WO2019180748A1

WO2019180748A1 - Probiotic formulation and uses thereof

Info

Publication number: WO2019180748A1
Application number: PCT/IN2019/050234
Authority: WO
Inventors: Indu Pal KAUR; Garima SHARMA
Original assignee: Panjab University; Unique Biotech Ltd
Priority date: 2018-03-21
Filing date: 2019-03-21
Publication date: 2019-09-26

Abstract

The present invention provides a probiotic formulation for topical application having low water activity in the range of about 0.2 to about 0.75 for maintaining skin and/or mucous membrane microbiome balance, maintaining vaginal health, vaginal mucosal integrity, inhibiting one or more pathogens, and for anti-acne, immunomodulatoy, antiinflammatory, calming and other cosmetic effects. The probiotic formulation includes at least one probiotic bacterial spore in the range of about l0⁶cfu to about l0¹⁴ cfu, an aqueous phase and a hydrophobic agent. The 10 bacterial spore is dispersed in a hydrophobic medium and is then constituted into an aqueous phase. The formulation enables release and germination of probiotic spores only on contact with the epidermal surface and provides for slow sustained release.

Description

PROBIOTIC FORMULATION AND USES THEREOF

FIELD OF INVENTION

The invention generally relates to probiotic formulation for maintaining skin or mucous membrane, specifically vaginal mucosa, micro biome balance. In particular the invention relates to probiotic formulation for application to skin or mucous membrane, specifically vaginal mucosa for inhibiting one or more pathogens, and for anti-acne, immunomodulatoy, antiinflammatory, calming and other cosmetic effects.

BACKGROUND

Probiotics are emerging as a potential therapy due to their ability to grow in any environment by colonizing and thus inhibiting the growth of harmful or pathogenic microorganisms. Probiotics produce their effect by multiple mechanisms, such as production of antimicrobial substances, inhibition of pathogen adhesion, competitive colonization and stimulation of immune response. These properties coupled with their capacity to colonise the skin and mucosal surface can be used to control the invasion of healthy or traumatized skin including vaginal mucosa by pathogenic organisms.

The human body contains trillions of cells and routinely harbours millions of bacteria. The microbial flora normally associated with the human body has an important influence on human development, physiology, immunity, and nutrition. Pathogenic organisms cause multiple diseases in various parts on the human body when they overgrow and replace the beneficial micro flora. Bacterial infections of the skin, affects the skin micro biome, and various diseases or disorders are caused by an imbalance in the skin micro biome. The microbial flora of the urogenital tract, changes specifically in women due to factors such as age, vaginal pH and hormonal levels, making it easier for the pathogenic organisms to colonize. Bacterial vaginosis, and Vulvovaginal candidiasis, are the two commonly occurring vaginal infections due to changes in the vaginal micro flora.

The extensive use of broad spectrum antibiotics and anti fungal against pathogenic microorganisms has resulted in serious consequences since these agents destroy the beneficial micro biome, thus causing imbalance in micro flora. The occurrence of multidrug resistant strains of microorganisms poses a further threat for the treatment of pathogenic diseases. The impairment of immunity in individuals due to variety of reasons such as AIDS, therapeutic treatments such as cancer therapy, chemotherapy, radiation treatment and drug-mediated immunosuppression following organ transplant results in the inability to fight the infections caused by the growth of pathogenic microorganisms.

Thus, there is a need for alternate methods to control the growth of pathogenic microorganisms without the use of antibiotics. Probiotics are known to restore the normal micro flora health and can be applied either internally or externally to restore the balance of beneficial microorganisms to pathogens. Many studies have shown that inflammatory skin diseases can be suitably manipulated by oral administration of probiotics, however this pathway is majorly immune system mediated. Thus it may have a limited scope for the control of infections, while a direct manipulation or modification of skin flora to restore its robustness may be a more favourable option. The transiently administered oral probiotic may not survive adverse conditions of the gut, and also fail to establish themselves in the gut for long-term effects. Furthermore immune homeostasis of the skin and its response to infection is controlled autonomously by the skin flora, independent of the gut flora. Ecological niche of skin commensals is different and independent of gut. Studies to recolonize the beneficial micro flora have focused on use of probiotics, especially lactobacillus strains. Sudha et al., have demonstrated the use of Bacillus coagulans. Topical route has been disclosed as a suitable and easily acceptable route of application for any medication. Probiotics are also known as an effective mode of treatment for treating skin as well as vaginal infections and other diseases.

There are creams and lotions that include a certain bacterial species or bacterial components, as a probiotic for topical application. One significant disadvantage of the creams and lotions, known to exist in the art, is the presence of high water content, which has a high risk of triggering the growth of added probiotics in the formulation itself. Probiotics available in suspended form is prone to variability of cfus/ dose administered; aerosols, sprays and powders lack the ability to adhere to the skin for longer duration. Hence, there is a need for a topical application, containing a probiotic that has a lower water content/ activity and sustained efficacy.

BRIEF DESCRIPTION OF DRAWINGS

So that the manner in which the recited features of the invention can be understood in detail, some of the embodiments are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

FIG. la shows onset of melting of suppositories at the end of 10 minutes, when placed at 37 C, according to an embodiment of the invention.

FIG. lb shows complete melting of suppositories at the end of 2 hours, when placed at 37 C, according to an example of the invention.

FIG. 2a shows germination of probiotics on abraded skin samples of rabbit at 6h post topical application, according to an embodiment of the invention.

