RU2812223C2 - Use of totarol and pharmaceutical composition containing totarol - Google Patents

Abstract

FIELD: gynecology.

SUBSTANCE: use of totarol as an active substance in an amount from 0.001 to 5 wt.pts for obtaining a drug for the treatment of inflammation of the vaginal mucosa of bacterial origin, for the symptomatic relief during such treatment and for the prevention of relapse of such inflammation, wherein this inflammation refers to anaerobic bacterial vaginosis (BV) and aerobic vaginitis (AV). The group of inventions also relates to a pharmaceutical composition for the treatment of inflammation of the vaginal mucosa of bacterial origin, for the symptomatic relief during such treatment and for the prevention of relapse of such inflammation, wherein this inflammation refers to anaerobic bacterial vaginosis (BV) and aerobic vaginitis (AV).

EFFECT: high therapeutic utility and safety, as well as high antibacterial effectiveness against bacteria that are the main cause of both BV and AV, without adverse effects on anaerobic bacteria of the genus Lactobacillus.

3 cl, 4 dwg, 6 tbl, 6 ex

Description

Field of technology to which the present invention relates

The present invention relates to the use of totarol for the prevention of disturbances in the composition of the vaginal bacterial flora and to a pharmaceutical composition containing totarol as an active ingredient. The composition is intended for use in gynecology and obstetrics during antibacterial, antifungal, antiprotozoal and mixed therapy, and as a means of regulating the acidity of the vaginal environment. The composition can be used as a pharmaceutical preparation or a medical product.

State of the art

Bacterial vaginosis (BV), a disorder of the natural bacterial flora, is an inflammatory condition most commonly found in the female birth canal. According to available estimates, the incidence of this nosological entity in women during puberty is 10-40%. During the course of BV, a decrease in the number or even a complete reduction in the population of rod-shaped bacteria of the genus Lactobacillus occurs, which leads to the uncontrolled proliferation of strictly anaerobic bacteria. The type of rod-shaped bacteria of the genus Lactobacillus, their content and the amount of glycogen stored in the epithelial cells of the vagina, converted by these bacteria into lactic acid, regulate and are responsible for maintaining the proper pH level of the vaginal environment. The response of the vaginal environment depends on the total amount of organic acids produced by these bacteria, the most important of which is lactic acid, mentioned above. The controlled production of these acids causes a pH value of the vaginal environment below 4.5, which is necessary for the adhesion of bacteria of the genus Lactobacillus to the vaginal epithelium and allows maintaining the correct microflora in qualitative and quantitative terms. Thus, the correct microflora suppresses the growth of microorganisms that require an environment with a pH value>4.5. Thus, physiological microflora protects against infection by pathogenic microorganisms, including by competing for nutrients and receptor sites on the cell surface, and stimulates the reproductive tract immune system to produce antibodies that cross-react with other microorganisms.

The most common pathogen that is the main cause of BV is Gardnerella vaginalis, which is found in almost all women with this condition, as is another anaerobe, Atopobium vaginae, which is also found in 50% of cases of BV caused by the rod-shaped bacteria Mobiluncus spp. The ability for pathogenic bacteria to multiply to form a multibacterial biofilm that adheres to the vaginal epithelium is a particularly important characteristic that prevents therapy during the course of BV. Colonies of bacteria that form a biofilm containing Gardnerella vaginalis and Atopobium vaginae as the main components have the ability to attach to wet surfaces and to each other. The purpose of forming a biofilm matrix is to protect the microorganisms that form it from the destructive effects of natural factors in the vaginal environment and from the effects of pharmacological agents, including antibiotics. The compact structure of biofilm is very difficult to remove from the vaginal walls. In women suffering from BV, biopsies showed the presence of a biofilm attached to epithelial cells, in which the bacteria Gardnerella vaginalis accounted for 90%.

The phenomenon of biofilm formation by pathogenic microorganisms, the absence of signs of inflammation in BV, cases of incorrect diagnosis of BV, reaching 61% (identification of BV according to the Amsel criteria is inexpensive, simple and can be carried out in almost every gynecological office; however, it has disadvantages associated with the subjectivity of the assessment and lower recurrence rate, depending on the experience of the physician), and a group of patients in whom BV is asymptomatic, lead to relapses in the majority of treated patients after 12 months.

The characteristic signs and mechanisms that occur during BV described above are extremely important from a medical point of view, due to the fact that bacterial vaginal infection is considered one of the etiological factors of spontaneous abortion, premature birth, intrauterine infections, premature rupture of amniotic fluid, intrauterine growth retardation ( IUGR), inflammation of the mucous membrane of the uterus and pelvic organs. In addition, BV can cause other perinatal infections that are equally dangerous for the infected mother and newborn.

