Formulation of Clindamycine hydrochloride vaginal suppository containing Lacto bacillus spores.

S.C. Shivhare¹*, Dr. U.D.Shivhare², Dr. Preeti Srivastav¹, K.G. Malviya¹

¹MJRP college of heath Care and Allied Sciences, MJRP university, Jaipur India.

²Sharad Pawar College of Pharmacy, Nagpur India.

*Corresponding Author E-mail: sshivhare82@gmail.com

ABSTRACT

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 Clindamycine Hydrochloride with micro organism lactic acid bacillus spores in a pharmaceutical formulation as suppository.

KEYWORDS: Vaginosis, Lactic acid Bacillus Spores, Clindamycine hydrochloride, formulation, viability stability.

INTRODUCTION:

Bacterial vaginosis (BV):

BV is a clinical syndrome associated with a group of pathogenic microorganisms rather than

a 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 glycole: carbopol base.^[^7-20]

Clindamycine Hydrochloride:

Clindamycin is a lincomycin-class antibiotic It is synthesized from microbially fermented lincomycin by replacing a hydroxyl group at the 7-position of lincomycin by a chlorine group, that significantly increases its activity. Clindamycine medication prescribed for the treatment of bacterial infections including those resulting in bacterial vaginosis. Clindamycin exhibits bacteriostatic effects on bacterial organisms by inhibiting the biosynthesis of proteins required for bacterial growth. According to MedlinePlus, clindamycin is commonly prescribed for the treatment of bacterial vaginosis in the form of a vaginal suppository or vaginal cream.

Mechanism of action:

The effect of clindamycin, which is primarily bacteriostatic, is exerted by its binding to the 50S ribosomal subunit and the consequent inhibition of bacterial protein synthesis. It is active against aerobic Gram-positive and anaerobic bacteria, mycoplasmas, some protozoa, yeast infection (candidiasis) or infection with Trichomonas vaginalis (trichomoniasis) 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^[21-25]

MATERIAL AND METHOD:

Clindamycine hydrochloride 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, clindamycine hydrocloride 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 Clindamycine Suppository

S No	Ingredients	Qty taken in gms	Actual qty to be taken for 1 suppository
1	Clindamycine Hydrochloride I.P	0.2 gm	200 mg
2.	Lactobacillus Spore 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. ^[26-28]observation shown in table.2

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 ^[27-38]observation shown in table.3

Table 2: Viability of Lactobacillus sporogenes from Clindamycin HCl. Suppositories

Time Period	CFU (Colony Forming Unit)
Time Period	Ambient temperature			2-8°C (Cool Storage)
0 Day	5.92 X 10⁵	6.07 X 10⁵	5.94 X 10⁵	5.92 X 10⁵	6.07 X 10⁵	5.94 X 10⁵
1^st week	5.31 X 10⁵	5.27 X 10⁵	5.23 X 10⁵	5.78 X 10⁵	5.72 X 10⁵	5.81 X 10⁵
2^nd week	4.73 X 10⁴	4.97 X 10⁴	4.61 X 10⁴	5.11 X 10⁵	5.04 X 10⁵	5.08 X 10⁵
3^rd week	3.21 X 10⁴	2.92 X 10⁴	3.11 X 10⁴	4.78 X 10⁵	4.52 X 10⁵	4.71 X 10⁵
4^th Week	1.21 X 10³	1.52 X 10³	1.55 X 10³	2.28 X 10⁵	2.21 X 10⁵	2.12 X 10⁵

Table 3: Stability Study of Clindamycin Hcl 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	98.90 ± 0.46	No significant changes were Seen	98.53 ± 0.20
2	15	No significant changes were Seen	97.58 ± 0.90	No significant changes were Seen	97.24 ± 0.89
3	30	No significant changes were Seen	96.82 ± 0.31	No significant changes were Seen	94.60 ± 0.51
4	45	No significant changes were Seen	94.52 ± 0.54	No significant changes were Seen	91.54 ± 0.21

RESULT AND DISCUSSION:

In the current study, successful attempts were made to develop a stable lactic acid spore containing clindamycine hydrochloride 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 1. 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 2. 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.

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Received on 29.12.2012 Accepted on 10.02.2013

Asian J. Res. Pharm. Sci. 3(1): Jan.-Mar. 2013; Page 05-07