RU2073520C1 - Medicinal preparation and method of urogenital infection prophylaxis and treatment - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: preparation has an active agent - microbial mass of living bifidobacteria taken at effective amount and pharmaceutically acceptable additions. Preferable amount of bifidobacteria is 10³-10⁸ living cells per one dose. Bifidobacteria that can be used: strains Bifidobacterium bifidum N 1, N 791 or "ЛБА"-3. Additionally, agent can contain one or some biologically active components showing immunomodulating, regenerating or protective action at amount 0.1-100.0 mg per one dose. Other additional biologically active components can be: hyaluronic acid, lysozyme, interferons, antiviral and antimycotic substances. The special additions can be: confectionery, food or friture fat, butyrum-cacao, paraffin, Vaseline, lanolin and emulsifier taken at amount 1.5-2.2 g per one dose. Preparation can be made as suppository, ointment or cream. Method of prophylaxis and treatment of urogenital infections involves intravaginal and/or rectal administration to patient of medicinal preparation containing 10³-10⁸ living cells of bifidobacteria per one dose for 10-30 days; for prophylaxis - 1-2 doses and for treatment - 2 doses per a day. Before, simultaneously or after administration of preparation antibacterial, antimycotic or eubiotic agents can be administrated also. EFFECT: enhanced effectiveness of preparation. 9 cl, 13 tbl

Description

 The invention relates to medicine, and more specifically relates to a new drug for the prevention and treatment of urogenital infections.

 Diseases caused by urogenital infections are among the most frequent causes of morbidity in the world (Krasnopolsky V.I. Bulletin of the Russian Association of Obstetricians and Gynecologists, vol. 1. N 2, 1994, p.12). In recent years, both in the Russian Federation and abroad, there has been a tendency toward an increase in the number of diseases such as chlamydia, bacterial vaginosis, mycoplasmosis, candidiasis, ureoplasmosis, gardnerellosis, genital herpes, etc.

 In medical practice, antimicrobials are widely used to treat urogenital infections. However, these drugs are toxic, have a non-specific bactericidal effect and cause the occurrence of pathogenic microorganisms that are resistant to the action of this antibacterial agent, as a result of which antibiotic therapy requires at least restoration of the microbiocenosis of the body.

 In connection with the foregoing, the search for new drugs that are completely safe, have no contraindications and do not violate the microbiocenosis of the body, for the prevention and treatment of urogenital infections, is of particular importance.

 The closest analogue of the invention is a pharmaceutical composition comprising, as an active principle, a microbial mass of live acidophilic lactobacilli and an antimicrobial agent. as well as the growth medium and growth factors of lactobacilli, for the prevention of urogenital infections (WO 93/09793). A known preparation may contain pharmaceutically acceptable excipients and be vaginal suppositories, ointment, cream, paste or gel.

 A distinctive feature of the known composition is the use of obligate components of the normal urogenital microflora of the body. The mechanism of action of the composition is to destroy part of the pathogenic urogenital microflora using an antimicrobial agent; blocking the reproduction and development of the remaining pathogenic and conditionally pathogenic urogenital microflora due to the adhesion of the microbial component lactobacilli on it; intensive growth and development of lactobacilli caused by growth factors and nutrient components; creating, as a result, normal urogenital microflora, which provides natural urogenital homeostasis, which in turn is a barrier to infection by pathogenic and conditionally pathogenic microflora.

A disadvantage of the known composition is, first of all, the use of an antimicrobial agent of a number of antibiotics or sulfonamides. In addition, the use of the composition is contraindicated in the presence of mycoses. And finally, the known composition requires a high concentration of lactobacilli (1.6 • 10 ⁹ living cells per dose), which increases its cost.

 The inventive drug is new and is not described in the literature.

 The aim of the invention is to create a new, highly effective, non-toxic, non-side effects and contraindications drug for the prevention and treatment of urogenital infections of a bacterial, viral and mycotic nature, localized mainly in the genitourinary tract and rectum.

This is achieved by the fact that the preparation includes, as an active principle, the microbial mass of live bifidobacteria. Preferably, it contains 10 ³ -10 ⁶ living cells of Bifidobacterium bifidum strains N 1, N 791 or LVA-3 per dose.

 As biologically active ingredients of the immunomodulating, regenerating and protective action, the preparation may contain lysozyme, interferons, immunoglobulins. hyaluronic acid, biologically active peptides, growth factors of bifidobacteria, as well as antimicrobial antiviral or antimycotic agents.

