RU2622980C1 - Means for effective cupping of acute and/or chronic pain syndrome and method of its application - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine and relates to an injectable and/or nasal agent based on the tetrapeptide 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 the active substance a therapeutically effective amount of the tetrapeptide sequence of the H-Tyr-D-Arg-Phe-Gly-NH₂ or H-Tyr-D-Arg-Phe-Sar-OH and at least one auxiliary substance. The group of inventions also relates to a method of preventing and/or treating an acute or chronic pain syndrome comprising administering said agent in a therapeutically effective amount; method for preparing mentioned agent comprising mixing the tetrapeptide with at least one suitable auxiliary substance.

EFFECT: effective cupping of acute and chronic pain syndrome, including on the late stages of cancer.

31 cl, 9 ex, 1 tbl

Description

Technical field

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

State of the art

Classical approaches to the relief of acute pain are represented by therapy based on the search for ligands that interact with opioid receptors (ORs) 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 is quite diverse in clinical manifestations, but they also have common properties - they are constant and, as a rule, are progressive in nature. 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 antitumor treatment. About 3.5 million patients daily suffer 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 of both a periodic and chronic nature, first-line 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 phenomena of physical and mental dependence. However, their analgesic activity is manifested mainly with neuralgic, muscle, joint pain, headache and toothache, and with severe pain associated with injuries, abdominal surgery, malignant neoplasms, etc., they are practically ineffective. Another undesirable side effect of these drugs is a negative effect on the gastrointestinal tract, hematopoietic system, excretory system.

Over time, cancer patients begin a period in which such relatively weak drugs cease to have a pronounced effect. In such cases, opioid-based preparations are used. The group of low-molecular narcotic analgesics (morphine and its derivatives) is characterized by a strong analgesic effect, which makes it possible to use them for injuries and diseases accompanied by severe pain (malignant neoplasms, myocardial infarction, and others). Despite this, this group has a big drawback - they affect 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 makes it possible to use their unconscious elements as drugs 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 negative effects, such as respiratory depression and consciousness, can lead to serious consequences, including fatal ones.

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

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

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

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

Known heptapeptide having analgesic activity, combined with thermoregulatory and / or vasomotor activity and / or with an influence on the behavioral response of the test subject, and a method for changing the physiological activity of dermorphin [Patent No. 2134121, issued August 10, 1999], having 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 was changed 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 dermorphin sequence with respect to its analgesic activity were represented by the N-terminal tetrapeptide, in which the presence of a D-Ala residue is mandatory [Montecucchi et al., 1981 ].

In addition, it was shown that B-Am £ 2-dermorphin and its N-terminal tetrapeptide analogs were resistant to peptide cleavage and have potent antinociceptive effects [Sasaki et al., 1985]. It was noted that the tetrapeptide H-Tyr-D-Ala-Phe-Gly-OH was most resistant to cleavage.

Given the potential importance of this class of tetrapeptides for clinical use, a number of applications were filed for inventions: JPS58213743 (A), published 12.12.1983, and JPS6054400 (A), published 03/28/1985.

Application JPS58213743 (A) concerns the possibility of producing tetrapeptides, in particular H-Tyr-D-Arg-Phe-Gly-NH ₂ and H-Tyr-D-Arg-Phe-Sar-OH, and their potential opioid activity is indicated receptors.

JPS6054400 teaches the analgesic activity of tetrapeptides. In particular, the presence of a pronounced analgesic effect in the tetrapeptide Tyr-D-Arg-Phe-Gly-NH ₂ (more than 6 times the activity of morphine) is indicated. This peptide was injected subcutaneously in mice on a model of acute pain, while Ringer's solution was used as a solvent. Including, the application describes the potential possibility of preparing a composition for other administration without giving any examples of such a composition.