Another condition associated with disturbances in the qualitative and quantitative composition of the vaginal microflora is inflammation of the vagina caused by microorganisms existing under aerobic conditions, called aerobic vaginitis (AV). With AB, there is also a decrease in the number or absence of bacteria of the genus Lactobacillus, as well as an increase in mixed vaginal flora, which is dominated by spherical bacteria, gram-positive streptococci of group B: Streptococcus agalactiae, Enterococcus fecalis, Staphylococcus aureus, and rod-shaped gram-negative bacteria: Escherichia coli. As in the case of BV, in the case of aerobic vaginitis, identical changes are also observed. The concentration of lactates in the vaginal microflora decreases, the pH value of the vaginal environment increases to >6.0, and the concentration of proinflammatory cytokinins also increases. Due to the lack of clear diagnostic indicators and the simultaneous occurrence of other infections, such as mycosis and trichomoniasis, the incidence of AV is not precisely determined.

The occurrence of disturbances in the composition of the vaginal microflora causes inflammation in the genital tract, which can be caused by two completely different types of pathogenic bacteria that exist under completely disparate oxygen conditions. Abnormal biocenosis and inflammation can also lead to the development of inflammatory postoperative complications, in particular gynecological, obstetric and urological. They also pose a huge threat to the fetus, whose body is deprived of contact with microorganisms during intrauterine development, however, when passing through the birth canal, it is colonized by the mother’s vaginal microflora.

Most currently used treatment regimens are based primarily on metronidazole, tinidazole and, in the case of BV, clindamycin. These substances are commonly used orally or intravaginally according to common practice. The fundamental advantages of metronidazole are its activity against both aerobic and anaerobic bacteria, and the fact that anaerobic lactic acid rod-shaped bacteria of the genus Lactobacillus are completely resistant to this antibiotic. In addition, this drug is inexpensive, easily accessible and generally well tolerated by patients. Unfortunately, reports are increasingly appearing in the world scientific literature about the resistance of Gardnerella vaginalis to the action of this chemotherapeutic agent, which also indicates an increase in the percentage of resistant strains of this species. Studies conducted by Goldstein et al. in 1993, showed the percentage of strains resistant to metronidazole at 20%, while already in 2002 the same group of researchers found an increase in the percentage of resistant strains to a level of 29%. The most recent reports indicate that the process of acquisition of resistance to metronidazole is accelerating at a significant rate. In drug resistance studies conducted and published in 2007, resistance of Gardnerella vaginalis strains to metronidazole was observed to be as high as 70%. Studies conducted in Poland on a collection of 67 strains isolated from a group of 604 women confirmed that 68.7% (46 out of 67) of the studied strains were resistant to metronidazole. The alarming increase in the number of anaerobic strains associated with BV exhibiting the described mechanism of resistance to metronidazole may lead to the need for changes in treatment regimens for bacterial vaginosis in a few years.

Compared to metronidazole, clindamycin is more effective against anaerobic gram-negative rod-shaped bacteria and, at the same time, has limited activity against bacteria existing under aerobic conditions. In addition, the negative consequences of the use of this drug are associated with its high activity against bacteria of the genus Lactobacillus and the ability to inhibit the growth of bacteria of the genus Lactobacillus, which can lead to disruption of the regeneration process of the normal vaginal ecosystem and, as a result, lead to secondary fungal infections of the external female genitalia organs and vagina, caused by Saccharomyces species Candida albicans and Candida glabrata.

Amoxicillin with clavulanic acid has been proposed as a second-line drug for the treatment of BV. Unfortunately, there is no data in the literature assessing the effectiveness of this antibiotic in the treatment of vaginal infections caused by anaerobic bacteria.

Despite the high effectiveness of the described antibiotics, relapses of vaginal infections and infections of the birth canal of bacterial origin are observed quite often. This phenomenon is particularly observed in patients with BV. It is estimated that about 30% of women experience a recurrence of clinical symptoms of BV within 3 months and about 80% within the first year after completion of therapy. One of the main reasons for relapses is the resistance of anaerobic bacterial strains to the antibiotics used, which at the same time have high antibacterial activity against lactic acid rod-shaped bacteria of the genus Lactobacillus, which naturally exist in the vaginal microflora and are necessary for its normal functioning.

An important problem is changes in the structures of bacteria, which are the main cause of infections of the vaginal mucosa. The scale of changes occurring in pathogenic microorganisms aimed at the development of new resistance mechanisms is multidirectional and multi-level. Often, a single pathogen uses multiple mechanisms to defend against a wide range of commonly used therapeutics.

Thus, it is currently difficult to predict the spectrum of antibacterial activity of a substance active against pathogenic microorganisms with such a variable structure, despite its well-known activity against pathogenic microorganisms already tested. It has been observed that the activity of a therapeutic substance can change over time, and in some cases even fade under the influence of newly emerging defense mechanisms of the pathogenic microorganism.

Therefore, changes in activity, including its complete attenuation, and limitation of the spectrum of activity against pathogenic microorganisms of various types are serious problems with currently used therapies, both in the case of BV and AB infections. The increasing incidence of Gardnerella vaginalis resistance to metronidazole limits effective treatment for BV and is likely contributing to the increased rate of recurrence of clinical symptoms.

