ITMI20112352A1 - VAGINAL COMPOSITION BASED ON PROANTOCYANIDINS - Google Patents

Description

Title: VAGINAL COMPOSITION BASED ON PROANTHOCYANIDINS

Description

Field of the invention

The present invention relates to a vaginal composition based on proanthocyanidins, in particular for the treatment of Streptococcus agalactiae infections.

State of the art

Group B streptococcus, or Streptococcus agalactiae (GBS), is the causative agent of severe neonatal infections in developed nations and is found in 15-35% of pregnant women. According to Blond et al. (1), analysis of 8 published studies showed that GBS was found in mothers from 7.6 to 22.8% of pregnancies. This percentage varies according to ethnicity and sampling sites, which can be the vagina alone or the vagina and rectum. The asymptomatic vaginal presence of GBS varies during pregnancy. Its presence in the vagina can be associated with vaginitis, urinary infection and increases the risk of chorioamniot ite. After childbirth, the consequences of GBS infection can be manifold, ranging from chorioamniotitis and postpartum endometritis, to bacteremia and septicemia. Infection in pregnant women can also lead to premature birth, premature rupture of membranes and low birth weight of the newborn. Infection in the contaminated infant can also cause septicemia accompanied by shock, pneumonia, acute respiratory distress syndrome and neurological infections such as meningitis which can lead to permanent handicaps and even death.

Antibiotic treatment of asymptomatic infection during pregnancy is not recommended. Pregnant women who are asymptomatic carriers of GBS should not be treated before labor as the latter does not reduce the level of bacteria found at the time of labor. Furthermore, a typical problem related to the administration of antibiotics is the onset of resistance phenomena that can nullify the effectiveness of the treatment at the time of delivery.

Therefore, the normal therapy is to treat the woman during childbirth with intravenous antibiotics, which however does not ensure the total elimination of the risks to the unborn child.

The rate of infected newborns corresponds to 3 to 12% of pregnancies.

Publicly available data reports that 12,000 infants are infected and about 2,000 die each year in the US.

Bacterial vaginosis (BV) is also a vaginal infection that can affect pregnant women. BV is characterized by profound modification of the normal vaginal flora with disappearance of Lactobacilli and abnormal development of a polymorphic flora, including Gardnerella vaginalis, Atopobium vaginae and anaerobic microorganisms.

BV can cause miscarriage and premature birth and is associated with an increased risk of contracting HIV. All cases of BV should be treated during pregnancy.

Again, antibiotic treatment of BV is not sufficient to eradicate the infection.

BV and GBS cause high risks that impact the outcome of pregnancy, both for the newborn and for the mother.

Summary of the invention

It is therefore an object of the present invention to provide a treatment of vaginal infections that is safe and effective, so much so that it can be proposed both for the treatment of asymptomatic and symptomatic infections even during pregnancy.

This treatment is carried out according to the invention, by means of a bactericidal or bacteriostatic agent, alone or in combination with other active ingredients. The bactericidal or bacteriostatic agent of the invention belongs to the class of proanthocyanidins.

In particular, the treatment according to the invention is aimed at the prevention and treatment of Streptococcus agalactiae infections.

A further object of the invention is therefore a bactericidal or bacteriostatic vaginal formulation containing one or more active ingredients according to the invention, among which at least one is selected from the class of proanthocyanidins, together with pharmaceutically acceptable excipients and vehicles.

Particular objects of the invention are those recited in the attached claims, the definitions of which form an integral part of the present description.

Detailed description of the invention

The present invention relates to a compound or mixtures of several compounds belonging to the class of proanthocyanidins for use as bacteriostatic or bactericide in the prevention and treatment of bacterial infections of the vaginal tract for separate, sequential or combined administration.

Proanthocyanidins, also known as condensed tannins or procyanidins, are natural plant metabolites widely available in fruits, vegetables, nuts, seeds, flowers and bark of various plant species. Typical sources of proanthocyanidins are grapes and red wine, cranberry berries and the leaves of blueberry, birch, gingko and hawthorn. Proanthocyanidins are responsible for the blue-violet and red colors in plants.

