Field of Invention:
The invention relates to a formulation for the treatment of Abnormal Vaginal Discharge Syndrome (AVDS).
Background of the Invention:
A small amount of vaginal discharge is usually normal. The discharge consists of secretions (mucus) produced mainly by the cervix but also in the vagina. The discharge is usually thin and clear, milky white, or yellowish. Its amount and appearance vary with age. Typically, the discharge has no odor. It is not accompanied by itching or burning.
During a woman's reproductive years, the amount and appearance of the normal vaginal discharge vary with the menstrual cycle. For example, at the middle of the cycle (at ovulation), more mucus is usually produced and the mucus is thinner. Pregnancy, use of oral contraceptives, and sexual arousal also affect the amount and appearance of the discharge. After menopause, the estrogen level decreases, often reducing the amount of normal discharge.
A vaginal discharge is considered abnormal if it is
• heavier than usual
• thicker than usual
• puslike
• white and clumpy (like cottage cheese)
• grayish, greenish, yellowish, or blood-tinged
• foul-smelling (fishy)
• accompanied by itching, burning, a rash, or soreness.
Vaginal infections are a type of vaginitis, or inflammation of the lining (mucosa) of the vagina. Inflammation may result from chemical or mechanical irritants, such as hygiene products, bubble bath, laundry detergents, contraceptive foams and jellies, and synthetic underwear, as well as from bacterial, yeast, and viral infections.
Abnormal vaginal discharge syndrome (AVDS) is a very common disorder worldwide. It has been reported that 30 % of the urban women and 62 % of rural women in India suffer from this syndrome. Several women worldwide suffer from a variety of reproductive tract infection, which cause AVD, itching, irritation, etc.
Several microorganisms are constituents of the normal vaginal flora. Their production of bacteriocins, acids, and hydrogen peroxide are mechanisms, which keep pathogenic colonies from proliferating.
In women, anything that reduces the acidity (increases the pH) of the vagina increases the likelihood of infection. Many bacteria normally reside in the vagina. One type, called lactobacilli, normally maintains the acidity of the vagina. By doing so, lactobacilli help

keep the lining of the vagina healthy and prevent the growth of bacteria or yeasts that cause infections.
Bacterial vaginosis, the most common vaginal infection, results when the number of protective lactobaciili decreases and the number of other normally occurring bacteria (such as Gardnerella and Peptostreptococcus) increases. The reason for these changes is unknown. In women, yeast infections due to the fungus Candida albicans (candidiasis) are particularly common.
Antibiotics taken by mouth tend to kill the bacteria in the vagina that normally suppress the growth of yeast. Thus, the use of antibiotics increases the risk of developing vaginal infections.
Typically, vaginal infections produce a vaginal discharge and irritation in the genital area. The appearance and amount of the discharge vary depending on the cause. Rarely, the folds of skin around the vaginal and urethral openings become stuck together. However, vaginal infections sometimes produce minimal or no symptoms.
A white, gray, or yellowish cloudy discharge with a foul or fishy odor is typically caused by bacterial vaginosis. A thick, white, and clumpy discharge is typically caused by candidiasis, a yeast infection. A heavy, greenish yellow, frothy discharge that may have a bad odor is typically caused by trichomoniasis, a protozoan infection.
Treatment varies according to the cause.
Bacterial vaginosis is treated with an antibiotic taken by mouth or applied as a vaginal gel or cream. Propionic acid jelly may be used to make the vaginal secretions more acidic and thus discourage the growth of bacteria.
Yeast infections are treated with antifungal drugs applied as a cream to the affected area, inserted into the vagina as a suppository, or taken by mouth. A single dose of an antifungal drug taken by mouth is usually as effective as vaginal creams and suppositories. However, if infections recur often, several doses may be needed.
For trichomoniasis, a single dose of metronidazole cures up to 95% of women. However, a single dose is more likely to cause nausea and vomiting than treatment with several smaller doses. During sexual intercourse, condoms should be used until the infection resolves.
Women who are at high risk of a yeast infection, such as those who have an impaired immune system, who have diabetes, or who are taking antibiotics for a long time (as for a urinary tract infection), may need to take an antifungal drug to prevent other infections from developing.
US patent application no. US20030064103A1 and US patent application no. US20030017207A1, which is continuation in part of US patent application no. US20030064103A1 relates to compositions for treating vulvovaginitis and vaginosis by the use of antifungal agents that include azoles and a buffering system that include gluconodeltalactone. However, said patent does not mention the type of formulation. The drawback of the composition of said patent is that it may cause some side effects.
WO04105662A1 talks of microbicidal compositions and methods for preventing the transmission of or treating sexually transmitted infections and/or common vaginal

