INTRODUCTION:

The present research and study is directed to Anti-microbial and lactic acid bacillus combination in a comprising pharmaceutical acceptable carrier and the methods for treating fungal, bacterial, protozoal and yeast infection. Some of the most common pathogens associates with invasive fungal infections are the opportunistic yeast, such as Candida spp. and Asppergillus spp. thousands of Candida spp cells can be present in an individual, primarily in the gastrointestinal tract, as a harmless commensal organism. However, Candida spp., such as C.albicans, cause oppotunistics fungal infections. Infections can be localized such as a vaginal infection or an oral infection, both of which cause a considerable degree of discomfort. The objective of this study was to develop a vaginal suppository containing lacti acid bacillus spores. Further the present research study provided the combination of anti infective drug clotrimazole with micro organism lactic acid bacillus spores in a pharmaceutical formulation as suppository.

Bacterial vaginosis (BV):

BV is a clinical syndrome associated with a group of pathogenic microorganisms rather than specific pathogen. It is a very common manifestation amongst the women population. Though the exact causative pathogen has not been figured out, it has been observed that there is a corresponding decrease in the population of the lactobacilli species. This results in the increase in the pH of the vaginal lumen due to the reduction in the lactic acid production. Apart from the lactic acid, the production of lactocin and H2O2 also receives a setback. In general, the lactobacilli are replaced with the increased population of pathogenic gram negative anaerobic bacteria like E. coli, G. vaginalis, M. hominis and M. Curtisii. Bacterial vaginosis (BV) is characterized by an alteration of normal vaginal microflora in which a mixed anaerobic bacterial flora becomes prevalent over the population of lactobacilli. The common organisms causing a vaginosis as Gardnerella vaginalis, Candida albicans. (candidiasis, genital candidiasis, or vulvovaginal candidiasis), Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae, the herpes simplex virus, the human papilloma virus (HPV), Gardnerella vaginalis, Mobiluncus, Bacteroides, and Mycoplasma ^[1-6].

Lacto bacillus spores:

Lactobacillus refers to a group of lactic acid producing bacteria that make up many of the 400 normal probiotic species in the human body. Lactobacilli are “friendly” bacteria, meaning that they normally occur in the human gastrointestinal and genitourinary tracts and play important roles in promoting good health. The presence and dominance of Lactobacillus in the vagina is associated with a reduced risk of bacterial vaginosis and urinary tract infections. The mechanisms appear to involve anti-adhesion factors, by-products such as hydrogen peroxide and bacteriocins lethal to pathogens. In the present study, lactic acid bacillus spores since it gives better releasing rate in a conventional suppository of Water Soluble/Water Miscible Bases polyethylene gycol: carbopol base ^[7-20].

Clotrimazole:

An imidazole derivative with a broad spectrum of antimycotic activity. Clotrimazole is an antifungal medication commonly used in the treatment of fungal infections of both humans and animals such as vaginal yeast infections, oral thrush, and ringworm. It is also used to treat athlete's foot and jock itch, body ringworm. It can also be used to prevent oral thrush in certain patients. Clotrimazole demonstrates activity both in vitro and in clinical infections against the following yeast, protozoa, fungus: Candida, Trichomonas vaginalis, Giardia duodenalis (also termed G. lamblia), and Entamoeba histolytica. Clotrimazole does not appear to have activity against most strains of vaginal lactobacilli. It has been used for trichomoniasis, amoebiasis and giardiasis. Clotrimazole is active against a wide range of yeast bacterial infection including candida Bacteroides spp. Clostrium spp. and gardnerella, vaginalis.

Mechanism of Action

The primary mechanism of action of clotrimazole is against the division and growing of fungi. Clotrimazole alters the permeability of the fungal cell wall and inhibits the activity of enzymes within the cell. Studies show minimal concentrations of clotrimazole cause leakage of intracellular phosphorus compounds into the ambient medium, along with the breakdown of cellular nucleic acids and an accelerated. This leads eventually to the cell's death. It does not appreciably spread through the user's body, but remains at the point of application. It inhibits biosynthesis of the sterol ergostol, an important component of fungal cell membranes. Its action leads to increased membrane permeability and apparent disruption of enzyme systems bound to the membrane ^[21-25].

