EA040799B1 - AGENCY FOR EFFECTIVE RELIEF OF ACUTE AND/OR CHRONIC PAIN SYNDROME AND METHOD OF ITS APPLICATION - Google Patents

Abstract

The invention relates to medicine and veterinary science, in particular to means for the effective relief of acute and chronic pain syndrome and can be used in extreme medicine and the treatment of acute and chronic pain syndrome, including in the late stages of oncological diseases. Claimed is an agent for injection and/or nasal administration based on a tetrapeptide of the sequence H-Tyr-D-Arg-Phe-Gly-NH2 or H-Tyr-D-Arg-Phe-Sar-OH, for the prevention and/or treatment of acute or chronic pain syndrome, which contains as an active substance a therapeutically effective amount of a tetrapeptide of the sequence H-Tyr-D-Arg-Phe-Gly-NH2 or H-Tyr-D-Arg-PheSar-OH and at least one auxiliary substance in the following ratio of components, wt.%: tetrapeptide of the sequence H-Tyr-D-Arg-Phe-Gly-NH2 or H-Tyr-D-ArgPhe-Sar-OH - 0.01-0.5; auxiliary substance - up to 100. Also claimed is a method of using this agent for the prevention and/or treatment of acute or chronic pain syndrome.

Description

Technical field

The invention relates to medicine and veterinary medicine, in particular to agents for effective relief of acute and chronic pain syndrome and can be used in extreme medicine and therapy of acute and chronic pain syndrome, including in the late stages of oncological diseases.

Prior Art

Classical approaches to the relief of acute pain are represented by therapy based on the search for ligands that interact with opioid receptors (OR) in the human body. There are three main groups of OR - mu-, delta- and kappa-receptors. It is believed that mu receptors are mainly responsible for the analgesic effect, while delta and kappa have various physiological manifestations: analgesic, euphorogenic, dysphorogenic, respiratory depression, etc.

Often in patients with a generalized stage of cancer, there is a combination of several types of pain and the causes that caused them. Therefore, pain sensations are quite diverse in clinical manifestations, but they also have common properties - they are constant and, as a rule, are progressive. In some patients, pain appears as a result of tumor growth or is a consequence of the spread of the process to other organs. In other cases, pain may occur due to complications of anticancer treatment. About 3.5 million patients suffer daily from pain of varying intensity. According to statistics, about 40% of patients with intermediate stages of the process and 60-87% with generalization of the disease experience pain in the range from moderate to severe.

For cases of minor or moderate manifestations of pain, both periodic and chronic, first- and second-line drugs are used - non-narcotic drugs (neopids), non-steroidal anti-inflammatory drugs or weak opiates (codeine, dionine, tramal). Non-narcotic analgesics do not affect the respiratory center, do not cause euphoria and the phenomena of physical and mental dependence. However, their analgesic activity is manifested mainly in neuralgic, muscle, joint pain, headache and toothache, and in case of severe pain associated with trauma, abdominal surgery, malignant neoplasms, etc., they are practically ineffective. Another undesirable side effect of these drugs is the negative impact on the gastrointestinal tract, the hematopoietic system, the excretory system.

Over time, there comes a period in cancer patients when such relatively weak drugs cease to have a pronounced effect. In such cases, drugs based on opioids are used. A group of low molecular weight narcotic analgesics (morphine and its derivatives) are characterized by a strong analgesic effect, which makes it possible to use them for injuries and diseases accompanied by a pronounced pain syndrome (malignant neoplasms, myocardial infarction, and others). Despite this, this group has a big drawback - they have an effect on the central nervous system of a person, manifested in the development of euphoria, and with their repeated use, addiction occurs, syndromes of mental and physical dependence are observed. It is no secret that it is precisely this property that allows them to be used as drugs by irresponsible elements and limits the widespread use of drugs by the rules for the circulation of narcotic drugs. Another negative factor is the rather narrow therapeutic range of doses used. The danger of manifestation of negative effects, such as respiratory depression and consciousness, can lead to serious consequences up to lethal.

The search for new highly effective analgesics is an urgent problem of modern pharmacology, since painkillers used in the treatment of pain syndrome do not have the necessary balance in terms of efficiency and safety.

The search for peptide analgesics is an interesting and promising problem of modern experimental pharmacology. Being, in fact, the quintessence of natural endogenous peptides, this group of drugs potentially has high efficiency and selectivity in determining analgesic and euphorogenic activities. Features of the chemical structure of several amino acids does not allow the use of a peptide drug as a drug or to make its chemical modifications in order to impart euphorogenic activity.

It is known to study various peptide substances for the presence of analgesic activity. Among endogenous analgesic peptides, beta-endorphin, met-enkephalin, leuenkephalin, dynorphin A can be mentioned. However, they are not convenient to use as drugs due to their biochemical characteristics and large molecular size.

In the 80s of the 20th century, a natural peptide with affinity for opioid receptors, dermorphin, was discovered. Various variants and modifications of dermorphin-derived peptides have been investigated.

Known heptapeptide having analgesic activity, combined with thermoregulatory and/or vasomotor activity and/or influence on the behavioral response of the subject, and a method of changing the physiological activity of dermorphin patent No. 2134121, issued August 10

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1999], which has the properties of endogenous analgesics.

The use of this peptide provides a change in the level of analgesic and thermoregulatory activity of the original dermorphin.

However, the level of analgesic activity of dermorphin, which has been modified in this way, is also not high enough.

At the same time, according to early studies on this issue, it was found that the minimum requirements for the complete sequence of dermorphin in relation to its analgesic activity were represented by an N-terminal tetrapeptide, in which the presence of a DAla residue is mandatory [Montecucchietal., 1981].

In addition, it was shown that D-Arg 2-dermorphin and its N-terminal tetrapeptide analogs were resistant to peptide cleavage and had a potent antinociceptive effect [Sasakietal., 1985]. It was noted that the tetrapeptide H-Tyr-D-Ala-Phe-Gly-OH was the most resistant to cleavage.

