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 vaginalis, Giardia 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]
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 |
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.
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. and Gwanzura, L. et al. 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. and Leiberman, J.R. (1997) The relationship
between bacterial vaginosis and preterm birth. Arch.
Gynecol. Obstet.1997;259: 51-58.
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. Lossick JG, Kent HL. Trichomoniasis: trends in diagnosis and management. Am J Obstet Gynecol 1991;165:1217-22.
22. Cotch MF, Pastorek
JG, Nugent RP, et al. Vaginal Infections and Prematurity Study Group:
demographic and behavioral predictors of Trichomonas vaginalis infection among pregnant women. Obstet Gynecol 1991;78:1087-92.
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. Laga M, Manoka
A, Kikuvu M. Non-ulcerative sexually transmitted
diseases as risk factors for HIV-1 transmission in women: results from a cohort
study. AIDS 1993; 7:95-102.
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 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