Formulation of Tinidazole Vaginal Suppository Containing Lacto Bacillus Spores.

 

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

1.MJRP College of Heath Care and Allied Sciences, MJRP University, Jaipur India.

2.Sharad Pawar College of Pharmacy, Nagpur India.

 

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 tinidazole. The second objective was to establish a stable tinidazole suppositiry alone with Lactic acid Bacillus Spores for vaginosis.

 

KEYWORDS: Vaginosis, Lactic acid Bacillus Spores, Tinidazole, formulation, viability, stability test.

 

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 tinidazole 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 ethylene: carbopol base [7-20].

 

Tinidazole:

Tinidazole demonstrates activity both in vitro and in clinical infections against the following protozoa: Trichomonas vaginalisGiardia duodenalis (also termed G. lamblia), and Entamoeba histolytica. Tinidazole does not appear to have activity against most strains of vaginal lactobacilli. It has been used for trichomoniasis, amoebiasis and giardiasis. tinidazole is active against a wide range of bacteria including Bacteroides spp. Anaerobic cocci, fuso bacterium spp. Clostrium spp. and gardnerella, Vaginalis. It is also effective against  protozoa including Trichomonas spp. Entamoeba histolytica and Glardia lamblia. 

 

Mechanism of Action:

Tinidazole is a prodrug and antiprotozoal agent. The nitro group of tinidazole is reduced in Trichomonas by a ferredoxin-mediated electron transport system. The free nitro radical generated as a result of this reduction is believed to be responsible for the antiprotozoal activity. It is suggested that the toxic free radicals covalently bind to DNA, causing DNA damage and leading to cell death. [21-25]

 

MATERIAL AND METHOD:

Tinidazole 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. Tinidazole 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]

 

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]

 


 

Table 1: Formulation of  Tinidazole Suppository

S.No

Ingredients

Qty taken in gms

Actual qty to be taken for 1 suppository

1.

Tinidazole I.P

0.2gm

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

 

Table. 2: Viability of  Lactobacillus Sporogenes From Tinidazole Suppositories

Time Period

CFU (Colony Forming Unit)

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

1st  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

2nd  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

3rd  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

4th 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  Tinidazole Suppository

S.

No

Days

Freeze and Thaw (Six Cycles)

Accelerated Temperature

Physical Changes

% drug Content ± S.D.

Physical Changes

% drug Content ± S.D.

1

0

No significant changes were Seen

98.70 ± 0.55

No significant changes were Seen

98.26 ± 0.10

2

15

No significant changes were Seen

97.64 ± 0.42

No significant changes were Seen

96.77 ± 0.62

3

30

No significant changes were Seen

96.28 ± 0.88

No significant changes were Seen

93.78 ± 1.30

4

45

No significant changes were Seen

94.95 ± 1.57

No significant changes were Seen

91.37 ± 1.06

 

 


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]

 

RESULT AND DISCUSSION:

In the current study, successful attempts were made to develop a stable lactic acid spore containing tinidazole 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 2-80 C temperature respectively, was observed when grown on a standard MRS medium plate as shown in the table 2. 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 3. 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 13.02.2013

Modified on 04.03.2013

Accepted on 20.03.2013     

© A&V Publication all right reserved

Research Journal of Pharmaceutical Dosage Forms and Technology. 5(2): March- April, 2013, 75-78