Given the potential importance of this class of tetrapeptides for clinical use, a number of patent applications have been filed JPS58213743 (A), published 12/12/1983 and JPS6054400 (A), published 03/28/1985.

Application JPS58213743 (A) concerns the possibility of obtaining tetrapepids, in particular H-Tyr-D-ArgPhe-Gly-NH ₂ and H-Tyr-D-Arg-Phe-Sar-OH, and indicates their potential activity associated with opioid receptors.

The application JPS6054400 indicates the analgesic activity of tetrapeptides. In particular, the tetrapeptide Tyr-D-Arg-Phe-Gly-NH ₂ has a pronounced analgesic effect (more than 6 times the activity of morphine). This peptide was administered subcutaneously to mice in an acute pain model using Ringer's solution as a diluent. In particular, the application describes the potential for preparing a composition for other administration without giving any examples of such a composition.

A certain disadvantage of these tetrapeptides was the presence of a certain physical dependence and the development of tolerance to them, which prevented their further promotion as a medicinal substance on the market [Chakietal., 1988b]. Thus, systemic administration of Tyr-DArg-Phe-Gly- _NH2 tetrapeptide to mice led to the development of tolerance to the analgesic effect of the peptide [Chakietal., 1990]. With chronic subcutaneous administration of this tetrapeptide, rats also developed physical dependence, but the signs of dependence were less pronounced than with the development of physical dependence on morphine [Chakietal., 1988]. This is due to the similar mechanism of action of tetrapeptides and small molecules of morphine-like substances.

The high selectivity of the proposed tetrapeptides for different types of opioid receptors, as well as the close-to-natural metabolism of biochemical transformations of receptors internalized into the cell, provides milder effects of the development of tolerance and dependence when used in therapeutic dosages.

One of the most important features of the biological action of the peptides H-Tyr-D-Arg-Phe-GlyNH2 and H-Tyr-D-Arg-Phe-Sar-OH is the absence of such serious side effects as the effect on the central nervous system in the form of impaired consciousness and development euphoria, as well as the lack of influence on the respiratory center. Another positive feature of the claimed tetrapeptides is a wide therapeutic range of application without the development of pronounced side effects. These features allow the use of drugs based on these tetrapeptides in a much broader field of therapy outside hospitals, in the field or at home by low-level personnel or by the patient himself.

Disclosure of invention

Thus, the objective of the present invention is to develop a composition, methodology and method of using an agent containing, as an active ingredient, the tetrapeptides H-Tyr-D-Arg-PheGly-NH2 and H-Tyr-D-Arg-Phe-Sar-OH, which is a highly active analgesic means for injection, infusion and / or nasal administration, intended for the prevention and / or treatment of acute or chronic pain syndrome and which can be stored for a long time in the form of a solution and emulsion for direct administration, as well as in a dry powder form, for example in the form of a dry powder or lyophilized form.

The problem is solved by the fact that the proposed tool for injection and/or nasal administration based on the tetrapeptide sequence H-Tyr-D-Arg-Phe-Gly-NH ₂ or H-Tyr-D-Arg-PheSar-OH, for the prevention and/or treatment of acute or chronic pain syndrome, which contains as an active substance a therapeutically effective amount of a tetrapeptide of the sequence H-Tyr-D-Arg-Phe-Gly-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH, and at least one excipient selected from the group including stabilizers, prolongators, buffering agents, solubilizing emulsifiers, solvents, fillers, preservatives and other excipients approved for medical use. The proposed composition contains mainly the following ratio of components, wt.%: tetrapeptide sequence H- 2 040799

Tyr-D-Arg-Phe-Gly-NH2 or H-Tyr-D-Arg-Phe-Sar-OH - 0.01-0.5, excipients up to 100.

At the same time, as a stabilizer, the composition contains at least one substance selected from the group including Trilon B, sodium metabisulfite, sodium thiosulfate, glycine, arginine, histidine, lysine, or their physiologically acceptable salts, for example, such as hydrochloride, sulfate, acetate , glutamate, aspartate and maleate.

As a prolongator, at least one substance selected from the group including polyvinylpyrrolidone with a molecular weight of 10-60 kDa, dextran with a molecular weight of 10-100 kDa, for example, polyglucin or rheopolyglucin, polyvinyl alcohol, sodium carboxymethylcellulose.

As a buffering agent, at least one substance selected from the group consisting of sodium chloride, sodium/potassium hydro-and/or dihydrogen phosphate, sodium or ammonium acetate.

As an emulsifier-solubilizer - at least one substance selected from the group including soy lecithin for injection, polysorbate-20, polysorbate-60, polysorbate-80, span-20, span-40, span-60, span-85, sodium dodecyl sulfate.

As a solvent - at least one substance selected from the group including water for injection, sterile saline solution, olive oil, peach oil, sunflower oil.

As a filler - at least one substance selected from the group including sorbitol, mannitol, mannitol, xylitol, lactose, sucrose, dextrose, a copolymer of lactic and glycolic acids.

As a preservative, at least one substance selected from the group including chlorobutanol hydrate, ethyl alcohol, benzyl alcohol, phenol, metacresol, chlorocresol, benzoic acid, sorbic acid, merthiolate, nipagin; nipazole, benzalkonium chloride or bromide, benzethonium chloride, cetylpyridinium chloride, dimethyldodecylbenzylammonium chloride, methylparaben, propylparaben.

The proposed means for injection can be made in the form of a dry powder, solution for intramuscular or intravenous administration, subcutaneous or intradermal administration, or solution for infusion.

The proposed agent for nasal administration can be made in the form of a dry powder, emulsion, liquid solution.

The proposed tool in liquid form should have a pH of 4 to 8. Preferably 4.5-5.5.

The essence of the invention lies in the fact that, as it was experimentally established, adding to the tetrapeptide the sequence H-Tyr-D-Arg-Phe-Gly-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH of one of the above auxiliary substances allows to improve the biological bioavailability of the drug, reduce the development of undesirable effects, expressed in the disorder of the functions of the central nervous system and respiratory failure, the development of tolerance to the treatment and obtain a more pronounced pharmacological result.