Compounds of natural origin can play an important role in the treatment of inflammation of the mucous membranes of the vagina, especially inflammation of a mixed nature: BV+AV or BV together with a concomitant secondary fungal infection. A broad spectrum of antibacterial activity against both aerobic and anaerobic bacteria is a prerequisite for therapeutic utility in the treatment of resistant cases or mixed infections. In addition, it is necessary to take into account the effect of the new substance on the physiological component of the natural bacterial microflora of the vagina, namely lactic acid bacteria of the genus Lactobacillus.

A tricyclic diterpene with the nonproprietary name “totarol”, which has a chemical structure represented by formula 1, has antibacterial activity.

It is a naturally occurring compound found in the endemic tree species Podocarpus totara (mahogany) found throughout New Zealand.

The antibacterial activity of totarol has been disclosed in a number of scientific papers and patent publications. In the publication "Antibacterial activity of totarol and its potentiation" Journal of Natural Products Vol.55, No. 10, pp.1436-1440, October 1992, describes the high effectiveness of totarol-containing extracts against gram-positive bacteria: Bacillus subtilis, Brevibacterium ammoniagenes, Streptococcus mutans, Propionibacterium acnes and Staphylococcus aureus. In the publication "Clinical report on the Efficacy of Totarol and Totarol in Combination with Tea Tree Oil as an Antimicrobial Against Gram-negative Bacteria" prepared for Mende-DEK Ltd. November 2003, the effectiveness of totarol was confirmed against four gram-negative bacteria: Salmonella menston, Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa, and one gram-positive bacterium: Enterococcus fecalis. The publication "The synthesis and antibacterial activity of totarol derivatives. Part 1: modifications of ring-C and pro-drugs" Bioorganic & Medicinal Chemistry 1999, 7(9): 1953-1964 describes its activity against the Gram-positive bacterium Streptococcus pneumoniae. The publications cited above disclose information about the bacteria against which totarol is active, and also indicate a fairly large group of pathogenic bacteria against which totarol does not exhibit any antibacterial activity. The latter are often pathogenic microorganisms of the same genus against which totarol activity was discovered, which indicates that the effect on the corresponding microbial strains is not obvious and individual.

According to the description of Japanese patent application No. JP 01311019, the activity of totarol against gram-positive bacteria in preparations intended for external use is disclosed. In turn, the description of application WO2005073154 presents a method for extracting totarol from plant materials, and a preparation obtained from totarol obtained by the described method. In this description, it was proven that totarol also acts against gram-negative bacteria. The composition of the drug, its form and use were disclosed in this application in a very general manner, without indicating its specific purpose. Application specification WO2014137231 discloses the use of totarol-containing preparations for the treatment of breast inflammation, uterine inflammation and wounds. Application specification CN104027260 discloses the use of preparations in the form of toothpaste or mouthwash for the treatment of wounds in the oral cavity.

At the moment, the antibacterial activity of totarol has not been proven against pathogenic bacteria, which are the main causes of infections of the vaginal mucosa. They are the Gram-variable anaerobic bacterium Gardnerella vaginalis, which causes BV infections, and the Gram-positive aerobic bacterium Streptococcus agalactie, which causes AB infections.

As a result of studies using clinical strains isolated from the female birth canal conducted by the authors of the present invention, it turned out that totarol exhibits unusually high activity against these pathogenic microorganisms at a very low minimum inhibitory concentration (MIC). Activity against both a clinical strain of the pathogenic anaerobic bacterium Gardnerella vaginalis and a clinical strain of the aerobic bacterium Streptococcus agalactie leads to the fact that - which is very valuable - the spectrum of activity of totarol overlaps with the spectrum of activity of metronidazole, for which there is a sharply increasing resistance of the above-mentioned microorganisms, which limits or even eliminates the effective medical use of the latter.

Until now, the effect of totarol on lactic acid rod-shaped bacteria of the genus Lactobacillus was also unknown. For the first time such a study was carried out by the authors of the present invention. Quite unexpectedly, it was discovered that although rod-shaped bacteria of the genus Lactobacillus, such as Gardnerella vaginalis, are anaerobic bacteria, there is a significant approximately 10-fold difference in the MIC concentration values for these microorganisms, which is best illustrated in the following Table.

This non-obvious and at the same time crucial for the subject of the present invention difference between effective concentrations for individual bacteria of the same genus determines the uniqueness and exceptional quality of totarol in the described application.

The new and unique properties of totarol, first discovered by the authors of the present invention, determine its enormous medical potential for use, which is the object of the present invention. Low MIC concentrations, along with high antibacterial effectiveness against bacteria that are the main cause of both BV and AV, do not show activity against anaerobic bacteria of the genus Lactobacillus and, thus, do not have an adverse effect on anaerobic bacteria of the genus Lactobacillus, which are the main component physiologically correct vaginal microflora. This unique mechanism of action of totarol determines its enormous therapeutic utility in the treatment of vaginal infections of bacterial origin. The lack of activity against lactic acid rod bacteria in the appropriate MIC range prevents additional sterilization of the vaginal microflora, while at the same time creating optimal conditions for the growth of these bacteria. Increasing the concentration of lactic acid rod bacteria ensures the natural restoration of the protective barrier of the vagina, thus preventing the recurrence of clinical symptoms and protecting the vaginal environment from secondary fungal infections that accompany classical therapy with clindamycin.

