Evaluation of frog as an animal model to study the effective permeability co-efficient: Application to CYP3A4 substrates 

 

By Neelima Yerasi Krishna Devarakonda, Himabindu Vurimindi 

ABSTRACT

To evaluate the reliability of using in situ frog intestinal perfusion technique for permeability assessment of drugs which are substrates for CYP3A4 enzymes. Felodipine was used as the probe drug. Single Pass Intestinal perfusion (SPIP) studies were performed in frogs of the species Rana tigrina using established method for rats with some modifications. Initially the contents of the intestine were flushed out with blank perfusion solution, then with the test solution and then perfused with test solutions at a flow rate of 0.2ml/min using syringe pump for 90 min after 20 min of equilibration. The perfusate was collected at every 10 min. Water flux was quantified using phenol red, a non-absorbable inert marker. Permeability was determined in 6 frogs and the results were presented as mean ± SD. Effective permeability coefficient (Peff) of felodipine was calculated in the presence and absence of inhibitors (ketoconazole and verapamil) using the parallel-tube model. Peff of felodipine when perfused alone was 1.31± 0.04× 10-4cm/s, which was increased to 2.06± 0.37× 10-4cm/s in presence of ketoconazole but was almost unaffected (1.28± 0.14× 10-4cm/s) in presence of verapamil. Mass spectrum of a test sample from perfusion studies showed M+2H peak corresponding to pyridine metabolite of felodipine (mol wt-382) in humans. The results obtained in this study lead us to conclude that it is possible to determine the Peff value for compounds which are substrates of CYP3A4 using in situ frog intestinal perfusion technique. 

Key words: Intestinal permeability, CYP3A4enzymes, Frog perfusion model, Effective permeability co-efficient. 

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