Related application:

This application claims priority from our Indian Patent Applications No. 945/MUM/2010 dated 29/03/2010 and No. 94/MUM/201 1 dated 1 1/01 /201 1 . The contents of which may be treated as incorporated herein by reference.

Field of Invention:

The present invention relates to a synergistic liquid vaginal spray composition. More particularly, the present invention relates to a synergistic vaginal spray composition containing lactic acid and at least one antifungal agent for the treatment of vaginal fungal infections such as vulvovaginal candidiasisor "yeast" infection bacterial vaginosis. Trichomoniasis, Chlamydia vaginitis. Gonococcal vaginitis. Viral vaginitis. Noninfectious vaginitis. The present invention also relates to process for preparation of the said composition.

Background of Invention:

The normal ecosystem of the vagina involves many different organisms that are involved in a system of checks and balances. No other concept in vaginal health is as important as the state of the ecosystem of the vagina. The flora that colonizes the vagina takes place in the birth canal during delivery, and the flora that is established in the newborn girl must therefore consist -of the same strains as in the mother. The vaginal environment of a newborn changes during the first month, then again at prepuberty, puberty, during the reproductive years and post-menopausally. Additionally, the cyclic hormonal changes of the menstrual cycle also influence the vaginal ecosystem. It is a variable state throughout a woman's lifetime, but nothing is more key to this ecosystem than lactobacil!us. The range of bacterial types isolated is immense, including Staphylococcus species, Garnerella vaginalis. Streptococcus species, Bacteriodes species, Lactobacillus species, Mobiluncus, Candida species, (most commonly Candida albicans), and more. The predominant organisms isolated from the normal vagina are members of the Lactobacillus genus. One species of bacteria inhibits another and an elegant combination of pH, vaginal immunity, hormonal activity and the community of organisms that occupy the vagina interact in a manner in which bacterial overgrowth is controlled, unless the well- established mechanism of balance is thrown off by one factor or another.

A flora that inhabits vaginal surface can be categorized as "resident" flora and "transient" flora.

"Resident" flora generally refers to microflora which generally "colonizes" a particular tissue surface. In case of vagina the flora mainly comprises Lactobacilli genus. "Transient" flora generally refers to the flora which may inhabit a tissue surface, may be for days to months, but never more than that. The transient flora is mainly comprises pathogenic organism, which are, in healthy people, kept under check by the resident flora. Resident flora, in effect, acts as a first line of defense against variety of pathogens and pathologies.

Lactobacillus helps in maintaining the environment of vagina. Lactic acid is produced by the metabolism of lactobacillus and although there may be other ways in which the vagina maintains its normal acidic environment, the role of lactobacilli seems evident. Lactobacilli thrive at an acidic pH of 3.5-4.5 and these values are indeed found in the normal vagina throughout the menstrual cycle.

Lactobacilli have also been shown to interfere with how pathogenic bacteria adhere and colonize the cells of the vagina. Lactobacilli also act directly as antibacterials and may function as an immune stimulant locally in controlling microbial levels in the vagina.

As seen from above, role of lactic acid, produced by Lactobacilli by fermentation of glycogen, in maintaining the status quo has been very well documented, for example, in article by Maria Silvina Juarez Tomas et al, published as "Growth and lactic acid production by vaginal Lactobacillus acidophilus CRL 1259, and inhibition of uropathogenic Escherichia colf Journal of Medical Microbiology (2003), 52, 1 1 17- 1 124, states effect of lactic acid produced by L. acidophilus, which is generally found as a part of vaginal flora. Further, an article by Gupte, Pushpaet et al. published in Indian Journal of Sexually Transmitted diseases and AIDS 2009, Vol. 30, No. 2 clearly states the importance of the lactic acid in maintaining normal health of vulva.

In an event that the natural defenses of body fail, the person needs to be treated by a pharmaceutical agent which can control the fungal infections, usually an antifungal agent. Antifungal drugs/agents are medication used to treat fungal infections such as athlete's foot, ringworm, candidiasis (thrush), serious systemic infections such as cryptococcal meningitis, and others. Such drugs are usually obtained on doctor's prescription or purchased over-the-counter.

Antifungals work by exploiting differences between mammalian and fungal cells to kill the fungal organism without dangerous effects on the host. Unlike bacteria, both fungi and humans are eukaryotes. Thus fungal and human cells are similar at the molecular level. This makes it more difficult to find or design drugs that target ffhgi without affecting human cells. Consequently, many antifungal drugs cause side-effe!ets. Some of these side-effects can be life-threatening if the drugs are not used properly.