FIG. 2b shows germination of probiotics on non-abraded skin samples of rabbit at 6h post topical application, according to an embodiment of the invention.

FIG. 2c shows germination of probiotics on abraded skin samples of rabbit at l2h post topical application, according to an embodiment of the invention.

FIG. 2d shows germination of probiotics on non-abraded skin samples of rabbit at l2h post topical application, according to an embodiment of the invention.

Fig 2e shows germination of probiotics on abraded skin samples of rabbit at 24h post topical application, according to an embodiment of the invention.

FIG. 3a shows germination of probiotics 2 hours post vaginal administration, according to an embodiment of the invention.

FIG. 3b shows germination of probiotics 4 hours post vaginal administration, according to an embodiment of the invention.

FIG. 3c shows germination of probiotics 6 hours post vaginal administration, according to an embodiment of the invention.

FIG. 3d shows germination of probiotics 24 hours post vaginal administration, according to an embodiment of the invention. FIG. 4 shows the plot between shear rate versus viscosity and shear rate versus shear stress of the formulation, according to an embodiment of the invention.

FIG. 5 shows graphical plot between shear stress and shear strain of the formulation of the present invention, according to an embodiment of the invention. FIG. 6a shows TEM image of probiotic loaded formulation, according to an embodiment of the invention.

FIG. 6b shows FESEM image of probiotic loaded formulation, according to an example of the invention.

FIG. 7a shows the histology of stratified epithelial layer of the female reproductive tract of control group, according to an embodiment of the invention.

FIG. 7b shows the histology of stratified epithelial layer of the female reproductive tract after 14 days of regular vaginal administration of probiotic loaded suppository, according to an example of the invention.

FIG. 7c shows the histology of the cervix area of the control group, according to an embodiment of the invention.

FIG. 7d shows the histology of the cervix area of the treated group after 14 days of regular vaginal administration of suppositories, according to an example of the invention.

FIG. 7e shows the histology of the uterus area of the treated group after 14 days of regular vaginal administration of suppositories, according to an embodiment of the invention.

FIG. 7f shows the histology of the epithelial layer of the treated group after 14 days of regular vaginal administration of suppositories, according to an embodiment of the invention.

Fig. 8 shows a comparative chart of extent of wound healing, provided by the topical gel formulation, in rats according to an embodiment of the invention.

Fig. 9 shows a graph of total number of colony forming units remaining, when treated with the suppository formulation of the invention, according to an example of the invention. SUMMARY OF THE INVENTION

One aspect of the invention provides a probiotic formulation for topical application having low water activity in the range of about 0.2 to about 0.75 for maintaining skin and/or mucous membrane microbiome balance. The probiotic formulation includes at least one probiotic bacterial spore in the range of about 10⁶ cfu to about 10¹⁴ cfu, an aqueous phase and a hydrophobic agent. The bacterial spore is dispersed in a hydrophobic medium and is then constituted into an aqueous phase. The formulation thus obtained is a formulation for application to skin or mucous membrane, specifically vaginal mucosa for maintaining vaginal health, vaginal mucosal integrity, inhibiting one or more pathogens, and for anti-acne, immunomodulatoy, antiinflammatory, calming and other cosmetic effects. The formulation enables release and germination of probiotic spores only on contact with the epidermal surface and provides for slow sustained release. In another aspect the Water activity (a_w) is kept as low as < 0.60-0.75 a_w that allows for greater shelf-life of probiotic products. The formulation absorbs water when it comes in contact with the skin or mucosal surface to allow for the germination of the spores and ensure sustained release of the probiotic bacteria.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments of the invention provide for a probiotic formulation for topical application having low water activity in the range of about 0.2 to about 0.75. The probiotic formulation includes at least one probiotic bacterial spore in the range of about 10⁶ cfu to about 10¹⁴ cfu, an aqueous phase and a hydrophobic agent. The bacterial spore is dispersed in a hydrophobic medium and is then constituted into an aqueous phase. The formulation described herein briefly shall be explained in detail, below.

Accordingly, the subject matter described herein is directed to a formulation. The formulation for topical application having low water activity in the range of about 0.2 to about 0.75, includes one or more probiotic bacterial strain, including but not limited to Bacillus clausii, Bacillus coagulans, Bacillus Laevolacticus, Bacillus laterosporus, Bacillus mesentericus, Bacillus subtilis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum In particular, the formulation includes of B. coagulans, PTA-11748. Alternatively, variants of the bacterial strain can also be used. The phrase "variant thereof’ as used herein in is referred to the bacterial strains of the invention, especially those which have been deposited, is defined as a bacteria belonging to the cluster having a RAPD similarity of at least 80 % to said probiotic bacterial strains; or having the ability to colonize the skin or mucous membrane. The formulation also includes pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include but not limited to a gelling agent, a dispersing agent, a humectant, oil, wax, fats or lipids, components rich in linoleic acid, a diluent, a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a surfactant, an organic solvent, water, film forming polymer, opacifier, plasticizer a modified release polymer and/or combinations thereof. As used herein, the term “probiotic” refers to microorganisms examples of which include but are not limited to bacteria, yeast, viruses, and/or fungi, which form, at a minimum, a part of the transient or endogenous flora and, thus, possess a beneficial prophylactic or therapeutic effect upon the host organism. Probiotics are generally known to be clinically-safe and are considered non-pathogenic by those skilled in the art. Although, not bound by any particular mechanism, the probiotic activity of Bacillus species is hypothesized to result from competitive inhibition of growth of pathogens due to superior colonization, lactic and organic acid production or other extracellular products having anti-microbial, anti-inflammatory, antioxidant, and anti-ageing, hydrating, and detoxifying activity skin clarifying, skin brightening, calming or combinations thereof.