 The preparation may contain pharmaceutically applicable target additives and can be used in the form of suppositories, ointments, creams, pastes or gel.

 In contrast to the known composition, the preparation uses bifidobacteria microorganisms unusual for the genitourinary tract, which are obligate components of the intestinal microflora.

 The mechanism of action of the drug is as follows. Bifidobacteria, possessing increased antagonistic activity against pathogenic and conditionally pathogenic urogenital microflora, including fungi, compete with it for binding sites on the urinary tract epithelial cells and food chains. In this regard, pathogenic and conditionally pathogenic urogenital microflora, including fungi, are displaced by bifidobacteria, which are then eliminated, and the normal genitourinary tract takes their place.

 The drug has no contraindications and is used to treat mycoses, in particular candidiasis. In addition, the drug is completely safe, significantly more effective and has lower cost, since it is used without an antimicrobial agent, growth factors of bifidobacteria and nutrient components in significantly (10 1000000 times) lower concentrations of living bacterial cells per dose.

 It should be noted that bifidobacteria in the compositions or are independently used to create and maintain intestinal normoflora, to regulate intestinal microflora, to treat intestinal dysbiosis, dysentery, colic in newborns, reactive post-infectious arthritis, atopic dermatitis, to obtain immunomodulator, to reduce urea concentration in the body , for regulating plasma cholesterol, for biological deodorization, etc. (DE 2755037 Al, EP 0482530 A3, SU 1286212 Al, SU 1258414 Al, DE 3716938, SU 1553132 Al, SU 1816215 A3, RU 2023445, DE 3406772 Al, EP 0181170 Bl, EP 0208818 Bl, EP 0228861Bl). However, the use of bifidobacteria as a means for the prevention and treatment of urogenital infections is unknown.

 Thus, the proposed drug has a highly effective antibacterial, antimycotic and antiviral effect, is completely safe, has no contraindications and does not detect side effects and is used for the prevention and treatment of a wide range of urogenital infections.

 The proposed preparation is a microbial mass of living lyophilized dried in a cultivation medium. antagonistically active strains of Bifidobacterium bifidum. It is an amorphous product with a yellowish tint, odorless and tasteless, readily soluble in water with the formation of a suspension of bacterial cells. The drug is used in the form of suppositories, ointments, creams, pastes or gel.

 Example 1. The preparation in the form of suppositories includes the ingredients for one suppository are shown in table. 1-9.

 Example 2. The preparation in the form of an ointment includes the ingredients in a single dose, are given in table. 10 and 11.

 Example 3. The preparation in the form of a cream includes the ingredients in a single dose, are given in table. 12 and 13.

 Clinical trials of the drug for the prevention and treatment of urogenital infections in the genitourinary tract and rectum were carried out in district women's clinics, in clinics of the Moscow Regional Research Institute of Obstetrics and Gynecology, the Center for Obstetrics, Gynecology and Perinatology of the Ministry of Health of the Russian Federation, and the Central Skin and Venereological Research Institute, Coloproctology Center and a number of city and regional hospitals for 902 patients and 65 healthy people.

 Example 4. Clinical trials of the drug and its antibacterial analogues for the prevention and treatment of bacterial vaginosis were carried out in district women's clinics and in the Center for Obstetrics, Gynecology and Perinatology of the Ministry of Health of the Russian Federation on 138 women of reproductive age with identified bacterial vaginosis and 30 pregnant women.

 For the diagnosis of bacterial vaginosis, the following symptoms are necessary: homogeneous, milky consistency of vaginal discharge; the pH of the vaginal discharge is more than 4.5; positive amine test; the presence of "key" cells in the vaginal discharge.

 The vaginal discharge during bacterial vaginosis is liquid, whitish in color, homogeneous discharge is evenly distributed along the walls of the vagina. Unlike vaginal candidiasis or trichomoniasis, there is no inflammatory reaction of the walls of the vagina, there is no hyperemia and edema of the vulva or walls of the vagina. Irritation, edema and soreness in patients with bacterial vaginosis are rarely pronounced, more often these complaints are mild.

 The pH of the vaginal contents can be determined using paper tests or a pH meter after diluting the vaginal discharge with an isotopic sodium chloride solution. This test has a high sensitivity (80-98%). but not very specific (50-70%).