The imidazole and triazole drugs are synthetic antifungal drugs that inhibit the enzyme cytochrome P450 14a-demethylase. This enzyme converts lanosterol to ergosterol, and is required in fungal cell membrane synthesis. These drugs also block steroid¾synthesis in humans.

Since, both lactic acid and an antifungal agent inhibits proliferation of pathogenic organisms, particularly fungi, by making the environment unfavorable for the pathogens to thrive, a combined, synergistic use may also be envisaged.

US Publication No. 20070286812 discloses a nasal formulation comprising pharmaceutical antifungal agents along with and natural antifungal agents (Lactobacillus species) in a solid, liquid or aerosol form.

US Patent No. 7125708 relates to a composition comprising Lactobacillus strain NCIMB 41 1 14 and an antifungal agent for the treatment of yeast infection. Indian Patent Application No. 675/KOL/2004 (EPl 506781 ) disclosed a vaginal care composition comprising viable Lactobacillus and/or Bifidobacterium microorganisms; non-viable Saccharomyces cultures; vitamin A/retinol, and zinc.

For instance, European Patent No. 1764100, filed by Johnson & Johnson Consumer Companies, Inc. discloses a buffered composition for lowering vaginal pH. The said Patent suggests that lactic acid and an antifungal agent for lowering and maintaining pH of vaginal surface in range of 3 to 4.5. The invention is directed to reducing self- perceived vaginal odor by maintaining the pH in normal range. Further, the composition in the said Patent is in the form of suspensions, emulsions, clear and opaque gels, semisolid systems, including ointments, pastes, oil-in-water (o/w) creams, semisolid emulsions with solid internal phases, semisolid emulsions with fluid internal phases, vaginal suppositories, insertable tablets, soft or hard gelatin capsules and the like.

US Publication No. 2010173965 relates to use of an azole compound with an organic acid with a view to enhance absorption.

Although suppositories have attained some success, they have some disadvantages. Most of the current commercial vaginal suppositories, either melt or dissolve in the vaginal tract into an oily or aqueous liquid. This resulting liquid in turn tends to leak out or is expelled out of the vaginal cavity resulting either in soiled clothing and/or inferior efficacy. The applicator is very long, e.g. suppository (approx. 1 1 cm) and gel (approx. 12 cm) which produces more risk and fear to the patients.

In view of the above, none of the above prior arts discloses the combination of lactic acid with an antifungal agent in a liquid vaginal spray dosage forms. Hence, the current inventors ^' have come up with a novel synergistic liquid vaginal spray dosage form comprising lactic acid in combination with at least one antifungal agent in unique blend of solvents that provide relatively better effect than other dosage forms like vaginal tablets or capsules and messy application of gel and creams. Summary of Invention:

In accordance with above, the present invention provides a synergistic combination of lactic acid and at least one antifungal agent in liquid vaginal spray dosage form for treatment of vaginal fungal infection such as vulvovaginal candidiasis or "yeast" infection bacterial vaginosis, Trichomoniasis, Chlamydia vaginitis, Gonococcal vaginitis, Viral vaginitis, Non-infectious vaginitis.

In an aspect, the composition of the present invention, afford a better patient compliance, better bioavailability, when compared to painful intramuscular injections, suppositories such as vaginal capsules or tablets and messy application of gel and creams, as the present formulation is in solution form that can administered by spraying method.

In another aspect, the antifungal drugs of the present invention give the local as well as systemic effect via vaginal route whereas lactic acid maintains the desired vaginal pH levels. Maintenance of vaginal pH helps to cover the loss of lactic acid due to infection, which is otherwise naturally produced by lactobacilli into vagina.

In yet another aspect, the present invention provides a process for preparing the said liquid vaginal spray composition.

Detailed Description of Invention:

The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

For purpose of current invention, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. In an embodiment, the invention provides a synergistic liquid vaginal spray dosage form to provide local as well as systemic effect, comprising therapeutically effective amount of at least one antifungal agent along with lactic acid, useful for the treatment of vaginal fungal infections like vulvovaginal. Candidiasis or "yeast" infection bacterial vaginosis, Trichomoniasis, Chlamydia vaginitis, Gonococcal vaginitis, Viral vaginitis, Noninfectious vaginitis.

In another embodiment, the invention provides novel mode of delivery for treatment of vaginal fungal infections like Candidiasis or "yeast" infection Bacterial Vaginosis, Trichomoniasis, Chlamydia vaginitis, Gonococcal vaginitis, Viral vaginitis, Noninfectious vaginitis which provides better patient compliance as compare to painful intramuscular injections, as it is in solution form.

