RU2367467C2 - Peptide pharmaceutical composition normalising urination, and method of application thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medical agents normalising urination, and can be used as means for correction of dysuric disorders of various geneses, including caused by infectious-inflammatory and neuroendocrinological factors. There is declared pharmaceutical composition normalising urination, containing as a reactant, peptide glutamyl- aspartic acid in effective amount of general formula: H-Glu-Asp-OH of a sequences 1 [SEQ ID NO:1] and a pharmaceutically acceptable carrier. There is declared agent containing peptide- aspartic acid of general formula: H-Glu-Asp-OH of a sequence 1 [SEQ ID NO:1] showing biological activity with regard to normalising urination. There is disclosed method of detrusor-sphincter continence consisting in introduction to the patient of the pharmaceutical composition containing as a reactant, peptide glutamyl- aspartic acid in effective amount of general formula: H-Glu-Asp-OH of a sequence 1 [SEQ ID NO:1].

EFFECT: enabled normalising of urination by detrusor-sphincter continence.

5 cl, 6 ex, 3 tbl, 2 dwg

Description

The invention relates to drugs that normalize urination, and can be used as a means for the correction of dysuric disorders of various origins, including due to exposure to infectious-inflammatory and neuroendocrine factors.

The most common manifestations of urination disorders are difficulty and delay in urination, frequent urination, frequent and imperative urges to urinate, involuntary urination and urinary incontinence, which significantly worsen people's quality of life and create great psychological problems. Any type of urination dysfunction is determined by abnormal changes in the anatomical and functional safety of the urinary tract, especially the bladder and its sphincters, prostate gland, and also the nervous structures that regulate the urination act.

For medical treatment of urination disorders, drugs of various pharmacological groups are known, among which α-adrenergic blockers, such as alfuzosin, doxazosin, tamsulosin and terazosin, are mainly used (Encyclopedia of Medicines. - M .: Publishing House LLC RLS-2004. - P.68, 314, 827, 839); anticholinesterase drugs, such as ubretide (Encyclopedia of drugs. - M .: Publishing house LLC “RLS-2004” - S.887), Kalimin (Medicinal products in Russia: Vidal Handbook. - M.: Publishing house ZAO AstraFarmServis, 2003. - S.451); 5-α reductase inhibitors, for example finasteride (Encyclopedia of drugs. - M.: Publishing House LLC “RLS-2004. - P.919); as well as phytotherapeutic agents, such as Serenoa repens lipidosterol extract, extracts of the root Pygeum africanum, Hypoxis rooperi, Cucurbita pero, Populus tremula, Echinacea purpurea and Secale cereale (Alyaev Yu.G., Grigoryan V.A., Hajiyeva Z.K. Ras. urination. - M .: Publishing house Litterra, 2006. - P.208).

However, it should be noted that the above medicines to one extent have either side effects or low efficiency.

For the medical treatment of dysuric disorders in women, therapy based on the use of estrogen drugs is used. Estrogen has been found to be effective in increasing intraurethral pressure and altering the structure of the mucous membrane and blood vessels. It should be noted that the side effects observed during estrogen treatment are well known.

Known compositions comprising peptides used for the medical treatment of dysuric disorders (WO 9933482, publ. 08/07/1999; WO 9803189, publ. 01/29/1999 and WO 2004004701, publ. 15.01.2004). It should be noted that the known means allow to solve this problem only in a limited number of cases (urinary incontinence). Due to the fact that the treatment of dysuric disorders is a difficult and largely unresolved task, the development of effective drugs, including compounds with biological activity, manifested in the regulation of the detrusor-sphincter system of the bladder, remains urgent.

A peptide of the formula H-Glu-Asp-OH of various configurations is known under registration numbers RN 3918-84-1 and RN 3914-66-7 (STN int. BD Registry Chemical abstract). Glutamyl-aspartic acid peptide (The unitcell dimensions and the space group of L-glutaminyl-L-aspagine. Pasternak R.A. et.al. March 1954-D2) is known in the art.

The present invention solves the problem of obtaining a peptide agent with biological activity, manifested in the regulation of the detrusor-sphincter system of the bladder, as well as a pharmaceutical composition containing this peptide as an active substance, the use of which normalizes the urination of a subject in need of it.

"Subject in need of this" means a mammal, for example a human (patient), having one or more manifestations of dysuric disorders, in which the use of the proposed tool is advisable from the standpoint of modern biomedical knowledge.

The technical result of the invention is to create a tool with biological activity, manifested in the normalization of urination by regulating the function of the detrusor-sphincter system of the bladder, as well as a pharmaceutical composition containing the indicated peptide as an active substance, which can be used to regulate the function of the detrusor-sphincter system Bladder.

