Characterization of novel dihydrothienopyridinium and thienopyridinium metabolites of ticlopidine in vitro: role of peroxidases, cytochromes p450, and monoamine oxidases.

Ticlopidine is an agent that inhibits adenosine diphosphate-induced platelet aggregation. Metabolic studies with ticlopidine have indicated that the principal routes of metabolism are N-dealkylation, N-oxidation, and oxidation of the thiophene ring. However, ticlopidine shares some structural features that are similar to those of cyclic tertiary amines such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and tetrahydroisoquinolines, which are converted to neurotoxic pyridinium metabolites, via the iminium (dihydropyridinium) species. The current in vitro studies examined the potential of ticlopidine to undergo a similar conversion by cytochrome P450 (P450), peroxidases, and monoamine oxidase (MAO). The results from these studies have suggested that ticlopidine undergoes an overall 4-electron oxidation to the novel thienopyridinium metabolite (M6) via the intermediate 2-electron oxidation product, the thienodihydropyridinium metabolite (M5) by P450, horseradish peroxidase, and myeloperoxidase and, to a lesser extent, by MAO. The structures of these metabolites were characterized by liquid chromatography (LC)-tandem mass spectrometry and LC-NMR. Qualitative studies with baculovirus-expressed P450s revealed the involvement of P450 3A4 in this conversion. Interestingly, M5 was the primary metabolite in the peroxidase-mediated reactions and was quite stable to air oxidation or disproportionation. It was less electrophilic and did not form cyanide, glutathione, or N-acetylcysteine adducts. On the other hand, M6 was the major metabolite in P450-catalyzed oxidation of ticlopidine. The results from this study have revealed that in addition to metabolism of the thiophene ring of ticlopidine, the tetrahydropyridine moiety of the compound is susceptible to a 2-electron and a 4-electron oxidation like other cyclic tertiary amines.     

Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity.

Cytochrome P450 (P450) enzymes catalyze a variety of reactions and convert chemicals to potentially reactive products as well as make compounds less toxic. Most of the P450 reactions are oxidations. The majority of these can be rationalized in the context of an FeO(3+) intermediate and odd electron abstraction/rebound mechanisms; however, other iron-oxygen complexes are possible and alternate chemistries can be considered. Another issue regarding P450-catalyzed reactions is the delineation of rate-limiting steps in the catalytic cycle and the contribution to reaction selectivity. In addition to the rather classical oxidations, P450s also catalyze less generally discussed reactions including reduction, desaturation, ester cleavage, ring expansion, ring formation, aldehyde scission, dehydration, ipso attack, one-electron oxidation, coupling reactions, rearrangement of fatty acid and prostaglandin hydroperoxides, and phospholipase activity. Most of these reactions are rationalized in the context of high-valent iron-oxygen intermediates and Fe(2+) reductions, but others are not and may involve acid-base catalysis. Some of these transformations are involved in the bioactivation and detoxication of xenobiotic chemicals.     

Mechanism-based inactivation of human cytochromes p450s: experimental characterization, reactive intermediates, and clinical implications

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.     

Toxicological significance of mechanism-based inactivation of cytochrome p450 enzymes by drugs

Cytochrome P450 (P450) enzymes oxidize xenobiotics into chemically reactive metabolites or intermediates as well as into stable metabolites. If the reactivity of the product is very high, it binds to a catalytic site or sites of the enzyme itself and inactivates it. This phenomenon is referred to as mechanism-based inactivation. Many clinically important drugs are mechanism-based inactivators that include macrolide antibiotics, calcium channel blockers, and selective serotonin uptake inhibitors, but are not always structurally and pharmacologically related. The inactivation of P450s during drug therapy results in serious drug interactions, since irreversibility of the binding allows enzyme inhibition to be prolonged after elimination of the causal drug. The inhibition of the metabolism of drugs with narrow therapeutic indexes, such as terfenadine and astemizole, leads to toxicities. On the other hand, the fate of P450s after the inactivation and the toxicological consequences remains to be elucidated, while it has been suggested that P450s modified and degraded are involved in some forms of tissue toxicity. Porphyrinogenic drugs, such as griseofulvin, cause mechanism-based heme inactivation, leading to formation of ferrochelatase-inhibitory N-alkylated protoporphyrins and resulting in porphyria. Involvement of P450-derived free heme in halothane-induced hepatotoxicity and catalytic iron in cisplatin-induced nephrotoxicity has also been suggested. Autoantibodies against P450s have been found in hepatitis following administration of tienilic acid and dihydralazine.Tienilic acid is activated by and covalently bound to CYP2C9, and the neoantigens thus formed activate immune systems, resulting in the formation of an autoantibodydirected against CYP2C9, named anti-liver/kidney microsomal autoantibody type 2, whereas the pathological role of the autoantibodies in drug-induced hepatitis remains largely unknown.     

