Mechanism-based Cytochrome P450 Research Articles

Differences in xanthotoxin metabolites in seven mammalian liver microsomes.

Xanthotoxin, abundantly occurring in fruits, vegetables, grapefruit juice and oils, is widely used in medicine for the treatment of psoriasis and vitiligo. Xanthotoxin possesses the ability to inhibit mechanism-based cytochrome P450 (CYP450)-mediated activities in rats and mice. Furthermore, it time-dependently obstructs a number of CYP450-mediated functions in humans. CYP450 enzymes are most abundant in the liver and induce metabolic activation of numerous xenobiotic compounds. The present study aimed to identify the similarities and differences in xanthotoxin metabolism in liver microsomes of 7 mammalian species, including human liver microsomes (HLM), Rhesus monkey liver microsomes (RMLM), Cynomolgus monkey liver microsomes (CMLM), Sprague Dawley rat liver microsomes (RLM), mouse liver microsomes (MLM), Dunkin Hartley guinea pig liver microsomes (PLM) and Beagle dog liver microsomes (DLM). Ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometric analysis was used to determine the metabolites. A total of 3 metabolites were detected in RMLM, CMLM and RLM. Furthermore, two metabolites were observed in MLM, HLM, PLM and DLM. By analyzing the type and quantity of metabolites, the metabolism of xanthotoxin in MLM was indicated to be most similar to that in HLM. The metabolic transformations of xanthotoxin in the liver microsomes of the 7 species were analyzed in further detail. On the whole, the results of the present study provide a deeper understanding of the metabolic patterns of xanthotoxin in liver microsomes of different species, which may prove to be advantageous regarding the metabolic mechanisms of action of xanthotoxin. Further insight into drug metabolism with respect to different species will also aid in the selection of appropriate animal models for further research.

Progress Curve Analysis of CYP1A2 Inhibition: A More Informative Approach to the Assessment of Mechanism-Based Inactivation?

Mechanism-based cytochrome P450 inactivation is defined as a time- and NADPH-dependent inactivation that is not reversible upon extensive dialysis. Current methodologies use dilution approaches to estimate the rate of inactivation and offer limited mechanistic insight and are significantly influenced by experimental conditions. We investigated the potential of progress curve analysis because this experimental design allows investigation of both the reversible (K(iapp)) and irreversible (K(i), K(inact)) components of the reaction mechanism. The human liver microsomal CYP1A2 inactivation kinetics of resveratrol, oltipraz, furafylline, and dihydralazine (Fig. 2) were evaluated. The inactivation results for furafylline (K(i), 0.8 microM; K(inact), 0.16 min(-1)) are within 2-fold to published data (K(i), 1.6 microM; K(inact), 0.19 min(-1)). Resveratrol and dihydralazine results are within a 4.3-fold range of published data, which compares well with ranges of estimates of these parameters across publications (e.g., furafylline has estimates ranging of K(i) from 1.6 to 22.3 microM and K(inact) from 0.19 to 0.87 min(-1)). This range of estimates highlights the potential caveats surrounding the existing methodologies that have been previously discussed in depth. In addition to these inactivation parameters, we have been able to demonstrate a variation in balance of reversible versus irreversible inhibition within these inactivators. Oltipraz and resveratrol have K(iapp) values similar to their K(i), indicating a significant early onset reversible inhibition, whereas furafylline and dihydralazine are dominated by irreversible inactivation. This approach allows a more mechanistic investigation of an inactivator and in the future may improve the prediction of clinical drug-drug interactions.

Drug-Drug Interactions via Mechanism-Based Cytochrome P450 Inactivation: Points to Consider for Risk Assessment from In Vitro Data and Clinical Pharmacologic Evaluation

This commentary discusses the approaches to, and key considerations in the in vitro-in vivo extrapolation of drug-drug interactions (DDI) resulting from mechanism-based inactivation (MBI) of cytochrome P450 (CYP) enzymes and clinical pharmacologic implications. In vitro kinetic assessment and prediction of DDI produced via reversible inhibition and MBI rely on operationally and conceptually distinct approaches. DDI risk assessment for inactivators requires estimation of maximal inactivation rate (k(inact)) and inactivator potency (KI) in vitro, that need to be considered in context of the biological turnover rate of the enzyme (kdeg) and clinical exposures of the inactivator (I), respectively, to predict interaction magnitude. Risk assessment cannot be performed by a simple comparison of inactivator potency against in vivo exposure since inactivation is both concentration and time-dependent. MBI contour plots tracking combinations of I:KI and k(inact):k(deg) resulting in identical fold-reductions in intrinsic clearance are proposed as a useful framework for DDI risk assessment. Additionally, substrate-specific factors like fraction of the total clearance of the object drug via the enzyme being inactivated (f(m(CYP) )) and the bioavailability fraction across the intestine for CYP3A substrates (F(G)) are important determinants of interaction magnitude. Sensitivity analysis of predicted DDI magnitude to uncertainty in input parameters is recommended to inform confidence in predictions. The time course of reversal of DDI resulting from CYP inactivation is determined by the half-life of the enzyme which is an important consideration in the design and interpretation of clinical DDI studies with inactivators.

