Formulation of Povidone Iodine 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:

Human vagina represents a dynamic ecosystem dominated by certain species of Lactobacillus. This microorganism restricts the growth of pathogens by using properties of steric exclusion and inhibitory substance production. Serious complications including bacterial vaginosis and vaginal cancer are often determined in women with reduced numbers of lactobacilli. Local application of Lactobacillus is consequently promising to keep the vagina colonized by this strain, which consequently reduces the infections. The first objective of this research was to develop a local application pharmaceutical formulation of a vaginal suppository containing lyophilized culture of Lactobacillus with anti microbial agent Povidone iodine. The second objective was to establish a stable clindamycine suppositiry alone with Lactic acid Bacillus Spores for vaginosis.

KEYWORDS: Vaginosis, Lactic acid Bacillus Spores, Povidone Iodine, formulation, viability, stability.

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 Povidone Iodine 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 glycole: carbopol base. ^[7-20]

Povidone iodine:

Povidone-iodine (PVP-I) is a chemical complex of polyvinylpyrrolidone (povidone, stable PVP) and elemental iodine. It contains from 9.0% to 12.0% available iodine, calculated on a dry basis. Gardnerella, Bacteroides and Enterobacteria , Proteus, Staphylococcus, Streptococcus, Escherichia, Salmonella, Candida, Serralia Spores-baccillus, clostridium, Shigella, Clostridium, Corynebacterium, Mycobacterium, Diplococcus, Citrobacter, Microsporum. Povidone iodine does not appear to have activity against most strains of vaginal lactobacilli. For many years iodine has been recognized as an effective broad- spectrum biocidal agent. Using PVP-iodine has significantly reduced the irritancy and toxicity associated with its use. Because of the products wide usage and established efficiency, it is presently included as an antiseptic agent in the United States pharmacopoeia as well as in many other national compendia.

Mechanism of action:

Elemental iodine has a very broad antimicrobial spectrum: bacteria, viruses, bacterial endospores, fungi and protozoas are destroyed through oxidative interaction and direct iodination of biological macromolecules. However, there have been reports of certain resistant germs. Povidone-iodine (synonym: PVP iodine) is an iodophor, i.e. it is a labile complex of iodine with the polyvinyl pyrrolidone polymer, from which iodine is continually delivered. Only this free iodine has antimicrobial activity.^[21-25]

MATERIAL AND METHOD:

Povidone Iodine 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, Povidone Iodine 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 Povidone Iodine Suppository

S.No	Ingredients	Qty taken in gms	Actual qty to be taken for 1 suppository
1	Povidone Iodine I.P	0.1gm	100 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. Shown in table. 2. ^[26]

Table.2: Viability of lactobacillus sporogenes from povidone iodine suppositories.

*Time Period*	*CFU (Colony Forming Unit)*
*Time Period*	*Ambient temperature*			*2-8°C (Cool Storage)*
0 Day	6.31 X 10⁵	6.12 X 10⁵	6.16 X 10⁵	6.31 X 10⁵	6.12 X 10⁵	6.16 X 10⁵
1^st week	5.74 X 10⁵	5.87 X 10⁵	5.61 X 10⁵	6.22 X 10⁵	5.98 X 10⁵	6.02 X 10⁵
2^nd week	6.13 X 10⁴	6.77 X 10⁴	6.21 X 10⁴	5.61 X 10⁵	5.28X 10⁵	5.76 X 10⁵
3^rd week	4.35 X 10⁴	4.72 X 10⁴	4.81 X 10⁴	4.67 X 10⁵	4.52 X 10⁵	4.81 X 10⁵
4^th Week	5.21 X 10³	5.32 X 10³	5.05 X 10³	4.18 X 10⁵	4.21 X 10⁵	4.16 X 10⁵

Table 3: Stability Study of Povidone Iodine 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	98.07 ± 0.54	No significant changes were Seen	97.61 ± 0.39
2	15	No significant changes were Seen	97.15 ± 0.90	No significant changes were Seen	96.06 ± 0.16
3	30	No significant changes were Seen	95.44 ± 1.30	No significant changes were Seen	93.07 ± 0.86
4	45	No significant changes were Seen	92.58 ± 0.98	No significant changes were Seen	87.62 ± 1.46

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, observation shown in table.3. ^[26-28]

RESULT AND DISCUSSION:

In the current study, successful attempts were made to develop a stable lactic acid spore containing Povidone Iodine 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.

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Received on 20.01.2013 Accepted on 12.02.2013

Asian J. Pharm. Res. 3(1): Jan.-Mar. 2013; Page 06-09