Toxicological Significance of Mechanism-Based Inactivation of Cytochrome P450 Enzymes by Drugs

Cytochrome P450 (P450) enzymes oxidize xenobiotics into chemically reactive metabolites or intermediates as well as into stable metabolites. If the reactivity of the product is very high, it binds to a catalytic site or sites of the enzyme itself and inactivates it. This phenomenon is referred to as mechanism-based inactivation. Many clinically important drugs are mechanism-based inactivators that include macrolide antibiotics, calcium channel blockers, and selective serotonin uptake inhibitors, but are not always structurally and pharmacologically related. The inactivation of P450s during drug therapy results in serious drug interactions, since irreversibility of the binding allows enzyme inhibition to be prolonged after elimination of the causal drug. The inhibition of the metabolism of drugs with narrow therapeutic indexes, such as terfenadine and astemizole, leads to toxicities. On the other hand, the fate of P450s after the inactivation and the toxicological consequences remains to be elucidated, while it has been suggested that P450s modified and degraded are involved in some forms of tissue toxicity. Porphyrinogenic drugs, such as griseofulvin, cause mechanism-based heme inactivation, leading to formation of ferrochelatase-inhibitory N-alkylated protoporphyrins and resulting in porphyria. Involvement of P450-derived free heme in halothane-induced hepatotoxicity and catalytic iron in cisplatin-induced nephrotoxicity has also been suggested. Autoantibodies against P450s have been found in hepatitis following administration of tienilic acid and dihydralazine.Tienilic acid is activated by and covalently bound to CYP2C9, and the neoantigens thus formed activate immune systems, resulting in the formation of an autoantibodydirected against CYP2C9, named anti-liver/kidney microsomal autoantibody type 2, whereas the pathological role of the autoantibodies in drug-induced hepatitis remains largely unknown.     

Heterotropic cooperativity in oxidation mediated by cytochrome p450

Cytochrome P450s (P450 or CYPs) comprise a superfamily of enzymes that catalyze the oxidation of a wide variety of xenobiotic chemicals. Although most of P450 inhibitors decrease the metabolic activities mediated by the corresponding P450 forms, unexpected phenomena, which are called as activation or heterotropic cooperativity, have been often observed. We summarize Michaelis-Menten constants (K(m)), maximal velocities (V(max)), V(max)/K(m) (intrinsic clearance) values, and/or metabolic activities for 22 activators and 24 substrates (30 reactions) mainly mediated by CYP3A4 among human P450 forms. Although an allosteric mechanism has been invoked to explain the cooperativity, the activation patterns or phenomena are dependent on substrates and selected enzyme sources in vitro. Interestingly, recent studies have been shown that human P450 forms other than CYP3A4, such as CYP1A2, CYP2C8, CYP2C9, CYP2D6, and CYP3A7, are also activated by some compounds, whereas there are few reports on CYP3A5. Several models describing interaction among substrates, effectors, and enzymes have been proposed, however, the detailed mechanism for the activation is still generally unknown even though some crystal structures have been shown. A few cases of the cooperativity of CYP3A in experimental animals have been presented, whereas the clinical significance of P450 cooperativity is still unclear. The collective findings provide fundamental and useful information for the activation of P450s by chemicals despite some contradictive kinetic parameters for the same reactions reported. To understand causal factor(s) and mechanism(s) for such different reports summarized here is still one of the hot research topics to be solved in current activation reactions.     