To solve the problem and achieve the technical result, a group of inventions is proposed, united by a common inventive concept.

In one of its aspects, the present invention relates to a pharmaceutical composition that normalizes urination, containing as active ingredient an effective amount of a glutamyl-aspartic acid peptide of general formula H-Glu-Asp-OH of sequence 1 [SEQ ID NO: 1] and a pharmaceutically acceptable carrier. In this case, the pharmaceutical composition is in a form suitable for parenteral administration.

In another aspect, the present invention relates to a glutamyl-aspartic acid peptide of general formula H-Glu-Asp-OH of sequence 1 [SEQ ID NO: 1].

In another aspect, the present invention relates to an agent comprising a glutamyl-aspartic acid peptide of general formula H-Glu-Asp-OH of sequence 1 [SEQ ID NO: 1], which has a biological activity that manifests itself in normalizing urination by regulating the function of the detrusor-sphincter urinary system the bubble.

A further aspect of the present invention relates to a method for regulating the function of the detrusor-sphincter system of the bladder, which method comprises administering to the patient a pharmaceutical composition containing, as active substance, an effective amount of the glutamyl-aspartic acid peptide of the general formula H-Glu-Asp-OH sequence 1 [SEQ ID NO: 1] at a dose of 0.01 μg / kg to 100 μg / kg of body weight once daily for 10 days. In this case, the administration is carried out intramuscularly.

The glutamyl-aspartic acid peptide of the general formula H-Glu-Asp-OH is obtained by the classical method of peptide synthesis in solution.

An agent containing the glutamyl-aspartic acid peptide of the general formula H-Glu-Asp-OH exhibits biological activity expressed in normalizing urination, namely, in regulating the function of the detrusor-sphincter system of the bladder, having a normalizing effect on the nature of urination. The possibility of an objective manifestation of a technical result when using the invention is confirmed by reliable data given in the examples, which contain experimental and clinical information obtained in the process of conducting research according to the methods adopted in this field.

The study of the biological activity of the peptide H-Glu-Asp-OH was carried out according to its effect on the growth of bladder wall explants, on smooth muscle cells of the detrusor, the nature of urination, uroflow metrics in elderly and senile patients with dysuric disorders, as well as patients with chronic prostatitis.

The normalizing effect of an agent containing the peptide H-Glu-Asp-OH on the character of urination was revealed during its experimental and clinical study.

In order to obtain a pharmaceutical composition of the invention, an effective amount of the H-Glu-Asp-OH peptide as an active ingredient is mixed with a pharmaceutically acceptable carrier according to pharmaceutical compounding methods.

The term "effective amount" means the use of such an amount of active substance, which, in accordance with its quantitative indicators of activity and toxicity, as well as on the basis of specialist knowledge should be effective in this dosage form. As a carrier, various substances may be used depending on the dosage form of the preparation desired for administration to the body. For parenteral administration, saline or sterile water is usually used as a carrier.

The invention is illustrated by drawings and illustrated in table 1-3.

Figure 1 shows the effect of the peptide H-Glu-Asp-OH on the growth of bladder wall explants.

Figure 2 shows the effect of the H-Glu-Asp-OH peptide on in vitro detrusor smooth muscle cells.

Table 1 shows the effect of a pharmaceutical composition containing H-Glu-Asp-OH peptide as an active substance on the morphological and biochemical parameters of the peripheral blood of guinea pigs in the study of chronic toxicity.

Table 2 shows the effect of the peptide H-Glu-Asp-OH used in the pharmaceutical composition as an active substance on the urodynamics of patients with chronic prostatitis.

Table 3 shows the effect of the pharmaceutical composition containing the peptide H-Glu-Asp-OH as the active substance on the urodynamics of elderly and senile patients with dysuric disorders.

The invention is illustrated by an example of the preparation of a peptide by the classical method for the synthesis of the H-Glu-Asp-OH peptide in solution (Example 1), examples confirming the peptide's biological activity (examples 2, 3), an example of the study of the toxicity of the H-Glu-Asp-OH peptide used as an active substance in a pharmaceutical composition (example 4). In addition, examples of the results of clinical use of a pharmaceutical composition containing H-Glu-Asp-OH peptide as active substance (examples 5 and 6), demonstrating its pharmacological properties and confirming the possibility of achieving a therapeutic effect.