In addition to the above-mentioned new and unexpected properties of totarol, its enormous therapeutic potential is determined by the absence of a resistance mechanism that occurs in the case of metronidazole.

The present inventors have further demonstrated the high therapeutic utility and safety of gynecological applications through pioneering tissue studies evaluating the effects of totarol on human vaginal epithelial cells using the A-431 cell line. The tests performed did not confirm the apoptotic and necrotic effect of the test substance on human vaginal epithelial cells A-431 over a wide range of incubation periods (2-24 hours).

The test results confirming a broad spectrum of antibacterial activity for specific strictly defined concentrations of MIC of totarol, along with the absence of activity against lactic acid bacteria of the genus Lactobacillus and the absence of adverse effects on human vaginal epithelial cells, first obtained by the authors of the present invention, became the basis for the use of totarol according to the present invention. Use in accordance with the present invention is the result of studies conducted by the inventors of the present invention showing that totarol is active against clinical strains of Gardnerella vaginalis and Streptococcus agalactie isolated from the female birth canal. The studies were conducted based on the methodology described in the document "Determination of the Minimum Concentration of Totarol & Antibiotics Required to Inhibit and Kill Growth of Staphylococcus aureus in Broth Cultures" (Project NZC-MEND-1312). The results and methods of conducting the described studies are presented in more detail in the examples of the present invention.

However, totarol has limitations due to the physicochemical properties of the substance. The hydrophobic nature of its molecule, leading to a complete lack of solubility in water, is a fundamental technological problem that prevents the effective use of the substance in gynecological compositions. Due to the target area of application of the pharmaceutical composition, namely the vaginal mucosa, the use of most organic solvents to dissolve totarol has not been possible. Solvents related to alcohols, such as ethanol, or polyhydric alcohols, such as propylene glycol, are highly irritating, and the addition of water to them leads to the precipitation of dissolved totarol. The use of halogen derivatives, such as methylene chloride or chloroform, as solvents is also impossible due to their toxic nature. The embryotoxic and teratogenic properties of N,N-dimethylformamide do not allow its use as a component of pharmaceutical compositions. DMI, used in in vitro studies, is used in the pharmaceutical industry, but only in the production of topical drugs such as steroid ointments.

Since the composition of the vaginal microflora may change under the influence of antibiotics, endocrine or immunologically active agents during pregnancy or menopause, it is recommended to use lactic acid in the form of a vaginal gel, especially after completion of pharmacological treatment for BV.

Polish patent No. 166898 discloses a method for producing a methylcellulose-lactic acid complex, which involves solvation of methylcellulose with lactic acid in the form of a solution consisting of 5-25 wt. parts of lactic acid and 10-20 wt. parts of 95% ethyl alcohol. The solution is sprayed at 75-95 wt. parts of methylcellulose with continuous stirring, and then the product is dried, removing ethyl alcohol. The resulting powder is granulated. Lactic acid binds methylcellulose, forming a binary complex. Methylcellulose, solvated with lactic acid, has rapid water-fixing properties, forming a gel with viscosity, adhesive and coating properties similar to those of the mucus formed in the vagina under physiological conditions. Compositions using only lactic acid, which have an effect based on the regulation of the pH value, are insufficient in the treatment of vaginal infections and can only be used for prophylactic purposes or to maintain good hygiene of the birth canal of healthy women.

From Polish patents No. 194437, 201868 and 201869, a method of production and compositions are known in which a methylcellulose-lactic acid complex is used, where individual strains of lactic acid bacteria Lactobacillus crispatus, Lactobacillus jenseni, Lactobacillus rhamnosus, Lactobacillus gasseri, Lactobacillus helveticus are additionally used as active substances in the form of a lyophilisate. The composition contains from 75 to 95 wt. parts of methylcellulose, from 0.5 to 5 wt. parts of lactic acid, from 0.5 to 5 wt. parts of the main polymer, preferably Eudragit or chitosan, or polyvinylpyrrolidone, or mixtures thereof, and from 0.5 to 5 wt. parts of the bacterial lyophilisate of Lactobacillus, and the stoichiometric ratio of lactic acid to the main polymer is in the range from 1:1 to 8:1. Preferably, the composition contains a culture medium in an amount of from 5 to 10 wt. parts, in the form of monosaccharides or polysaccharides. The production method of this known composition involves obtaining a binary component by dissolving the main polymer in a solution of lactic acid at a stoichiometric ratio to the free amino groups in the main polymer in the range from 1: 1 to 8: 1, stirring until the main polymer is completely dissolved, and then adding ethyl alcohol to quantity of at least 5 wt. parts, stirring until completely homogeneous and spraying the mixture thus obtained onto the methylcellulose, and stirring until it is uniformly wetted. The resulting product is subjected to drying, during which the ethyl alcohol evaporates, and the Lactobacillus bacterial lyophilisate is added to the dry powder thus obtained.