The formation of reactive intermediates in the MAO-catalyzed oxidation of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Oxidation of MPTP by monoamine oxidase (MAO), leading to the formation of reactive metabolites, is a critical step in the expression of the nigrostriatal toxicity of this molecule. A catalytic mechanism for the 2-electron oxidation of MPTP to MPDP+ and for the further 2-electron oxidation of MPDP+ to MPP+ is proposed, involving the formation of carbon-centered radical intermediates. These radical species appear to be involved in the mechanism-based inactivation of MAO by MPTP, possibly by generating 1,4-dihydropyridine adducts with the enzyme apoprotein or its coenzyme FAD. The pathways of metabolism of MPTP in brain and peripheral tissues and the active accumulation of metabolites of MPTP in dopaminergic neurons are discussed in terms of their possible contribution to the selective cytotoxicity of the compound.     

P450 subfamily CYP2J and their role in the bioactivation of arachidonic acid in extrahepatic tissues

Historically, there has been intense interest in P450 metabolic oxidation, peroxidation, and reduction of xenobiotics. More recently, there has been a growing appreciation for the role of P450s in the oxidation of lipophilic endobiotics, such as bile acids, fat-soluble vitamins, and eicosanoids. This review details the emerging CYP2J subfamily of P450s and their role as catalysts of arachidonic acid metabolism.     

Mechanism-based inactivation and reversibility: is there a new trend in the inactivation of cytochrome p450 enzymes?

Recent studies with cytochrome P450 (P450) enzymes from the 2E and 2B subfamilies have shed light on what may be a new trend in the mechanism-based inactivation of P450s: reversibility. The reversible inactivation of P450-type enzymes was first reported in the mid-1990s by Dexter and Hager [Dexter AF and Hager LP (1995) J Am Chem Soc 117:817-818], who studied the transient heme N-alkylation of chloroperoxidase by allylbenzene and 1-hexyne. While characterizing small tert-butyl acetylenes as mechanism-based inactivators of P450s 2E1 and 2B4, Hollenberg and coworkers observed the reversible inactivation of an acetylene-inactivated T303A mutant of P450 2E1. The mechanism of reversibility was a combined product of the structure of the inactivator and the positioning of conserved amino acid residues, threonine 303 (alanine in the mutant) and glutamate 302, in the enzyme active site. Reversibility was also observed with both wild-type P450 2B4 and the T302A mutant of 2B4, although this inactivation and reversibility did not seem to depend on the T302 residue. Subsequent studies have attempted to elucidate the chemical/structural requirements of the inactivator in determining reversibility and have shown that both the size and the chemical nature of functional groups play an important role. At this time, reversibility has only been observed with P450 2E and 2B enzymes during their mechanism-based inactivation by terminal alkynes. Future studies with P450s from other subfamilies and structurally distinct inactivators will greatly aid our understanding of the molecular and chemical determinants of reversibility.     

N-hydroxyarylamines

Aryl and heterocyclic amines are of particular interest because of their carcinogenicity. The N-hydroxy derivatives are formed by oxidation, usually by the cytochrome P450 (P450) enzymes and most often by P450 family 1. The mechanism of oxidation appears to resemble that of other P450 reactions. The N-hydroxy products can be conjugated to yield esters, which are unstable and form nitrenium ions. Reaction with DNA is most common at the N2 atom and particularly at the C8 atom of guanine. A mechanism involving initial formation of an N7-guanyl adduct can be utilized in explaining the C 8-guanyl adducts plus several other side reactions. The high mutagenicity of N-hydroxy heterocyclic amines in bacterial systems has provided a useful tool for the development of models useful for screening and chemoprevention and for the generation of P450 enzymes with altered properties.     

Catalytic activity of cytochrome P450 isozymes purified from rat liver in converting 11-oxo-delta 8-tetrahydrocannabinol to delta 8-tetrahydrocannabinol-11-oic acid.