Example 1. The synthesis of the peptide H-Glu-Asp-OH

1. Name of the compound: glutamyl-aspartic acid.

2. The structural formula of H-Clu-Asp-OH

3. Gross formula without counterion: C ₉ H ₁₄ N ₂ O ₇ .

4. Molecular weight without counterion: 262.21.

5. Counterion: acetate.

6. Appearance: odorless white amorphous powder.

7. Synthesis method: the peptide is obtained by the classical method of synthesis in solution according to the scheme:

BOC - tert-butyloxycarbonyl group,

OSu - N-oxysuccinimide ester,

DCC-N'N'-dicyclohexylcarbodiimide,

OBzl - benzyl ether,

TFA - trifluoroacetic acid,

8. Characteristics of the finished product:

- content of the main substance: 98.33% (by HPLC, 220 nm);

- TLC - individual, R _f = 0.24 (acetonitrile-water 2: 1).

- moisture content: 6%.

- pH 0.01% solution: 4.3.

- Specific optical rotation: [α] D ²² : + 31 ° (c = 1, H ₂ O), "Polamat A", Carl Zeiss Jena.

Synthesis Example:

1) BOC-Glu (OBzl) -OSu, N-tert-butyloxycarbonyl- (γ-benzyl) glutamic acid N-oxysuccinimide ester (I)

N-tert-butyloxycarbonyl- (γ-benzyl) glutamic acid BOC-Glu (OBzl) -OH (33.7 g, 0.1 mol) was dissolved in 50 ml of N, N'-dimethylformamide, cooled to minus 10 ° С , solutions of N, N'-dicyclohexylcarbodiimide (23.0 g, 0.11 mol) in 30 ml of N, N'-dimethylformamide and N-hydroxysuccinimide (13.0 g, 0) cooled to a temperature of 4-6 ° C were added , 11 mol) in 20 ml of N, N'-dimethylformamide. The reaction mixture was stirred for 12 hours while cooling with ice and then for one day at room temperature. The precipitated N, N'-dicyclohexylurea was filtered off and the resulting activated ester solution was used without isolation in the next step.

2) BOC-Glu (OBzl) -Asp (OBzl) -OH, N-tert.butyloxycarbonyl- (γ-benzyl) glutamyl- (β-benzyl) aspartate (II)

(β-Benzyl) aspartic acid H-Asp (OBzl) -OH (28.0 g, 0.12 mol) and 36 ml (0.12 mol) of triethylamine were suspended in 50 ml of N, N'-dimethylformamide and stirred for 1 h. Then, a solution of the activated BOC-Glu (OBzl) -OSu (I) ester obtained in the previous step was added in portions. The reaction mixture was stirred at room temperature for 2 days. Then acidified with 0.5 N. a solution of sulfuric acid to pH 2-3 and was extracted with ethyl acetate 4 × 50 ml. Hoods were combined and washed sequentially with 0.5 N. a solution of H ₂ SO ₄ 3 × 50 ml, water 2 × 50 ml, 5% NaHCO ₃ 2 × 50 ml, water 2 × 50 ml, saturated NaCl 2 × 50 ml. The organic layer was dried over Na ₂ SO ₄ , the solvent was evaporated in vacuo, the residue was crystallized under hexane. Received 50 g of the product (92%). R _f = 0.34 (benzene-acetone 2: 1).

3) H-Glu-Asp-OH (III), glutamyl-aspartic acid

The protected dipeptide BOC-Glu (OBzl) -Asp (OBzl) -OH (II) (5.4 g) (0.01 mol) was dissolved in a mixture of methyl alcohol - water (4: 1) (50 ml) and hydrogenated over the catalyst Pd / C (5%) for 4 hours. The catalyst was filtered off, the solvent was evaporated in vacuo, and the residue was dried in vacuo over KOH and P ₂ O ₅ . Next, the product was dissolved in 25 ml of a mixture of methylene chloride-trifluoroacetic acid (5: 1) and kept at room temperature for 2 hours. The completion of the release reaction was monitored by TLC in an acetonitrile-water system (1: 3). The solvent was evaporated in vacuo, the residue was dried in vacuo over KOH.

For purification, 320 mg of the preparation was dissolved in 4 ml of a 0.01% trifluoroacetic acid solution and subjected to high performance liquid chromatography on a 50 x 250 mm reverse phase column Diasorb-130-C16T, 7mkm. Chromatograph Beckman System Gold, 126 Solvent Module, 168 Diode Array Detector Module. Chromatography conditions - A: 0.1% TFA; B: MeCN / 0.1% TFA, gradient B 0 → 50% in 100 min. The sample volume is 5 ml, detection at a wavelength of 215 nm, scanning at wavelengths of 190-600 nm, a flow rate of 10 ml / min. The main peak fraction was taken. The solvent was evaporated in vacuo at a temperature not exceeding 40 ° С, evaporation was repeated (5 times) with 10 ml of 10% acetic acid solution.

Finally, the residue was dissolved in 20 ml of deionized water and lyophilized.

Received 250 mg of a purified preparation in the form of an odorless white amorphous powder.