Thus, the developed agent allows its use in drug therapy for the treatment or prevention of acute or chronic pain, facilitating the administration of the required therapeutic dose and ensuring the accuracy of dosing the required therapeutic dose.

In addition to the therapeutic advantages listed above, the proposed tool has stability at room temperature, long shelf life (at least 2 years).

We have experimentally selected and studied compositions for injection and/or nasal use for the prevention and/or treatment of acute and chronic pain of various origins containing the following components.

Liquid dosage form for injection

Range, mass% Buffer 0.01-0.2 Filler 0-6 Stabilizer 0-4 Tetrapeptide substance 0.01-0.5 Solvent Up to 100

pH range 4-8. This form has a shelf life of 2 years at +4-8°C.

Powder form (composition after dilution with a solvent to 1 ml)

Range, mg/ml Buffer 0.01-0.2 Filler 0-6 Prolongator 0-8 Stabilizer 0-4 Tetrapeptide substance 0.01-0.5

pH range 4-8 after dissolution. This form has a shelf life of more than 2 years at +418°C.

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For the nasal form, a liquid composition with the addition of preservatives is used.

Range, mass% Buffer 0.01-0.2 Filler 0-1.5 stabilizer 0-1.5 Tetrapeptide substance 0.01-0.5 preservative 0-0.5 Solvent Up to 100

pH range 4-8.

The compositions can be used for the following diseases, which are accompanied by acute and chronic pain:

Oncology.

Pain in oncological diseases, usually 3-4 stages or in the hospice mode.

Burns of various origins.

Traumatic and surgical pain.

Injuries, postoperative prescription of analgesics, use in extreme medicine.

Childbirth.

Cardiology.

The tool can be used for myocardial infarction, unstable angina pectoris - that is, conditions accompanied by severe pain. Due to the fact that cardiac diseases are among the most common, the need for such pain relief therapy arises quite often.

Veterinary.

The tool can be used for surgical and postoperative use for pain relief of domestic and farm animals, for the treatment of pain in horses with injuries, sterilization and artificial insemination operations, etc.

A special case of the claimed agent are the following possible compositions, developed by us experimentally, agents for injection and/or nasal use for the prevention and/or treatment of acute and chronic pain of various origins, containing the following components.

Liquid dosage form for injection

pH range 4-8. This form has a shelf life of 2 years at +4-8°C.

Powder form (composition after dilution with water for injection to 1 ml)

Range, mg/ml sodium acetate 0.01-0.2 Mannitol 0-6 Polyvinylpyrrolidonemedium daymolecular 0-8 Glycine 0-4 Tetrapeptide substance 0.1-0.5

pH range 4-8 after dissolution. This form has a shelf life of more than 2 years at +418°C.

For the nasal form, a liquid composition with the addition of preservatives is used.

Range, mass% sodium acetate 0.01-0.2 sodium chloride 0-1 Mannitol 0-1.5 Glycine 0-1.5 Metacresol 0-0.5 Tetrapeptide substance 0.05-0.5 Water for injections Up to 100

pH range 4-8.

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Also proposed is a method for preparing an agent for injection and / or nasal administration based on a tetrapeptide of the sequence H-Tyr-D-Arg-Phe-Gl y-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH, for the prevention and /or treatment of acute or chronic pain syndrome by mixing a tetrapeptide of the sequence H-Tyr-D-Arg-Phe-Gl y-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH with at least one appropriate excipient and, if necessary, sterilization of the product; pouring the solution into ampoules, vials or containers for infusion solutions; drying solutions in a sublimation unit; sealing of ampoules or capping of vials with finished products.

Also proposed is a method for preventing and/or treating acute or chronic pain syndrome, including the introduction of the above-mentioned agent for injection and/or nasal administration based on a tetrapeptide of the sequence H-Tyr-D-Arg-Phe-Gly-NH2 or H-Tyr-D- Arg-Phe-Sar-OH in a therapeutically effective dose.

Depending on the characteristics of a particular clinical case and the physiological parameters of the patient, 0.5-10 mg of a sterile solution of the agent is injected, preferably subcutaneously or intradermally or intranasally. Before using the product in powder form, it is diluted with water for injection in an amount of 1-2 ml.

Short description

Examples of the invention.

For a better understanding of the invention, examples of the specific preparation of the proposed dosage forms are given below, possible compositions of each type of means of the form and a description of the properties of the resulting drug are given.

Example 1. Obtaining peptides.

Peptides H-Tyr-D-Arg-Phe-Gl y-NH ₂ and H-Tyr-D-Arg-Phe-Sar-OH were synthesized on an AapptecFocus XC III automatic peptide synthesizer by the solid phase method according to the Fmoc protocol on a Rink polymer carrier. based on a polystyrene matrix using the DEPCDI/HOBt method of amino acid activation. The peptides were cleaved from the polymer and deblocked with TFA:m-crezole. Purification of the peptide substance was carried out using reverse phase HPLC (Sigma-AldrichCo. LLCSUPELCO Ascentis C18 HPLC column, 10x250 mm) in an acetonitrile gradient. The identity of the peptides was determined by means of mass spectrometry analysis (MALDI). The purity of the obtained product is 98%.

Also, peptides can be synthesized by the method described in applications JPS58213743 (A) and JPS6054400 (A) or any other methods for the synthesis of peptides in the liquid phase and on a solid carrier.

Example 2. Obtaining a dry sterile powder for injection.

To obtain a stable injectable dosage form of tetrapeptides of the sequence H-Tyr-D-Arg-Phe-Gl y-NH ₂ and H-Tyr-D-Arg-Phe-Sar-OH, dry scattering under aseptic conditions prepares triturations from sterile tetra peptides and excipients. The above mentioned sorbitol, mannitol, mannitol, xylitol, lactose, sucrose, dextrose, copolymer of D, L-lactic and glycolic acids can be used as a filler in the composition for obtaining dry powder; substances that have a stabilizing effect on the tetrapeptide molecule (glycine, arginine, histidine, lysine or their physiologically acceptable salts) and other excipients approved for medical use.