The invention discloses probiotic strains in the formulation including but are not limited to Bacillus clausii, Bacillus coagulans, Bacillus Laevolacticus, Bacillus laterosporus, Bacillus mesentericus, Bacillus subtilis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum. In particular, the formulation includes, Bacillus species, in particular B. coagulans , PTA-11748, in therapeutic formulations as a probiotic, for the prevention or control of infections caused by pathogens including, but not limited to a microbial infection, yeast infection, fungal infection, or a viral infection and for anti-acne, immunomodulatoy, antiinflammatory, calming and other cosmetic effects. As will be discussed, these formulations can be made in a variety of configurations, specifically, the probiotic bacteria are included as spores in formulations suited for application on skin or mucous membrane for a plurality of actions.

In one embodiment of the invention, the probiotic formulation includes at least one probiotic bacterial spore the range of about 10⁶ cfu to about 10¹⁴ cfu, an aqueous phase and a hydrophobic agent. The bacterial spore is dispersed in a hydrophobic medium and is then constituted into an aqueous phase. In one embodiment of the invention, the hydrophobic medium is oil. Examples of oil used includes but is not limited to sunflower oil, soybean oil, safflower oil, hemp oil, salicornia oil, evening primrose oil, poppy seed oil, grape seed oil, com oil, wheat germ oil, cotton seed oil, walnut oil, sesame oil, rice bran oil, canola oil, almond oil, pistachio oil and combination thereof. In one example of the invention, sunflower oil is used as a hydrophobic medium.

In one embodiment of the invention, aqueous medium includes to humectant, surfactant, gelling agent and plasticizer. Examples of humectant includes but is not limited to Polyethylene glycol (PEG), Propylene glycol, hexylene glycol, butylene glycol, glycerol, glycerine. Examples of surfactant includes but is not limited to Tweens, Spans, gelatin, polyethylene glycols and their derivatives or a combination thereof. Examples of gelling agent includes but is not limited to gelatin, agar, bentonite, alginates, polyethylene glycol, acacia, alginic acid, Carbopols (now known as carbomers), carboxymethyl cellulose, ethylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, magnesium aluminum silicate (Veegum), methylcellulose, poloxamers (Pluronics), polyvinyl alcohol, sodium alginate, tragacanth, and xanthan gum or a combination thereof. In one example of the invention, Polyethylene glycol (PEG) is used as humectant, Tweens is used as surfactant and gelatin is used as the gelling agent. In an embodiment of the invention, the ratio of hydrophobic medium to aqueous medium is in the range of about 6: 1 to about 1 :3. The formulation of the invention is prepared by dissolving gelatin in an aqueous phase. The concentration of the gelatin is in the range of about 10% to about 20% (wt/volume). In one example of the invention, the concentration of the gelatin is 14.8% (wt/volume). The formulation may further include emulsifying, stabilizing, suspending, thickening and gelling agent. The gelling agents may comprise acacia, alginic acid, bentonite, Carbopols (now known as carbomers), carboxymethyl cellulose, ethylcellulose, gelatin, hydroxyethyl cellulose, hydroxypropyl cellulose, magnesium aluminum silicate (Veegum), methylcellulose, poloxamers (Pluronics), polyvinyl alcohol, sodium alginate, tragacanth, and xanthan gum. In an example, Pluronic F127 is used in the formulation in a concentration range of 5-30%, specifically about 15%. Carbopol is used in the range of 0.5-10%, more specifically 1-5%. In one example Carbopol (Ultrez 10), at concentrations ranging between 1% and 5%, is dispersed in 25 ml of PEG 400, and the dispersion is homogenised for 10-15min min at 500-1000 rpm. The aqueous phase comprises of a suitable surfactant, gelling agent, a humectant and a plasticizer. It is well known in the art that non inonic surfactants Sorbitan fatty acid esters such as Spans and the corresponding polyoxyethylene (POE) adducts such as Tweens are safe agents for biological tissues in general, and specifically skin. These non-ionic emulsifiers are compatible with various ingredients used in the preparation of emulsions and are not affected by pH. In one example of the invention, the surfactant chosen is Tween 80 or span 80. The two surfactants may be used individually at a concentration ranging from 5 - 30%. The surfactants may also be used in combination. Any commonly known humectants such as Polyethylene glycol (PEG) Propylene glycol, hexylene glycol, butylene glycol, glycerol or glycerine. The glycerine may be used in the formulation in the range of 10-80%. In one example of the invention, glycerin is used as humectant. The concentration of glycerin is in the range of about 20% to about 40%. The dissolution of gelatin in water is carried out at a temperature range from about 35°C to about 55°C. A hydrophobic agent generally used to disperse the probiotic culture in oil phase, maintained at same or lower temperature, is then added to the aqueous gel to form an emulgel formulation which is stirred continuously with cooling till it coalesces to a semi-solid mass. The coalesced mass obtained on mixing of the two phases is then subjected to extended stirring to result in a particulate gel structure, suitable for use for application on skin. The emulgel formulation can also be poured directly while warm into moulds to form suppositories or films on cooling.