4) Analysis of the finished product

- The content of the basic substance was determined by HPLC on a Phenomenex C 18 LUNA column 4.6 × 150 mm. A: 0.1% TFA; B: MeCN; grad.B 0-100% in 10 min. Flow rate 1 ml / min. Detection at a wavelength of 220 nm, scanning at wavelengths of 190-600 nm, a sample of 20µl. The content of the main substance is 98.33%.

- TLC: individual, R _f = 0.24 (acetonitrile-water 2: 1, Sorbfil PTCX-P-B-UV plates, CTX-1 VE silica gel 8-12 μm, manifestation of chlorine / benzidine).

- Moisture content: 6% (gravimetrically by weight loss during drying 20 mg at a temperature of 100 ° C).

- pH 0.01% solution: 4.3 (potentiometric).

- Specific optical rotation: [α] D ²² : + 31 ° (c = 1, H ₂ O), "Polamat A", Carl Zeiss Jena.

Example 2. The effect of the peptide H-Glu-Asp-OH on the development of bladder wall explants

The experiments were performed on 26 fragments of the bladder wall of Wistar rats weighing 150-200 g. The culture medium for explant cultivation consisted of 35% Needle solution, 25% fetal calf serum, 35% Hanks solution, 5% chicken embryonic extract, glucose (0.6%), insulin (0.5 units / ml), penicillin (100 units / ml), glutamine (2 mM) were added to the medium. Bladder wall fragments were placed in this medium and cultured in Petri dishes in an incubator at a temperature of 36.7 ± 0.2 ° C for 2 days. In order to determine the effective concentration, the peptide H-Glu-Asp-OH was added to the experimental medium at concentrations of 1, 10, 100, and 200 ng / ml. The criterion of biological activity was the area index (PI), the ratio of the area of the entire explant along with the growth zone to the outgoing area of the bladder wall fragment. The IP values were expressed as a percentage, the control IP value was taken as 100%.

Figure 1 shows the effect of the peptide H-Glu-Asp-OH on the development of bladder wall explants.

It was found that after 1 day of cultivation, the explants spread on a collagen substrate and the proliferation of proliferating and migrating cells began on the periphery of the explant. On the 3rd day of cultivation at a concentration of the peptide H-Glu-Asp-OH of 100 ng / ml, a significant increase in the explant PI by 25% was observed compared with the control PI values (Fig. 1). When studying bladder wall explants for longer periods of cultivation (7 days), a similar stimulating effect of the peptide H-Glu-Asp-OH at the same concentration was revealed.

Thus, with respect to the tissue of the wall of the bladder, the peptide H-Glu-Asp-OH had a tissue-specific effect, which manifests itself in stimulating the growth of explants.

Example 3. The effect of the peptide H-Glu-Asp-OH on smooth muscle cells of the bladder detrusor in vitro

The experiments were carried out on a strip of rat bladder detrusor mounted between a fixed rod and a force transducer placed in a special bath with Krebs solution. H-Glu-Asp-OH peptide was added to the solution at concentrations of 1, 5, 10, and 20 μg / ml, and then the authoristic contractile activity of the bladder strip was recorded.

Figure 2 shows the effect of the peptide H-Glu-Asp-OH on the strain of the rat bladder detrusor strip in vitro.

It was found that under the action of the peptide H-Glu-Asp-OH, a dose-dependent change in the phase-tonic contractions of the smooth muscle cells of the bladder detrusor occurs (Fig. 2).

The maximum tension developed by the muscle in response to the action of the peptide H-Glu-Asp-OH increased as its concentration in the perfusate increased to 100 μg / ml. So, if in the control solution the maximum voltage was 91.6 g / cm ² , then under the action of the peptide H-Glu-Asp-OH at a concentration of 100 μg / ml, the value of the indicator increased to 241.2 g / cm ² (figure 2) .

Example 4. The study of the toxicity of the peptide H-Glu-Asp-OH

The general toxic effect of the H-Glu-Asp-OH peptide was studied in accordance with the requirements of the Guidelines for the Experimental (Preclinical) Study of New Pharmacological Substances (2005): acute toxicity with a single administration of the drug, as well as subacute and chronic toxicity with prolonged administration of the peptide.

A study of acute toxicity was conducted on 60 white outbred male mice weighing 18-20 g. Animals were randomly divided into 6 equal groups. The drug was administered to animals once intramuscularly at doses of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg in 0.25 ml of sterile 0.9% NaCl solution. The control group animals were injected with the same volume in a sterile 0.9% NaCl solution.