A specific pharmaceutical composition was made in a solid sterile form, in the form of a dry powder. The drug in appearance is an amorphous powder or a porous mass of white with a yellowish tint or yellow color in an ampoule or vial.

The ampoule or vial contains the following composition after dilution with water for injection to 1 ml in (mg / ml):

sodium acetate 0.4 mg;

mannitol 5 mg;

glycine 5 mg;

tetrapeptide 1.5 mg;

pH up to 4.7.

As indicated above, when used, the contents of the ampoule or vial are dissolved in 1-2 ml of water for injection.

This form has a shelf life of more than 2 years at +4-18°C.

Example 3 Preparation of an injectable aqueous solution.

To prepare a stable injection solution in water for injection, first dissolve excipients from the groups of prolongators, stabilizers, etc. Polyvinylpyrrolidone with a molecular weight of 10-60 kDa, dextran with a molecular weight of 10-100 kDa can be used as prolongators in the composition for an injection aqueous solution. (polyglucin or rheopolyglucin), polyvinyl alcohol, sodium carboxymethylcellulose. Trilon B, sodium metabisulfite, sodium thiosulfate, glycine, arginine, histidine, lysine, or their physiologically acceptable salts (hydrochloride, sulfate, acetate, glutamate, aspartate, and maleate) can be used as stabilizers in the composition for an injectable aqueous solution. Then, the inventive tetrapeptide is dissolved in the resulting solution of excipients. The resulting solution is sterilized by membrane filtration under aseptic conditions, passing through a filter with a pore diameter of 0.22 μm, poured into ampoules or vials in an inert gas atmosphere, sealing the ampoules or capping and running the vials with finished products.

A specific pharmaceutical composition is made in liquid form, in appearance it is a transparent liquid packaged in ampoules or vials. One ampoule/vial contains the tetrapeptide H-Tyr-D-Arg-Phe-Gl y-NH ₂ or -Tyr-D-Arg-Phe-Sar-OH.

The ampoule or vial contains the following composition in wt.%:

sodium acetate - 0.04;

sodium chloride - 0.5;

mannitol - 0.5;

glycine - 0.5;

peptide - 0.15;

water for injection - up to 100;

pH up to 4.7.

The resulting preparation is ready for direct injection.

This form has a shelf life of 2 years at +4-8°C.

Example 4 Preparation of a solution for nasal administration.

To prepare a stable injection solution in water for injection, first dissolve excipients from the groups of prolongators, stabilizers, etc. Polyvinylpyrrolidone with a molecular weight of 10-60 kDa, dextran with a molecular weight of 10-100 kDa can be used as prolongators in the composition for an injection aqueous solution. (polyglucin or rheopolyglucin), polyvinyl alcohol, sodium carboxymethylcellulose. Trilon B, sodium metabisulphite, sodium thiosulfate, glycine, arginine, histidine, lysine or their physiologically acceptable salts (hydrochloride, sulfate, acetate, glutamate, aspartate and maleate) can be used as stabilizers in the composition for an injectable aqueous solution. To increase the antimicrobial stability of the drug, one of the above preservatives can be included in the composition. Then the claimed tetrapeptide is dissolved in the resulting solution of excipients.

The resulting solution is sterilized by membrane filtration under aseptic conditions, passing through a filter with a pore diameter of 0.22 μm, poured into polymer or glass vials in an inert gas atmosphere, and vials with finished products are sealed.

A specific pharmaceutical composition is made in liquid form, in appearance it is a transparent liquid packaged in bottles with a drop or spray nozzle.

The ampoule or vial contains the following composition in wt.%:

sodium acetate - 0.04;

sodium chloride - 0.5;

mannitol - 0.5;

glycine - 0.5;

tetrapeptide - 0.15;

metacresol - 0.1;

water for injection - up to 100;

pH up to 4.7.

This form has a shelf life of 1 year at +4-8°C.

Example 5. Obtaining a lyophilized powder for the preparation of a solution for injection.

To obtain a storage-stable and highly soluble lyophilized powder for solution for injection, auxiliary shaping substances from the groups of fillers, stabilizers, buffer additives, and prolongators are used.

The pharmaceutical composition based on tetrapeptides can be obtained by any known lyophilization method. One such method includes the following steps.

1. Preparation of solution for bottling.

Dissolution of the proposed tetrapeptide in water for injection.

Addition of excipients.

2. Sterilizing filtration, which includes pre- and sterilizing filtration on successive filters with a pore diameter of 0.5 and 0.22 microns.

3. Aseptic filling of the solution into ampoules or vials.

4. Freeze-drying of the solution in a freeze-dryer. The cassettes are placed in a freeze dryer and frozen for 4 hours to a temperature of (-45)°C. Drying is carried out at a residual pressure of 100 to 120 µm Hg. within 48 hours

5. Sealing of ampoules, capping and sealing of vials with the finished product.

6. Packaging and labeling.

The pharmaceutical composition is made in a sterile solid form, in appearance it is an amorphous powder or loose tablet in an ampoule or vial.

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The ampoule or vial contains the following composition after dilution with water for injection to 1 ml (in mg / ml):

sodium acetate - 0.4 mg;

mannitol - 5 mg;

glycine - 5 mg;

tetrapeptide - 1.5 mg;

pH up to 4.7.

As indicated above, when used, the contents of the ampoule or vial are dissolved in 1-2 ml of water for injection.

Example 6. Influence of the developed agent on peripheral opioid activity on the example of the tetrapeptide H-Tyr-D-Arg-Phe-Gly-NH ₂ in the in vivo model.