Low water activity of the probiotic composition significantly increases its shelf- life. Water activity also plays an important role on the viability of bacteria. Water activity reflects the part of moisture content of a product which can be exchanged between the product and its environment. Water activity determines the lower limit of "available" water for microbial growth. Since bacteria, yeast, and molds require a certain amount of "available" water to support growth, designing a product below a critical aw level provides an effective means to control germination of spores and hence prevent spoilage due to increase in number of probiotic though keeping them viable. Water may be present, even at high content levels, in a product, but if its energy level is sufficiently low the microorganisms cannot remove the water to support their growth. The water activity level that limits the growth of the vast majority of pathogenic bacteria is 0.90aw, 0.77aw for spoilage molds, and the lower limit for all microorganisms is 0.60aw. In one embodiment of the invention, the reactive amount of water is in the range of about 0 .4 to about 0.70 a_w.

The probiotic formulation, as described herein above can be administered in the form of emulsion, cream, lotion, gel, oil, serums, beads, emulgel, ointment, suspension, aerosol spray, powder, film, wound-dressing aerosol powder, semi solid formulation, pessaries or suppositories. In one example of the invention, the formulation is in the form of suppositories. The probiotic formulation of the has immunomodulatory, anti-inflammatory, wound healing, antioxidant, anti-ageing, anti-acne, skin clarifying, skin brightening, hydrating, calmimg and detoxifying activity. The probiotic formulation thus obtained inhibits growth of bacterial, yeast, fungal or viral pathogens.

Further, the bacillus spores can be formulated on flexible materials such as diapers, band aids, tampons, and personal articles, all directed to the objective of introducing the bacteria topically to skin or a mucous membrane tissue. The cell surface hydrophobicity of Bacillus coagulans is found to be 30.4 ± 5.34%. Hydrophobicity of less than 50 % indicates capacity of probiotic cells to adhere to the epithelial cells of the skin or the mucosal membrane. Viability during production and storage in the formulation and growth and establishment on skin/mucous membrane on application of the probiotics continues to be the main difficulty in the use of probiotics on skin or in mucosal membranes, such as vaginal mucosa. The topical gel, film or vaginal suppositories/ pessaries of the invention are thus formulated to ensure the viability and growth of the Bacillus , particularly the B. coagulans, especially in the adverse conditions posed by vaginal mucosal membranes or aerobic exposure on skin surface. Furthermore, suppositories/ pessaries tend to be ejected from the vaginal cavity if uncomfortable or irritant. The suppositories of the present invention swell sufficiently and gain weight by absorbing water from the vaginal cavity and thus fit into the vaginal cavity, so that they are not thrown out. Table I shows the percentage weight gain in suppository on swelling, according to an embodiment of the invention. It is found that the suppositories/ pessaries melt at body temperature. FIG. la shows onset of melting of suppositories at the end of 10 minutes, when placed at 37°C, according to an embodiment of the invention. The suppositories start to melt after lOmin but within 2 hours when placed at 37°C. FIG. lb shows complete melting of suppositories at the end of 2 hours, when placed at 37°C, according to an example of the invention. Fast melting can result in leaky suppository which have poor patient acceptance; and if the suppository does not dissolve or melt it will be ejected from the body without release.

TABLE I

The suppository formulation of the current invention is tested for the time when the suppository either melts or dissolves completely in the medium, specifically Simulated Vaginal Fluid, hereinafter referred to as SVF. Table II shows composition of simulated vaginal fluid, according to one example of the invention.

TABLE II

Extent of swelling/weight gain of probiotic suppository/ pessary formulation in the presence of SVF shows an almost 100% gain in the weight as shown in Table I. This is determined by placing a suppository in a 50 ml beaker at room temperature. Weight of the suppository is noted before start of the study. 0.75 ml of SVF is added to each suppository at regular intervals of 15 minutes up to 1 hour. After 1 hour the suppository is taken out and dried with help of filter paper to remove fluid present on its surface and reweighed. The same procedure is repeated up to 6 hours. The weight of the suppository is noted by drying it with filter paper every hour during the study, up to 6 hours. The beneficial effect of the probiotic on the skin surface or any mucosal membrane is credited to the enzymes and bacteriocins released by these cells in their active state. Therefore, in order to elicit physiological action of probiotic their successful germination to the active vegetative form following application is important. The probiotic loaded topical gel or suppositories of the invention are found to exhibit germination post application.

The germination of the probiotic following application of the probiotic loaded gel formulation on the skin surface is established on the abraded and non abraded shaved skin of rabbits. FIG. 2a shows germination of probiotics on abraded skin samples of rabbit at 6h post topical application, according to an embodiment of the invention. FIG. 2b shows germination of probiotics on non-abraded skin samples of rabbit at 6h post topical application, according to an embodiment of the invention. FIG. 2c shows germination of probiotics on abraded skin samples of rabbit at l2h post topical application, according to an embodiment of the invention. FIG. 2d shows germination of probiotics on non-abraded skin samples of rabbit at l2h post topical application, according to an embodiment of the invention. Fig 2e shows germination of probiotics on abraded skin samples of rabbit at 24h post topical application, according to an embodiment of the invention.

