted so that it HC is the first introduction of therapeutic compounds vertebral, includes encapsulating therapeutic compounds in the system of delivery of drugs that have slowed the rate of release of the compound from about 2 to about 7 days, and the introduction of this system of delivery of drugs in a single epidural dose of spinal and delivery system drug includes multivesicular liposomes obtained from the group consisting of phosphatidylcholines eggs, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine and their appropriate combinations.

2. The method according to p. 1, in which multivesicular liposome further includes at least one steroid.

3. The method according to p. 1, in which the vertebrate is a mammal.

4. The method according to p. 3, in which the mammal is a human.

5. The method according to p. 1, in which the delivery system drug is dispersed system.

6. The method according to p. 1 or 2, wherein therapeutic compound is an opioid.

7. The method according to p. 1 or 2, wherein therapeutic compound is a peptide or peptidomimetic.

8. The method according to p. 6, in which t is eating is hydromorphone.

10. The method according to p. 1 or 2, wherein therapeutic compound selected from the group consisting of codeine, hydrocodone, Levorphanol, oxycodone, Oxymorphone, diacetylmorphine, buprenorphine, nalbuphine, butorphanol tartrate, pentazocine, methadone, fentanyl, Sufentanil and Alfentanil.

11. The method according to p. 1 or 2, wherein therapeutic compound selected from the group consisting of enkephalins, endorphins, casomorphin, kyotorphin and their biologically active fragments.

12. The method according to p. 1 or 2, wherein therapeutic compound is an antagonist of opiate.

13. The method according to p. 12, in which the opiate antagonist selected from the group consisting of naloxone and naltrexone.

14. The method according to p. 1 or 2, wherein therapeutic compound is neurotropic factor.

15. The method according to p. 14, in which neurotropic factor selected from the group consisting of insulin-like growth factor, ciliary neurotropic factor, nerve growth factor, dopamine, epinephrine, norepinephrine, gamma-aminobutyric acid and neostigmine.

16. The method according to p. 1, in which the delivery system of the medicinal product form is injected through the epidural catheter.

17. The method according to p. 16, in which an epidural catheter is injected down from serum needle for subcutaneous injection introduced into the epidural space.

19. A method of reducing respiratory depression in patients receiving analgesic agent, including epidural introduction of this analgesic means, characterized in that a single dose encapsulated funds injected into multivesicular liposomal delivery system.

20. The method according to p. 19, in which the analgesic compound is an opioid.

21. The method according to p. 20, in which the opioid is martinslife.

22. The method according to p. 20, in which an opioid selected from the group consisting of hydromorphone, codeine, hydrocodone, Levorphanol, oxycodone, Oxymorphone, diacetylmorphine, buprenorphine, nalbuphine, butorphanol tartrate, pentazocine, methadone, fentanyl, Sufentanil and Alfentanil.

23. The method according to p. 21, in which the dose contains from about 1 to about 60 mg of martinslife.

24. The method according to p. 19, in which the new form is a disperse system.

 

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31 cl, 13 dwg, 4 tbl

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