The effect was assessed by the ability to inhibit the contractions of isolated organs caused by electrical stimulation - the ileum of guinea pigs (PKMS). The work was carried out on a special device for testing the opioid activity of substances on a model of the ileum of guinea pigs. PCMS taken from non-linear animals was placed in a 10 ml vessel with inserted electrodes containing Krebs solution at a temperature of 34°C in a thermostatically controlled water bath (HOECHST Organbad, Germany) to maintain a constant temperature in the working cells. The lower end of the organ was fixed motionless in the vessel near the bottom. The upper end was attached with a ligature to the force registration sensor (K-30 isometric registration sensors, HugoSachsElektronik KG, Germany). The initial tension of the organs was 1 g. The solution with the intestinal segments placed in it was constantly aerated. The PCMS preparations were equilibrated in Krebs solution for one hour.

Using plate electrodes, PCMS was stimulated by electrical stimulation with series of four rectangular pulses each 0.5 ms long with an interpulse interval of 1.5 ms and intervals between series of 7.5 s at a voltage of 80 V using an electrical pulse generator (type 215/I, HugoSachsElektronik , Germany). Contractions were recorded in isometric mode using K30 sensors through two-channel amplifiers on an MS 6601 recorder (Watanabe, Japan).

The test agent was injected into a solution washing the organs in a volume of 5 to 15 μl, at final concentrations of 1x10 ^-9 -1x10 ^-6 M. Each subsequent injection of the agent was carried out 1 min after the previous dose, without prior washing of the organs according to the cumulative principle. Based on the data obtained, dose-response curves were constructed for morphine and the test agent. The activity of the substances was expressed by pD2 (numerically equal to the negative decimal logarithm of the concentration of the substance, causing a 50% effect of the maximum value).

The study of the opioid activity of the peptide-based agent was carried out in comparison with a standard solution of morphine. During the experiment, the change in the amplitude of contractions of the PCMS was taken into account. We used a parallel study on 4 cells with PCMS for each drug.

According to the table. 1 according to the EU ₅₀ of morphine is 3.26x10 ^-7 M, and the pD2 is 6.48. For the agent based on the studied peptide, EC ₅₀ was 2.37x10 ^-7 M, and pD2 was 6.62.

The drug is superior to morphine in efficiency and activity when exposed to opioid μreceptors localized on PCMS.

Table 1. Dependence of the degree of inhibition of PMCL amplitude movements depending on the concentration of TP1, TP2 or morphine

Structure-1 peptide, mean values Structure-2 peptide, mean values morphine, mean values Concentration in-va, mol/l amplitude. mm oppress.% amplitude. mm oppress.% amplitude. mm oppress.% 0.0E+00 38.7 0.0 38.2 0.0 39.5 0.0 1.0E-09 38.7 0.0 38.2 0.0 39.5 0.0 4.0E-09 38.7 0.0 38.2 0.0 39.5 0.0 1.4E-08 36.0 7.0 35.3 7.6 36.0 8.7 4.4E-08 30.3 22.9 29.7 22.2 29.0 25.8 1.4E-07 23.0 41.1 22.0 42.4 23.8 39.2 4.4E-07 15.7 61.1 15.2 60.2 18.0 55.0 1.4Е-06 9.3 76.6 8.6 77.5 13.0 68.2

The inhibitory effect of morphine and the agent based on the studied peptide on electrical stimulation contractions of PCMS was prevented by the preliminary introduction of the opioid receptor antagonist naloxone at a concentration of 10 ^-6 M into the washing solution.

Example 7. Comparative study of the analgesic activity of an agent (in the form of an injection form according to example 3) based on the tetrapeptide H-Tyr-D-Arg-Phe-Gly-NH ₂ in comparison with a solution of morphine in equal molar concentrations in-vivo were carried out using the TailFlick and HotPlate methods on a rat model.

2 doses of the agent were studied - 0.1 and 5 mg / kg. Morphine at doses of 5 and 15 mg/kg was used as a control substance.

In the experimental and control groups, rats were taken from the same batch, approximately the same weight, about 160 g.

Peptide and morphine were injected intraperitoneally with a syringe in equal weight amounts to laboratory animals of the experimental and control groups, respectively. The introduction of the substance was carried out in the form dissolved in water for injection, so that the total volume of the injected solution did not exceed 0.5 ml. The measurement was started 15 min after the administration of the substance.

Both tests were performed in pairs and sequentially one after the other. First, each animal was tested before the administration of substances, then 15 minutes after the administration. Substances were administered intraperitoneally in 0.5 ml of physiological saline separately in groups of morphine, peptide.

The TailFlick test is based on the spinal reflex of a rat's tail flicking when irritated by heat.

The study was carried out on a German-made TailFlick apparatus. The device is a fixator for an animal with a groove for the tail. An incandescent lamp with a focusing lens is installed above the chute. The lamp turns on by pressing the button, and turns off when the animal's tail flicks. The device measures the time of pressing the button and fixes it on the display and prints it on a paper tape. With the introduction of substances that cause an analgesic effect, the time to tail flick increases. Since animals differ in individual pain sensitivity, two measurements are taken on each animal - before and after the administration of the substance. The threshold value of complete analgesia is considered to be exposure time longer than 10 s (tail tissues begin to break down). The study was carried out on male rats weighing 160-180 g, 6 animals for each dose.

The HotPlate test is based on the paw-licking reflex when an animal hits a hot surface. The device is a metal surface with a diameter of 30 cm, the temperature of which is controlled by software. The platform is closed with a transparent fence 40 cm high. The temperature of the metal platform is set to 52°C. When placing an animal on the site, the time is measured until the moment when the animal licks its paws. With the introduction of analgesics, the time to licking of the paws increases.

Studies have shown the presence of a pronounced analgesic activity of the proposed agent in comparison with control animals treated with placebo and the reference drug morphine. The effect of complete analgesia was found at a dose of peptide analgesic 5 mg/kg. The effectiveness of analgesia in the proposed drug at a dose of 0.1 mg/kg corresponds to that of 5 mg/kg of morphine. The work carried out confirms the data obtained about a significantly higher activity of the agent in comparison with morphine by approximately 10-20 times.