Presence of vegetative cells, vegetative cells with spores and released spores are observed at different time intervals. The germination of the probiotic following application of probiotic loaded suppositories inserted into vagina is established by the swabs collected from rat’s vagina. FIG. 3 generally shows germination of probiotics at different time intervals, post vaginal administration, according to an embodiment of the invention. FIG. 3a shows germination of probiotics 2 hours post vaginal administration, according to an embodiment of the invention. FIG. 3b shows germination of probiotics 4 hours post vaginal administration, according to an embodiment of the invention. FIG. 3c shows germination of probiotics 6 hours post vaginal administration, according to an embodiment of the invention. FIG. 3d shows germination of probiotics 24 hours post vaginal administration, according to an embodiment of the invention. Presence of vegetative cell indicates the germination of probiotic from spores to active vegetative form, which is essential for any therapeutic effect. The performance of the topical formulation depends on parameters including but not limited to hardness, compressibility, adhesiveness, cohesiveness, rheology the total probiotic content of prepared formulation and/or combinations thereof. The mechanical properties of the topical gel formulation are assessed for ease of removal of product from the container, good spreadability and, good bioadhesion which ensures retention of the probiotic formulation at the site of application. The hardness test is performed to measure the force required to produce deformation of the prepared gel. Determination of hardness influences the ease of application of the gel to the desired site. The freshly prepared formulation exhibits a hardness of 0.310 ± 0.030 N. The values are acceptable for topical gel application and are in accordance with previous studies. Compressibility is required to achieve compaction of the product along a definite distance. A low compressibility value enables easy removal of the gel from the container and easy spreadibility on application. The compressibility of the freshly prepared formulation and the sample stored for 2 months is 1.379 ± 0.134 Nmm and 7.360 ± l.l2lNmm, respectively. The values are within the suitable limits.

Adhesiveness in texture profile analysis is commonly defined as the ability to overcome the attractive forces between the surface of the sample and the probe surface. The adhesiveness of the freshly prepared formulation is -0647±0.078 Nmm. The cohesiveness of the gel is important to determine the reconstruction ability of the gel after application. The high cohesiveness value indicates that the structure of the gel is maintained upon application. The Cohesiveness values are 0.946 and the values are indicative of a good performance of the product. FIG. 4 shows the plot between shear rate versus viscosity and shear rate versus shear stress of the formulation, according to an embodiment of the invention. From the nature of the curve obtained by plotting shear rate versus viscosity and shear rate versus shear stress it is inferred that the developed formulation comprises of a polydispersed system.

FIG. 5 shows graphical plot between shear stress and shear strain of the formulation of the invention, according to an embodiment of the invention. The curve obtained from the plot of shear stress and shear rate denotes the highly flocculated system. FIG. 6a shows TEM images of probiotic loaded formulation, according to an embodiment of the invention. FIG. 6b shows FESEM images of probiotic loaded formulation, according to an example of the invention. The oil/ emulgel floccules containing dispersed probiotic spores are present in a more uniform particulate gel matrix, as observed under TEM and FESEM.

Yield stress of a material is defined as the minimum stress above which flow can be observed. It is an indicator of spreadability and retention of semisolids. The yield value is calculated from the intercept obtained from the plot of initial points of shear stress and shear rate. The developed formulation shows a high yield value of 271.2 Pa. The high yield value is attributed to the rigidity of the gel structure which, requires high shear to initiate flow. High yield value signifies that the system has sufficient fluid strength and hence will exhibit appreciable contact time. Graph between log shear stress and log shear rate shows that the value of flow index (n) is less than 1, specifically 0.259, which signifies the shear thinning nature of the developed formulation. Consistency index for the developed formulation is 791.55 Pa.s, which is appreciable and will ensure sufficient contact time. Similarly, probiotic loaded suppositories are evaluated for Uniformity of Weight, disintegration and total probiotic content. All the results in table III comply with the standards described by Indian Pharmacopoeia 2014, hereinafter referred to as IP 2014, for ideal suppositories.

Safety studies: In vivo studies are performed in animal models. In one embodiment of the invention, 6 months old female rat, weighing about l25g-200g and 6 months old Albino female rabbit weighing about 1.4-1.7 kg are chosen to assess the safety (irritation/corrosion) of the formulation of the invention on topical on application.

Approximately 24 h before the test, fur of the animals is removed using hair clippers followed by the application of a hair removing cream from the dorsal area of the trunk of the animals. Care is taken to avoid abrading the skin, and only animals with healthy, intact skin are used. The test substance, the probiotic formulation of the current invention is applied onto a gauze patch which is applied and fixed on the shaved skin surface of approximately 6 cm², with a non-irritating tape for a period of 4 h. Care is taken to ensure good contact and uniform distribution of the substance on the skin surface. Area just below the neck is selected as the site of application so that the access of the animal to the patch and/or its ingestion or inhalation is not possible.

A small area of shaved skin area adjacent to the test area is kept uncovered to serve as control. At the end of the exposure period, gauze patch is removed, and the covered skin is examined for any reactions, as detailed in the Table IV, 1 h after the patch is removed.

Clinical observations and grading of skin reactions: All animals are examined for signs of erythema and oedema, and the response is scored at 60 minutes, and then at 24, 48 and 72 hours after removal of the patch. For the initial test in one animal, the test site is also examined immediately after the patch is removed. Dermal reactions are graded and recorded as elaborated in Table IV. Results establish the pre-clinical safety of the formulation with no visible signs of erythema or oedema in any animal at the end of the test.