Proanthocyanidins belong to the structural class of flavonoids, molecules with a polyphenolic structure. In particular, they are polymers or oligomers of tannins that derive from the oxidative condensation of the C4 carbon of a benzopyranic heterocycle of a tannin with the C6 or C8 carbon of another tannin. In one embodiment, the proanthocyanidins are in the form of cranberry extract (Vaccinium macrocarpon). Preferably, this extract is in the form of an aqueous solution having a concentration of active principle comprised between 60 and 75% w / v.

The term "bacterial infections of the vaginal tract" includes infections with Gardnerella vaginalis, Candida albicane, Neisseria gonorrheae, Atopobium vaginae, Chlamidia trachomatis, Trichomonas vaginalis, Mycoplasma genitalium, Treponema pallidum and Streptococcus agalactiae.

Bacterial infections of the vaginal tract are in particular Streptococcus agalactiae infections.

A further object of the invention consists of a bactericidal and / or bacteriostatic vaginal formulation comprising at least one compound belonging to the proanthocyanidin class, as defined above, possibly in association with other antibacterial agents, preferably but not exclusively selected from lauric acid, monolaurate and their blends.

Evaluation of the inhibitory and bactericidal activity of the_ compounds_ of the invention_ towards_ Streptococcus agalactiae

The experiments were conducted using a cranberry extract as an aqueous solution (substance E).

BACTERIAL STRAINS

Three strains of Streptococcus agalactiae isolated from vaginal-rectal swabs during pre-partum controls were used for the assay. The strains were named "strain 2", "strain 10" and "strain 11". Isolation was performed on blood agar medium and identification was achieved by API 20 Strep (Bio-Mérieux) biochemical tests and by identification of the Lancefield group.

The experiments were also conducted on an ATCC 12386 strain of Streptococcus agalactiae.

The strains were kept at a temperature of -80 ° C in a suspension of 20% glycerol blood broth.

CULTURAL LANDS AND SOLUTIONS

The "Brain Heart Infusion broth" (BHIb, Becton Dickinson) medium was used for the culture of Streptococcus agalactiae, "Mueller-Hinton broth" (M-Hb) medium with the addition of 2 % "lacquered" horse blood and "Mueller-Hinton agar" medium (M-Ha) with the addition of 5% defibrinated horse blood (Oxoid).

PREPARATION OF THE BACTERIAL INOCULUS

Strains of Streptococcus agalactiae were grown in BHIb broth for 24 hours at 37 ° C. Immediately prior to the assay, the bacterial suspensions were diluted until a turbidity equivalent to standard no. 0.5 from McFarland. 50 μΐ of an additional 1: 100 dilution in 2% blood broth was dispensed into the wells of the microtiter plates. The presumed titer of the inoculum performed with this procedure is approximately 5xl0 <4> cfu (colony forming units) / well.

The bacterial cultures were also serially diluted according to a value of 10 (up to a dilution value equal to 10 <7>) and an aliquot of 0.1 ml of each dilution was sown in duplicate on M-Ha blood 5% for the determination of the effective titer to be used, subsequently, for the determination of the MBC (Minimum Bactericidal Concentration).

PREPARATION OF THE DILUTIONS OF THE PRODUCT UNDER CONSIDERATION

The test substance was prepared for testing by dilution and sterilization as described below.

The concentration of substance E defined as "parent" represents the highest concentration at which it was possible to obtain complete solubilization and is 4 times higher than the highest concentration tested in the test.

Solvent substance cone, "mother" AND water 26.2% DETERMINATION OF THE MINIMUM INHIBITING CONCENTRATION

(MIC)

The assay was performed in 96-well microtiter plates. In the first well of each of the 2 rows, 50 μΐ of M-Hb at normal concentration were deposited. Starting from the first well of each row, volumes of 50 μΐ were transferred from each well to the next 10 times, thus obtaining a series of consecutive doubling dilutions. The 12 well of each row was kept free of substance as a positive bacterial growth control.

