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. hominisand 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 polyethylenegycol: 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 ofyeast 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

Evaluation of Vaginal Suppositories:

1. Physical evaluation:

Table2: Test of Appearance, Odour, Colour, Shape, Surface Condition:

Sr.No	Physical characteristics	Formulation
Sr.No	Physical characteristics	Clotrimazole suppository
1	Shape	Ogive
2	Surface condition	Smooth
3	Color	Half white
4	Odor	Odourless

Test of appearance: odour, colour, shape, surface condition:

Colour and the surface characteristics of the suppositories are relatively easy to assess. It is important to check for the absence of fissuring, pitting, fat blooming, exudation, sedimentation, and the migration of the active ingredients. ^[30-32]Suppositories can be observed as an intact unit and also by splitting them longitudinally, the result shown in table. 2

Weight Variation Test:

The weight variation test was determined according to the British Pharmacopoeia. Twenty suppositories were weighed individually and the average weights were determined. No suppositories should deviate from average weight by more than 5% except two, which may deviate by not more than 7.5%, shown in table 3. ^[32]

Table 3: Physico-Chemical Characterization formulations.

Parameters	Formulation
Weight variation *	5.0307 ± 0.1528
Hardness *	1.90 ± 0.12
Melting time*	37.6 - 41.6 ± 0.51
Softening time*	10.24 ± 0.04
Disintegration time*	13.34 ± 0.14
Content uniformity*	98 % ± 1.53

* All Values Represents Mean + sd, n=6

Test of physical strength:

Hardness (breaking) test:

The hardness of 10 suppositories from each batch was determined by cutting the middle portion of suppository. It was measured in its diametric direction using Monsanto hardness tester. Result shown in table 3.^[^32]

Test for melting range:

The ascending melting point method was used to determine the melting point of each type of suppositories. Capillary tubes, 10 cm in length, sealed at one end, were filled with the formulation to about 1cm height, and then it was dipped in gradually heated electro-thermal thermometer. The melting temperature was recorded when the suppositories started to melt. Results shown in table 3 ^[33-34]

Test of softening time:

It measures the time necessary for suppository to liquefy under pressure similar to those Bound in rectum/vagina in presence of water at body temperature.^[^32-35]shown in table 3.

Disintegration Test (tablet disintegrator):

Randomly six suppositories were selected from each batch for disintegration test. Disintegration test was performed without disc in citric acid/phosphate buffer solution pH 4.4 maintained at 37± 0.5°C using USP disintegration apparatus (Electrolab ED-2L). The suppository to be tested was placed in a cylindrical glass container with perforated ends and immersed in 1,000 ml of citric acid/phosphate buffer solution pH 4.4 maintained at 37 ± 0.5°C. The cylindrical glass container was moved up and down in the buffer. The time for disintegration was noted, which should not more than 60 minutes as per BP. shown in table 3.^[^30-32]

2. Chemical Evaluation:

Test for uniformity:

3. Release Studies/diffusion/dissolution rate:

Fig.1.Release profile of Clotrimazole from suppository

Three suppositories were randomly selected from each base and assayed individually for drug content. The suppository was melted with gentle heating in a water bath in the presence of 25 mL of phosphate buffer solution, pH 7.4. The volume was adjusted to 250 mL with phosphate buffer. The flask was agitated on a shaking water bath at 37°C for 4 h. After centrifugation and filtration, the UV absorbance of the solution was measured spectrophotometrically at λmax 314 nm against a blank solution prepared by treating plain suppositories in the same manner ^[29]shown in table 3. In-vitro dissolution/release rate profile were determined by spectrophotometrically at 260 nm shown in Fig1. The Cumulative Percentage release was noted for 12 hr as Carbopol 934 , one of the base of formulation giving some sustain released action of a drug which was found to be 98.60 % at acceptable limit.

4. Microbial Evaluation/Assay:

Table 4: Microbial Strains

S.No	Strain	Organism	Media
1	Staphylococcus aureus ATCC 25923	Gram Positive	Mueller Hinton Agar
2	Bacillus subtilis ATCC 13597	Gram Positive	Mueller Hinton Agar
5	Aspergillusniger ATCC 9029	Fungal Strain	Sabouraud Dextrose Agar
6	Candida albicans ATCC 24433	Fungal Strain	Sabouraud Dextrose Agar

A standardized inoculum of bacteria/fungus is swabbed onto the surface of a Mueller Hinton Agar/ Sabouraud Dextrose Agar plates. Sample of antimicrobial agents are loaded in well in the agar.