FIG. 7 generally shows the histology of the vaginal mucosa and reproductive tract, according to an embodiment of the invention. The developed suppository formulation does not show any signs of irritation etc or any histological changes in the vaginal mucosa or reproductive tract FIG. 7a shows the histology of stratified epithelial layer of the female reproductive tract of control group, according to an embodiment of the invention. FIG. 7b shows the histology of stratified epithelial layer of the female reproductive tract after 14 days of regular vaginal administration, according to an example of the invention. FIG. 7c shows the histology of the cervix area of the control group, according to an embodiment of the invention. FIG. 7d shows the histology of the cervix area of the treated group after 14 days of regular vaginal administration of suppositories, according to an example of the invention. FIG. 7e shows the histology of the uterus area of the treated group after 14 days of regular vaginal administration of suppositories, according to an embodiment of the invention. FIG. 7f shows the histology of the epithelial layer of the treated group after 14 days of regular vaginal administration of suppositories, according to an embodiment of the invention. The formulation of the invention is capable of preventing or treating bacterial, yeast, fungal or viral infection. The formulation can be applied topically to skin or a mucous membrane. The probiotic formulation of the invention is applied to a flexible article that is intended to be worn by or attached to skin or a mucous membrane to allow probiotic activity of the bacteria to occur adjacent to or on the skin or mucous membrane, for example suppository, diaper, pliable material for wiping skin or a mucous membrane, dermal patch, adhesive tape, absorbent pad, tampon or article of clothing. The probiotic formulation is impregnated into a fibrous or non-fibrous solid matrix. The efficacy of the probiotic formulation to preserve itself, is assessed in challenge studies against the test organisms listed in Table V.

Results inc icate that the prepared formulation has self preservative effects in the sense that even when challenged by specified number of recommended organisms as per Indian Pharmacopoeia listed in Table V, the formulation limits their growth to numbers below the limits set by the Indian Pharmacopoeia, results in table VI.

Example 1

Initially gelatin in the concentration range of about 15.2% is soaked in same quantity of water 15.2% for about an hour. Subsequent to soaking, the mixture is further heated at a temperature range of about 35 C to about 55 C to form a solution (A). Separately, Tween 80 at the concentration range of about 9%, glycerin at the concentration range of about 25.5 % and cyclomethicone at the concentration range of about 4.6% are mixed and heated at a temperature range of about 35 C to about 55 C to obtain an aqueous mixture (B). Further, the aqueous mixture (B) is added to the previously formed solution (A) and stirred continuously to obtain a subsequent mixture (C). The temperature of the mixture (C) is maintained at around 35 C to around 55 C. The probiotic spores at a range of about 10⁶ to 10¹² cfu/ unit dose are dispersed in sunflower oil at a concentration range of about 30.5% to form dispersion (D). This dispersion (D) is then slowly added to the previously formed mixture (C) and constantly stirred at about 200 to about 500 rpm until a milky white emulgel (E) is formed. Subsequent to the formation of emulgel (E), heating is discontinued, and it is allowed to cool with constant stirring at about 200 rpm to about 500 rpm to form a coalesced mass due to gelatinizing of the gelatin in the external phase of the emulgel matrix. The coalesced mass thus formed is then broken using a mechanical stirrer, at 2000 rpm for about 10 to about 15 min, maintaining the gel at around 4 C to around 8 C. Continuous stirring of the formed gel at about lOOOrpm to about 1500 rpm for about 15 minutes results in a nanoparticulate gel.

Example 2

Initially gelatin in the concentration range of about 14% to about 15% is soaked in an equal quantity of water (14-15%) for about an hour. Subsequent to soaking, the mixture is further heated at a temperature range of about 35 C to about 55 C to form a solution (A). Separately, Tween 80 at the concentration range of about 8.5% and glycerin at the concentration range of about 27% to about 40% are mixed and heated at a temperature range of about 35 C to about 55 C to obtain an aqueous mixture (B). Further, the aqueous mixture (B) is added to the previously formed solution (A) and stirred continuously to obtain a subsequent mixture (C). The probiotic spores at a range of about 10⁶ to 10¹² cfu/ unit dose are dispersed in sunflower oil at a concentration range of about 21.5% to about 34% to form dispersion (D). This dispersion (D) is then slowly added to the previously formed mixture (C) and constantly stirred at about 200 to about 500 rpm. The mixture thus obtained is then cooled down to about 35°C to about 45°C through constant stirring and poured into a previously cooled and lubricated suppository moulds. The mounds are then refrigerated overnight, removed from the formed suppositories, packed and stored under refrigeration. Oil and Tween in the composition allows easy release of samples from the moulds.

Example 3

Initially gelatin in the concentration range of about 14% to about 15% is soaked in an equal quantity of water 14-15% for about an hour. Subsequent to soaking, the mixture is further heated at a temperature range of about 55° C to form a solution (A). Separately, Tween 80 at the concentration range of about 8.5% and glycerin at the concentration range of about 27% to about 40% are mixed and heated at a temperature range of about 35°C to about 55°C to obtain an aqueous mixture (B). Further, the aqueous mixture (B) is added to the previously formed solution (A) and stirred continuously to obtain a subsequent mixture (C). The probiotic spores at a range of about 10⁶ to 10¹² cfu/ unit dose are dispersed in sunflower oil at a concentration range of about 21.5% to about 34% to form dispersion (D). This dispersion (D) is then slowly added to the previously formed mixture (C) and constantly stirred at about 200 to about 500 rpm. The mixture thus obtained is then cooled down to about 35°C to about 45°C through constant stirring and poured into a previously cooled and lubricated shallow mould to form films or sheet or dressings. The mounds are then refrigerated overnight, removed from the formed films or sheet or dressings, packed and stored under refrigeration. Oil and Tween in the composition allows easy release of samples from the moulds. Example 4