infections by the use of microbicides specifically Ciclopirox olamine.
WO0992635A1 relates to a pharmaceutical formulation for stimulating the growth of gram-positive bacilli and increasing the acidity of the vagina for the treatment of vaginitis and bacterial vaginal disease, by the use of certain sugar(s). WO0992635A1 does not envisage the use of probiotic microorganisms in the formulation for treating vaginitis and bacterial vaginal disease.
The object of the present invention is to develop a formulation for the treatment of abnormal vaginal discharge syndrome that does not consist of any chemical agents.
Another object of the present invention is to provide a consortium of microorganisms in dry form as powders / granules / coated or uncoated.
Therefore, the present invention provides a consortium of probiotic microorganisms in the formulation which when administered in the vagina multiply by themselves and produce and secrete organic acids and other metabolites which are inhibitory to pathogens causing abnormal vaginal discharge syndrome.
Summary of the invention:
The present invention relates to a formulation for treatment of the abnormal vaginal discharge syndrome (AVDS) or Vaginosis comprising a consortium of microorganisms belonging to Bacillus species and a carrier having a total strength of up to 150 billion CFU.
The microorganisms in the present formulation are selected from the group Lactobacillus sporogenes, Lactic acid Bacillus and Lactobacillus acidophilus. The selected microorganisms, viz. Lactobacillus sporogenes, Lactobacillus acidophilus and other lactic acid bacteria can independently grow and produce lactic acid in the vagina when applied. However, they grow synergistically together to bring the therapeutic action faster.
The carrier for making the formulation is selected from the group comprising of lactose, starch, magnesium stearate, talc, silicon-di-oxide etc.
The pharmaceutical adjuvants are selected from the group consisting of Lactose, Starch, Povidone, Talc, sodium starch glycollate, etc
The formulation is in the form of tablet, capsule, gel or powder for topical application.
The present invention further teaches the method of preparing the formulation for treatment of the abnormal vaginal discharge syndrome (AVDS) or Vaginosis comprising the steps of:
- maintaining the selected microorganism as pure slant cultures wherein each culture is manufactured individually in fermenters,
- harvesting the broth from the fermenters,
- centrifuging the said broth to obtain a biomass,
- spray drying the biomass, and

- blending the calculated amount of each culture in a Double Cone Blender along with the carrier to obtain the formulation.
Detailed Description of the invention:
It has been observed that women suffering from recurring episodes of abnormal vaginal discharge syndrome (AVDS) or Vaginosis are invariably depleted of probiotics microorganisms, which are present in healthy women's vagina.
These friendly probiotic microorganisms secrete organic acid, which maintain the vaginal pH of healthy women around 4. The acidic pH is a discouraging factor for several pathogens. They also secrete substances, which are inhibitory to the growth of pathogens. The reasons of depletion of these microorganisms is due to the use of antibiotics as these antibiotics destroy beneficial microorganisms along with the pathogens, stress, use of other topical applications, contraceptives etc also disturb the friendly microflora.
The present invention relates to a composition for broad based treatment and prevention of vaginal infections.
The composition of the present invention comprises a consortium of microorganisms such as Lactobacillus sporogenes, Lactic Acid Bacillus and Lactobacillus acidophilus in the form of a tablet for topical application. The said tablet is for tntravaginal application.
The selected microorganisms, viz. Lactobacillus sporogenes, Lactobacillus acidophilus and other lactic acid bacteria can independently grow and produce lactic acid in the vagina when applied.
These microorganisms together have a very good ability of lowering the pH in vagina and also secreting substances inhibitory to the growth of pathogens. It has been observed that the combined effect of these organisms is much better than the effect of any single organism from this combination. They grow synergistically together to bring the therapeutic action faster.
The formulation containing a total strength of up to 150 billion CFU.
The topical formulation in the form of suppository, gel, capsule, cream, tablet or any other liquid, semisolid, powder or solid form.
The coated tablet for sustained release of bacteria contained in the formulation for higher efficacy.
Manufacturing Process of Lactobacillus sporogenes (Lactic Acid Bacillus)
The process of manufacturing of Lactic acid bacillus {Lactobacillus sporogenes) is based on the property of bacteria to produce bacterial endospores. In the course of its growth, Lactobacillus sporogenes produces lactic acid by utilizing the nutrients present in the medium. When the nutrients are completely utilized, it is forced to form the spores. The biomass is harvested from the fermentation medium by means of centrifugation and spray dried to obtain a concentrated powder of Lactic acid bacillus.