A certain drawback 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 [Chaki et al., 1988]. Because systemic administration of the tetrapeptide Tyr-D-Arg-Phe-Gly -NH ₂ mice led to the development of tolerance to the analgesic effect of the peptide [Chaki et al., 1990]. With chronic subcutaneous administration of the indicated tetrapeptide to rats, physical dependence also developed, but the signs of development of dependence were less pronounced than with the development of physical dependence on morphine [Chaki et al., 1988]. This is explained by a similar mechanism of action of tetrapeptides and small molecules of morphine-like substances.

The greater selectivity of the claimed tetrapeptides to different types of opioid receptors, as well as the close to natural metabolism of the biochemical transformations of receptors internalized into the cell, provide milder effects of tolerance and dependence when applied in therapeutic dosages.

One of the most important features of the biological effect of peptide H-Tyr-D-Arg- Phe-Gly-NH ₂ and H-Tyr-D-Arg- Phe-Sar-OH is the lack of serious side effects, such as effects on the CNS in a violation consciousness and development of euphoria, as well as the lack of influence on the respiratory center. Another positive feature of the claimed tetrapeptides can be called a wide therapeutic range of use without the development of pronounced side effects. These features allow the use of drugs based on tetrapeptide data in significantly wider areas of therapy outside hospitals, in the field, or at home by low-skilled personnel or by the patient alone.

Disclosure of invention

Thus, the objective of the present invention is to develop a composition, methodology and method of using an agent containing H-Tyr-D-Arg-Phe-Gly-NH ₂ and H-Tyr-D-Arg-Phe-Sar-OH tetrapeptides as active ingredient, which is a highly active painkiller for injection, infusion and / or nasal administration, intended to prevent and / or treat 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 dry powder m form, for example in the form of dry powder or lyophilized form.

The problem is solved in that a means for injecting and / or nasal administration based on a tetrapeptide of the sequence H-Tyr-D-Arg-Phe-Gly-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH, to prevent and / or treatment of an acute or chronic pain syndrome that contains 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 consisting of stabilizers, prolongators, buffering additives, emulsifiers - solubilizers, solvents, fillers, preservatives and other auxiliary substances that are approved for medical use. The proposed composition contains mainly the following ratio of components, wt.%:

The tetrapeptide of the sequence H-Tyr-D-Arg-Phe-Gly-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH is 0.01-0.5 auxiliary substances up to 100.

Moreover, as a stabilizer, the composition contains at least one substance selected from the group comprising 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 consisting of polyvinylpyrrolidone with a molecular weight of 10-60 kDa, dextran with a molecular weight of 10-100 kDa, for example, polyglukin or reopoliglukin, polyvinyl alcohol, sodium carboxymethyl cellulose;

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 comprising soya 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 comprising water for injection, sterile saline, olive oil, peach oil, sunflower oil;

As a filler, 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 preservative, 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; nipazole, benzalkonium chloride or bromide, benzethonium chloride, cetylpyridinium chloride, dimethyldodecylbenzylammonium chloride, methyl paraben, propyl paraben.

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

The proposed tool 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 from 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 of H-Tyr-D-Arg-Phe-Gly-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH of one of the above auxiliary substances can improve the biological bioavailability of the drug, reduce the development of unwanted effects, expressed in the dysfunction of the central nervous system and respiratory failure, the development of tolerance to treatment and to obtain a more pronounced pharmacological result.

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

In addition to the above therapeutic benefits, the proposed tool has stability at room temperature, a 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

PH range 4-8.

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

Powdered form (composition after dilution with a solvent up to 1 ml).

The pH range is 4-8 after dissolution.

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

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

PH range 4-8.

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

Oncology.

Pain in cancer, usually 3-4 stages or in hospice mode.

Burns of various genesis.

Traumatic and surgical pain.

Injuries, postoperative administration 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 cardiological diseases are one of the most common, the need for such anesthetic therapy arises quite often.

Veterinary science

The tool can be used in surgical and postoperative use for the anesthesia 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 funds are the following possible compositions that we have developed experimentally, means 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.

Powdered form (composition after dilution with water for injection up to 1 ml).

The pH range is 4-8 after dissolution.

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

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

PH range 4-8.