The purpose of vaginal administration of the individual strains mentioned above is to provide optimal conditions for the complete restoration of the physiological bacterial flora of the vagina. At the same time, the authors of the mentioned patent descriptions emphasize that the reproductive organs, like other anatomical areas of a person, have numerous “ecological niches”, and, therefore, the vaginal vault, the cervical canal, the outer wall of the cervix and the anterior part of the vagina all have different bacterial flora . The described differences in the composition of the bacterial flora cause a decrease in the effectiveness of the described compositions containing one strain of rod-shaped bacteria of the genus Lactobacillus. The use of more strains in one composition is not preferable in light of current research, as this may lead to too high a concentration of these bacteria, resulting in the cause of cytolytic vaginosis, which is often misdiagnosed and treated as vaginal candidiasis.

Raw materials in the form of bacterial lyophilisates have numerous serious limitations due to the specificity of the material containing living organisms, and its use in technological conditions is associated with a number of difficulties. The parameters of probiotic raw materials are characterized by high variability, strictly depending on numerous parameters of the fermentation process. Obtaining final raw materials of high purity requires the use of substances that increase the viability of microorganisms and stabilize the fermentation process, as well as complex and expensive purification and lyophilization methods. The number of colony-forming units (CFU) in the bulk mass of the resulting raw material strictly depends on both the type of bacterial strain and the method and parameters of the entire production process. This amount continually decreases over time as the stability and viability of probiotic strains depend on temperature, storage conditions and water content. This negatively affects the effectiveness and stability of bacterial properties during storage and distribution of probiotic products.

The production process of ready-to-use pharmaceutical products containing probiotic raw materials must be carried out on separate production lines using specialized equipment, since the quality control systems and GMP (Good Manufacturing Practice) guidelines required by the pharmaceutical industry classify these raw materials as a significant source of microbiological pollution. The need for specialized production equipment is a consequence of the high risk of colonization and the fact that regularly used cleaning procedures do not guarantee adequate cleanliness. This can only be achieved through the use of advanced and expensive cleaning and sterilization methods, which involve the dismantling of individual working elements that come into contact with the product being produced. Cleaning and sterilization procedures undergo extensive validation to ensure that they are effective and reproducible, and that even small amounts of aggressive cleaning agents are not used in the cleaning equipment.

Disclosure of the Invention

The objective of the present invention is to solve the problems described above.

The essence of the present invention is the use of totarol to obtain a drug for the treatment of inflammation of the vaginal mucosa of bacterial origin and to alleviate symptoms during such treatment. In particular, the use according to the present invention relates to bacterial vaginosis (BV) and aerobic vaginitis (AV).

Preferably, totarol when used according to the present invention is used in the form of a composition containing from 75 to 95 wt. parts of a cellulose derivative, preferably methylcellulose, from 0.5 to 5 wt. parts of lactic acid, from 0.5 to 5 wt. parts of a base polymer, preferably an acrylic polymer, most preferably a copolymer of methacrylic acid and ethyl acrylate or chitosan or polyvinylpyrrolidone, or mixtures thereof, wherein the stoichiometric ratio of lactic acid to base polymer is in the range from 1:1 to 8:1. According to the present invention, the active substance in the composition is totarol in an amount of 0.001 to 5 wt. parts

The composition may also contain a culture medium for probiotic bacteria in an amount of 5 to 10 wt. parts in the form of monosaccharides or polysaccharides. The composition may also contain additional active components in an amount from 0.001 to 10 wt. %, depending on the type of component.

The composition according to the present invention is obtained by a method known from the prior art (PL 194437, PL201868, PL201869), according to which totarol is added to alcohol. In more detail, the main polymer is dissolved in a lactic acid solution at a stoichiometric ratio to the free amino groups in the main polymer in the range from 1:1 to 8:1, the solution is stirred until the main polymer is completely dissolved, then the primary alcohol is added in an amount of at least 5 wt. parts together with dissolved totarol, mix until completely homogeneous, and the separated mixture is applied to the surface of the cellulose derivative particles, stirring until uniform wetting. The resulting product is dried, during which the primary alcohol is removed, and the product is then dispensed into containers or further processed into the desired form.

"Totarol is extremely active against clinical strains of pathogenic anaerobic bacteria Gardnerella vaginalis and Enterococcus faecalis, as well as a clinical strain of aerobic bacteria Streptococcus agalactia with a very low minimum inhibitory concentration (MIC)."

Brief description of drawings

Fig. 1 - on the left side: necrotic cells (magnification 400×) after 8 hours of incubation with H ₂ O ₂ ; on the right side: necrotic cells (magnification 400×) after 24 hours of incubation with H ₂ O ₂ .

Fig. 2nd left side: absence of apoptosis and necrosis in epithelial cells (magnification 400×) after 8 hours of incubation - negative control; right side: absence of apoptosis and necrosis in epithelial cells (magnification 400×) after 24 hours of incubation - negative control.