Subsequently, in all wells of the plate, with the exception of the 11th of each row, 50 μΐ of double concentration (4%) bacterial M-Hb blood inoculum was deposited. In the 11th well, only double concentration M-Hb blood was deposited but without bacteria, in order to have a negative control for each row. The scheme described was used for the assay of each of the three strains of Streptococcus agalactiae.

After incubation at 37 ° C for 24 hours, the microtiter plates were examined for the presence or absence of bacterial growth in each well. For each substance, the minimum inhibitory concentration was determined, defined as the lowest concentration capable of inhibiting bacterial growth or preventing clouding of the liquid inside the well.

DETERMINATION OF THE MINIMUM BACTERICIDE CONCENTRATION (MBC)

Immediately after determining the MIC value for each substance, a volume equal to 50 μΐ was taken from each well and seeded on the surface of plates containing 5% M-Ha blood. After incubation at 37 ° C for 24 hours, the number of colonies grown on the surface of the medium was counted, thus determining the number of bacteria surviving after 24 hours of contact with the substance at the concentration present in the well. By comparing the number of surviving bacteria in each well and that of the bacterial inoculum initially deposited, the MBC value was determined, defined as the lowest concentration of each substance capable of reducing the bacterial load by 99.9%, in the previously defined test conditions.

DETERMINATION OF THE MINIMUM INHIBITING CONCENTRATION

(MIC) ON AGARIZED SOIL

The assay was performed on 5% blood M-Ha medium. The medium was prepared at a concentration 33% higher than the final one used in the test which corresponds to that indicated by the supplier. After sterilization in an autoclave, the medium was equilibrated to a temperature of 48 ° C and added with horse blood at a concentration of 6.6% (33% higher than the final one of the assay). Aliquots of 15 ml of M-Ha blood medium were transferred into 50 ml falcon tubes kept at 48 ° C, added with 5 ml of the dilutions of the substances to be tested, mixed, poured into Petri dishes and allowed to solidify. Twenty-five bacterial inocula, each approximately approximately 5 μΐ in volume and containing approximately 10 <5> cfu, were deposited on the surface of the M-Ha blood medium of each plate. After incubation at 31 C for 18 hours, the reading of the minimum inhibitory concentration (MIC) was carried out, defined as the minimum concentration of substance capable of preventing bacterial growth detectable with the naked eye in correspondence with the inoculum deposition area.

RESULTS

Due to the turbidity of the solutions containing the substances, it was not possible to determine with sufficient reliability the minimum inhibitory concentrations in the assay in liquid medium for which only the minimum bactericidal concentration values are reported. To obtain the MIC values we proceeded with the "Determination of the Minimum Inhibitory Concentration on Agarized Medium".

Table I shows the bacterial concentration values of the cultures used to prepare the inocula and the concentrations of the inoculum suspensions.

Tables II and III show the MIC and MBC values of the test substances respectively against the bacterial strains tested.

Table I Concentration of Streptococcus agalactiae bacterial cultures and suspensions used as inoculum

Table II - Bactericidal activity of substance E expressed as MBC

E = cranberry extract (69% native extract), aqueous solution at 26.2% w / v (stock solution)

Table III - Inhibitory activity of the test substances expressed as MIC on AGARISED medium

CONCLUSIONS

The MBC values appear similar between the ATCC 12386 strain and the bacterial clinical isolation strain # 2, while they are slightly higher for the isolates n. 10 and n. il (Tables II and III). The higher values observed with strain 10 are attributable to a greater resistance shown by strain 10 towards the antibacterial agents tested, as this strain was found to be more resistant towards the reference antibiotic treatment, ampicillin. Observations similar to those reported for the MBC test are also valid for comments on the results of the MIC test (table III).