Cytochrome P450 isozymes purified from rat hepatic microsomes were able to catalyse the oxidation of 11-oxo-delta 8-tetrahydrocannabinol (11-oxo-delta 8-THC) to delta 8-THC-11-oic acid in the presence of NADPH, cytochrome P450 reductase and dilauroylphosphatidylcholine. The catalytic activities (nmol/min/nmol P450) of cytochrome P450s, UT-2 (IIC11), UT-4 (IIA2), UT-5 (IIC13), PB-1, PB-2 (IIC6), PB-4 (IIB1), MC-1 (IA2), MC-5 (IA1) and IF-3 (IIA1), were 0.69, 0.08, 0.07, 0.23, 0.46, 0.02, 0.06, 0.07 and 0.34, respectively, whereas the activities of cytochrome P450s, PB-5 (IIB2) and DM (IIE1), were less than 0.02 nmol/min/nmol P450. Cytochrome P450 IIC11 showed the highest catalytic activity of the cytochromes examined. The mechanism for the oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid by cytochrome P450 IIC11 was established as being an oxygenation since one atom of oxygen-18 was exclusively incorporated into the carboxylic acid formed under 18O2. The antibody raised to cytochrome P450 IIC11 inhibited by 60% the hepatic microsomal oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid in male rats. These results indicate that cytochrome P450 IIC11 is a major form of the cytochrome to catalyse the oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid in the hepatic microsomes of male rats and that the oxidation of aldehyde to carboxylic acid is a catalytic activity common to most isozymes of P450.     

Different mechanisms for inhibition of human cytochromes P450 1A1, 1A2, and 1B1 by polycyclic aromatic inhibitors

We have previously shown that several polycyclic aromatic hydrocarbons (PAHs) strongly inhibit their own and other PAH metabolism catalyzed by cytochrome P450 (P450) 1A1, 1A2, and 1B1 [Shimada, T., and Guengerich, F. P. (2006) Chem. Res. Toxicol. 19, 288-294]. In the present study, we examined mechanisms of how PAHs inhibit these P450 enzymes by using 7-ethoxyresorufin O-deethylation (EROD) as a model reaction. First, we examined mechanisms of inhibition of P450 1A1, 1A2, and 1B1 by the synthetic model inhibitors 1-(1-propynyl)pyrene (1PP), 1-ethynylpyrene (1EP), 2-ethynylpyrene (2EP), and 4-(1-propynyl)biphenyl (4Pbi). Both 1PP and 1EP inhibited P450 1A1 in a mechanism-based manner, but P450 1B1 and 1A2 were directly inhibited by 1PP and 1EP. Interestingly, P450 1B1 inactivated 1PP and 1EP to products that were not inhibitory to P450 1B1. 4Pbi was a mechanism-based inhibitor of P450 1A1 and 1B1, but 2EP directly inhibited these P450s. All four of the inhibitors directly inhibited P450 1A2. We also found that benzo[a]pyrene and seven other PAH compounds tested inhibited P450 1A2 in a mechanism-based manner, but fluoranthene directly inhibited P450 1A2. All of the nine PAHs examined were direct inhibitors of P450 1A1 and P450 1B1. These results suggest different mechanisms of inhibition of P450 1A1, 1A2, and 1B1 by PAHs and related chemicals and that interactions between P450 enzymes and PAH inhibitors are involved in differences in inhibition of the enzymes.     

Role of human cytochrome P-450 IIE1 in the oxidation of many low molecular weight cancer suspects.

The role of human cytochrome P-450 IIE1 (P-450 IIE1) in the oxidation of a number of suspect carcinogens was examined by using a variety of approaches, including (1) selective inhibition of catalytic activity in human liver microsomes by diethyldithiocarbamate, which was found to be a selective mechanism-based inactivator of P-450 IIE1, (2) correlation of rates of different catalytic activities with each other and with chlorzoxazone 6-hydroxylation, an indicator of P-450 IIE1, in human liver microsomes, (3) demonstration of catalytic activity in reconstituted systems containing purified human P-450 IIE1, and (4) immunoinhibition of catalytic activity in human liver microsomes with rabbit anti-human P-450 IIE1. The results collectively indicate that P-450 IIE1 is a major catalyst of the oxidation of benzene, styrene, CCl4, CHCl3, CH2Cl2, CH3Cl, CH3CCl3, 1,2-dichloropropane, ethylene dichloride, ethylene dibromide, vinyl chloride, vinyl bromide, acrylonitrile, vinyl carbamate, ethyl carbamate, and trichloroethylene. Levels of P-450 IIE1 can vary considerably among individual humans--the availability of chlorzoxazone as a noninvasive probe of human P-450 IIE1 and of disulfiram (oxidized diethyldithiocarbamate) as an inhibitor may facilitate discernment of the in vivo significance of human P-450 IIE1 as a factor in the bioactivation and detoxication of these cancer suspects. Further, many investigations with diethyldithiocarbamate, disulfiram, and ethanol in humans and experimental animals may be interpreted in light of mechanisms involving P-450 IIE1.     