 A positive amine test consists in the smell of rotten fish when a 10% KOH solution is added to a drop of vaginal discharge. A positive test is associated with the appearance of volatile amines (putrescine, cadaverine, trimethylamine), which are the product of the activity of obligate anaerobes. In the absence of Trichomonas, this test is highly sensitive and specific (more than 90%). However, in cases of absolute prevalence of gardnerella, the test is negative.

 The “key” cells are mature epithelial cells (the surface layer of the vaginal epithelium), onto which small gram-stick bacilli stick tightly and in large quantities over the entire surface. Cells are easily found in unpainted native smears and in fixed smears of the vaginal discharge during any staining method. However, this method achieves the greatest specificity and sensitivity (approaching 100%) when stained according to Gram, when it is easy to exclude "false-key" cells, which may be associated with the adhesion of lactobacilli.

 The first group of 30 patients was administered intravaginally the claimed drug for 20 days at the rate of 2 doses per day. At the end of the course of treatment in 17 patients, bacterial vaginosis was not detected, which corresponds to 56.7% of the cure.

 With an increase in the course of treatment to 30 days, bacterial vaginosis was not detected in another four patients, which corresponds to 70% of the cure.

 The second group of 68 patients was injected intravaginally with CIP (complex immunoglobulin preparation) in the form of suppositories at the rate of 2 suppositories per day for five days, then the intravaginally declared preparation was administered at the rate of 2 doses per day for ten days, after which acylact in the form of candles for 10 days, two candles per day.

 At the end of the course of complex therapy in 51 patients, bacterial vaginosis was not detected, which corresponds to 75% of the cure.

 A third group of 20 patients was injected intravaginally with the antibacterial drug dalacin in the form of a cream at the rate of 1 dose per day according to the instructions for 7 days. At the end of the course of antibiotic therapy in 12 patients, bacterial vaginosis was not detected, which corresponds to 60% of the cure.

 The fourth group of 20 patients was administered orally the antibacterial drug metronidazole in the form of tablets at the rate of 500 mg 2 times a day for 7 days. At the end of the course of antibiotic therapy in 12 patients, bacterial vaginosis was not detected, which corresponds to 60% of the cure.

 Thus, the comparative clinical studies have shown greater efficacy of the claimed drug for the treatment of bacterial vaginosis compared with antibacterial agents.

It should be noted that recurrence of the disease in the third and fourth group of patients 7-10 months after the course of treatment was observed in 50-70% of women, while in the first and second groups only 25-35%
In addition, an examination of 30 pregnant women at risk group showed that prophylactic use of the drug for 20-30 days in the early stages of pregnancy at the rate of 1-2 doses per day reduces the risk of bacterial vaginosis by 60%
PRI me R 5. Clinical trials of the claimed drug and its antibacterial analogues for the prevention and treatment of mycoses, in particular, candidiasis was carried out in the district women's clinics and clinic of the Moscow regional research institute of obstetrics and gynecology in 246 women of reproductive age with identified candidiasis and 30 women at risk.

 Candidiasis was diagnosed by smear candida.

 The first group of 28 patients was administered intravaginally pimafucin in the form of suppositories for 3 days, 1 suppository per day. At the end of the course of antimycotic therapy, candidiasis was not detected in 8 patients, which corresponds to 29% of the cure.

 The second group of 40 patients was injected intravaginally with gyno-pevaryl-150 in the form of suppositories for 3 days, 1 suppository per day. At the end of the course of antimycotic therapy in 26 patients, candidiasis was not detected, which corresponds to 65% of the cure.

 The third group of 82 patients was administered intravaginally the claimed drug for 15 days at the rate of 2 doses per day. At the end of the course of treatment, candidiasis was not detected in 46 patients, which corresponds to 56% of the cure.

 A fourth group of 96 patients was administered intravaginally gyno-pevaryl-150 in the form of suppositories for 3 days, one suppository per day, after which the inventive preparation was similarly administered for 10 days at the rate of 2 doses per day. The course of complex therapy provides 100% cure.

 Thus, the claimed drug in terms of effectiveness of antimocotic action surpasses pimafucin and is slightly inferior to gyno-pevaril-150, and the best results are demonstrated by complex therapy.

Examination of 35 women at risk (with a decrease in the titer of lactobacilli in the vagina and lower acidity of the vaginal environment) showed that the prophylactic use of the claimed drug for 10-20 days intravaginally at a dose of 1-2 doses per day reduces the risk of candidiasis by 65%
Example 6. Clinical trials of the claimed drug for the treatment of chlamydia were carried out in a clinic of the Moscow Regional Research Institute of Obstetrics and Gynecology, the Central Skin and Venereological Research Institute and a number of regional hospitals for 300 women of reproductive age with identified chlamydia.