Also provided is a method of preparing an injectable and / or nasal tetrapeptide-based preparation of the sequence H-Tyr-D-Arg-Phe-Gly-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH to prevent and / or treating an acute or chronic pain syndrome by mixing a tetrapeptide of the sequence H-Tyr-D-Arg-Phe-Gly-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH with at least one suitable adjuvant, 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 ampoules or corking finished product bottles.

Also provided is a method for the prevention and / or treatment of acute or chronic pain syndrome, comprising administering the aforementioned agent for injection and / or nasal administration based on a tetrapeptide of the sequence H-Tyr-D-Arg-Phe-Gly-NH ₂ or H-Tyr-D -Arg-Phe-Sar-OH at a therapeutically effective dose.

Depending on the particular clinical case and physiological parameters of the patient, 0.5-10 mg of a sterile solution of the drug is injected, mainly 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.

Examples of carrying out the invention.

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

Example 1

Peptide Production

The peptides H-Tyr-D-Arg-Phe-Gly-NH ₂ and H-Tyr-D-Arg-Phe-Sar-OH were synthesized on an Aapptec Focus XC III automated peptide synthesizer by the solid phase method according to the Fmoc protocol on a Rink polymer carrier on based on a polystyrene matrix using the DEPCDI / HGBt amino acid activation method. The peptides were cleaved from the polymer and released with a mixture of TFA: m-crezole. The peptide substance was purified using reverse phase HPLC (Sigma-Aldrich Co. LLC SUPELCO Ascentis C18 HPLC column, 10 × 250 mm) in an acetonitrile gradient. The identity of the peptides was determined by mass spectrometry (MALDI) studies. The purity of the obtained product is 98%.

Also, peptides can be synthesized by the method described in the applications JPS58213743 (A) and JPS6054400 (A), or by 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, the sequences H-Tyr-D-Arg-Phe-Gly-NH ₂ and H-Tyr-D-Arg-Phe-Sar-OH are prepared by dry crushing under aseptic conditions using sterile tetra peptide and auxiliary substances. The above 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 auxiliary substances approved for medical use.

The specific pharmaceutical composition was in 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 in an ampoule or vial.

An 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. Obtaining an injectable aqueous solution.

To prepare a stable injection solution in water for injection, first dissolve auxiliary substances 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 injection aqueous solution. (polyglucin or reopoliglukin), polyvinyl alcohol, sodium carboxymethyl cellulose. 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 injectable aqueous solution. Then, the inventive tetrapeptide is dissolved in the resulting excipient solution. 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, ampoules are sealed or sealed and run-in bottles with finished products.

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

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

Sodium Acetate 0.04

Sodium Chlorine 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. Obtaining a solution for nasal administration.

To prepare a stable injection solution in water for injection, first dissolve auxiliary substances 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 injection aqueous solution. (polyglucin or reopoliglukin), polyvinyl alcohol, sodium carboxymethyl cellulose. 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 injectable aqueous solution. To increase the antimicrobial stability of the drug, one of the above preservatives may be included in the composition. Then, the inventive tetrapeptide is dissolved in the resulting excipient solution. 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 bottles in an inert gas atmosphere, and corking the finished product bottles is carried out.

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

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

Sodium Acetate 0.04

Sodium Chlorine 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 lyophilized powder for the preparation of a solution for injection.

To obtain a stable during storage and well soluble lyophilized powder for the preparation of a solution for injection, auxiliary excipients from the groups of fillers, stabilizers, buffering additives, and prolongators are used.

The tetrapeptide-based pharmaceutical composition can be prepared by any known lyophilization method. One of these methods involves the following steps:

1. Preparation of the filling solution.

- Dissolution of the claimed tetrapeptide in water for injection.

- Addition of excipients.

2. Sterilizing filtration, which includes preliminary and sterilizing filtration on sequentially installed filters with pore diameters 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-drying unit. Cassettes are placed in a freeze-drying unit and frozen for 4 hours to a temperature of (- 45) ° С. Drying is carried out at a residual pressure of from 100 to 120 μm Hg. within 48 hours.