Figure 3 left side: absence of apoptosis and necrosis in epithelial cells (magnification 400×) after 8 hours of incubation - solvent control; on the right side:

absence of apoptosis and necrosis in epithelial cells (magnification 400×) after 24 hours of incubation - solvent control.

Fig. 4th left side: absence of apoptosis and necrosis in epithelial cells (magnification 400×) after 8 hours of incubation - totarol; on the right side: absence of apoptosis and necrosis in epithelial cells (400× magnification) after 24 hours of incubation - totarol.

Carrying out the invention

The composition according to the present invention is intended for use in the prevention and treatment of inflammation of the vaginal mucosa. It is characterized by high efficiency and selectivity of antibacterial activity, thanks to the use of a strictly defined amount of totarol as an active substance. In addition, the composition may contain substances exhibiting antifungal and antiprotozoal activity.

The most important unique advantage of the composition according to the present invention in its described form is that the highly selective antibacterial activity of totarol, directed against pathogenic bacteria, does not disrupt the correct biocenosis and does not destroy lactic acid rod-shaped bacteria of the genus Lactobacillus, which are the most significant component of the physiological flora. An additional agent that stimulates the growth and restoration of the correct vaginal flora is lactic acid, which, due to its regulating function of lowering the pH value, forms a barrier against microbes sensitive to an acidic environment and creates favorable conditions for the growth of bacteria of the genus Lactobacillus.

In solutions currently used, the therapeutic substance in the form of an antibiotic disrupts the pathogenic flora, while simultaneously disrupting the physiological flora and lactic acid rod-shaped bacteria, which are its main component. The pharmaceutical composition according to the present invention, due to the presence of an antibacterially active concentration of totarol, determined experimentally, acts in a specific and selective manner only on pathogenic bacteria that are the cause of inflammation caused by BV or AV. This concentration was determined as a result of a research process first carried out by the inventors of the present invention. The additional presence of lactic acid in the base polymer buffered composition acidifies the environment and supports the growth and development of existing Lactobacillus bacteria. Thanks to this, the composition exhibits very high efficiency with the simultaneous absence of adverse effects on the physiological biocenosis, which limits relapses of the disease recorded in most patients after treatment of BV or AV, which occur when classical methods of treatment are used. At the same time, the composition can utilize the known ability to precisely regulate the pH value by changing the ratio of lactic acid to the base polymer. The composition adheres well to the entire surface of the mucous membranes, which prolongs its residence time in the vagina and ensures uniform contact with the antibacterial agent in the form of totarol.

The composition to be used in the case of mixed infections can be obtained by supplementing the main composition of the composition with substances that have profiled antifungal or antiparasitic activity, when choosing a pH value suitable for treatment. The composition can also be used for prophylactic purposes or as a product for daily intimate hygiene. The composition in the form of a free-flowing powder can be used for direct gynecological treatment in the form of a powder or for the preparation of preparations in other forms such as gels or vaginal solutions after adding an appropriately selected amount of purified water, or for the preparation of tablets after mixing with known substances that promote tablets, or vaginal suppositories. The target composition of all these final preparations can be supplemented with substances exhibiting antifungal or antiprotozoal activity.

The method according to the present invention produces a composition with selective antibacterial activity directed only against pathogenic bacteria. Thanks to the uniform application of totarol on particles of a chemically modified cellulose derivative, the effect of developing the contact surface of the active substance with the mucous membrane is achieved throughout the entire volume of the drug used. In addition, this solution significantly limited, and in some cases eliminated, the need to use large amounts of water-miscible organic solvents in the final products to dissolve the hydrophobic totarol. At the same time, as a result of the buffering effect of the main polymer, the effect of gradual and prolonged release of the active substance is achieved along with lactic acid, which regulates the pH value of the vaginal environment. The described activity could not be obtained using the complex known from the descriptions of Polish patents Nos. 166898, 194437, 201868 and 201869. The composition obtained by the method according to the present invention exhibits its good adhesion to the entire surface of the mucous membranes, which prolongs the residence time inside the vagina and ensures uniform contact with the antibacterial agent in the form of totarol.

The present invention also relates to a composition as defined above for the preparation of a preparation for the maintenance treatment and relief of symptoms in the treatment of inflammation of the vaginal mucosa of bacterial origin. The composition is particularly useful in cases of recurrent infections of the vaginal mucosa. The present invention is presented in more detail in the examples.

Example 1.

Testing the antibacterial activity of totarol using DMI (dimethylisosorbide) as a solvent.

A suspension of 0.5 McF in physiological solution (suspension density 10 ⁸ CFU/ml) was obtained from a 24-hour culture of the standard strain. 1 ml of suspension was collected in 9 ml of TSB broth (10 ^-1 dilution). Suspensions with a dilution of 10 ^-6 were prepared as described. For further studies, a suspension with a cell density of 10 ⁵ CFU/ml was chosen. For each strain, two 96-well plates were prepared: the first was intended for measuring OD, the second for quantitative cultivation on solid medium.