Description of the individual steps of production:
Characteristics of the strain:
The bacterial strain Bacillus coagulans (also known as Lactobacillus sporogenes) is a gram-positive spore former and produces lactic acid during its growth.
Lactobacillus sporogenes is a gram-positive, spore-forming, lactic acid producing bacillus. The organism requires a complex mixture of organic substrates for growth, including fermentable carbohydrates and peptide
It is aerobic to microaerophilic in nature. It produces L (+) Lactic acid homofermentatively. The growth temperature range of Lactobacillus sporogenes is 35 to 50°C and the optimum pH is 5.5 to 6.5.
Lactobacillus sporogenes is a new bacterium that corrects the problems of depletion of flora of the intestines. Vaginal administration of L. sporogenes was investigated in nonspecific vaginitis. Lactobacillus sporogenes remains viable and healthy for years, so they have a very long shelf life. This bacterium does not even require refrigeration. The spores also pass through the stomach's acidic environment with no problem. For these reasons, Lactobacillus sporogenes is considered to be an excellent supplement to aid in replenishing normal intestinal flora. Lactobacillus sporogenes commonly referred as a beneficial bacterium, also known as a "probiotic."
Isolation and storage:
Pure culture is isolated and its ability to produce lactic acid and spore formation is checked and selected cultures are stored in refrigerator as slants.

Inoculum preparation:
The culture, which is previously selected, is inoculated into flask containing seed medium. The inoculum is cultivated on a shaker for about 10-24 hours at 37°C. The inoculum is also checked for its purity in a phase contrast microscope before inoculating into a batch.
Fermentation:
The fermentation medium is constituted in the fermenter, the pH is adjusted, the medium is sterilized and cooled. The medium is inoculated with the seed culture under aseptic conditions.
The fermentation medium consists of sugars as carbon source; proteins e.g. peptones as nitrogen source; minerals and vitamins.
Cultivation runs at 37°C for about 24 - 65hours. Samples are drawn and the pH is measured and microscopic observations are done. After the completion of growth and attaining maximum sporulation, the batch is harvested by centrifugation.
Centrifugation:
After completion of the fermentation the batch is harvested, the broth is removed through the harvest line and centrifuge to obtain biomass.
Spray drying:
The biomass that is obtained in the centrifugation is spray dried to obtain concentrated spray dried Powder.
Blending:
The concentrated powder, which is obtained in the spray drying, is analyzed. The batch is further diluted with a diluent of choice to a desired strength and analyzed and packed as required.
Formulation:
The product is again blended with other Active Ingredients like Lactobacillus acidophilus and other pharmaceutical adjuvants like binding agents and disintegrants for formulation into suitable dosage forms like tablets (for intra-vaginal application) by standard pharmaceutical practices using suitable pharmaceutical machinery. The product is analyzed as per the specifications and packed suitably.
Manufacturing Process of Lactobacillus acidophilus:
The process of manufacturing of Lactobacillus acidophilus is based on the property of bacteria to proliferate and multiply when supplied with nutrients. In the course of its growth, Lactobacillus acidophilus consumes the nutrients present in the medium and multiplies. When the nutrients are completely utilized the biomass is harvested from the fermentation medium by means of centrifugation and lyophilized to obtain a concentrated powder of Lactobacillus acidophilus