5. Sealing of ampoules, capping and filling of bottles with the finished product.

6. Packaging and labeling.

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

An 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. The effect of the developed tool on peripheral opioid activity on the example of the tetrapeptide H-Tyr-D-Arg-Phe-Gly-NH ₂ in an in vivo model.

The effect was evaluated by the ability to inhibit the contraction of isolated organs - the ileum of the guinea pigs (PCMS) caused by electrical stimulation. The work was carried out on a special installation for testing the opioid activity of substances on a guinea pig ileum model. PCMS taken from nonlinear animals was placed in a 10 ml vessel with introduced electrodes containing Krebs solution at 34 ° С in a water thermostatic bath (HOECHST Organbad, Germany), a thermostat to maintain a constant temperature in the working cells. The lower end of the organ was fixed in a vessel at the bottom. The upper end was fixed with a ligature to a force registration sensor (K-30 isometric registration sensors, Hugo Sachs Elektronik KG, Germany). The initial organ tension was 1 g. The solution with intestinal segments placed in it was constantly aerated. PCMS preparations were equilibrated in Krebs solution for an hour.

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

The test agent was injected into a solution washing organs in a volume of 5 to 15 μl, in final concentrations of 1 × 10 ^-9 - 1 × 10 ^-6 M. Each subsequent administration of the agent was made 1 minute after the previous dose, without preliminary washing of the organs by cumulative principle. Based on the obtained data, dose-response curves were constructed for morphine and the test agent. The activity of substances was expressed by pD2 (numerically equal to the negative decimal logarithm of the concentration of the substance, causing 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. A parallel study on 4 cells with PCMS for each preparation was used in the work.

According to the data in Table 1, which shows the dependence of the degree of suppression of the amplitude movements of the PMCS depending on the concentration of TP1, TP2 or morphine, the EU ₅₀ morphine is 3.26 × 10 ^-7 M, and the indicator pD2-6.48. For an agent based on the studied peptide, the EC50 was 2.37 × 10 ^-7 M, and pD2-6.62.

The agent is superior to morphine in efficiency and activity when exposed to opioid μ receptors localized to PCMS.

The inhibitory effect of morphine and the peptide-based agent on the reduction of PCMS caused by electrical irritation was prevented by the preliminary introduction of a naloxone antagonist of opioid receptors at a concentration of 10 ^-6 M.

Example 7. A comparative study of the analgesic activity of the agent (in the form of an injection 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 was carried out by the Tail methods Flick "and" Hot Plate "on rat models.

2 doses of the drug were investigated - 0.1 and 5 mg / kg. Morphine in doses of 5 and 15 mg / kg was used as a control substance.

In the experimental and control group of rats were taken from one batch, about the same weight about 160 g.

The peptide and morphine were injected intraperitoneally with a syringe in equal weight amounts to laboratory animals of the experimental and control groups, respectively. The substance was administered 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 minutes after the administration of the substance.

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

The Tail Flick test is based on the spinal reflex of the tail of the rat tail when irritated by its high temperature.

The study was conducted on a German-made Tail Flick apparatus. The device is a latch for the animal with a gutter for the tail. An incandescent lamp with a focusing lens is installed above the gutter. The lamp turns on at the touch of a button, and turns off when the animal pulls its tail. The device measures the time the button is pressed 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 withdrawal increases. Since animals differ in individual pain sensitivity, two measurements are made on each animal - before and after the introduction of the substance. The threshold value of complete analgesia is the exposure time longer than 10 seconds (tail tissue begins to break down). The study was performed on male rats weighing 160-180 g, 6 animals per dose.

The Hot Plate 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 regulated by software. The area is closed by a transparent fence 40 cm high. The temperature of the metal area is set at 52 ° C. When placing the animal on the platform, the time is measured until the animal lays its paws. With the introduction of analgesics, the time before licking of the legs increases.