Studies on the study of subacute toxicity were carried out on 50 white outbred male rats weighing 180-220 g. Daily, once a day, animals of the experimental groups were injected intramuscularly with doses of 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg in 0.5 ml of a sterile 0.9% NaCl solution. The animals of the control group were injected in the same volume with a sterile 0.9% NaCl solution. Before administration of the drug, on the 30, 60 and 90 days after the start of drug administration, the morphological composition and properties of peripheral blood were studied in animals. At the end of the experiment, biochemical and coagulological blood parameters were examined.

Studies of chronic toxicity were carried out for 6 months, based on the duration of the recommended clinical purpose of the drug, in 76 male guinea pigs with a body weight of 290-320 g. Animals of the experimental groups received daily intramuscularly a peptide for 6 months in doses of 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg in 0.5 ml of a sterile 0.9% NaCl solution. In the control group, animals were injected in a similar fashion with a sterile 0.9% NaCl solution in the same volume. In animals, peripheral blood was determined by conventional methods: the number of red blood cells, hemoglobin, reticulocytes, platelets, white blood cells, white blood cell count, erythrocyte sedimentation rate (ESR), and erythrocyte resistance. Along with this, the content of total protein in blood serum was determined by the method of Lowry, potassium and sodium by plasma spectrophotometry. After the experiment was completed, a pathomorphological study of the brain and spinal cord, spinal ganglia, thyroid gland, parathyroid glands, adrenal glands, testes, pituitary gland, heart, lungs, aorta, liver, kidney, bladder, pancreas, stomach, small intestine, colon, thymus, spleen, lymph nodes, bone marrow.

When studying acute toxicity, it was found that a single administration of the studied peptide to animals at a dose exceeding the therapeutic recommended for clinical use by more than 5000 times does not cause toxic reactions, which indicates a large therapeutic breadth of the drug.

The study of subacute and chronic peptide toxicity indicates the absence of side effects with prolonged use of the drug in doses exceeding the therapeutic by 100-1000 times. In the study, there was no significant effect of the studied peptide on morphological, biochemical parameters in the peripheral blood, as well as on ESR and erythrocyte resistance (Table 1).

Table 1 shows the effect of the peptide H-Glu-Asp-OH on the morphological and biochemical parameters of the peripheral blood of guinea pigs in the study of chronic toxicity.

When assessing the general condition of animals, morphological and biochemical parameters of peripheral blood, the morphological state of internal organs, the state of the cardiovascular and respiratory systems, and liver and kidney function, pathological changes in the body were not detected.

The absence of a general toxic effect allows us to recommend a pharmaceutical composition containing the peptide H-Glu-Asp-OH as an active substance for clinical trials.

Example 5. The effectiveness of the pharmaceutical composition containing the peptide H-Glu-Asp-OH, in patients with chronic prostatitis

The study involved 56 patients with chronic prostatitis in the acute stage at the age of 24 to 45 years. The main manifestations of the disease were aching pains of typical localization, various disorders of urination (pain during urination, rapid and / or difficult urination), disorders of the ability to have sexual intercourse and signs of a neurotic condition.

All patients were examined according to the following diagnostic algorithm: digital rectal examination; transrectal ultrasound of the prostate; suprapubic ultrasound of the bladder to determine residual urine; general analysis of urine and blood; microscopy of prostate secretion; bacteriological examination of secretions and urine; calculation of the symptom index of chronic prostatitis - NIH - CPSI, 1999; determination of uroflowmetric index (urination rate). The listed parameters were determined in all patients before and after treatment.

Prior to prescribing antibacterial drugs, all patients underwent PCR diagnostics to detect sexually transmitted flora. Patients with a specific infection (chlamydia, ureoplasma, mycoplasma, trichomonads, etc.) were not included in this clinical trial.

Patients by randomization were divided into 2 groups. The control group consisted of 20 people who received conventional treatment, including antibacterial drugs (antibiotics), antispasmodics (no-spa, baralgin), metabolic stimulants (raveron), drugs that improve blood microcirculation (trental, escusan).

36 patients of the main group were divided into 3 subgroups depending on the severity of dysuric disorders.

Patients who noted the most pronounced urination disorders in the form of pain and increased urination up to 15-20 times, especially at night (up to 10 times), in addition to the above conventional treatment, were prescribed an intramuscular pharmaceutical composition containing H-Glu peptide as an active substance -Asp-OH, at a dose of 5 mg in 1.0 ml of physiological saline once daily for 10 days.

With a moderate severity of dysuric disorders (moderate soreness and increased urination up to 10 times a day), patients received, in addition to conventional means, an intramuscular pharmaceutical composition containing H-Glu-Asp-OH peptide as an active ingredient in a dose of 100 μg in 1.0 ml of physiological saline once daily for 10 days.

Patients who noted moderate urinary disorders, expressed in the frequency of nighttime urination up to 5 times, were prescribed, in addition to conventional treatment, an intramuscular pharmaceutical composition containing H-Glu-Asp-OH peptide as an active ingredient, at a dose of 1 μg in 1.0 ml saline once daily for 10 days.