Description of the individual steps of production:
Characteristics of the Strain:
Lactobacillus acidophilus is one of several bacteria in the genus Lactobacillus. It is commonly used commercially together with Streptococcus salivarius in the production of yogurt. The bacterial strain Lactobacillus acidophilus is a Gram-positive rod shaped bacteria and produces lactic acid during its growth.
Lactobacillus acidophilus gets its name from lacto- meaning milk, -bacillus meaning rodlike in shape, and acidophilus meaning acid loving.
This bacterium thrives in acidic environment (pH 5.5-6.5 or lower) and grows best at 35-45°C. Lactobacillus acidophilus occurs naturally in a variety of foods, including dairy, grain, meat, and fish. It is also present in human (and animal) intestines, mouths, and vaginas. L. acidophilus (and all lactic acid bacteria) absorbs lactose and metabolizes it into lactic acid. Certain related species (known as heterofermentive) also produce ethanol, carbon dioxide, and acetic acid this way. Lactobacillus acidophilus itself (a homofermentative microorganism) produces only lactic acid. Like many bacteria, L. acidophilus can be killed by excess heat, moisture, or direct sunlight.
L acidophilus may provide additional health benefits, including improved gastrointestinal function, a boosted immune system, and a decrease in the frequency of vaginal yeast infections. Some people report L acidophilus provides relief from indigestion and

diarrhea. It has been found that feed supplemented with L acidophilus and fed to cattle resulted in a 61% reduction of Escherichia coli. Research at Oklahoma State University has indicated L. acidophilus may be helpful reducing serum cholesterol levels. Studies at the University of Kentucky indicated a treatment with L acidophilus resulted in a 10 to 12% reduced risk of coronary heart disease in persons with high blood serum cholesterol levels.
L acidophilus is part of the normal vaginal flora. The acid produced by L acidophilus in the vagina helps to control the growth of the fungus Candida albicans, helping to prevent vaginal yeast infections. Certain spermicides and contraceptive creams can kill L. acidophilus in the vagina, clearing the path to possible yeast infections.
Isolation and storage:
Pure culture was isolated and its ability to produce lactic acid and is checked and selected cultures are stored in refrigerator as slants.
Inoculum preparation:
The culture, which is previously selected, is inoculated into flask containing seed medium.
The inoculum is cultivated on a shaker for about 20 - 24 hours at 37°C.
The inoculum is also checked for its purity in a phase contrast microscope before inoculating into a batch.
Fermentation:
The fermentation medium is constituted in the fermenter, the pH is adjusted, the medium is sterilized and cooled. The medium is inoculated with the seed culture under aseptic conditions.
The fermentation medium consists of sugars as carbon source; proteins eg. peptones as nitrogen source; minerals and vitamins.
Cultivation runs at 37°C for about 36-60hours. Samples are drawn and the pH is measured and microscopic observation is done. After the completion of growth the batch is harvested by centrifugation.
Centrifugation:
After completion of the fermentation the batch is harvested, the broth is removed through the harvest line and centrifuged to obtain biomass.
Spray drying:
The biomass that is obtained in the centrifugation is lyophilized to obtain concentrated dried Powder.
Blending:
The concentrated powder, which is obtained in the spray drying, is analyzed.