Studies have shown the presence of pronounced analgesic activity in the claimed drug compared with control animals receiving placebo and the comparison drug morphine. The effect of complete analgesia was detected with a peptide analgesic dose of 5 mg / kg. The effectiveness of analgesia in the claimed drug in a dose of 0.1 mg / kg corresponds to that for 5 mg / kg of morphine. The performed work confirms the obtained data on a significantly higher activity of the drug compared to morphine, approximately 10-20 times.

Example 8. Investigation of the effect of the injection agent based on the tetrapeptide (according to example 2) H-Tyr-D-Arg-Phe-Gly-NH ₂ on the behavioral reactions of animals, the study of the toxicity of large doses in the test "Open field" and the test to maintain equilibrium on a horizontal bar.

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

Investigated doses and routes of administration:

1 - control without injection

2 - injection of 300 μl of physiological saline (stress control)

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

4 - agent 1.5 mg / kg

5 - agent 5 mg / kg

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

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

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

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

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

Substances were injected intraperitoneally with a syringe in equal weight quantities to laboratory animals of the experimental and control groups, respectively. The substance was administered 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 minutes after the administration of the substance.

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

Open Field Test

An animal was released into a round, not brightly lit arena. The floor is laid out on squares with a side of 10 cm. The intersection of the side of the square with animals during movement is a conditional unit by which locomotor activity of mice is measured. Mouse movement in the center of the arena and on the periphery is recorded separately. The number of stands on the hind legs of the “rack” and peeping into holes in the “mink” floor was recorded. Observation was carried out visually with simultaneous data entry into the computer via the keyboard (semi-automatic registration).

Horizontal Balance Test

The animals were placed on a wooden rod with a diameter of 2 cm and a length of 40 cm, located horizontally at a height of at least 60 cm. The equilibrium retention time on the rod was recorded.

Processing of the results was carried out using a one-way ANOVA analysis of variance.

As studies have shown, large doses of the drug do not lead to a significant loss of orientation of the animals or to a decrease in behavioral activity. Even at a dose of 100 mg / kg, mouse products continued to actively move around the field, albeit with slightly reduced activity (by 20-25% at a dose of 20-100 mg / kg). By 20-30% there is a decrease in research activity (indicator "stand-mink").

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, the heartbeat remains stable.

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

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

Thus, the drug in the proposed dosage form slightly affects the behavioral characteristics of animals. The use of the drug in doses of 200 EC ₅₀ does not lead to toxic effects.

The above examples demonstrate that the preparation of the tetrapeptide H-Tur-D-Arg-Phe-Gly-NH ₂ has analgesic activity significantly superior to morphine. Moreover, unlike morphine H-Tyr-D-Arg-Phe-Gly-NH ₂ does not cause toxic effects and pronounced psychophysiological and behavioral disorders.

Under the conditions ensuring the stability of the peptide during storage, the 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 danger of overdose and allows with high safety to prescribe doses of the drug, corresponding to the severity of the pain syndrome.

By extrapolation, it was determined that the recommended dose for a person would be a range of 0.5-10 mg / dose, depending on the particular case of the 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 prototype of the drug in patients in the mode of information consent. The prototype of the drug was used in cases where the patient was shown treatment with morphine group painkillers (stage 3 according to the WHO system), but for one reason or another the drug was not available. When a regular morphine drug appeared, therapy with an experimental drug prototype was discontinued.

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

The following parameters were recorded:

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

The dynamics of pain 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 administration.

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

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

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

Patient 1. Male 48 years old.

Pancreatic cancer stage IV, metastases. Pain 6-9 on a scale. Prescribed narcotic analgesics.

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

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

Subjective sensations: during the first injections, a very mild dysphoric effect is felt for 10-15 minutes, then the effect passes. Violations of consciousness, activity were not detected. The patient takes care of himself.

The degree of anesthesia is 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, is unable to look after herself, is unable to maintain a normal conversation, practically does not sleep.

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

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

Subjective sensations: the intensity of the pain was significantly reduced, the ability to communicate was restored, the patient supported the conversation, was able to get up and walk, slept normally at night. No negative effects were detected.

The degree of anesthesia is 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. Prescribed narcotic analgesics.