In conclusion, the experiments show that the tested substance E has bacteriostatic and bactericidal activity both on ATCC strains of Streptococcus agalactiae, and on bacterial strains isolated from patients, and even more importantly on the ATCC strain representing serotype III responsible for at least the 60% of early neonatal infections, also called Early Onset Diseases, which are associated with the highest morbidity rate and higher risk of neonatal mortality.

Although an antibacterial activity has already been reported for proanthocyanidins, it has been related only to an inhibition of bacterial adhesion, thus suggesting the use of such substances in preventive treatments. On the other hand, no direct bactericidal or bacteriostatic activity has ever been reported. This evidence allows the use of proanthocyanidins in the treatment and eradication of an existing vaginal infection.

According to the present invention, the dose of the compounds proposed for administration to a woman (with a body weight of about 70 kg) ranges from 0.01 mg to 5 g and, preferably from 1 mg to 1 g of the active ingredient per dose unit. The dose unit can be administered, for example, 1 to 4 times a day. It should be considered that continuous dose adjustments may need to be made depending on the severity of the clinical condition to be treated. The exact dose is at the discretion of the treating physician.

The treatment according to the invention may comprise the administration of formulations containing the above compounds starting from the 32nd or 35th week of pregnancy until delivery or, if necessary, even afterwards. In fact the compounds of the invention are endowed with a very low toxicity and do not give resistance phenomena.

The bactericidal and / or bacteriostatic vaginal formulations according to the invention can for example be in the form of a vaginal gel, a vaginal douche, a vaginal cream, ointment, vaginal foam, vaginal tablets, hard and soft vaginal capsules. The pharmaceutical forms mentioned above can be immediate release or modified release according to the need.

The introduction of the posology unit into the vaginal cavity can be facilitated by the use of suitable specific techniques (vaginal applicators, syringes, tampons, etc ...).

To facilitate the incorporation of the active principles object of the present invention, a solvent is always provided. Although several compounds can be used for this purpose, water is always the solvent of choice due to its greater biocompatibility. For this purpose, non-aqueous compounds such as glycols, such as propylene glycol, butylene glycol, ethylene glycol, hexylene glycol, polyethylene glycol, etc can also be considered; and alcohols, such as ethanol, propanol, isopropanol; and related mixtures.

Typically the solvent is present in quantities higher than about 75%, in some formulations it can be higher than about 90%, finally in other cases it can be between about 90% and about 99.99% of the final formulation.

EXAMPLE 1 - vaginal gel

Bibliographical references

1.Blond MH, Poulain P Gold F, Bingen E, Watier H Quentin R. Infection bactérienne maternal-fetal EMC 2004 Obstétrique you 2, page 14.

Claims (7)

CLAIMS 1. Compound or mixture of several compounds belonging to the class of proanthocyanidins for use as bacteriostatic or bactericide in the prevention and treatment of bacterial infections of the vaginal tract for separate, sequential or combined administration. Compound according to claim 1, wherein said bacterial infections of the vaginal tract include infections with Gardnerella vaginalis, Candida albicane, Neisseria gonorrheae, Atopobium vaginae, Chlamidia trachomatis, Trichomonas vaginalis Mycoplasma genitalium, Treponema pallidum and Streptococcus agalactiae. Compound according to claim 1, wherein said bacterial infections of the vaginal tract are Streptococcus agalactiae infections. Compound according to any one of claims 1 to 3, wherein said compound or mixture of several compounds is an extract of Vaccinium macrocarpon. Compound according to any one of claims 1 to 4, wherein said treatment provides for the administration of said compound starting from the 32nd or 35th week of pregnancy. 6. Bactericidal and / or bacteriostatic vaginal formulation comprising at least one compound according to claim 1, optionally in association with an active principle selected from lauric acid, monoglycerine ester of lauric acid and their mixtures. 7. Formulation according to claim 6, wherein said formulation is selected from a vaginal gel, a vaginal douche, a vaginal cream, ointment, vaginal foam, vaginal tablets, hard and soft vaginal capsules.