Impact of the Herbal Medicine Sophora flavescens on the Oral Pharmacokinetics of Indinavir in Rats: The Involvement of CYP3A and P-Glycoprotein

Sophora flavescens is a Chinese medicinal herb used for the treatment of gastrointestinal hemorrhage, skin diseases, pyretic stranguria and viral hepatitis. In this study the herb-drug interactions between S. flavescens and indinavir, a protease inhibitor for HIV treatment, were evaluated in rats. Concomitant oral administration of Sophora extract (0.158 g/kg or 0.63 g/kg, p.o.) and indinavir (40 mg/kg, p.o.) in rats twice a day for 7 days resulted in a dose-dependent decrease of plasma indinavir concentrations, with 55%–83% decrease in AUC0-∞ and 38%–78% reduction in Cmax. The CL (Clearance)/F (fraction of dose available in the systemic circulation) increased up to 7.4-fold in Sophora-treated rats. Oxymatrine treatment (45 mg/kg, p.o.) also decreased indinavir concentrations, while the ethyl acetate fraction of Sophora extract had no effect. Urinary indinavir (24-h) was reduced, while the fraction of indinavir in faeces was increased after Sophora treatment. Compared to the controls, multiple dosing of Sophora extract elevated both mRNA and protein levels of P-gp in the small intestine and liver. In addition, Sophora treatment increased intestinal and hepatic mRNA expression of CYP3A1, but had less effect on CYP3A2 expression. Although protein levels of CYP3A1 and CYP3A2 were not altered by Sophora treatment, hepatic CYP3A activity increased in the Sophora-treated rats. All available data demonstrated that Sophora flavescens reduced plasma indinavir concentration after multiple concomitant doses, possibly through hepatic CYP3A activity and induction of intestinal and hepatic P-gp. The animal study would be useful for predicting potential interactions between natural products and oral pharmaceutics and understanding the mechanisms prior to human studies. Results in the current study suggest that patients using indinavir might be cautioned in the use of S. flavescens extract or Sophora-derived products

Expression of cytochrome P450 3A7 in Escherichia coli: Effects of 5' modification and catalytic characterization of recombinant enzyme expressed in bicistronic format with NADPH-cytochrome P450 reductase

Cytochrome P450 3A7 is the major P450 form present in fetal liver tissue and may be responsible for the detoxification of many drugs that reach the fetal circulation, We report the development of bacterial expression systems for P450 3A7, Maximal yields (up to 50 nmol P450/liter culture) were obtained with a construct in which the 5'-terminus of the 3A7 cDNA was modified to include the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, Proc. Natl. Acad. Sci, USA 88, 5597-5601, 1991) and to incorporate several downstream amino acid substitutions derived from the P450 3A5 sequence. This sequence also appeared optimal for expression of P450 3A4 and 3A5, Recombinant P450 3A7 was partially purified using ion-exchange and hydroxylapatite chromatography and reconstituted with NADPH-cytochrome P450 reductase, cytochrome bat and lipids, Activity comparable to that of P450 3A4 was demonstrated toward a number of procarcinogens. An alternative approach was used to further characterize recombinant 3A7 due to low yields of recombinant protein in the expression and poor recovery in the purification, P450 3A7 was subcloned into a bicistronic vector containing human NADPH-cytochrome P450 reductase and expressed in bacteria, Recombinant P450 3A7 coexpressed in bacterial membranes with NADPH-cytochrome P450 reductase showed similar levels of activity toward erythromycin (N-demethylation) and ethylmorphine (N-demethylation) to P450 3A4 and 3A5 expressed in the same system, whereas 3A7 was less active toward midazolam (1'- and 4-hydroxylation) and nifedipine (oxidation). (C) 1997 Academic Press

Expression of Cytochrome-P450-3A5 in Escherichia-Coli - Effects of 5' Modification, Purification, Spectral Characterization, Reconstitution Conditions, and Catalytic Activities (Vol 317, Pg 374, 1995)

Cytochrome P450 (P450) 3A5 is a human enzyme with 85% amino acid sequence identity to the more predominantly expressed P450 3A4 and has been reported to have overlapping catalytic specificity. The 5'-terminus of a P450 3A5 cDNA was modified for optimal expression in Escherichia coli using the vector pCW, by aligning the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, (1991) Proc. Natl. Acad. Sci. USA 88, 5597-5601) to the 3A5 cDNA. Two constructs were made, differing by their identity with the modified 3A4 N-terminal sequence (E. M. J. Gillam, T. Baba, B-R. Rim, S. Ohmori, and F. P. Guengerich, (1993) Arch. Biochem. Biophys. 305, 123-131). The first modified sequence (3A5#1) was identical to recombinant P450 3A4 up to codon 15, the 3A5 sequence being introduced thereafter. In the other (3A5#2), the successful 3A4 N-terminal nucleotide sequence was attached to codon 30. The yield was greater than fourfold higher in the first construct [up to 260 nmol (liter culture)(-1)]. The recombinant P450 3A5 (construct 1) was purified to electrophoretic homogeneity using a variation of a three-step procedure developed previously for P450 3A4, with an overall yield of similar to 40%. Purified P450 3A5 was active in nifedipine oxidation, testosterone 6 beta-hydroxylation, aflatoxin 3 alpha-hydroxylation and 8,9-epoxidation, N-ethylmorphine N-demethylation, erythromycin N-demethylation, and d-benzphetamine N-demethylation. The reconstitution of nifedipine oxidation, testosterone 6 beta-hydroxylation, and the aflatoxin oxidation activities showed dependence upon the presence of cytochrome b(5), divalent cations, phospholipid mixtures, glutathione, and cholate similar to that previously found for purified P450 3A4. However, rates of the N-demethylations of N-ethylmorphine, erythromycin, and d-benzphetamine were as high or higher for P450 3A5 than P450 3A4 and were not particularly dependent upon modifications of reconstitution systems. (C) 1995 Academic Press, Inc