When comparing the performance of patients in the control and the main groups, more pronounced positive dynamics was noted in the group of patients receiving a pharmaceutical composition containing H-Glu-Asp-OH peptide as an active ingredient, as a component of complex therapy.

When assessing the dynamics of subjective indicators, all patients who received, in addition to conventional means, a pharmaceutical composition containing H-Glu-Asp-OH peptide as an active substance, rated their condition as "improved." According to the scale of symptoms, this corresponds to the positions “good” - 72% and “satisfactory” - 28%. There were no patients who did not notice improvement in the main group.

At the same time, out of 20 patients in the control group, only 38% considered their condition “satisfactory”, 32% of patients noted a good effect, and 30% of the patients in the control group noted “no obvious improvement” when evaluating the effect of treatment.

At the end of treatment using a pharmaceutical composition containing the peptide H-Glu-Asp-OH as the active ingredient, pain during urination completely disappeared in 62.0% of the patients of the main group and significantly decreased in 34.7% of patients who complained ( in the control group, 46.2% and 32.4%, respectively). In 3.3% of patients, treatment did not produce the expected effect, and pain dynamics were not observed (in the control group, 21.4% of patients).

Pollakiuria (increased urination) completely ceased to bother 82.3% of patients in the main group, in addition, they also disappeared the need for nightly urination.

Stranguria (difficulty urinating) ceased to bother 74.8% of patients in the main group, 15.9% noted a marked increase in urine flow and relief of urination.

It should be noted that patients of the control group of pollakiuria and stranguria, especially the need for nocturnal urination, continued to be disturbed even after the end of the course of treatment of chronic prostatitis with conventional means.

The data presented in table 2, which shows the effect of the pharmaceutical composition containing the peptide H-Glu-Asp-OH as the active substance on the urodynamics of patients with chronic prostatitis, allow us to draw the following conclusions.

In patients of the control group receiving conventional therapy, the uroflowmetric index remained almost unchanged after treatment.

In the group of patients of the main group who received a pharmaceutical composition containing H-Glu-Asp-OH peptide as an active ingredient as a component of complex therapy, the uroflowmetric index increased almost 2 times, which confirms the positive effect of the peptide as an active substance on the nature of urination.

It should be noted that uroflograms recorded after treatment in patients with stage I and II chronic prostatitis showed restoration of the main parameters of urination to normal values under the influence of a pharmaceutical composition containing H-Glu-Asp-OH peptide as an active substance in a dose of 1 μg or 100 μg in 1 ml of physiological saline, respectively (Table 2). In stage III disease, the complete normalization of urination indices was prevented by a decrease in the elasticity of the neck of the bladder due to sclerotic changes in the tissue of the prostate gland, but nevertheless, in these patients who received a pharmaceutical composition containing H-Glu-Asp-OH peptide as an active substance, dose of 5 mg in 1 ml of saline, there was a noticeable increase in the stream of urine and a decrease in the need for night urination up to 3-4 times.

Thus, the results of the study indicate the therapeutic efficacy of the pharmaceutical composition containing the active substance peptide H-Glu-Asp-OH at a dose of 5 mg, 100 μg and 1 μg (depending on the severity of urination disorders) in 1.0 ml physiological saline once daily for 10 days and the appropriateness of its use in the complex treatment of inflammatory diseases of the prostate, accompanied by dysuric disorders.

Example 6. The effectiveness of the pharmaceutical composition containing the peptide H-Glu-Asp-OH, in elderly patients with dysuric disorders

The study involved 67 patients suffering from impaired urination, including 43 men aged 68 to 82 years and 24 women aged 58 to 84 years. All elderly and senile patients complained of frequent urination up to 10-20 times a day, especially from night hours (up to 5-10 times), which significantly reduced the quality of life of older people and made their social adaptation difficult.

The effectiveness of treatment was evaluated based on the dynamics of complaints of patients and indicators of urodynamics. Determined the maximum, average speed and time of urination, the time to reach the maximum speed of urination, evaluated uroflowmetric index.

Patients by randomization were divided into 2 groups. The control group consisted of 25 people (17 men, 8 women) who received conventional treatment, including antispasmodic drugs (spazmeks, spazmol, Neo spazmoveralgin) and anticholinergic (detrusitol) action.

Of the main group, 42 patients (26 men, 16 women) were divided into 3 subgroups depending on the severity of urination disorders. Patients who noted the most pronounced dysuric disorders in the form of increased urination up to 15-20 times a day, especially at night (up to 10 times), were prescribed an intramuscular pharmaceutical composition containing H-Glu-Asp-OH peptide as an active ingredient in a dose 5 mg in 1.0 ml of saline once daily for 10 days.