In preferred embodiment, the invention provides a liquid vaginal spray dosage form to provide local as well as systemic effect, comprising therapeutically effective amount of at least one antifungal agent along with lactic acid in association with a unique blend of solvents and co-solvents, useful for the treatment of vaginal fungal infections like Candidiasis or "yeast" infection bacterial vaginosis, Trichomoniasis, Chlamydia vaginitis, Gonococcal vaginitis, Viral vaginitis, Non-infectious vaginitis. The blend will be of solvents and co-solvents in their suitable ratio, the ratio of solvent and co-solvent will depends on the drug.

The composition of the present invention further comprises other pharmaceutically acceptable polymers mainly used for bio adhesion and to maintain proper viscosity of formulation. Suitable polymer are selected from ethyl cellulose, polyvinylpyrrolidone, hydroxyl propyl methyl cellulose, hydroxyl propyl cellulose, hydroxyl ethyl cellulose, ethyl cellulose either alone or in combination is present in an amount of 0.5 to 10%. ,

The composition of the present invention has better patient compliance than vaginal tablets or capsule or painful intramuscular injection and messy application of gel and creams, as the current formulation is dispensed in a mist form to cover a larger area of vaginal cavity with very small amount of dispensed volume, thus increasing the bioavailability of the said composition. Suitable antifungal agents are selected from the group of Miconazole, Ketoconazole, Clotrimazole, Econazole, Bifonazole, Butoconazole, Fenticonazole, Isoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Griseofulvin, Fluconazole Fosfluconazole, Itraconazole, Posaconazole, Voriconazole, Amorolfine, Butenafine, Naftifine, Terbinafine, Terbinafine, Natamycin, Nystatin, Amphotericin B, Thiabendazole, Anidulafungin, Caspofungin, MicafunginFlucytosineor their pharmaceutically acceptable salts alone or in combination thereof. The said antifungal agent(s) is present in the range of 5% to 25% to achieve the desired therapeutic benefit.

The said lactic acid is present in range of 2-10 mg preferably 4 mg i.e. approximately 1 to 10%. The ratio of antifungal agent and lactic acid is 25: 1 .

Suitable solvents and co-solvents are selected from group of polyethylene glycol, diethylene glycol monoethyl, glycofurol, benzyl alcohol, labrasol, Benzyl benzoate and Ethyl oleate, propylene glycol, Diethylene Glycol Monoethyl Ether, ethyl alcohol, cremophore ELP, Solutol, transcutol, capmul, captex and their combinations like in. desired amount of about 5% to 90%.

The composition of the present invention can be prepared by the conventional methods such as simple mixing wherein solvents are mixed in the vessel followed by addition of polymer and an antifungal agent. The mixture is heated under continuous stirring to achieve complete dissolution of the active agent and the excipients and then the said mixture cooled. After cooling the mixture lactic acid is added under continuous stirring and the -formulation so obtained is stored in airtight closed container in light resistant area.

In another embodiment, the invention provides a spray dispenser comprising therapeutically effective amount an antifungal agent with lactic acid in association with a unique blend of solvents and co-solvents. The composition is filled in a convenient container that can give the formulation in form of fine droplets or mist. The container assembly is specifically designed to provide drug dispensing directly into vagina in unit dosage form. Hence, it is easy to deliver in to vaginal orifice. The composition of the present invention is administered via metered dosing spray nozzle to deliver accurate dose of drug.

The invention provides a composition which is in a liquid vaginal spray dosage form comprising therapeutically effective amount of an antifungal agent with lactic acid in association with a unique blend of solvents and co-solvents, wherein the said composition does not crystallize at very low temperature during storage.

In the most preferred embodiment, the present invention provides a liquid vaginal spray dosage comprising therapeutically effective amount of Clotrimazole in combination with lactic acid in unique blend of solvent, useful for treatment of infections such as valvovaginal candidiasisor "yeast" infection, bacterial vaginosis, Trichomoniasis, Chlamydia vaginitis, Gonococcal vaginitis, Viral vaginitis, Noninfectious vaginitis.

Total volume of the formulation is 5 ml and Clotrimazolein its base form is present in amount of 25% in combination with lactic acid 1 %. Ratio of the both actives in the » - formulation is 25: 1 . Other excipients such as polyethylene glycol is present in an amount ;, _; of 23.9%, diethylene glycol monoethyl ether in an amount of 23.8%, glycofurol in an amount of 23.8%, benzyl alcohol in ^' an amount of 0.5% and ethyl cellulose in an amount >, of 2%.