Example 8 Investigation of the effect of the tetrapeptide-based injectable (according to example 2) HTyr-D-Arg-Phe-Gly-NH ₂ on the behavioral responses of animals, a study of toxicity of high doses in the open field test and the test for maintaining balance on a horizontal rod.

The purpose of the experiment was to identify an approximate effective dose of a substance that affects the behavioral performance of the animal. As a result, several animals were studied after administration of various doses of the drug.

Investigated doses and routes of administration:

- control without injection;

- injection of 300 µl of saline (stress control);

- agent 0.5 mg/kg (EC ₅₀ for analgesic effect);

- agent 1.5 mg/kg;

- agent 5 mg/kg;

- agent 10 mg/kg (20 EU ₅₀ doses for analgesic effect);

- agent 20 mg/kg (40 EU ₅₀ doses for analgesic effect);

- agent 100 mg/kg (200 EU ₅₀ doses for analgesic effect)%;

- morphine 120 mg/kg (20 EU ₅₀ doses for analgesic effect).

In the experimental and control groups, male mice were taken from the same batch, approximately the same weight (22-24 g).

Substances were injected intraperitoneally with a syringe in the same weight amounts to laboratory animals of the experimental and control groups, respectively. The introduction of the substance was carried out in a dosage form dissolved in water for injection so that the total volume of the injected solution did not exceed 0.5 ml. The measurement was started 15 min after the administration of the substance.

15 minutes after injection into the abdominal wall without the introduction of a substance, the introduction of saline solution, morphine solution or the studied peptide, or without any influences, sequential testing was carried out in the following order.

Test Open field.

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An animal was released into a round, not brightly lit arena. The floor is drawn into squares with a side of 10 cm. The crossing of the side of the square by the animal during movement is a conventional unit by which the locomotor activity of mice is measured. Mouse movement in the center of the arena and on the periphery are recorded separately. The number of standing up on the hind legs of the rack and looking into the holes in the floor of the mink was recorded. Observation was carried out visually with simultaneous data entry into the computer through the keyboard (semi-automatic registration).

Balance test on a horizontal rod.

Animals were placed on a wooden rod 2 cm in diameter and 40 cm long, located horizontally at a height of at least 60 cm. The time to maintain balance on the rod was recorded.

The results were processed using one-way ANOVA.

Studies have shown that large doses of the drug do not lead to a significant loss of orientation of animals or to a decrease in behavioral activity. Even at a dose of 100 mg/kg, the mice continued to actively move around the field, although with somewhat reduced activity (by 20-25% at a dose of 20100 mg/kg). There is a decrease in exploratory activity by 20-30% (counter-mink index).

In the experiment, the dose causing the toxic effect was not reached. Even at a dose of 100 mg/kg, the animals continue to move actively, breathing is not disturbed, and the heartbeat remains stable.

It should be noted that with the introduction of the control substance morphine (20 EU ₅₀ doses), motor activity becomes significantly reduced by 50-70%.

In the test for maintaining balance on a horizontal rod, no significant differences were found between the experimental and control individuals. Experienced animals keep well on the rod. Some difference between the experimental animals and the control can be considered their reduced activity during horizontal movement along the perch by 10-20% for high doses of the drug 20-100 mg/kg.

Thus, the agent in the proposed dosage form has little effect on the behavioral characteristics of animals.

The use of the drug in doses of 200 EU ₅₀ does not lead to toxic effects.

The given examples demonstrate that the preparation of the tetrapeptide Н-Tyr-D-Arg-Phe-Gl·y-NH ₂ has an analgesic activity significantly superior to that of morphine. At the same time, unlike morphine, H-Tyr-D-Arg-Phe-Gly-NH ₂ does not cause toxic effects and pronounced psychophysiological and behavioral disorders.

Under conditions that ensure the stability of the peptide during storage, its analgesic activity is ten times higher than that of morphine. An important conclusion is the wide therapeutic range of this method, which exceeds 200 therapeutic doses. This means that this drug does not have a pronounced risk of overdose and allows you to prescribe doses of the drug corresponding to the severity of the pain syndrome with high safety.

By extrapolation, it was determined that the recommended dose for humans will be in the range of 0.5-10 mg/dose, depending on the specific case of pain syndrome.

The same results were obtained for the agent based on H-Tyr-D-Arg-Phe-Sar-OH.

Example 9 Clinical Application

The use of the drug prototype in patients in the informed consent mode. The prototype of the drug was used in cases where the patient was indicated for treatment with painkillers of the morphine group (stage 3 according to the WHO system), but for one reason or another, the drug was not available. When a regular morphine drug appeared, the therapy with the experimental prototype of the drug was stopped.

Based on human dosage extrapolation data, 0.7-3 mg of active tetrapeptide was used in a single dose by subcutaneous administration under the supervision of a specialist. The drug was packaged in a lyophilic form of 3 mg per ampoule and was dissolved in 1-3 ml of water for injection before use.

The following parameters were registered.

The time of onset of the effect of anesthesia, the duration of the effect.

Dynamics of pain syndrome on a digital scale 0-10: 0 - no pain, 10 - unbearable pain. The patient regularly notes on the scale the intensity of pain during treatment to assess the analgesic effect. Separately, the intensity of the pain syndrome was noted before taking the experimental prototype of the drug and during the reception.

Physical activity of the patient. Measured in points: 1 - normal activity, 2 - reduced activity; the patient is able to visit a doctor on his own, 3 - bed rest less than 50% of the daytime, 4 - bed rest more than 50% of the daytime, 5 - full bed rest.

Subjective sensations of the patient - a change in perception, mood, the presence / absence of euphoria / dysphoria, discomfort in the body, gastrointestinal tract.

Basically, the study of patients with types of pain corresponding to somatic and internal was carried out, although it is often not possible to accurately systematize the type of pain.

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Patient 1. Man, 48 years old.

Pancreatic cancer stage IV, metastases. Pain 6-9 on a scale. Narcotic analgesics were prescribed.