MATERIAL AND METHOD:

Clotrimazole I.P was a gift sample from Alpa Laboratory Ltd., Indore, Madhya Pradesh. Poly Ethylene Glycol 6000-8000 and carbopol 934 purchased from Central Drug House (P) Ltd., New Delhi. Lacto bacillus spores also were gifted from Sanzyme Ltd Banjara Hill, Hyderabad. All other chemicals and reagents were used of analytical grade.

Preparation of Suppositories:

The 20 vaginal suppository were prepared with the same combination as lactic acid bacillus spores, Clotrimazole and bases Polyethelen glycol (PEG 6000-8000), Carbapol 934 (1%) as shown in table 1.The conventional suppositories were prepared by fusion method. The Carbapol 934 (1%) was used as a muco-adhesive agent and PEG (6000-8000) as the suppository base which was melted over the water bath, then carbapol 934, followed by drug was added to the melted base with continuous stirring. Finally, lyophilized Lactobacillus Spore was added in the melted base at the temperature about 40-45°C with gentle stirring until a homogeneous mass was produced. After that the mixture was poured into a metal suppository mold at a temperature just above the congealing point of the suppository base and cooled over the ice bath. The mold was then allowed to solidify for 1 hour at room temperature and finally all the prepared suppositories were kept in the refrigerator for further studies ^[26-28].

Table 1: Formulation of Clotrimazole Suppository

S. No	Ingredients	Qty taken in gms	Actual qty to be taken for 1 suppository
1.	Clotrimazole I.P	0.1gm	100 mg
2.	Lactobacillus SporeS 150 million	1 gm	1000 mg
3.	Carbapol 934	1%	50 mg
4.	Poly Ethylene Glycol 6000-8000	q.s	q.s
	Total	5 gm	5000 mg

Viability Test and Stability of spores

The vaginal suppositories containing Lactobacillus Sporogenes were kept in glass containers at ambient temperature (30±2°C) and 2-8°C for 3 months. At appropriate time intervals, 0, 1 week, 2 week, 3 week and 4 week, the survival of lactobacillus was determined by plate method using MRS agar medium result shown in table.2. ^[26-28]

Stability Studies

Suppositories were wrapped in the aluminum foil and kept in stressed condition by six cycles of freeze (2-8°C) and thaw (25°C) process. Suppositories were also kept in accelerated condition temperature (30°C) for 45 days. Suppositories were examined visually and drug content as per the procedure of content uniformity, result shown in table.3 ^[26-28]

Table 2: Viability of Lactobacillus Sporogenes From Clotrimazole Suppositories

Time Period	CFU (Colony Forming Unit)
Time Period	Ambient temperature			2-8°C (Cool Storage)
0 Day	5.72 X 10⁵	5.61 X 10⁵	5.84 X 10⁵	5.72 X 10⁵	5.61 X 10⁵	5.84 X 10⁵
1^st week	3.12 X 10⁵	4.87 X 10⁵	5.23 X 10⁵	4.31 X 10⁵	4.42 X 10⁵	4.41 X 10⁵
2^nd week	4.13 X 10⁴	3.97 X 10⁴	4.21 X 10⁴	4.11 X 10⁵	4.04 X 10⁵	4.18 X 10⁵
3^rd week	1.61 X 10⁴	1.92 X 10⁴	1.81 X 10⁴	3.67 X 10⁵	3.52 X 10⁵	3.81 X 10⁵
4^th Week	3.91 X 10³	3.82 X 10³	4.05 X 10³	3.18 X 10⁵	3.21 X 10⁵	3.32 X 10⁵