Inhibition of renal arachidonic acid omega-hydroxylase activity with ABT reduces blood pressure in the SHR.

The mechanism-based cytochrome P-450 (CYP) inhibitor 1-aminobenzotriazole (ABT) was characterized as an inhibitor of renal arachidonic acid metabolism and administered to spontaneously hypertensive rats (SHRs) to determine the effect of reduced eicosanoid production on mean arterial pressure (MAP). A single intraperitoneal dose of ABT to Sprague-Dawley rats caused a dose-dependent loss of renal CYP content, arachidonic acid metabolism, and CYP4A protein. In the cortex and outer medulla, ABT showed a high degree of selectivity for the CYP4A enzymes, reflected by the potent inhibition of 19- and 20-hydroxyeicosatetraenoic acid (19- and 20-HETE) formation. A 50 mg/kg dose of ABT reduced cortical 20-HETE formation to 16.1 +/- 0.82% of control and outer medullary 20-HETE formation to 23.8 +/- 0.45% of control. In contrast, there was no inhibition of renal epoxygenase activity at this dose. Renal CYP content, arachidonic acid omega- and (omega-1)-hydroxylase activity, and CYP4A protein levels gradually return to control levels by 72 h after a single dose of ABT. Cortical 20-HETE formation recovered from 17.9 +/- 3.15% of control at 6 h to 84.8 +/- 4.67% of control at 72 h after ABT administration. A single injection of ABT to 7-wk-old SHRs caused an acute reduction in MAP, which remained suppressed for at least 12 h. The effect was maximal within 4 h and averaged 17-23 mmHg during the 4- to 12-h period after administration. 20-HETE formation was inhibited 85% in the cortex and 70-80% in the outer medulla during the period when MAP was reduced. A structurally related ABT analog 1-hydroxybenzotriazole had no effect on blood pressure or renal arachidonic acid metabolism. These results identify ABT as a selective inhibitor of renal CYP4A activity and provide further support for a role for 20-HETE in the regulation of blood pressure.

Herbicide-insecticide interaction in maize: Malathion inhibits cytochrome P450-dependent primisulfuron metabolism

Tolerance of maize to sulfonylurea herbicides such as primisulfuron has recently been reported to be impaired by the use of some organophosphorus insecticides. In an effort to elucidate the mechanism of this interaction, the effect of the insecticide, malathion, on the metabolism of primisulfuron was studied in whole plants, in excised leaves, and in a microsomal in vitro system from maize. Foliar application of malathion to 7-day-old plants had no influence on leaf uptake and translocation of primisulfuron, but caused a decrease in the rate of herbicide metabolism. In excised leaves, malathion increased the metabolic half-life of primisulfuron. In microsomal preparations, malathion inhibited cytochrome P450-dependent primisulfuron phenyl- and pyrimidinering hydroxylation. Loss of primisulfuron phenyl-ring hydroxylase activity was time-dependent, saturable with respect to malathion concentration, and attenuated in the absence of NADPH. The kinetic data suggest a mechanism-based cytochrome P450 inactivation by malathion. The oxoanalogue of malathion, malaoxon, did not influence the metabolic half-life of primisulfuron in excised leaves and was a poor inhibitor of microsomal primisulfuron hydroxylation. Neither insecticide had any effect in vitro on total microsomal cytochrome P450 content. From the present results it may be concluded that malathion affects primisulfuron tolerance of maize due to the inhibition of cytochrome P450 monooxygenases involved in herbicide metabolism.

Effect of monooxygenase inhibitors on bentazon uptake and metabolism in maize cell suspension cultures

Maize “black Mexican sweet” (BMS) cell suspension cultures were used to study the effects of various cytochrome P450 monooxygenase inhibitors on the uptake and metabolism of the herbicide bentazon. Maize cells rapidly absorbed bentazon and metabolized it via aryl hydroxylation and glycosylation to a glycosyl conjugate of 6-hydroxybentazon. BMS cells accumulated bentazon to levels approximately 20-fold greater than those in the external medium. When BMS cells were incubated in an external medium containing 25 μ M bentazon, the formation of the glycosyl conjugate (ca. 2 nmol/min/g fresh wt) was rate-limited by aryl hydroxylation. Tetcyclacis, a plant growth retardant, phenylhydrazine, a mechanism-based cytochrome P450 inhibitor, and piperonyl butoxide, an insecticide synergist, inhibited bentazon metabolism with I 50 values of approximately 0.1, 1.0, and 1.0 μ M, respectively. Other mechanism-based cytochrome P450 inhibitors, 3(2,4-dichlorophenoxy)-1-propyne and aminobenzotriazole, also inhibited bentazon metabolism but were less effective. The results obtained with selected inhibitors are consistent with the hypothesis that aryl hydroxylation of bentazon is catalyzed by a cytochrome P450 monooxygenase.