The batch is further diluted with a diluent of choice to a desired strength and analyzed and packed as required.
Formulation:
The product is again blended with other active Ingredients like Lactobacillus sporogenes and other pharmaceutical adjuvants like binding agents and disintegrants for formulation into suitable dosage forms like tablets (for intra-vaginal application) by standard pharmaceutical practices using suitable pharmaceutical machinery. The product is analyzed as per the specifications and packed suitably.
The tablet specifically prepared for vaginal application is inserted into the vagina with the help of clean fingers or with the help of special applicators provided with the formulation. The microorganisms present in the formulation multiply in the vagina and produce lactic acid and other metabolites, which are inhibitory to the growth of pathogenic microorganisms present in the vagina and cure the disease.
The invention will now be explained with the help of examples without limiting the scope of the invention.
Example 1:
Granules of starch are prepared by making a paste of soluble starch and drying it a Fluid Bed Drier. The starch granules are sifted to get granules of suitable size. The sifted starch granules are blended with the powders of Lactobacillus acidophilus and Lactobacillus sporogenes, Lactose, Starch, Magnesium stearate and talc in a double cone blender. The blended product is made into tablets in a Rotary Tablet Machine using suitable punches to get the required size and shape of the vaginal tablet. The temperature during the above blending process is at ambient temperature. The temperature during compression of tablets may be about 45°C to 50°C for a short period. Appropriate overages of active ingredients are added to overcome any losses during the manufacturing process of the formulation.
The Concentration of the active ingredients can be adjusted as per the requirements. Some proposed formulae of the vaginal tablets:
Formula 1:
Each uncoated I coated vaginal tablet contains: Lactobacillus acidophilus: 1 Billion CFU Lactobacillus sporogenes: 1 Billion CFU
Formula 2:
Each uncoated I coated vaginal tablet contains: Lactobacillus acidophilus: 5 Billion CFU Lactobacillus sporogenes: 5 Billion CFU
Formula 3:
Each uncoated / coated vaginal tablet contains: Lactobacillus acidophilus: 15 Billion CFU Lactobacillus sporogenes: 15 Billion CFU

Formula 4:
Each uncoated I coated vaginal tablet contains: Lactobacillus acidophilus: 20 Billion CFU Lactobacillus sporogenes: 10 Billion CFU
Formula 5:
Each uncoated I coated vaginal tablet contains: Lactobacillus acidophilus: 30 Billion CFU Lactobacillus sporogenes: 20 Billion CFU
Formula 6:
Each uncoated I coated vaginal tablet contains: Lactobacillus acidophilus: 0.5 Billion CFU Lactobacillus sporogenes: 0.5 Billion CFU
It will readily be appreciated by those skilled in the art that the present invention is not limited to the specific embodiments herein shown. Thus variations may be made within the scope and spirit of the accompanying claims without sacrificing the principal advantages of the invention.

We Claim:
1. A formulation for treatment of the abnormal vaginal discharge syndrome (AVDS)
or Vaginosis comprising a consortium of microorganisms belonging to Bacillus
species, carrier and other pharmaceutical adjuvants like binding agents and
disintegrants.
2. A formulation as claimed in claim 1 wherein the microorganisms are selected
from the group Lactobacillus sporvgenes, Lactic acid Bacillus and Lactobacillus
acidophilus.
3. A formulation as claimed in claim 1 wherein carriers are selected from the group
consisting of lactose, starch, magnesium stearate, silicon-di-oxide, talc etc.
4. A formulation as claimed in claim 1 wherein the pharmaceutical adjuvants are
selected from the group consisting of Lactose, Starch, Povidone, Talc, sodium
starch glycollate, etc
5. A formulation as claimed in claim 1 wherein the formulation has a total strength
of up to 150 billion CFU.
6. A formulation as claimed in claim 1 wherein formulation is in the form of a tablet,
capsule, gel or powder for topical application.
7. A formulation as claimed in claim 1 wherein said formulation is for intravaginal
application.
8. The method of preparing the formulation for treatment of the abnormal vaginal
discharge syndrome (AVDS) or Vaginosis as claimed in claim 1 comprising the
steps of:
- maintaining the selected microorganism as pure slant cultures wherein each culture is manufactured individually in fermenters,
- harvesting the broth from the fermenters,
- centrifuging the said broth to obtain a biomass,
- spray drying the biomass, and
- blending the calculated amount of each culture in a Double Cone Blender
along with the carrier to obtain the formulation.
9. A formulation for treatment of the abnormal vaginal discharge syndrome (AVDS)
or Vaginosis substantially as herein described with reference to the foregoing
examples.
10. The method of preparing the formulation for treatment of the abnormal vaginal
discharge syndrome (AVDS) or Vaginosis substantially as herein described with
reference to the foregoing examples.