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Tinidazole is a versatile anti-amoebic and anti-anaerobic drug used in treatment of intestinal infection. The aim of present study was to develop and evaluate a guar gum based novel target release Tinidazole matrix tablet in animal models and healthy human volunteer using Gamma Scintigraphy technique. Anti-anaerobic and anti-protozoal activity of the developed formulation was studied in vitro against Bacteroides fragilis and Dentamoeba fragilis. Tinidazole was successful radiolabelled with 99mTc-pertechnetate using stannous chloride as a reducing agent and stable up to 24 h in normal saline and serum. Radiolabeled formulation was evaluated in 6 Newzealand white rabbits by gamma Scintigraphy in static manner up to 24 h for its retention in gastrointestinal tract (GIT). Similar set of study was conducted in 12 healthy human volunteers for similar objective Scintigraphy images of healthy human volunteer showed retention of optimized formulations in stomach up to 60 min, from where it moved to duodenum further and reached ileum in around 5 h. However, initiation of drug release was observed from intestine at 7 h. Complete dissociation and release of drug was observed at 24 h in colon due to anaerobic microbial rich environment. Results drawn from Scintigraphy images indicate that radiolabeled 99mTc-Tinidazole tablet transit through upper part of GI without disintegration. Hence the developed matrix tablet may have a role in treatment of intestinal infection caused by anaerobic bacteria.