 Diagnosis of chlamydia was carried out on the basis of an anamnesis, an assessment of the clinical signs of the disease, and laboratory research methods. Due to the fact that urogenital chlamydia is more characteristic of a low-symptom and latent course, the laboratory diagnosis of this infection was of primary importance. Laboratory diagnosis of chlamydia was carried out by polymerase chain reaction (PCR).

 Clinical material for PCR was collected in test tubes with buffer solution, stored in freezers. Repeated freezing-thawing was not allowed.

Material Processing:
Clinical material in a volume of 100 μl was mixed with 100 μl of lysis buffer (1% tween-20, 1% NP-40, 60 μg / ml proteinase K, phosphate buffer). The mixture was incubated at 56 ^° C for 2 hours, after which it was inactivated at 95 ^° C for 10 minutes (heat treatment is necessary to inactivate proteinase K and disinfect the clinical sample). Inactivated samples were centrifuged at 10,000 g for 10 minutes. The supernatant was collected and used for PCR.

 Carrying out PCR.

The reagents were added to the reaction tube in the following sequence: bidistilled water up to 30 μl of the final volume of the mixture; reaction buffer 10% 2.5 μl; 650 mM MgCl ₂ solution 1.2 μl; dNTP's mixture of 2.5 μl; Taq polymerase 1-1.5 units. oligonucleotide primers 15 pmol; control DNA sample or clinical sample 1-2 μl.

 To protect against evaporation, 30 μl of mineral oil was layered onto the reaction mixture. The tubes were placed in an amplifier.

PCR was carried out in the following temperature conditions: 96 ^o C 4 min, 55 ^o C 10 min 1 cycle; 95 ^o C 1 min, 55 ^o C 1 min, 72 ^o C 2 min 35 cycles.

 After completion of PCR, the amplification products were fractionated by electrophoresis in a 1.5% agarose gel. The presence of a specific DNA fragment was determined using the control DNA of the studied pathogen amplified simultaneously with clinical samples.

 The result of the analysis was considered positive in the following case: the size of the DNA product in the clinical sample exactly corresponded to the DNA product of the control positive sample; in the sample containing the clinical sample and the internal standard, the specific DNA product of the internal standard was identified; in a sample containing instead of a clinical sample an aliquot of water (negative control), DNA products were not detected.

 The result of the analysis was considered negative in the following case: in the sample containing the clinical sample, specific DNA products were not detected; PCR results in both control samples are similar to the previous ones.

 It should be noted that in patients with urogenital chlamydia there is a significant contamination by yeast of the genus Candida. So, in 135 of 300 patients with identified chlamydia, candidiasis was also noted. Studies have shown that in this case it is advisable to begin treatment with the elimination of mycoses. Therefore, patients with identified candidiasis were prescribed complex therapy according to example 5.

 Then, all patients were injected orally with the antibacterial drug Vilprafen (yosamycin) for 15 days at the rate of 500 mg 2 times a day, after which the intravaginally claimed drug was administered for 10 days at the rate of 2 doses per day, and finally, acylact in the form of suppositories was similarly administered for 10 days, 1 candle per day.

 2 weeks after the end of the course of treatment, all patients underwent clinical and laboratory control by PCR. However, in 285 patients, chlamydia was not detected, which corresponds to 95% of the cure. Fifteen patients with newly diagnosed chlamydia were prescribed a similar course of complex therapy with the oral administration of another antibiotic zanocin-200 or zanocin-100 at a rate of 200 mg 2 times a day, which provided 100% cure.

 Thus, the cure of chlamydia provides the use of a combination of the claimed drug with antibacterial and eubiotic agents.

 Example 7. In recent years, there has been a tendency to increase the number of mixed urogenital infections, which, according to G. Torrigiani, account for 50-90% of the number of sexually transmitted infections.

 Clinical trials of the claimed drug for the treatment of mixed urogenital infections were carried out in the clinic of the Moscow Regional Research Institute of Obstetrics and Gynecology, the Center for Obstetrics, Gynecology and Perinatology of the Ministry of Health of the Russian Federation and the Central Skin and Venereal Research Institute on 218 women of reproductive age with identified mixed urogenital infections. At the same time, chlamydial ureoplasma was detected in 55 patients, chlamydial-gardnerellosis in 32 patients, chlamydial candidiasis in 23 patients, chlamydial-viral (herpes virus type II and type I, cytomegalovirus), in 20 patients, chlamydial-mycoplasma infection, in 23 patients had a combination of 3 infections, and 20 patients had a combination of 4 and 5 infections.