Carbopol at a concentration range of about 4% is added to PEG used in the range of about 51%, to form a mixture. This mixture is then subjected to two rounds mixing, first at around 800 rpm to around 1000 rpm for about 10 minutes and second mixing at around 1000 rpm to around 1500 rpm for about 15 to 20 minutes. The mixture is then kept aside for about two to three days to form Carbopol-PEG gel. Separately, Span at a concentration range of about 7% and oil at a concentration range of about 15% are mixed and heated at around 60°C to obtain a mixture (A). Further, Tween at a concentration range of about 8% and about 15% water is mixed separately to obtain a second mixture (B) and formed mixture is then maintain at 60°C. The contents of mixture (A) and mixture (B) are then mixed at a temperature of 60°C using magnetic stirrer at around 600 rpm to around 1000 rpm for about 10 to 15 minutes to form an emulsion. Once the emulsion thickens, the stirring is continued until the mixture cools down to room temperature. The emulsion thus formed is then added to the Carbopol-PEG gel with slow stirring to form an emulgel. The probitic spores at the range of about 106 to about 1012 cfu/ unit dose is then added into the emugel and stirred well to ensure homogenous mixing.

Efficacy Studies: The in vivo therapeutic effect of the probiotic gel formulation is confirmed in terms of wound healing. Superficial wound is generated in adult Laca mice. Animals are divided into six groups, Group I (naive control) consisted of animals with unabraded skin and not receiving any treatments and served as negative control. Superficial wound is generated in Group II- VI animals. Group II (positive control) receive no treatment, Group III, IV and V animals are treated with blank formulation, probiotic loaded developed gel formulation of the invention and free probiotic suspended in 1% CMC respectively. Group VI receive treatment with marketed formulation Soframycin® Skin cream, Sanofi India Ltd. B.No. E5103.

Wound healing is measured in terms of % reduction in wound size, with the size of wound on day 0 taken as 100% for each animal. FIG. 8 generally shows wound healing in rats, according to an embodiment of the invention. Wound healing (%) is achieved with the probiotic loaded formulation it means that the effect achieved with the gel of the invention has effect similar to an antibiotic cream available in the market and generally prescribed for treating wounds is significantly higher than that obtained with control, free probiotic, and blank formulation. No statistically significant (p<0.05) difference is observed between the latter three treatments at all days. It can be concluded from the results that the free probiotic applied without formulation does not produce a significant effect while the probiotic formulation of the invention can be used not only as an adjuvant therapy but as a main line treatment, because no statistically significant difference was found between the marketed formulation (Soframycin®) and probiotic loaded formulation. Results of the study show a higher therapeutic efficacy than that of the marketed formulation Vulvovaginal Candidasis (VVC) in rats:

Similarly, efficacy of probiotic suppositories are tested for Vulvovaginal Candidasis, hereinafter referred to as VVC, in rats. Animals are subcutaneously injected with hormonal injections of b-Estradiol every day for 6 days for establishment of pseudo-estrus state. The 20 mΐ (cfu/ml) of the standardized Candida albicans suspension (maximum volume for the vagina, without leakage) is introduced into the vagina 1 week after hormonal induction for induction of infection. The hormone injections are continued throughout the study period, but on alternate days. After confirmation of infected vagina, one suppository is administered intravaginally every day, to the treatment group. Animals are divided into 6 groups as shown in table VII.

Marketed formulation Candid V® gel, free probiotic dispersion, probiotic loaded suppositories and blank suppositories, are inserted in infected rats and fungal burden is monitored every day for 7 days.

Fig. 8 shows a comparative chart of extent of wound healing, provided by the topical gel formulation, in rats according to an embodiment of the invention. Five different topical applications are chosen, namely A=Control, B=Free probiotic, C= Blank gel formulation, D=Marketed formulation, and E= probiotic gel formulation. The redness of vaginal opening of rat infected with Candida albicans is observed and indicates presence of infection in positive control group. Correspondingly probiotic loaded vaginal suppositories treated animals showed no signs of inflammation or redness, as evident by the clear skin around the vaginal opening. Fig. 9 shows a graph of total number of colony forming units present, when treated with the suppository formulation of the invention, according to an example of the invention. Probiotic loaded suppositories treated animals show significant improvement during the experimental period; the positive control and blank suppositories group show no statistically significant decrease (with p<0.00l) in the fungal load. In the initial phase of treatment no significant (p<0.00l) difference is observed between marketed formulation and free probiotic group whereas the probiotic loaded suppositories group showed significantly better results. However, on seventh day of treatment both the free probiotic group and probiotic loaded suppositories group show significantly better effects than the marketed formulation. Though the free probiotic dispersion show significant effects but its application is not clinically possible or patient acceptable as it will leak out following application. It can be concluded from the results that the probiotic formulations can be used as a main treatment for Vulvovaginal Candidasis (VVC), as found to elicit better results as compared to marketed formulation Candid V® gel.

The formulation has advantages over the prior art in that the formulation preserves probiotics viability for extended storage preferably under refrigerated conditions but also at room temperature, by maintaining the probiotics in a dormant state under storage conditions. This is achieved by formulating a system which has a low water activity, less than 0.9, more appropriately < 0.6. The system also assigns a self-preservative capacity to the formulation. This is tested by inoculating the formulation with known standard bacterial and fungal strains and ensuring that they do not grow sufficiently in the formulation as per the pharmacopoeial challenge test. This helps to avoid addition of any preservative to the formulation. Preservatives are usually toxic and also irritating in nature and may not be advisable for use on already traumatized or irritated skin/mucosal membranes in atopic dermatitis, acne, eczema and infection or wound. The probiotics of the formulation further elicits the physiological action by allowing successful germination to the vegetative form following application to skin or mucosal membrane, such as vaginal mucosa. The formulation of the current invention ensures sufficient contact area and adherence at the application site. The inclusion of a humectant in the formulation absorbs moisture from the atmosphere or the internal layers of the skin to increase the moisture at the site of application helping in the germination of dormant bacterial cells including spores.