7-Ethynylcoumarins: selective inhibitors of human cytochrome P450s 1A1 and 1A2

To discover new selective mechanism-based P450 inhibitors, eight 7-ethynylcoumarin derivatives were prepared through a facile two-step synthetic route. Cytochrome P450 activity assays indicated that introduction of functional groups in the backbone of coumarin could enhance the inhibition activities toward P450s 1A1 and 1A2, providing good selectivity against P450s 2A6 and 2B1. The most potent product 7-ethynyl-3,4,8-trimethylcoumarin (7ETMC) showed IC(50) values of 0.46 μM and 0.50 μM for P450s 1A1 and 1A2 in the first six minutes, respectively, and did not show any inhibition activity for P450s 2A6 and 2B1 even at the dose of 50 μM. All of the inhibitors except 7-ethynyl-3-methyl-4-phenylcoumarin (7E3M4PC) showed mechanism-based inhibition of P450s 1A1 and 1A2. In order to explain this mechanistic difference in inhibitory activities, X-ray crystallography data were used to study the difference in conformation between 7E3M4PC and the other compounds studied. Docking simulations indicated that the binding orientations and affinities resulted in different behaviors of the inhibitors on P450 1A2. Specifically, 7E3M4PC with its two-plane structure fits into the P450 1A2's active site cavity with an orientation leading to no reactive binding, causing it to act as a competitive inhibitor.     

Revisiting the peroxidase oxidation of 2,4,6-trihalophenols: ESR detection of radical intermediates

The peroxidase oxidation of 2,4,6-trichlorophenol (TCP) has been clearly shown to result in 2,6-dichloro-1,4-benzoquinone (DCQ). DCQ is a 2-electron oxidation product of TCP that has undergone para dechlorination. Many peroxidases show similar oxidation of the substrate, TCP, to yield the quinone, DCQ. Depending on the substrate, peroxidases are thought to carry out both 1- and 2-electron oxidations; the mechanism can be confirmed by the detection of both enzyme and substrate intermediates. This article presents ESR evidence for the transient 2,4,6-trichlorophenoxyl radical intermediate (TCP?), which exists free in solution, i.e., is not enzyme associated. These data are best explained as a 1-electron peroxidase oxidation of TCP to form TCP?, followed by enzyme-independent radical reactions leading to the 2-electron oxidized product. Also presented are data for the peroxidase oxidation of 2,4,6-trifluorophenol and 2,6-dichloro-4-fluorophenol.     

Mechanism-based inactivation of human liver microsomal cytochrome P-450 IIIA4 by gestodene

A series of 17 alpha-acetylenic steroids was examined with regard to ability to inactivate human liver microsomal cytochrome P-450 (P-450) IIA4, an enzyme involved in the oxidation of a number of drugs, carcinogens, and steroids, including estrogens and progestogens. Of the eight compounds tested, gestodene was found to be particularly active as a mechanism-based inactivator of P-450 IIIA4. Inhibition of both microsomal nifedipine oxidation and 17 alpha-ethynylestradiol (EE) 2-hydroxylation was dependent upon NADPH and gestodene concentration. Rates of inactivation were pseudo first order-values of kinactivation = 0.4 min-1 and Ki = 46 microM and a partition ratio of 9 were calculated. The kinactivation is approximately 50-fold greater than estimated for EE and is one of the highest reported for P-450 mechanism-based inactivators. Spectrally detectable P-450 was also destroyed in microsomes, but several experiments indicate that little covalent binding to amino acid residues of P-450 IIIA4 occurs. Microsomal inactivation of P-450 could be blocked by the presence of other P-450 IIIA4 substrates, and several activities catalyzed by other P-450s were not inhibited under conditions in which greater than 90% of P-450 IIIA4 was inactivated. Consideration of structure/activity relationships among the 17 alpha-acetylenic steroids examined indicates that the delta 15 double bond is critical but is not in itself sufficient for the inactivation process, which is postulated to result from attack of P-450 on the substituted acetylenic carbon and lead to porphyrin N-alkylation. The effectiveness of this mechanism-based inactivator may account for reports of increased estrogen and steroid levels in some women using gestodene in oral contraceptives.     

