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Grapefruit juice has been found to significantly increase oral bioavailability of several drugs metabolized by cytochrome P450 3A4 (P450 3A4) through inhibiting the enzymatic activity and decreasing the content of intestinal P450 3A4. HPLC/MS/MS and HPLC/UV analyses of ethyl acetate extracts from grapefruit juice revealed the presence of several furanocoumarins of which bergamottin (BG) is the major one. BG was shown to inactivate P450 3A4 in a reconstituted system consisting of purified P450 3A4, NADPH-cytochrome P450 reductase, cytochrome b5, and phospholipids. Inactivation was time- and concentration-dependent and required metabolism of BG. The loss of catalytic activity exhibited pseudo-first-order kinetics. The values of kinactivation and KI calculated from the inactivation studies were 0.3 min-1 and 7.7 microM, respectively. While approximately 70% of the erythromycin N-demethylation activity was lost during incubation with BG in the reconstituted system, P450 3A4 retained more than 90% of the heme as determined either by UV-visible spectroscopy or by HPLC. However, approximately 50% of the apoP450 in the BG-inactivated P450 3A4 incubation mixture could not be recovered from a reverse-phase HPLC column when compared with the -NADPH control. The mechanism of the inactivation appears to involve modification of the apoP450 in the active site of the enzyme instead of heme adduct formation or heme fragmentation. These results indicate that BG, the primary furanocoumarin extracted from grapefruit juice, is a mechanism-based inactivator of P450 3A4. BG was also found to inhibit the activities of P450s 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4 in human liver microsomes.

Abuse of synthetic cannabinoids (SCs), such as [1-naphthalenyl-(1-pentyl-1H-indol-3-yl]-methanone (JWH-018) and [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone (AM2201), is increasing at an alarming rate. Although very little is known about the metabolism and toxicology of these popular designer drugs, mass spectrometric analysis of human urine specimens after JWH-018 and AM2201 exposure identified monohydroxylated and carboxylated derivatives as major metabolites. The present study extends these initial findings by testing the hypothesis that JWH-018 and its fluorinated counterpart AM2201 are subject to cytochrome P450 (P450)-mediated oxidation, forming potent hydroxylated metabolites that retain significant affinity and activity at the cannabinoid 1 (CB 1 ) receptor. Kinetic analysis using human liver microsomes and recombinant human protein identified CYP2C9 and CYP1A2 as major P450s involved in the oxidation of the JWH-

Oxidation of the mechanistic probes trans,trans-2-methoxy-3-phenylmethylcyclopropane and methylcubane by six cytochrome P450 isozymes has been studied. The probes differentiate between radical and cationic species in that different structural rearrangements occur for the two types of intermediates. The P450 isozymes are the phenobarbital-inducible hepatic isozymes P450 2B1 (from rat) and P450 2B4 (from rabbit), the expressed truncated isozymes P450 ∆2B4 and P450 ∆2E1 (ethanol-inducible, from rabbit), and mutants of the latter two in which an active site threonine was replaced with alanine, ∆2B4 T302A, and ∆2E1 T303A. Cationic rearrangement products were found from both probes. Oxidations of trans,trans-2-methoxy-3-phenylmethylcyclopropane gave small amounts of radical-derived rearrangement products indicating that hydroxylation occurs via insertion reactions with transition state lifetimes in the 80-200 fs range. A mechanistic description of cytochrome P450-catalyzed hydroxylations that is in accord with the present and previous radical probe results is presented. This description incorporates the recent demonstrations that two electrophilic oxidants are produced in the natural course of P450 oxidation reactions and that both electrophilic oxidant forms can effect hydroxylation reactions. Following production of a peroxo-iron species, protonation gives a hydroperoxoiron species. Protonation of the hydroperoxo-iron species gives an iron-oxo species and water. Hydroxylations by both the hydroperoxo-iron and iron-oxo species occur by insertion reactions. The hydroperoxo-iron species inserts the elements of OH + producing protonated alcohol products that can react in solvolysis-type reactions to give cationic rearrangement products. The iron-oxo species reacts by insertion of an oxygen atom.

ABSTRACT:Silybin, a major constituent of the milk thistle, is used to treat several liver disorders. Silybin inactivated purified, recombinant cytochromes P450 (P450) 3A4 and 2C9 in a mechanism-based manner. The inactivations were time-, concentration-, and NADPHdependent. The inactivation of the 7-benzyloxy-4-(trifluoromethyl-)coumarin O-debenzylation activity (P450 3A4) was characterized by a K I of 32 M, a k inact of 0.06 min ؊1 , and a t 1/2 of 14 min.Testosterone metabolism to 6-␤-hydroxytestosterone (P450 3A4) was also inactivated with a K I of 166 M, a k inact of 0.08 min ؊1, and a t 1/2 of 9 min. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified human P450 2C9 was inactivated with a K I of 5 M, a k inact of 0.14 min ؊1, and a t 1/2 of 7 min. Parallel loss of heme was observed with both P450s. Activity of both P450 enzymes was not recovered after removal of silybin either by dialysis or by spin gel filtration. In addition, silybin inhibited the glucuronidation of 7-hydroxy-4-trifluoromethylcoumarin catalyzed by recombinant hepatic UDP-glucuronosyltransferases (UGTs) 1A1, 1A6, 1A9, 2B7, and 2B15, with IC 50 values of 1.4 M, 28 M, 20 M, 92 M, and 75 M, respectively. Silybin was a potent inhibitor of UGT1A1 and was 14-and 20-fold more selective for UGT1A1 than for UGT1A9 and UGT1A6, respectively. Thus, careful administration of silybin with drugs primarily cleared by P450s 3A4 or 2C9 is advised, since drug-drug interactions cannot be excluded. The clinical significance of in vitro UGT1A1 inhibition is unknown.

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The P450 type cytochromes are responsible for the metabolism of a wide variety of xenobiotics and endogenous compounds. Although P450-catalyzed reactions are generally thought to lead to detoxication of xenobiotics, the reactions can also produce reactive intermediates that can react with cellular macromolecules leading to toxicity or that can react with the P450s that form them leading to irreversible (i.e., mechanism-based) inactivation. This perspective describes the fundamentals of mechanism-based inactivation as it pertains to P450 enzymes. The experimental approaches used to characterize mechanism-based inactivators are discussed, and the criteria required for a compound to be classified as a mechanism-based inactivator are outlined. The kinetic scheme for mechanism-based inactivation and the calculation of the relevant kinetic constants that describe a particular inactivation event are presented. The structural aspects and important functional groups of several classes of molecules that have been found to impart mechanism-based inactivation upon metabolism by P450s such as acetylenes, thiol-containing compounds that include isothiocyanates, thiazolidinediones, and thiophenes, arylamines, quinones, furanocoumarins, and cyclic tertiary amines are described. Emphasis throughout this perspective is placed on more recent findings with human P450s where the site of modification, whether it be the apoprotein or the heme moiety, and, at least in part, the identity of the reactive intermediate responsible for the loss in P450 activity are known or inferred. Recent advances in trapping procedures as well as new methods for identification of reactive intermediates are presented. A variety of clinically important drugs that act as mechanism-based inactivators of P450s are discussed. The irreversible inactivation of human P450s by these drugs has the potential for causing serious drug-drug interactions that may have severe toxicological effects. The clinical significance of inactivating human P450s for improving drug efficacy as well as drug safety is discussed along with the potential for exploiting mechanism-based inactivators of P450s for therapeutic benefits.

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