In a further embodiment, the present invention provides a method of treating fungal infections such as Vulvovaginal Candidiasis or "yeast" infection, bacterial vaginosis, Trichomoniasis, Chlamydia vaginitis, Gonococcal vaginitis, Viral vaginitis, Noninfectious vaginitis, which comprises administering 'an effective amount' of the 'liquid vaginal spray composition comprising at least one antifungal agent with lactic acid' to the subject suffering from the said fungal infections. The subject mentioned herein is human.

The invention further discloses use of the 'spray composition of the invention comprising at least one antifungal agent with lactic acid' intended to treat fungal infections such as vulvovaginal Candidiasis or "yeast" infection, bacterial vaginosis, Trichomoniasis, Chlamydia vaginitis, Gonococcal vaginitis, Viral vaginitis, Noninfectious vaginitis. Clinical Trial was conducted to study improved efficacy of vaginal spray comprising Clotrimazole and lactic acid of present invention (described in example 3) on human subjects with vulvovaginal candidiasis.,

The aforesaid trial has been performed according to ICH - GCP guidelines and schedule Y. Total 1 1 patients were screened and 10 patients were enrolled into study out of which nine patients completed the study. Patients were informed about the spray, dose and the way of administration at the time of enrollment. There was no serious or adverse event occurred throughout the trial. Patients Case Report Forms (CRF) was filled during and after completion of the dosage regimen of three alternative days for each patient respectively. The data was collected, compared and analyzed from the Case Report Form (CRF).

Patients received Clotrimazole 100 mg/0.8 gm with lactic acid 4 mg/0.8 gm (4 sprays) . ^■ ? vaginal Spray once daily at bed time for six consecutive days. Efficacy was evaluated by ^■■ ■ ^■ suppression of symptoms. Most of the symptoms were suppressed within three days _: although the recommended therapy with existing formulation is for six days. All patients have completed the study successfully, except one which was considered as treatment s failure. No adverse event occurred dnring the study.

The study shows that Clotrimazole with lactic acid liquid vaginal spray is highly effective in patients suffering from the infection of valvovaginal candidiasis.

The following example, which includes preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

Example: Example: 1

Manufacturing Process:

Step 1 : Weighing accurately Polyethylene Glycol 400, Diethylene Glycol Monoethyl Ether, Glycofurol, and Benzyl Alcohol in S.S.Vessel and mixing all by continuous stirring;

Step 2: Weighing accurately Ethyl cellulose and dissolving into step 1 ; : Step 3: Dissolving Clotrimazole IP into mixture of Polyethylene Glycol 400, Diethylene Glycol Monoethyl Ether, Glycofurol, and Benzyl Alcohol, Ethyl cellulose by heating the mixture at 70°C in Manufacturing Vessel with continuous stirring;

Step 4: Then, immediately transferring in to another clean vessel and allowing it to cool; Step 5: After cooling adding lactic acid with stirring;

Step 6: Store the formulation in airtight closed container in light resistant area.

Example: 2

SR.NO INGREDIENTS LABEL CALIM

(200 mg/spray)

1 Fluconazole 100 .

2 Lactic Acid 4

3 Propylene Glycol 40

4 Glycofurol 34

5 Labrasol 15 6 PVP-K 30 3

7 Benzyl Alcohol 4

Total volume 200mg/spray

Manufacturing process:

Step 1 : Weighing accurately Polyethylene Glycol 400, Labrasol, Glycofurol, and Benzyl

Alcohol in S.S. Vessel and mixing all by continuous stirring;

Step 2: Weighing accurately PVP-K 30 and dissolving into step 1;

Step 3: Dissolving Fluconazole into mixture of Polyethylene Glycol 400, Labrasol,

Glycofurol, and Benzyl Alcohol, PVP-K 30 by heating the mixture at 70°C in

Manufacturing Vessel Avith continuous stirring;

Step 4: Then, immediately transferring in to another clean vessel and allowing it to cool; Step 5: After cooling adding lactic acid with stirring;

Step 6: Store the formulation in airtight closed container in light resistant area.