Table 3: Stability Study of Clotrimazole Suppository

S.No	Days	Freeze and Thaw (Six Cycles)		Accelerated Temperature
S.No	Days	Physical Changes	% drug Content ± S.D.	Physical Changes	% drug Content ± S.D.
1	0	No significant changes were Seen	98.71 ± 0.55	No significant changes were Seen	98.24 ± 0.10
2	15	No significant changes were Seen	97.65 ± 0.42	No significant changes were Seen	96.77 ± 0.62
3	30	No significant changes were Seen	96.26 ± 0.88	No significant changes were Seen	93.77 ± 1.30
4	45	No significant changes were Seen	94.94 ± 1.57	No significant changes were Seen	91.38 ± 1.06

RESULT AND DISCUSSION:

In the current study, successful attempts were made to develop a stable lactic acid spore containing Clotrimazole suppositories for the treatment of vaginosis.

Viability Test and Stability of spores, sufficient growth of the Lactobacillus (10⁵ colony-forming units/ml) on 0,1,2,3,4 weeks at ambient and 2-8⁰C temperature respectively, was observed when grown on a standard MRS medium plate as shown in the table 4. Colony characteristics and gram staining confirmed the presence of Lactobacillus. This indicates that the viability of the Lactobacillus was not affected during preparation of the formulation.

Stability studies of suppositories were examined on the day 0,15,30,45 at freeze and at accelerated temperature for percent drug content and physical changes, shown in table 8. It was noted that there were no significant changes in physical and percent drug content seen in the formulation unit respectively.

CONCLUSION:

It was concluded that the bioactive dosage formulation containing anti microbial agent with L. sporogenes appears to be a good candidate for probiotic prophylaxis and treatment of vaginal infections. The developed assembly was satisfactory in simulating the application site. The viability of L. sporogenes was not affected during preparation of the suppository. Thus, the suppository formulation containing Lactobacillus in this research work may be beneficial in preventing bacterial vaginosis. Further investigations have to be carried out in antimicrobial activity with lacto bacillus spore in the bacterial viginosis treatment is needed.

ACKNOWLEDGEMENTS:

Researchers are very much thankful to the Alpa Laboratory Ltd., Indore, Madhya Pradesh, Central Drug House (P) Ltd., New Delhi, Sanzyme Ltd Banjara hill, Hyderabad, MJRP College of Heath Care and Allied Sciences, MJRP University Jaipur, Sharad Pawar College of Pharmacy, Nagpur, for providing necessary facilities.

REFERENCES:

1. Penn RL. Gynecological and obstetrical infections. In: Reese RE, Gordon Douglas Jr. R, 2nd ed. A practical approach to infectious diseases. Boston: Little, Brown and Company; 1986.p.385-421.

2. Thadepalli H, Gorbach SL, Keith L. Anaerobic infections of the female genital tract; bacteriologic and therapeutic aspects. Am-J Obstet Gynecol 1973;117:1034-40.

3. Ledger WJ. Micro-organisms that cause infection. In: Ledger WJ, ed. Infections in the female. 2nd ed. Philadelphia: Lea & Febiger;1986.p.9-34.

4. Ledger WJ. Antimicrobial agents. In: Ledger WJ,2nd ed. Infections in the female. Philadelphia: Lea & Febiger;1986.p.95-126.

5. Bleker OP, Folkertsma K, Dirks-Go SIS. Diagnostic procedures in vaginitis. Eur J Obstet Gynecol Reprod Biol 1989;31:10-11.

6. Bleker OP, Folkertsma K, Dirks-Go SIS. Diagnostic procedures in vaginitis. Eur J Obstet Gynecol Reprod Biol 1989;31:179-83.

7. McFarland LV. Beneficial microbes: health or hazard? Eur J Gastroenterol Hepatol 2000;12:1069-1071.

8. Gionchetti P, Rizzello F, Venturi A, Campieri M. Probiotics in infective diarrhoea and inflammatory bowel diseases. J Gastroenterol Hepatol 2000;15:489-493.