After overnight incubation, the diameter of zone of inhibition is measured around each disk, and the experiment was carried out with help of Indian Pharmacopeia, NCLS Guidelines^[^27-28]. Stains used, shown in table 4 and observation in table 5 and 6.

5. Stability Studies:

Suppositories were wrapped in the aluminium 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 ^[28]. Suppositories were examined visually and drug content as per the procedure of content uniformity, result shown in table 7.

Table 5: Summary of Results of Antimicrobial Activity

S. No	Formulations	MIC Concentration (µg/mL)
S. No	Formulations	*Staphylococcus aureus*	*Bacillus subtilis*	*Aspergillus* *niger*	*Candida albicans*
1	Clotrimazole suppositories	16	8	>64	>64

Table 6:.Antimicrobial Activity of Clotrimazole Suppositories

S.No	Dose of Formulations (µg/mL)	Zone of inhibition in diameter (mm)
S.No	Dose of Formulations (µg/mL)	*Staphylococcus aureus*	*Bacillus subtilis*	*Aspergillus* *niger*	*Candida albicans*
1	64	14	16	4	3
2	32	15	16	0	2
3	16	12	15	0	0
4	8	7	12	0	0
5	4	8	7	0	0
6	2	8	8	0	0
7	1	6	8	0	0
8	0.5	0	6	0	0
9	0.25	0	6	0	0

RESULT AND DISCUSSION:

In the current study, successful attempts were made to develop lactic acid spore containing Clotrimazole suppositories for the treatment of vaginosis. The formulations were tested under in vitro conditions on fungal and bacterial culture taken as a model causative organism, microbial assay, test as well as evaluated for the physicochemical parameters such as appearance, physical properties, drug content, in-vitro dissolution and stability studies. All the physical characteristics of 20 suppository formulation shown in table 2, appearance was give shape, odourless with smooth surface and half white colour because of PEG 6000-8000:carbapol base used. The other Physico-chemical characterization of the formulations as shown in table 3, weight variation of all suppositories were within the acceptable limit of 100% ± 5%. The breaking strength of all suppositories shown in table 3, were between 1.7- 2.1 kg/cm which was good for the expected results. The average melting range 37.6 - 41.6^oC shown in table 3, which was satisfactorily to melt at normal body temp 37^oC. Product’s liquefaction time was measured and the average softening time was 9.24 ± 0.29 minutes which is up to the mark, shown in table 3, since in general liquefaction should take no longer than about 30 minutes. The mean disintegration time as shown in table 3, was 13.34 ± 0.14 minutes which was matching with acceptable limits, according to BP the disintegration time of each suppository should be less than 60 minutes. The drug content of all the suppositories was determined spectrophotometrically at 260 nm shown in table 3. It varied from 98.22 to 99.41 % which was at acceptable limit of 85% -115% of the label claim for suppository.

In-vitro dissolution/release rate profile were determined by spectrophotometrically at 260 nm shown in Fig1, the Cumulative Percentage release was noted for 12 hr as Carbapol 934 , one of the base of formulation giving some sustain released action of a drug which was found to be 98.60 % at acceptable limit.

Microbial test and stability studies were found to be well within the limits and official standards Microbial Evaluation/Assay as shown in table 4,5 and 6, was determined by minimum inhibitory concentration (µg/mL) and by measuring the zone of inhibition (diameter in mm), the formulation was found to be the effective against the Staphylococcus aureus, Bacillus subtilis, Aspergillus niger, Candida albicans, microorganisms which are the primary causative organism for bacterial vaginosis.

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 7. 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 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 inhibitory action of the Clotrimazole was satisfactorily. The methods of its evaluation developed in this research work may be beneficial in preventing bacterial vaginosis. Further investigations have to be carried out in antimicrobial activity 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, BM college of Pharmaceutical Education and Research for providing necessary facilities.

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Received on 05.08.2014 Modified on 10.09.2014

Res. J. Pharm. Dosage Form. and Tech. 6(4):Oct.- Dec.2014; Page 230-234

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	98.70 ± 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