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

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

Subjective sensations: The intensity of the pain is significantly reduced, the patient supports the conversation, was able to get up and walk. No negative effects were detected.

The degree of anesthesia is 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. Prescribed narcotic analgesics.

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

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

Subjective sensations: There is no euphoria and dysphoria, the intensity of pain is significantly reduced, the patient supports the conversation. No negative effects were detected.

The degree of anesthesia is 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. Prescribed narcotic analgesics.

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

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

Subjective sensations: There is no euphoria and dysphoria, the intensity of pain is significantly reduced, the patient supports the conversation. No negative effects were detected.

The degree of anesthesia is 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 the vertebra in the lumbar region. Pain 7-8 on a scale. It cannot move independently.

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

The development time of the anesthetic effect 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 were detected.

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

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

Common to all these 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, impaired heartbeat were found. Patients suffering from severe pain after using the prototype of the drug were able to communicate normally with their relatives.

Claims (44)

1. A tool for injectable and / or nasal administration based on a tetrapeptide of the sequence H-Tyr-D-Arg-Phe-Gly-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH, for the prevention and / or treatment of acute or chronic pain syndrome which contains 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 auxiliary substance, in the following ratio of components, wt.%: tetrapeptide sequence H-Tyr-D-Arg-Phe-Gly-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH  0.01-0.5 excipient up to 100 2. The tool according to p. 1, characterized in that the tool contains at least one excipient selected from the group comprising stabilizers, prolongators, buffering additives, emulsifiers - solubilizers, solvents, fillers, preservatives and other excipients permitted for medical use. 3. The tool according to claim 2, characterized in that the stabilizer contains at least one substance selected from the group consisting of 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 tool according to claim 2, characterized in that at least one substance selected from the group consisting of polyvinylpyrrolidone with a molecular weight of 10-60 kDa, dextran with a molecular weight of 10-100 kDa, for example, polyglucin or reopoliglukin , polyvinyl alcohol, sodium carboxymethyl cellulose. 5. The tool according to p. 2, characterized in that as a buffering additive is at least one substance selected from the group consisting of sodium chloride, sodium / potassium hydro- and / or dihydrogen phosphate, sodium or ammonium acetate. 6. The tool according to p. 2, characterized in that as an emulsifier - solubilizer - at least one substance selected from the group comprising soya lecithin for injection, polysorbate-20, polysorbate-60, polysorbate-80, span-20, Span-40, Span-60, Span-85, sodium dodecyl sulfate. 7. The tool according to claim 2, characterized in that the solvent is at least one substance selected from the group comprising water for injection, sterile saline, olive oil, peach oil, sunflower oil. 8. The tool according to claim 2, characterized in that at least one substance selected from the group consisting of sorbitol, mannitol, mannitol, xylitol, lactose, sucrose, dextrose, a copolymer of lactic and glycolic acids is used as a filler. 9. The agent according to claim 2, characterized in that 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 ; nipazole, benzalkonium chloride or bromide, benzethonium chloride, cetylpyridinium chloride, dimethyldodecylbenzylammonium chloride, methyl paraben, propyl paraben. 10. The tool according to paragraphs. 1-9, characterized in that the tool can be made in the form of a dry powder, in liquid form in the form of a solution for intramuscular or intravenous administration, subcutaneous or intradermal administration, or a solution for infusion, a solution for nasal administration. 11. The tool according to p. 10, characterized in that the tool in liquid form should have a pH from 4 to 8, preferably 4.5-5.5. 