With a moderate severity of dysuric disorders (increased urination up to 10 times a day, including up to 5 times at night), patients received an intramuscular pharmaceutical composition containing H-Glu-Asp-OH peptide as an active substance at a dose of 100 μg 1.0 ml of saline once daily for 10 days. Patients who noted moderate urinary disorders, expressed in the frequency of urination at night up to 5 times, were prescribed intramuscularly a pharmaceutical composition containing H-Glu-Asp-OH peptide as an active ingredient, at a dose of 1 μg in 1.0 ml of physiological saline once daily within 10 days.

When comparing the performance of patients in the control and the main groups, pronounced positive dynamics was noted in the group of patients receiving a pharmaceutical composition containing the peptide H-Glu-Asp-OH as the active substance.

When assessing the dynamics of subjective indicators, all patients who received a pharmaceutical composition containing H-Glu-Asp-OH peptide as an active ingredient rated their condition as "improved." Pollakiuria (increased urination) completely ceased to bother 86.8% of patients in the main group, in addition, they also disappeared the need for nightly urination. In 13.2% of patients of the main group, pollakiuria significantly decreased to 7-8 times a day, and the need for night urination was reduced to 2-3 times.

The data given in Table 3, which shows the effect of the pharmaceutical composition containing the peptide H-Glu-Asp-OH as the active substance on the urodynamic state in elderly and senile patients with dysuric disorders, allow us to draw the following conclusions.

It should be noted that in the patients of the control group there were no positive dynamics of the bladder function both in subjective and objective indicators, despite the use of generally accepted means.

In patients of the control group receiving conventional therapy, the uroflowmetric index remained almost unchanged after treatment.

In the group of patients of the main group receiving a pharmaceutical composition containing H-Glu-Asp-OH peptide as an active ingredient, the uroflowmetric index increased almost 2 times, which confirms the positive effect of the peptide as an active substance on the nature of urination.

It should be noted that uroflograms recorded after treatment in elderly and senile patients with moderate dysuric disorders showed the restoration of the main parameters of urination to normal values under the influence of a pharmaceutical composition containing H-Glu-Asp-OH peptide as an active substance in a dose of 1 mcg or 100 mcg in 1 ml of saline, respectively.

In patients of senile age with severe manifestations of dysuric disorders, the full normalization of urination indices was hindered by an age-related decrease in the elasticity of the bladder neck, but nevertheless, in these patients who received a pharmaceutical composition containing H-Glu-Asp-OH peptide as an active ingredient in a dose of 5 mg in 1 ml of physiological solution, there was a noticeable increase in the stream of urine and a decrease in the need for daily urination up to 7-8 times, and at night - up to 2-3 times.

Thus, the results of the study indicate the therapeutic efficacy of the pharmaceutical composition containing H-Glu-Asp-OH peptide in doses of 5 mg, 100 μg and 1 μg (depending on the severity of dysuric disorders) in 1.0 ml saline once daily for 10 days and the appropriateness of its use in the treatment of patients with dysuric disorders of various origins.

It should also be noted that examples of the clinical use of a pharmaceutical composition containing H-Glu-Asp-OH peptide as an active substance in patients with dysuric disorders of various origins, clearly confirm that, in contrast to the agents known from the prior art, the proposed pharmaceutical composition is characterized by the ability to its introduction to provide improved activity against the detrusor-sphincter system of the bladder at low concentrations of the active substance, as well as the absence m side effects.

