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

Effect of <i>Sophora</i> extract on the expression of intestinal and hepatic P-gp.

<p>(A) Statistical data analysis of Western blotting results, (B) image of Western blotting results. P-gp levels were measured using the anti-rat C219 antibody as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031312#s2" target="_blank"><i>Materials and Methods</i></a>. ERp29 was used as control for the normalization of P-gp density. Bars represent mean ± S.E.M. of fold relative to the values in control group (n = 7–8). Statistical significance is indicated as * <i>P</i><0.05, *** <i>P</i><0.001, compared to control.</p

Effect of <i>Sophora</i> extract on the expression of intestinal and hepatic CYP3A1 and CYP3A2.

<p>(A) Statistical data analysis of Western blotting results, (B) image of Western blotting results. CYP3A1 and CYP3A2 levels were measured respectively using the specific anti-rat CYP3A1 and CYP3A2 antibodies. ERp29 was used as control for the normalization of CYP3A density. Bars represent mean ± S.E.M. of fold relative to the values in the control group (n = 7–8). No significant difference in the expression of either intestinal or hepatic CYP3A was observed.</p

Effect of <i>Sophora</i> extract on the output of urine and faeces, and on indinavir excreted via urine and faeces.

<p>(A) Urine output, (B) Faeces output, (C) Indinavir excreted in urine, (D) Indinair excreted in faeces. The urine and faeces were collected from 8 p.m. on day 6 (after the second daily treatment) to 8 p.m. on day 7 (before the second daily treatment). Values are expressed as mean ± S.E.M. (n = 8–9).</p

Time course of the plasma concentration of indinavir.

<p>Animals received oral administration of indinavir (40 mg/kg) together with (A) 1.5% Tween 80 (vehicle), 70% ethanol extract of <i>Sophora flavescens</i> (0.158 g/kg and 0.63 g/kg), (B) Oxymatrine capsule (45 mg/kg of oxymatrine equivalent), or ethyl acetate (EA) fraction of <i>Sophora</i> extract (0.082 g/kg), twice a day for 7 days, respectively. On the experimental day (day 8), animals were pretreated with corresponding vehicle or tested drugs at 1 h prior to the last dose of indinavir (40 mg/kg) given to all rats for pharmacokinetic study. Values are expressed as mean ± S.E.M. for each data point (n = 6–9).</p

Effect of <i>Sophora</i> extract on the CYP3A activity in liver.

<p>The CYP3A activity was measured using a luminescent assay (P450-Glo). Bars represent mean ± S.E.M. of fold relative to the values in the control group (n = 9). Statistical significance is indicated as * <i>P</i><0.05, compared to control.</p

Pharmacokinetic parameters of indinavir (40 mg/kg orally) after multiple dosage.

<p>The rats received oral administration of indinavir (40 mg/kg) together with 1.5% Tween 80 (vehicle), <i>Sophora</i> extract (0.158 g/kg or 0.63 g/kg), Oxymatrine capsule (45 mg/kg of oxymatrine equivalent), or ethyl acetate (EA) fraction of <i>Sophora</i> extract (0.082 g/kg), twice a day for 7 days, respectively. On the experimental day (day 8), animals were pretreated with corresponding vehicle or tested drugs at 1 h prior to the last dose of indinavir given to all rats for pharmacokinetic study. Values are expressed as mean ± S.E.M. (n = 6–9). Significance is indicated as</p><p>*<i>P</i><0.05,</p><p>**<i>P</i><0.01,</p><p>***<i>P</i><0.001, compared to vehicle control.</p

Effect of <i>Sophora</i> extract on the intestinal and hepatic mRNA levels encoding CYP3A1, CYP3A2, mdr1a and mdr1b.