9. Larsen B. Vaginal flora in health and disease. Clin Obstet Gynecol 1993; 36:107-121.

10. Redondo-Lopez V, Cook RL, Sobel JD. Emerging role of lactobacilli in the control and maintenance of the vaginal bacterial microflora. Rev Infect Dis 1990; 12:856-872.

11. Hiller SL. The vaginal microbial ecosystem and resistance to HIV. AIDS Res Hum Retroviruses 1998; 14(suppl 1):17-21.

12. Boris S, Barbes C. Role played by lactobacilli in controlling the population of vaginal pathogens. Microbes Infect 2000;2:543-546.

13. Sobel JD. Vaginitis. N Engl J Med 1997; 337:1896-1903.

14. McGregor JA, French JI. Bacterial vaginosis in pregnancy. Obstet Gynecol Surv 2000;55(suppl 5):1-19.

15. Sweet RL. Role of bacterial vaginosis in pelvic inflammatory diseases. J Infect Dis 1995; 20(suppl 2):271-275.

16. Sooper DE. Bacterial vaginosis and postoperative infections. Am J Obstet Gynecol 1993; 169:467-469.

17. Van de Wijgert, J.H.H.M, Mason P.R, Gwanzura L. Intravaginal practices, vaginal £ora disturbances, and acquisition of sexually transmitted diseases in Zimbabwean women. J. Infect. Dis. 2000;181,587-594.

18. Chaim W, Mazor M, Leiberman J.R.The relationship between bacterial vaginosis and preterm birth. Arch. Gynecol. Obstet.1997;259: 51-58.

19. 19.Reid G. and Bruce, A.W. Selection of Lactobacillus strains for urogenital probiotic applications. J. Infect. Dis. 2001;183 (Suppl.1),77-80.

20. Reid G, Bruce, A.W. and Taylor, M. Instillation of Lactobacillus and stimulation of indigenous organisms to prevent recurrence of urinary tract infections. Microecol Ther 1995;23:32-45.

21. Ritter. W. Pharmacokinetic fundamentals of vaginal treatment with clotrimazole. Am J Obstet Gynecol 1985;152:5-7.

22. Gachotte D, Pierson CA, Lees ND, Barbuch R, Koegel C, Bard M. A yeast sterol auxotroph (erg25) is rescued by addition of azole antifungals and reduced levels of heme. Proc Natl Acad Sci U S A. 1997; 94(21):11173-8

23. Pastorek JG, CotchMF, Martin DH, Eschenbach DA. Vaginal Infections and Prematurity Study Group: clinical and microbiological correlates of vaginal trichomoniasis during pregnancy. Clin Infect Dis 1996;23:1075-80.

24. Warrilow AG, Martel CM, Parker JE, Melo N, Lamb DC, Nes WD, Kelly DE, Kelly SL. Azole binding properties of Candida albicans sterol 14-alpha demethylase (CaCYP51). Antimicrob Agents Chemother. Epub 2010 Oct;54(10):4235-45.

25. Sorvillo F, Kerndt P. Trichomonas vaginalis and ampliﬁcations of HIV- 1 transmission. Lancet 1998; 351(9097):213-4.

26. Sanae K, and Nattha K, Formulation and Evaluation of Vaginal Suppositories Containing Lactobacillus, World Academy of Science, Engineering and Technology 2009;(31):630-633

27. Formulation development and release studies of indomethacin suppositories, ML sah, TR Saini, short communications, Ind. J. pharm. Sciences 2008;70(4):498-501.

28. Naikwade Sonali R, Kulkarni Pratibha P, Jathar Shripad R, Bajaj Amrita N. DARU 2008;16(3):119-127.

Received on 25.01.2013 Accepted on 13.02.2013

Asian J. Pharm. Tech. 3(1): Jan.-Mar. 2013; Page 05-08