A/ 100 pl of broth was added to wells B1-D1, B2-D2 and B-D 4-11; B/ 200 pl of broth was added to wells B3-D3; C/ 1 pl of stock solution I was added to wells B1-D1; D/ 1 pl of stock solution II was added to wells B2-D2; E/ 2 pl of stock solution III was added to wells B3-D3.

Using a multichannel pipette, 100 pL of sample was collected from each of wells B3-D3 and placed in wells B4-D4 (mixing by sucking and releasing the contents 5 times). Pipette tips were replaced and 100 μl of contents were collected from wells B4-D4 and placed in wells B5-D5, mixing as described previously. These operations were repeated until wells B11-D11. The contents in wells B11-D11 were mixed, and 100 μl of liquid was collected and discarded. In this manner, a series of dilutions of totarol samples in DMI ranging from 0.32% to 0.0004% were obtained. Then 100 pl of the corresponding bacterial culture was added to wells B, C, D 1-11. As a result, a change in the final concentration range of up to 0.16% -0.0002% was achieved.

Gaining control:

Negative control: 100 pl of broth + 1 pl of DMI was added to well A1. Positive control (I): 100 pl of bacterial culture broth was added to wells E1-G1.

Positive control diluent (II): 100 pl of bacterial culture broth + 1 pl of DMI was added to wells E2-G2.

The OD value in individual wells was obtained at a wavelength of 620 nm (reading time 0). From a second 96-well plate, the culture was quantitatively inoculated from each dilution into a well of appropriate solid medium and incubated for 24 hours at 37°C under aerobic or anaerobic conditions, depending on the strain. The plates were incubated under aerobic conditions or in an atmosphere with an increased concentration of CO ₂ (for strains that require it) in an incubator at 37°C. OD values were obtained after 8, 16 and 24 hours, respectively. Before each OD measurement, the contents of the wells were mixed gently. In parallel with the reading of OD values, cultures were inoculated from a second 96-well plate, from each dilution, into plates with a suitable solid medium and incubated for 24 hours at 37°C under aerobic or anaerobic conditions.

To analyze the experimental results, we took the average of OD measurements obtained for three repetitions at separate time points (0, 8, 16 and 24 h) for individual microbes (Gardnerella vaginalis, Streptococcus agalactiae, Enterococcus faecalis, Lactobacillus gasseri/plantarum). In addition, the number of bacteria grown on solid media was taken into account. The results are presented in tables 1, 2, 3, 4 and 5. The test results confirm the specific concentration-dependent selectivity and high antibacterial effectiveness of totarol against the strain of pathogenic microorganisms: Gardnerella vaginalis, Streptococcus agalactiae, Enterococcus faecalis, with low MIC values for bacteria of the genus Lactobacillus .

Example 2

Testing the effects of totarol on human vaginal epithelial cells of the A-431 cell line (ATCC® CRL-1555™)

The study was performed using the following test: Annexin-V-Fluos (Roche, Mannheim, Germany) during incubation for 2, 8 and 24 hours, in vitro, according to the instructions of the test manufacturer. A totarol sample was prepared by dissolving 3 mg of totarol in 3 ml of 100% DMI.

The cultivation period of the A-431 cell line was 20 days. Cultivation was carried out at a temperature of 37°C in an atmosphere containing 10% CO ₂ in DMEM culture medium (Institute of Immunology and Experimental Therapy PAS [IITD PAN], Wroclaw), supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) . The culture fluid was replaced regularly every 48 hours. Once a detachable culture or so-called “monolayer” had been achieved, the cells were passaged using (for approximately 10 min) 0.25% trypsin (Sigma-Aldrich). The resulting tissue line was then passaged into 24-well plates (TPP), adjusting their densities to 5 × 10 ⁵ per well. Tissue cultivation of the studied lines was carried out over the next 3 days on the surface of sterile microscope slides placed on the bottom of a 24-well plate until a detachable grown culture was obtained on the surface of the microscope slides. After obtaining the so-called “monolayer,” the cells were washed with PBS without Ca ²⁺ and Mg ²⁺ ions (IITD PAN, Wroclaw) and transfused into 700 μl of fresh DMEM with 10% FBS along with 300 μl of properly prepared totarol.

Negative control: 1000 µl fresh DMEM with 10% FBS.

Solvent control: 700 µl fresh DMEM with 10% FBS + 300 µl DMI solvent.

Necrosis positive control: 900 µl fresh DMEM with 10% FBS + 100 µl H ₂ O ₂ (30%).