 Candidiasis was detected by the definition of candida in a smear. Diagnosis of chlamydia and other urogenital infections was carried out by PCR according to example 6 using the corresponding test DNA of the pathogen as a control sample.

23 patients with chlamydial candidiasis infection were prescribed complex antimycotic therapy according to example 5, then complex anti-chlamydia therapy according to example 6. 2 weeks after the end of the course of treatment, all patients underwent clinical and laboratory control by PCR. The treatment efficiency was 95%
The remaining 195 patients were administered oral antibacterial drug vilprafen (yosamycin) for 15 days at the rate of 500 mg 2 times a day, after which the intravaginally claimed drug was administered for 10 days at the rate of 2 doses per day and, finally, acylact in the form of suppositories was similarly administered for 10 days, 1 candle per day. In this case, patients with chlamydial viral infection simultaneously with the claimed drug were injected intramuscularly with myelopid or realderone for 3-5 days at the rate of one dose per day. 2 weeks after the end of the course of treatment, all patients underwent clinical and laboratory control by PCR. The effectiveness of the treatment of chlamydial virus infection was 80% of all other mixed infections 90-95%
Thus, the cure of mixed urogenital infections provides the use of a combination of the claimed drug with antibacterial and eubiotic agents.

 Example 8. In 10% of all examined patients, diseases caused by urogenital infections were accompanied by proctitis of a similar etiology.

 Diagnosis of proctitis was performed by rectoscopy and clinical laboratory methods according to examples 4-6.

 Patients with identified proctitis against the background of the above course of treatment for a particular disease were administered the claimed drug rectally simultaneously with its intravaginal administration at the rate of 2 doses per day. At the same time, the course of treatment was completed by oral administration of the bacterial preparations of bifidumbacterin, colibacterin (bificol) and acylact (lactobacterin) in vials at the rate of 5 doses per dose 2 times a day for a month.

After treatment, all patients underwent clinical and laboratory control by the above methods (examples 4-6). The treatment efficiency was 90%
Prevention of proctitis was carried out simultaneously with the prophylaxis of a specific disease of a similar etiology by rectal administration of the inventive preparation at the rate of 1-2 doses per day. Moreover, the prophylactic use of the claimed drug reduces the risk of proctitis by 62.5%

Claims (9)

 1. A drug for the prevention and treatment of urogenital infections, containing as an active principle a microbial mass of live bacteria and pharmaceutically acceptable target additives, characterized in that it contains bifidobacteria in an effective amount as a microbial mass. 2. The drug according to claim 1, characterized in that it contains a microbial mass of bifidobacteria in an amount of 10 ³ 10 ⁸ living cells per 1 dose.  3. The drug according to claim 1 or 2, characterized in that it contains as bifidobacteria one or a mixture of strains of Bifidobacterium bifidum N 1, N 791 or LVA-3.  4. The drug according to claim 1, 2 or 3, characterized in that it additionally contains one or more biologically active ingredients with immunomodulatory, regenerating or protective effect, in the amount of 0.1 to 100.0 mg per 1 dose.  5. The drug according to claim 4, characterized in that the biologically active ingredients are substances selected from the group comprising hyaluronic acid, lysozyme, interferons, immunoglobulins, biologically active peptides, antimicrobial, antiviral or antimycotic substances.  6. The drug according to claim 1, characterized in that as the target additives it contains one or more ingredients from the group including confectionery, food or deep-frying fat, butter, cocoa, paraffin, petrolatum, lanolin and an emulsifier in an amount of 1.5 to 2 , 2 g per 1 dose.  7. The drug according to claim 1 or 6, characterized in that it is a suppository, ointment or cream. 8. A method for the prevention and treatment of urogenital infections by intravaginal and / or rectal administration of a drug to a patient, characterized in that the drug according to paragraphs is used as a drug. 1 7, containing 10 ³ 10 ⁸ living bifidobacteria cells per dose, the drug is administered for 10 30 days, while for prevention, 1 2 doses of the drug are administered per day, and for treatment, 2 doses per day.  9. The method according to claim 8, characterized in that before, simultaneously or after the administration of the drug according to claims 1 to 7, the patient is administered an antibacterial or antimycotic or eubiotic agent.

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