The invention thus offers a cost effective gelatin and other polymers based polydisperse particulate system, an emulgel formulation incorporating whole cell probiotics suitable for application to skin types, including vaginal mucosa, and diverse conditions. An emulgel combines the advantages of an emulsion and a gel in the same system. Gels provide a good spreadability and adherence, but contain large amount of water and are only suitable for delivery of hydrophilic components. Emulsion is biphasic water in oil or oil in water system prepared by dispersing fine globules of internal phase in a continuous phase using surfactants to reduce the interfacial tension between the two phases. When the water phase of an emulsion is gelled using a gelling agent, it is referred to as an‘EmulgeT. Emulgel improves its consistency, feel, adherence and release of the active. They can reduce availability of free water molecules and hence water activity of the formulation. Both hydrophilic and lipophilic actives can be incorporated into emulgels.

The formulation is composed of cost-effective ingredients, is easy to manufacture, and self-preservative. Low water activity of the formulation maintains incorporated live bacteria in their dormant form so that they do not overgrow in the pack during storage so as to foul the formulation. The formulation is stable in terms of maintaining the viability and number (cfu) of incorporated live probiotic. The formulation adheres to the skin surface for significant time and is not wiped off easily as are the spray or powder formulations. The aforesaid description is enabled to capture the nature of the invention. It is to be noted, however, that the aforesaid description illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to equally effective embodiments.

Claims

WE CLAIM:

1. A probiotic formulation for topical application having low water activity in the range of about 0.2 to about 0.75, wherein the formulation comprises of: a) at least one probiotic bacterial spore, in the range of about 10⁶ cfu to about 10¹⁴ cfu;

b) a hydrophobic medium; and

c) an aqueous medium wherein the probiotic bacterial spores are capable of germination on contact with surface of skin and/or mucosal surface for sustained release of the bacteria.

2. The probiotic formulation as claimed in claim 1, wherein the at least one probiotic bacterial spore comprises Bacillus clausii, Bacillus coagulans, Bacillus Laevolacticus, Bacillus laterosporus, Bacillus mesentericus, Bacillus subtilis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum or a combination thereof.

3. The probiotic formulation as claimed in claim 1, wherein the ratio of hydrophobic medium to aqueous medium is in the range of about 6: 1 to about 1 :3.

4. The probiotic formulation as claimed in claim 1, wherein the hydrophobic medium is oil.

5. The oil as claimed in claim 4, wherein the oil is chosen from the group comprising of sunflower oil, other such agents such as soybean oil, safflower oil, hemp oil, salicomia oil, evening primrose oil, poppy seed oil, grape seed oil, corn oil, wheat germ oil, cotton seed oil, walnut oil, sesame oil, rice bran oil, canola oil, almond oil, pistachio oil and combination thereof.

6. The probiotic formulation as claimed in claim 1, wherein the aqueous medium further comprises of a humectant, a surfactant, a gelling agent and a plasticizer.

7. The humectant as claimed in claim 6, wherein the humectant is chosen from the group comprising of Polyethylene glycol (PEG) Propylene glycol, hexylene glycol, butylene glycol, glycerol or glycerine

8. The humectants as claimed in claim 6, wherein the humectant is glycerin in the range 10% to 80%

9. The surfactant as claimed in claim 6, wherein the surfactant is chosen from the group comprising of Tweens, Spans, gelatin, polyethylene glycols and their derivatives or a combination thereof.

10. The gelling agent as claimed in claim 6, wherein the gelling agent is chosen from the group comprising gelatin, agar, bentonite, alginates, polyethylene glycol, acacia, alginic acid, Carbopols (now known as carbomers), carboxymethyl cellulose, ethylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, magnesium aluminum silicate (Veegum), methylcellulose, poloxamers (Pluronics), polyvinyl alcohol, sodium alginate, tragacanth, and xanthan gum or a combination thereof.

11. The probiotic formulation as claimed in claim 1, wherein the formulation inhibits growth of bacterial, yeast, fungal or viral pathogens.

12. The probiotic formulation as claimed in claim 1, wherein the formulation further has immunomodulatory, anti-inflammatory, wound healing, antioxidant, anti-ageing, anti-acne, skin clarifying, skin brightening, hydrating, calmimg and detoxifying activity.

13. The probiotic formulation as claimed in claim 1, wherein the formulation is in the form of emulsion, cream, lotion, gel, oil, serums, beads, emulgel, ointment, suspension, aerosol spray, powder, film, wound-dressing, aerosol powder, semi-solid formulation, pessaries or suppositories. REFERENCES:

1) RatnaSudha et al., 2010 Molecular typing and probiotic attributes of a new strain of Bacillus coagulans Unique IS-2 potential therapeutic agent; Genetic Engineering and Biology journal. GEBJ-7;

2) RatnaSudha et al., 2012 Clinical study of Bacillus coagulans Unique IS-2 in treatment of patients with bacterial vaginosis; Indian Journal of Microbiology.52(3): 396-399