<p>(A) CYP3A1, (B) CYP3A2, (C) mdr1a, (D) mdr1b. The mRNA contents were measured by real-time PCR and calculated as comparative levels over control using the 2^−ΔΔCT method (mean ± S.E.M., n = 7–9). Statistical significance is indicated as * <i>P</i><0.05, ** <i>P</i><0.01, compared to control.</p

Mitochondrial monoamine oxidase-A-mediated hydrogen peroxide generation enhances 5-hydroxytryptamine-induced contraction of rat basilar artery

BACKGROUND AND PURPOSE We evaluated the role(s) of monoamine oxidase (MAO)-mediated H2O2 generation on 5-hydroxytryptamine (5-HT)-induced tension development of isolated basilar artery of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. EXPERIMENTAL APPROACH Basilar artery (endothelium-denuded) was isolated for tension measurement and Western blots. Enzymically dissociated single myocytes from basilar arteries were used for patch-clamp electrophysiological and confocal microscopic studies. KEY RESULTS Under resting tension, 5-HT elicited a concentration-dependent tension development with a greater sensitivity (with unchanged maximum tension development) in SHR compared with WKY (EC50: 28.4 ± 4.1-nM vs. 98.2 ± 9.4-nM). The exaggerated component of 5-HT-induced tension development in SHR was eradicated by polyethylene glycol-catalase, clorgyline and citalopram whereas exogenously applied H2O2 enhanced the 5-HT-elicited tension development in WKY. A greater protein expression of MAO-A was detected in basilar arteries from SHR than in those from WKY. In single myocytes and the entire basilar artery, 5-HT generated (clorgyline-sensitive) a greater amount of H2O2 in SHR compared with WKY. Whole-cell iberiotoxin-sensitive Ca2+-activated K+ (BKCa) amplitude measured in myocytes of SHR was approximately threefold greater than that in WKY (at +60-mV: 7.61 ± 0.89-pA.pF-1 vs. 2.61 ± 0.66-pA.pF-1). In SHR myocytes, 5-HT caused a greater inhibition (clorgyline-, polyethylene glycol-catalase- and reduced glutathione-sensitive) of BKCa amplitude than in those from WKY. CONCLUSIONS AND IMPLICATIONS 5-HT caused an increased generation of mitochondrial H2O2 via MAO-A-mediated 5-HT metabolism, which caused a greater inhibition of BKCa gating in basilar artery myocytes, leading to exaggerated basilar artery tension development in SHR

Folic acid consumption reduces resistin level and restores blunted acetylcholine-induced aortic relaxation in obese/diabetic mice

Folic acid supplementation provides beneficial effects on endothelial functions in patients with hyperhomocysteinemia. However, its effects on vascular functions under diabetic conditions are largely unknown. Therefore, the effect(s) of folic acid (5.7 and 71 μg/kg/day for 4 weeks) on aortic relaxation was investigated using obese/diabetic (+db/+db) mice and lean littermate (+db/+m) mice. Acetylcholine-induced relaxation in +db/+db mice was less than that observed in +db/+m mice. The reduced relaxation in +db/+db mice was restored by consumption of 71 μg/kg folic acid. Acetylcholine-induced relaxation (with and without folic acid treatment) was sensitive to NG-nitro-l-arginine methyl ester, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, geldanamycin and triciribine. In addition, acetylcholine-induced relaxation was attenuated by resistin. The plasma level of resistin in +db/+db mice was sevenfold higher than that measured in +db/+m mice, and the elevated plasma level of resistin in +db/+db mice was reduced by 25% after treatment with 71 μg/kg folic acid. Folic acid slightly increased the ratio of reduced glutathione to oxidized glutathione in +db/+db mice. Moreover, folic acid caused a reduction in PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression, an increase in the phosphorylation of endothelial nitric oxide synthase (eNOSSer1177) and AktSer473, and an enhanced interaction of heat shock protein 90 (HSP90) with eNOS in both strains, with greater magnitude observed in +db/+db mice. In conclusion, folic acid consumption improved blunted acetylcholine-induced relaxation in +db/+db mice. The mechanism may be, at least partly, attributed to enhancement of PI3K/HSP90/eNOS/Akt cascade, reduction in plasma resistin level, down-regulation of PTEN and slight modification of oxidative state