The applied dose is 3 mg 2 times a day. The duration of therapy is 5 days.

The time for the development of the effect of anesthesia is 10-15 minutes.

Subjective sensations: at the first injections, a very slight dysphoric effect is felt for 10-15 minutes, then the effect disappears. Violations of consciousness, activity are not revealed. The patient takes care of himself.

The degree of anesthesia - up to 1-3 on a scale. The duration of the analgesic effect is 8-12 hours.

Patient 2. Woman, 72 years old.

Colon cancer stage IV, metastases. The pain does not stop, the patient screams in pain, she is not able to take care of herself, she is not able to maintain a normal conversation, she practically does not sleep.

The applied dose is 3 mg 2 times on the first day, then reduced to 1.5 mg 2 times a day. The duration of therapy is 4 days.

The time for the development of the effect of anesthesia is 10-15 minutes.

Subjective sensations: the intensity of pain is significantly reduced, the ability to communicate is restored, the patient maintains a conversation, was able to get up and walk, slept normally at night. No negative effects have been identified.

The degree of anesthesia - up to 2-3 on a scale.

The duration of the analgesic effect is 6-12 hours.

Patient 3. Woman, 56 years old.

Colon cancer stage IV, metastases. Pain 7-10 on a scale. Narcotic analgesics were prescribed.

The applied dose is 1.5 mg 2 times a day. The duration of therapy is 4 days.

The time for the development of the effect of anesthesia is 10-15 minutes.

Subjective sensations: the intensity of pain is significantly reduced, the patient maintains a conversation, was able to stand up and walk. No negative effects have been identified.

The degree of anesthesia - up to 1-3 on a scale.

The duration of the analgesic effect is 8-10 hours.

Patient 4. Woman, 61 years old.

Breast cancer stage IV, metastases. Pain 7-10 on a scale. Narcotic analgesics were prescribed.

The applied dose is 1.5 mg 2 times a day. The duration of therapy is 5 days.

The time for the development of the anesthesia effect is 10-20 minutes.

Subjective sensations: there is no euphoria and dysphoria, the intensity of pain is significantly reduced, the patient maintains a conversation. No negative effects have been identified.

The degree of anesthesia - up to 1-3 on a scale.

The duration of the analgesic effect is 8-10 hours.

Patient 5. Woman, 53 years old.

Breast cancer stage IV, metastases. Pain 7-8 on a scale. Narcotic analgesics were prescribed.

The applied dose is 0.7 mg 2 times in 1-3 days, 1 mg in 4-6 days. The duration of therapy is 6 days.

The time for the development of the effect of anesthesia is 10-15 minutes.

Subjective sensations: there is no euphoria and dysphoria, the intensity of pain is significantly reduced, the patient maintains a conversation. No negative effects have been identified.

The degree of anesthesia - up to 1-3 on a scale.

The duration of the analgesic effect is 8-10 hours.

Patient 6. Woman, 64 years old.

Compression fracture of a vertebra in the lumbar region. Pain 7-8 on a scale. Cannot move independently.

The applied dose is 1.5 mg 2 times a day. The duration of therapy is 2 days.

The time for the development of the effect of anesthesia is 10-15 minutes.

Subjective sensations: there is no euphoria and dysphoria, the intensity of pain is significantly reduced, the patient was able to move to another city to continue treatment. No negative effects have been identified.

The degree of anesthesia - up to 1-3 on a scale.

The duration of the analgesic effect is 8-12 hours.

Common to all the above cases can be considered a confirmed absence of negative reactions in the form of impaired consciousness, euphoria / dysphoria, problems with the gastrointestinal tract, no signs of respiratory depression, palpitations were found. Patients suffering from severe pain after using the prototype drug were able to communicate normally with their families.

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Claims (31)