Proper testing using the Annexin-V-Fluos test causes apoptotic cells to turn green. Propidium iodide (PI) was used to stain necrotic cells. This dye has the ability to freely penetrate into the cell only through a damaged cell membrane, staining it red inside. In the absence of damage to the cell membrane, propidium iodide remains on the cell surface, forming the characteristic red “halogen” environment of the cell. After the appropriate study time, that is, after 2, 8 and 24 hours, the culture liquid was removed and the contents were washed twice with PBS (preheated to 37°C). Then, 100 μl of the fluorescent stain mixture included in the Annexin-V-Fluos kit (Roche) was applied to the surface of each microscope slide (placed in the culture well) and additional Hoechst stain (which stains cellular DNA) was applied. The staining procedure was carried out in accordance with the manufacturer's recommendations. The culture along with the dyes applied to it was incubated at room temperature for 20 minutes. The intensity of light emitted by cells stained with individual stains was observed using a fluorescence microscope at a wavelength of 494-520 nm using 400x magnification. Cells were counted in five fields of view and the results were averaged. The reliability of the tests was confirmed by using hydrogen peroxide as a positive control, causing a clear necrosis process after only 2 hours of incubation. Data analysis was performed based on the average number of cells from five fields of view at individual time points (2, 8, and 24 h). The results are presented in Table 6 and in the form of photo documentation - Fig. 1, 2, 3, 4. Test results prove the absence of apoptotic and necrotic effects of totarol on human vaginal epithelial cells A-431 over a wide range of incubation times (2-24 hours).

Example 3. Preparation of the composition in powder form Composition ingredients:

1. totarol 0.1 wt. parts

2. lactic acid 1.0 wt. parts (2 moles)

3. Eudragit E-100 1.5 wt. parts (1 mol)

4. methylcellulose 97.4 wt. parts

The composition was prepared by dissolving Eudragit in lactic acid at the stoichiometric ratio specified in the composition in a mixer equipped with a mechanical stirrer, stirring for 20 minutes, then leaving the entire mixture for 24 hours. After this time, the resulting component was dissolved in 95% ethyl alcohol using 10 wt. parts of alcohol and 0.1 wt. part of totarol based on the total amount of ingredients. The entire mixture was stirred until the component dissolved, then the resulting solution was sprayed through a nozzle onto methylcellulose placed in the mixer of the spray nozzle, and stirred until the methylcellulose was evenly wetted, but for no less than 10 minutes. The wet product was dried at 25°C in a forced air dryer with a solvent condenser. During drying, ethyl alcohol was removed. A loose powder was obtained, which was mixed until completely homogenized and dosed into plastic or glass containers.

Example 4. Preparation of the composition in gel form Ingredients of the composition:

1. totarol 0.5 wt. parts

2. lactic acid 0.5 wt. parts (1 mol)

3. chitosan 0.83 wt. parts (1 mol)

4. methylcellulose 98.2 wt. parts

5. purified water 25 wt. parts based on the total number of ingredients

The composition was prepared similarly to that described in Example 3, but water was added with stirring to the resulting dry product in powder form and then, after obtaining a homogeneous mixture, left for 30 minutes without stirring. The resulting gel was dosed into plastic containers with applicators.

Example 5. Preparation of the composition in the form of tablets Ingredients of the composition:

1. totarol 0.08 wt. parts

2. lactic acid 0.5 wt. parts (1 mol)

3. polyvinylpyrrolidone-90 5.0 wt. parts (1 mol)

4. methylcellulose 94.4 wt. parts

The composition was prepared in a similar manner as in Example 3, but known tableting agents were added to produce a dry product in powder form, if necessary, and the product was then tableted in a known manner to obtain suitable therapeutic dosages.

Example 6. Preparation of the composition in the form of vaginal suppositories Ingredients of the composition:

1. totarol 0.5 wt. parts

2. lactic acid 4.0 wt. parts (8 moles)

3. chitosan 0.83 wt. parts (1 mol)

4. methylcellulose 94.4 wt. parts

Auxiliary products based on the total number of ingredients in the composition:

1. gelatin 16.5 wt. parts

2. purified water 83.5 wt. parts

The composition was prepared in a similar manner as in Example 3, but the resulting dry product in powder form was dissolved in a part of water to obtain a gel, to which was added an aqueous solution of gelatin (obtained from the remainder of the water) heated to a temperature of 90°C. The product was then mixed and poured into molds to form molded vaginal suppositories.

Claims (2)

1. The use of totarol as an active substance in an amount from 0.001 to 5 wt. parts for obtaining a drug for the treatment of inflammation of the vaginal mucosa of bacterial origin, for the relief of symptoms during such treatment and for the prevention and prevention of relapse of such inflammation, where said inflammation refers to anaerobic bacterial vaginosis (BV) and aerobic vaginitis (AV). 2. A pharmaceutical composition for the treatment of inflammation of the vaginal mucosa of bacterial origin, for the relief of symptoms during such treatment and for the prevention and prevention of relapses of such inflammation, where said inflammation refers to anaerobic bacterial vaginosis (BV) and aerobic vaginitis (AV), containing from 75 to 95 wt. parts of methylcellulose, from 0.5 to 5 wt. parts of lactic acid, from 0.5 to 5 parts by weight of a polymer selected from a copolymer of methacrylic acid and ethyl acrylate, a copolymer of dimethylaminoethyl methacrylate, butyl methacrylate, methyl methacrylate in a ratio of 2:1:1, chitosan, polyvinylpyrrolidone or mixtures thereof, where the stoichiometric ratio of lactic acid and the main polymer lies in the range from 1:1 to 8:1, characterized in that it contains totarol in an amount of from 0.001 to 5 wt. parts as active substance.

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