Table 1
Pharmaceutical composition based on a peptide that normalizes urination, and its use Indicators The control The introduction of the peptide H-Glu-Asp-OH for 6 months 1 mcg / kg 0.1 mg / kg 1 mg / kg Red blood cells, × 10 ¹² / l 5.3 ± 0.4 5.2 ± 0.4 5.3 ± 0.4 5.4 ± 0.6 Hemoglobin, g / l 14.1 ± 0.5 14.3 ± 0.5 14.7 ± 0.5 14.3 ± 0.9 Reticulocytes,% 1.12 ± 0.03 1.20 ± 0.06 1.15 ± 0.08 1.11 ± 0.01 Platelets × 10 ⁹ / L 143.2 ± 8.4 152.4 ± 7.5 147.3 ± 8.5 146.5 ± 10.2 White blood cells × 10 ⁹ / L 9.1 ± 0.4 10.1 ± 0.5 9.8 ± 0.4 9.6 ± 0.4 Band neutrons,% 0.42 ± 0.03 0.34 ± 0.03 0.35 ± 0.04 0.37 ± 0.08 Segmented neutrophils,% 45.3 ± 4.9 42.4 ± 3.7 45.7 ± 3.6 46.1 ± 3.9 Eosinophils,% 0.82 ± 0.04 0.54 ± 0.05 0.72 ± 0.07 0.68 ± 0.05 Basophils,% 0.81 ± 0.03 0.63 ± 0.05 0.62 ± 0.05 0.79 ± 0.06 Monocytes,% 2.52 ± 0.04 1.82 ± 0.03 1.94 ± 0.06 2.28 ± 0.03 Lymphocytes,% 46.3 ± 2.5 42.8 ± 3.9 47.4 ± 2.1 49.2 ± 1.9 ESR, mm / h 2.11 ± 0.03 1.93 ± 0.07 1.92 ± 0.06 2.17 ± 0.01 Erythrocyte resistance,% NaCl - maximum 0.42 ± 0.03 0.41 ± 0.05 0.43 ± 0.03 0.43 ± 0.02 - minimum 0.34 ± 0.03 0.27 ± 0.03 0.32 ± 0.05 0.31 ± 0.01 Total protein in serum, g / l 72.4 ± 3.9 72.5 ± 3.8 72.3 ± 3.8 72.1 ± 4.2 Sodium in serum, mmol / l 152.6 ± 7.6 152.1 ± 6.2 152.6 ± 7.1 152.3 ± 7.5 Serum potassium, mmol / l 5.2 ± 2.3 5.3 ± 2.7 5.2 + 1.4 5.3 ± 1.9

table 2
Pharmaceutical composition based on a peptide that normalizes urination, and its use Indicator Before treatment After treatment using conventional means After treatment using a pharmaceutical composition comprising an H-Glu-Asp-OH peptide Urinary retention time, min 4.8 ± 0.3 3.7 ± 0.4 2.6 ± 0.1 * # Urination count - in the daytime 8.3 ± 0.4 7.4 ± 0.5 6.1 ± 0.2 * - at night time 4.2 ± 0.3 3.7 ± 0.2 1.61 ± 0.01 * # The degree of abdominal pressure (points) 3.2 3.0 2.3 * The nature of the urine stream (points) 3.3 2.9 2.1 * Average urination rate (ml / s) 12.3 ± 1.1 14.8 ± 1.5 17.9 ± 1.4 * # Maximum urination rate (ml / s) 16.4 ± 2.7 18.8 ± 2.1 21.3 ± 2.9 Time to reach maximum urination rate (ml / s) 6.6 ± 0.2 5.8 ± 0.4 4.5 ± 0.2 * * - P <0.05 compared with the indicator before treatment; # - P <0.05 compared with the indicator in patients of the control group.

Table 3
Pharmaceutical composition based on a peptide that normalizes urination, and its use Indicator Before treatment After treatment using conventional means After treatment using a pharmaceutical composition comprising an H-Glu-Asp-OH peptide Urinary retention time (min) 5.4 ± 0.7 5.1 ± 0.6 2.8 ± 0.8 * # Urination count - in the daytime 9.8 ± 0.7 8.7 ± 0.8 6.4 ± 0.3 * # - at night time 5.8 ± 0.6 5.5 ± 0.5 1.4 ± 0.1 * # Average urination rate (ml / s) 11.6 ± 1.5 12.7 ± 1.1 17.2 ± 1.6 * # Maximum urination rate (ml / s) 14.2 ± 1.8 16.3 ± 1.7 22.6 ± 2.2 * # Time to reach maximum urination rate (ml / s) 7.4 ± 0.1 6.7 ± 0.6 5.1 ± 0.7 * * - P <0.05 compared with the indicator before treatment; # - P <0.05 compared with the indicator in patients of the control group.

Claims (5)

1. A pharmaceutical composition that normalizes urination, characterized in that the active substance contains an effective amount of a glutamyl-aspartic acid peptide of the general formula: H-Glu-Asp-OH of sequence 1 [SEQ ID NO: 1] and a pharmaceutically acceptable carrier. 2. The pharmaceutical composition according to claim 1, characterized in that it is in a form suitable for parenteral administration. 3. An agent containing a glutamyl-aspartic acid peptide of the general formula: H-Glu-Asp-OH of sequence 1 [SEQ ID NO: 1] having a biological activity that manifests itself in normalizing urination by regulating the function of the detrusor-sphincter system of the bladder. 4. A method of regulating the function of the detrusor-sphincter system of the bladder, which consists in administering to the patient a pharmaceutical composition containing as an active substance an effective amount of a glutamyl-aspartic acid peptide of the general formula: H-Glu-Asp-OH sequence 1 [SEQ ID NO: 1] at a dose of 0.01 to 100 mcg / kg body weight once daily for 10 days. 5. The method according to claim 5, characterized in that the introduction is carried out intramuscularly.

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