CLAIM 1. An agent for injection based on a tetrapeptide of the sequence H-Tyr-D-ArgPhe-Gly-NH2 or H-Tyr-D-Arg-Phe-Sar-OH for the prevention and / or treatment of acute or chronic pain syndrome, which in liquid form contains as an active substance a therapeutically effective amount of a tetrapeptide of the sequence H-Tyr-D-Arg-Phe-Gl y-NH ₂ or H-Tyr-DArg-Phe-Sar-OH and excipients in the following ratio of components, wt.% : tetrapeptide sequence H-Tyr-D-Arg-Phe-Gl·y-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH - 0.01-0.5; buffering additive - 0.01-0.2; solvent - up to 100, while the product must have a pH of 4 to 8. 2. The agent according to claim 1, characterized in that, as excipients, it additionally contains in wt.%: filler - 0-6; stabilizer - 0-4. 3. The agent according to claim 2, characterized in that, as a stabilizer, it contains at least one substance selected from the group including Trilon B, sodium metabisulfite, sodium thiosulfate, glycine, arginine, histidine, lysine, or their physiologically acceptable salts, for example such as hydrochloride, sulfate, acetate, glutamate, aspartate, and maleate. 4. The agent according to claim 1, characterized in that it contains at least one substance selected from the group consisting of sodium chloride, sodium/potassium hydro- and/or dihydrogen phosphate, sodium or ammonium acetate as a buffering additive. 5. The agent according to claim 1, characterized in that it contains at least one substance selected from the group consisting of water for injection, sterile saline, olive oil, peach oil, sunflower oil as a solvent. 6. The agent according to claim 2, characterized in that it contains at least one substance selected from the group including sorbitol, mannitol, mannitol, xylitol, lactose, sucrose, dextrose, a copolymer of lactic and glycolic acids as a filler. 7. The agent according to claims 1 to 6, characterized in that the agent can be made in liquid dosage form in the form of a solution for intramuscular or intravenous, subcutaneous or intradermal administration, or a solution for infusion. 8. An agent according to claim 7, characterized in that its pH is preferably 4.5-5.5. 9. The agent according to claim 8, characterized in that the solution for injection preferably contains in wt.%: sodium acetate - 0.04; sodium chloride - 0.5; mannitol - 0.5; glycine - 0.5; peptide - 0.15; water for injection - up to 100; pH up to 4.7. 10. An agent for injection based on a tetrapeptide of the sequence H-Tyr-DArg-Phc-Gl y-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH for the prevention and / or treatment of acute or chronic pain syndrome, which has a powder dosage form and contains as an active ingredient a therapeutically effective amount of a tetrapeptide of the sequence H-TyrD-Arg-Phe-Gl y-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH and excipients, which, after solvent dilutions to 1 ml in mg/ml are in the following ratio, wt.%: tetrapeptide sequence H-Tyr-D-Arg-Phe-Gly-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH - 0.01-0.5; buffering additive - 0.01-0.2. 11. Means but p. 10, characterized in that as excipients additionally contains in wt.%: filler 0-6; prolongator 0-8; stabilizer 0-4. 12. The agent according to claim 11, characterized in that at least one substance selected from the group including polyvinylpyrrolidone with a molecular weight of 1060 kDa, dextran with a molecular weight of 10-100 kDa is used as a prolongator, for example, polyglucin or rheopolyglucin, polyvinyl alcohol , sodium carboxymethyl cellulose. 13. The agent according to claim 11, characterized in that, as a stabilizer, it contains at least one substance selected from the group including Trilon B, sodium mstabisulfite, sodium thiosulfate, glycine, arginine, histidine, lysine, or their physiologically acceptable salts, for example such as hydrochloride, sulfate, acetate, glutamate, asyartate and maleate. - 11 040799 14. The agent according to claim 10, characterized in that it contains at least one substance selected from the group consisting of sodium chloride, sodium/potassium hydro and/or dihydrogen phosphate, sodium or ammonium acetate as a buffering additive. 15. The agent according to claim 10, characterized in that it contains at least one substance selected from the group consisting of water for injection, sterile saline solution, olive oil, peach oil, sunflower oil as a solvent. 16. The agent according to claim 11, characterized in that it contains at least one substance selected from the group including sorbitol, mannitol, mannitol, xylitol, lactose, sucrose, dextrose, a copolymer of lactic and glycolic acids as a filler. 17. An agent according to claims 10 and 11, characterized in that its pH after dilution is preferably 4.5-5.5. 18. An agent according to claims 10-17, characterized in that the powder form preferably contains (composition after dilution with a solvent to 1 ml in mg/ml): sodium phcetate - 0.4 mg; mannitol - 5 mg; glycine - 5 mg; tetrapeptide - 1.5 mg; pH up to 4.7. 19. An agent for nasal administration based on a tetrapeptide of the sequence H-Tyr-D-ArgPhe-Gly-NH2 or H-Tyr-D-Arg-Phe-Sar-OH for the prevention and/or treatment of acute or chronic pain syndrome, which has a liquid form and contains as an active substance a therapeutically effective amount of a tetrapeptide of the sequence H-Tyr-D-Arg-Phe-Gl y-NH ₂ or HTyr-D-Arg-Phc-Sar-OH and excipients, in the following ratio, wt .%: tetrapeptide sequence H-Tyr-D-Arg-Phe-Gl·y-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH - 0.01-0.5; buffering additive - 0.01-0.2; solvent - up to 100; while the product should have a pH of 4 to 8. 20. The agent according to claim 19, characterized in that, as excipients, it additionally contains in wt.%: filler - 0-1.5; stabilizer - 0-1.5; preservative - 0-0.5. 21. The agent according to claim 20, characterized in that, as a stabilizer, the agent contains at least one substance selected from the group including Trilon B, sodium metabisulfite, sodium thiosulfate, glycine, arginine, histidine, lysine, or their physiologically acceptable salts, for example, such as hydrochloride, sulfate, acetate, glutamate, aspartate and maleate. 22. The agent according to claim 19, characterized in that, as a buffering agent, it contains at least one substance selected from the group consisting of sodium chloride, sodium/potassium hydro and/or dihydrogen phosphate, sodium or ammonium acetate. 23. The agent according to claim 19, characterized in that it contains at least one substance selected from the group consisting of water for injection, sterile saline, olive oil, peach oil, sunflower oil as a solvent. 24. The agent according to claim 20, characterized in that it contains at least one substance selected from the group consisting of sorbitol, mannitol, mannitol, xylitol, lactose, sucrose, dextrose, a copolymer of lactic and glycolic acids as a filler. 25. The agent according to claim 20, characterized in that it contains at least one substance selected from the group consisting of chlorobutanol hydrate, ethyl alcohol, benzyl alcohol, phenol, metacresol, chlorocresol, benzoic acid, sorbic acid, merthiolate, nipagin as a preservative , nipazole, benzalkonium chloride or bromide, benzethonium chloride, cetylpyridinium chloride, dimethyldodecylbenzylammonium chloride, methylparaben, propylparaben. 26. An agent according to claims 19 and 20, characterized in that its pH is preferably 4.5-5.5. 27. The agent according to claims 19-26, characterized in that the liquid form for nasal administration preferably contains in wt.%: sodium acetate - 0.04; sodium chloride - 0.5; mannitol - 0.5; glycine - 0.5; tetrapeptide - 0.15; metacresol - 0.1; water for injection - up to 100; pH up to 4.7. 28. Method for prevention and/or treatment of acute or chronic pain syndrome, including - 12 040799 tea administration of an agent according to any of the preceding paragraphs in a therapeutically effective amount. 29. The method according to claim 28, characterized in that the agent is administered in an amount of 0.5-10 mg/dose. 30. A method for preparing the agent according to claims 1 to 19, including mixing a tetrapeptide of the sequence H-Tyr-D-Arg-Phe-Gly-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH with the appropriate excipients. 31. The method according to p. 30, characterized in that, if necessary, sterilize the agent; pouring the solution into ampoules, vials or containers for infusion solutions; drying solutions in a sublimation unit; sealing of ampoules or capping of vials with finished products. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2 -