Inhibition of cytochrome p450 enzymes by quinones and anthraquinones

In silico docking studies and quantitative structure-activity relationship analysis of a number of in-house cytochrome P450 inhibitors have revealed important structural characteristics that are required for a molecule to function as a good inhibitor of P450 enzymes 1A1, 1A2, 2B1, and/or 2A6. These insights were incorporated into the design of pharmacophores used for a 2D search of the Chinese medicine database. Emodin, a natural anthraquinone isolated from Rheum emodi and known to be metabolized by cytochrome P450 enzymes, was one of the hits and was used as the lead compound. Emodin was found to inhibit P450s 1A1, 1A2, and 2B1 with IC(50) values of 12.25, 3.73, and 14.89 μM, respectively. On the basis of the emodin molecular structure, further similarity searches of the PubChem and ZINC chemical databases were conducted resulting in the identification of 12 emodin analogues for testing against P450s 1A1-, 1A2-, 2B1-, and 2A6-dependent activities. 1-Amino-4-chloro-2-methylanthracene-9,10-dione (compound 1) showed the best inhibition potency for P450 1A1 with an IC(50) value of 0.40 μM. 1-Amino-4-chloro-2-methylanthracene-9,10-dione (compound 1) and 1-amino-4-hydroxyanthracene-9,10-dione (compound 2) both inhibited P450 1A2 with the same IC(50) value of 0.53 μM. In addition, compound 1 acted as a mechanism-based inhibitor of cytochrome P450s 1A1 and 1A2 with K(I) and K(inactivation) values of 5.38 μM and 1.57 min(-1) for P450 1A1 and 0.50 μM and 0.08 min(-1) for P450 1A2. 2,6-Di-tert-butyl-5-hydroxynaphthalene-1,4-dione (compound 8) directly inhibited P450 2B1 with good selectivity and inhibition potency (IC(50) = 5.66 μM). Docking studies using the 3D structures of the enzymes were carried out on all of the compounds. The binding modes of these compounds revealed the structural characteristics responsible for their potency and selectivity. Compound 1, which is structurally similar to compound 2 with the presence of an amino group at position 1, showed a difference in the mechanism of inhibition toward P450s 1A1 and 1A2. The mechanism-based inhibition seen for compound 1 may be attributed to the presence of the methyl group at the 2-position, in close proximity to the amino group. Compound 2, which is otherwise similar, lacks that methyl moiety and did not show mechanism-based inhibition.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin treatment alters eicosanoid levels in several organs of the mouse in an aryl hydrocarbon receptor-dependent fashion

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) adversely affects many mammalian organs and tissues. These effects are mediated by the aryl hydrocarbon receptor (AHR). CYP1A1, CYP1A2 and CYP1B1 are upregulated by the liganded AHR. These (and other) cytochromes P450 can metabolize arachidonic acid into a variety of bioactive eicosanoids. Towards investigating a potential role of eicosanoids in TCDD toxicity, arachidonic acid, two other unsaturated long-chain fatty acids, and up to twenty-five eicosanoids were measured in five organs/tissues of male and female wild-type and Ahr null mice treated or untreated with TCDD. TCDD generally increased the levels of the four dihydroxyeicosatrienoic acids (DHETs) and (where measured) 5,6-epoxyeicosatrienoic acid and 18-, 19- and 20-hydroxyeicosatrienoic acids (HETEs) in the serum, liver, spleen and lungs, but not the heart, of both sexes, and increased the levels in the serum, liver and spleen of several metabolites that are usually considered products of lipoxygenase activity, but which may also be generated by cytochromes P450. TCDD also increased the levels of the esterified forms of these eicosanoids in the liver in parallel with the corresponding free forms. The levels of prostanoids were generally not affected by TCDD. The above changes did not occur in Ahr null mice, and are therefore mediated by the AHR. TCDD increased the mRNA levels of Cyp1a1, Cyp1a2, Cyp1b1 and the Pla2g12a form of phospholipase A₂ to varying degrees in the different organs, and these increases correlated with some but not all the changes in eicosanoids levels in the organs, suggesting that other enzymes may also be involved.     