12. The tool according to p. 10, characterized in that the liquid dosage form for injection contains, wt.%: Buffer 0.01-0.2 Filler 0-6 Stabilizer 0-4 Substance 0.01-0.5 tetrapeptide Solvent Up to 100 13. The tool according to p. 10, characterized in that the powder form contains (composition after dilution with a solvent of up to 1 ml in mg / ml): Buffer 0.01-0.2 Filler 0-6 Prolongator 0-8 Stabilizer 0-4 Substance 0.01-0.5 tetrapeptide 14. The tool according to p. 10, characterized in that the liquid form for nasal administration contains, wt.%: Buffer 0.01-0.2 Filler 0-1.5 stabilizer 0-1.5 Substance 0.01-0.5 tetrapeptide Preservative 0-0.5 Solvent Up to 100 15. The tool according to p. 12, characterized in that the liquid dosage form for injection is preferably contains, wt.%: Sodium Acetate 0.04 Sodium Chlorine 0.5 Mannitol 0.5 Glycine 0.5 Peptide 0.15 Water for injections up to 100 pH up to 4.7 16. The tool according to p. 13, characterized in that the powder form preferably contains (composition after dilution with a solvent of up 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 17. The tool according to p. 14, characterized in that the liquid form for nasal administration preferably contains, wt.%: Sodium Acetate 0.04 Sodium Chlorine 0.5 Mannitol 0.5 Glycine 0.5 Tetrapeptide 0.15 Metacresol 0.1 Water for injections up to 100 pH up to 4.7 18. A method of preventing and / or treating an acute or chronic pain syndrome, comprising administering an agent according to any one of the preceding claims in a therapeutically effective amount. 19. The method according to p. 18, characterized in that the tool is administered in an amount of 0.5-10 mg / dose. 20. The method of preparation of funds for PP. 1-17, comprising mixing a tetrapeptide of the sequence H-Tyr-D-Arg-Phe-Gly-NH ₂ or H-Tyr-D-Arg-Phe-Sar-OH with at least one suitable adjuvant. 21. The method according to p. 20, characterized in that, if necessary, sterilize the product; pouring the solution into ampoules, vials or containers for infusion solutions; drying solutions in a sublimation unit; sealing ampoules or corking finished product bottles. 22. The method according to p. 20, characterized in that at least one excipient is selected from the group comprising stabilizers, prolongers, buffering additives, emulsifiers - solubilizers, solvents, fillers, preservatives and other auxiliary substances that are approved for medical use. 23. The method according to p. 22, characterized in that at least one substance selected from the group consisting of Trilon B, sodium metabisulfite, sodium thiosulfate, glycine, arginine, histidine, lysine or their physiologically acceptable salts, for example, is used as a stabilizer. such as hydrochloride, sulfate, acetate, glutamate, aspartate and maleate. 24. The method according to p. 22, characterized in that at least one substance selected from the group consisting of polyvinylpyrrolidone with a molecular weight of 10-60 kDa, dextran with a molecular weight of 10-100 kDa, for example, polyglucin or reopoliglukin , polyvinyl alcohol, sodium carboxymethyl cellulose. 25. The method according to p. 22, characterized in that as a buffering additive is at least one substance selected from the group comprising sodium chloride, sodium / potassium hydro- and / or dihydrogen phosphate, sodium or ammonium acetate. 26. The method according to p. 22, characterized in that as an emulsifier - solubilizer - at least one substance selected from the group comprising soya lecithin for injection, polysorbate-20, polysorbate-60, polysorbate-80, span-20, Span-40, Span-60, Span-85, sodium dodecyl sulfate. 27. The method according to p. 22, characterized in that the solvent is at least one substance selected from the group comprising water for injection, sterile saline, olive oil, peach oil, sunflower oil. 28. The method according to p. 22, characterized in that at least one substance selected from the group consisting of sorbitol, mannitol, mannitol, xylitol, lactose, sucrose, dextrose, a copolymer of lactic and glycolic acids is used as a filler. 29. The method according to p. 22, characterized in that 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 ; nipazole, benzalkonium chloride or bromide, benzethonium chloride, cetylpyridinium chloride, dimethyldodecylbenzylammonium chloride, methyl paraben, propyl paraben. 30. The method according to p. 21, characterized in that the tool in the preparation process can be performed in the form of a dry powder, in liquid form, in the form of a solution for intramuscular or intravenous administration, subcutaneous or intradermal administration or a solution for infusion, a solution for nasal administration . 31. The method according to p. 30, characterized in that the prepared product in liquid form should have a pH from 4 to 8, preferably 4.5-5.5.