Example: 3

Manufacturing process:

Step 1 : Weighing accurately Polyethylene Glycol 400, Labrasol, Glycofurol, and Benzyl Alcohol in S.S. Vessel and mixing all by continuous stirring;

Step 2: Weighing accurately PVP-K 30 and dissolving into step 1 ; Step 3: Dissolving Clotrimazole into mixture of Polyethylene Glycol 400, Labrasol, Glycofurol, and Benzyl Alcohol, PVP-K 30 by heating the mixture at 70°C in Manufacturing Vessel with continuous stirring;

Step 4: Then, immediately transferring in to another clean vessel and allowing it to cool ; Step 5: After cooling adding lactic acid with stirring;

Step 6: Store the formulation in airtight closed container in light resistant area.

Example: 4

Manufacturing process:

Step 1 : Weighing accurately Polyethylene Glycol 400, Labrasol, Glycofurol, and Benzyl

Alcohol in S.S. Vessel and mixing all by continuous stirring;

Step 2: Weighing accurately PVP-K 30 and dissolving into step 1 ;

Step 3: Dissolving Econazole into mixture of Polyethylene Glycol 400, Labrasol,

Glycofurol, and Benzyl Alcohol, PVP-K 30 by heating the mixture at 70°C in

Manufacturing Vessel with continuous stirring;

Step 4: Then, immediately transferring in to another clean vessel and allowing it to cool ; Step 5: After cooling adding lactic acid with stirring;

Step 6: Store the formulation in airtight closed container in light resistant area. Example: 5

Manufacturing process:

Step 1 : Weighing accurately Polyethylene Glycol 400, Labrasol, Glycofurol, and Benzyl Alcohol in S.S. Vessel and mixing all by continuous stirring;

Step 2: Weighing accurately PVP-K 30 and dissolving into step 1 ;

Step 3: Dissolving Miconazole into mixture of Polyethylene Glycol 400, Labrasol, * ^" Glycofurol, and Benzyl Alcohol, PVP-K 30 by heating the mixture at 70°C in Manufacturing Vessel with continuous stirring;

Step 4: Then, immediately transferring in to another clean vessel and allowing it to cool; Step 5: After cooling adding lactic acid with stirring;

Step 6: Store the formulation in airtight closed container in light resistant area. Example 6:

Clinical Trial to study improved efficacy of Clotrimazole with Lactic acid Vaginal spray (as described in Example 1 )

Investigational Product: Clotrimazole with Lactic acid Vaginal spray

Dose: Clotrimazole (100mg/0.8gm),with Lactic acid vaginal spray (4mg/0.8gm) (4spray) at night before sleep for six days.

No. of Patients: 10

Mode of administration: Vaginal

Duration of treatment: 6 days Suppression of symptoms was observed and percentage of individual unresolved symptoms was calculated.

Results:

Among all six symptoms of vulvovaginal candidiasis infection, itching or burning remained 1 1.1 1 % unresolved. Similarly, white discharge was also 1 1 .1 1 % unresolved, whereas Swelling, Yellow discharge, Edema& Redness were completely resolved at the end of the third day in compare to 6 days therapy as in the case of other formulations of vaginal clotrimazole (eg. cream, gel, suppository etc.)

Conclusions:

Hence, in view of the above table, it is concluded that vaginal spray preparation comprising Clotrimazole (100 mg/0.8 ml) with Lactic acid (4 mg/ 0.8 ml) has below mentioned advantages as compared to other vulvovaginal antifungal preparations in the market: The container having metered dosing spray nozzle delivers accurate dose of drug in mist form to cover large surface area of vagina and provide a local treatment which is not possible in case of cream or gel formulations which needs to be applied through applicators.

Length of nozzle is optimized to insert the nozzle of device (5.4 cm) which can deliver the drug at exact required site into vagina whereas in case of other formulations, the applicator is very long, e.g. suppository (approx. 1 1 cm) and gel (approx. 12 cm) which produces more risk and fear to the patients.

The intensity of burning sensation is very minor and comfort level is higher hence, patient compatible in case of the composition of present invention as compared to suppository, cream and gel formulations. Many a times, patients remove suppositories immediately after inserting into vagina. In case of cream and gel formations, many patients wash off drug after application because of unbearable burning sensation.

Patients show suppression of most of the symptoms within three days. However recommended full course of treatment is for six days. Thus, faster recovery period in comparison with other peer products.

The composition is non-sticky as compared to creams and gels. Therefore, patients feel easy to administer and more comfortable after application. Drug of the composition gets absorbed without leaving sticky inert particles behind.

Additional Lactic acid maintains the desired vaginal pH levels 4-4.5. This helps to cover the loss of lactic acid due to infection, which is otherwise naturally produced by Lactobacilli into vagina.