Studies to further investigate the inhibition of human liver microsomal CYP2C8 by the acyl-β-glucuronide of gemfibrozil

In previous studies, gemfibrozil acyl-β-glucuronide, but not gemfibrozil, was found to be a mechanism-based inhibitor of cytochrome P450 2C8. To better understand whether this inhibition is specific for gemfibrozil acyl-β-glucuronide or whether other glucuronide conjugates are potential substrates for inhibition of this enzyme, we evaluated several pharmaceutical compounds (as their acyl glucuronides) as direct-acting and metabolism-dependent inhibitors of CYP2C8 in human liver microsomes. Of 11 compounds that were evaluated as their acyl glucuronide conjugates, only gemfibrozil acyl-β-glucuronide exhibited mechanism-based inhibition, indicating that CYP2C8 mechanism-based inhibition is very specific to certain glucuronide conjugates. Structural analogs of gemfibrozil were synthesized, and their glucuronide conjugates were prepared to further examine the mechanism of inhibition. When the aromatic methyl groups on the gemfibrozil moiety were substituted with trifluoromethyls, the resulting glucuronide conjugate was a weaker inhibitor of CYP2C8 and mechanism-based inhibition was abolished. However, the glucuronide conjugates of monomethyl gemfibrozil analogs were mechanism-based inhibitors of CYP2C8, although not as potent as gemfibrozil acyl-β-glucuronide itself. The ortho-monomethyl analog was a more potent inhibitor than the meta-monomethyl analog, indicating that CYP2C8 favors the ortho position for oxidation and potential inhibition. Molecular modeling of gemfibrozil acyl-β-glucuronide in the CYP2C8 active site is consistent with the ortho-methyl position being the favored site of covalent attachment to the heme. Moreover, hydrogen bonding to four residues (Ser100, Ser103, Gln214, and Asn217) is implicated.     

Effects of 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine on hepatic cytochrome P-450 heme, apoproteins, and catalytic activities following in vivo administration to rats

Various 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) cause mechanism-based inactivation of cytochrome P-450 (P-450) via heme destruction. We have examined the time course of effects of DDC analogues on the catalytic activities and apoproteins of the major beta-naphthoflavone-, dexamethasone-, and phenobarbital-inducible isozymes of rat liver P-450 following in vivo administration. In beta-naphthoflavone-treated rats, all DDC analogues examined caused loss of the P-450 chromophore and dramatic loss of 7-ethoxyresorufin O-deethylase activity, a catalytic marker for P-450c. The isopropyl, hexyl, and isobutyl analogues caused the most pronounced loss/alteration of P-450c apoprotein levels, as revealed by two monoclonal antibodies (MAbs), 1-31-2 and 1-7-1. The apoprotein of P-450d was not altered. In dexamethasone-treated rats, all analogues except 4-hexyl-DDC caused loss of the P-450 chromophore and erythromycin N-demethylase activity, a catalytic marker for P-450p-related isozymes. Only 4-isopropyl-DDC caused significant loss/alteration of the apoprotein of P-450p-related forms, as revealed by MAb 2-13-1. In phenobarbital-treated rats, all analogues reduced the level of the P-450 chromophore, whereas only 4-hexyl-DDC and 4-isopropyl-DDC lowered 7-pentoxyresorufin O-dealkylase activity, a catalytic marker for P-450b. MAbs 2-66-3 and 2-8-1 revealed no change in the level of phenobarbital-inducible apoproteins recognized by these probes. In agreement with our previous in vitro studies [Mol. Pharmacol. 35;626-634 (1989)], P-450 c and p are targets for mechanism-based inactivation by DDC analogues. However, unlike the situation in vitro, loss of enzyme activity in vivo is, at least in some instances, accompanied by loss/alteration of the corresponding P-450 apoprotein.