Jan N. M. Commandeur

AMAP, the alleged non-toxic isomer of acetaminophen, is toxic in rat and human liver.

N-acetyl-meta-aminophenol (AMAP) is generally considered as a non-toxic regioisomer of the well-known hepatotoxicant acetaminophen (APAP). However, so far, AMAP has only been shown to be non-toxic in mice and hamsters. To investigate whether AMAP could also be used as non-toxic analog of APAP in rat and human, the toxicity of APAP and AMAP was tested ex vivo in precision-cut liver slices (PCLS) of mouse, rat and human. Based on ATP content and histomorphology, APAP was more toxic in mouse than in rat and human PCLS. Surprisingly, although AMAP showed a much lower toxicity than APAP in mouse PCLS, AMAP was equally toxic as or even more toxic than APAP at all concentrations tested in both rat and human PCLS. The profile of proteins released into the medium of AMAP-treated rat PCLS was similar to that of APAP, whereas in the medium of mouse PCLS, it was similar to the control. Metabolite profiling indicated that mouse PCLS produced the highest amount of glutathione conjugate of APAP, while no glutathione conjugate of AMAP was detected in all three species. Mouse also produced ten times more hydroquinone metabolites of AMAP, the assumed proximate reactive metabolites, than rat or human. In conclusion, AMAP is toxic in rat and human liver and cannot be used as non-toxic isomer of APAP. The marked species differences in APAP and AMAP toxicity and metabolism underline the importance of using human tissues for better prediction of toxicity in man.

A single active site mutation inverts stereoselectivity of 16-hydroxylation of testosterone catalyzed by engineered cytochrome P450 BM3.

Inversion of stereoselectivity: screening of a minimal mutant library revealed a cytochrome P450u2009BM3 variant M01u2009A82Wu2009S72I capable of producing 16u2009α-OH-testosterone. Remarkably, a single active site mutation S72I in M01u2009A82W inverted the stereoselectivity of hydroxylation from 16u2009β to 16u2009α. Introduction of S72I mutation in another 16u2009β-OH-selective variant M11u2009V87I, also resulted in similar inversion of stereoselectivity.

Active site substitution A82W improves the regioselectivity of steroid hydroxylation by cytochrome P450 BM3 mutants as rationalized by spin relaxation nuclear magnetic resonance studies.

Cytochrome P450 BM3 from Bacillus megaterium is a monooxygenase with great potential for biotechnological applications. In this paper, we present engineered drug-metabolizing P450 BM3 mutants as a novel tool for regioselective hydroxylation of steroids at position 16β. In particular, we show that by replacing alanine at position 82 with a tryptophan in P450 BM3 mutants M01 and M11, the selectivity toward 16β-hydroxylation for both testosterone and norethisterone was strongly increased. The A82W mutation led to a ≤42-fold increase in V(max) for 16β-hydroxylation of these steroids. Moreover, this mutation improves the coupling efficiency of the enzyme, which might be explained by a more efficient exclusion of water from the active site. The substrate affinity for testosterone increased at least 9-fold in M11 with tryptophan at position 82. A change in the orientation of testosterone in the M11 A82W mutant as compared to the orientation in M11 was observed by T(1) paramagnetic relaxation nuclear magnetic resonance. Testosterone is oriented in M11 with both the A- and D-ring protons closest to the heme iron. Substituting alanine at position 82 with tryptophan results in increased A-ring proton-iron distances, consistent with the relative decrease in the level of A-ring hydroxylation at position 2β.

Characterisation of human cytochrome P450s involved in the bioactivation of clozapine

Clozapine is known to cause hepatotoxicity in a small percentage of patients. Oxidative bioactivation to reactive intermediates by hepatic cytochrome P450s (P450s) has be proposed as a possible mechanism. However, in contrast to their role in formation of N-desmethylclozapine and clozapine N-oxide, the involvement of individual P450s in the bioactivation to reactive intermediates is much less well characterized. The results of the present study show that 7 of 14 recombinant human P450s were able to bioactivate clozapine to a glutathione-reactive nitrenium ion. CYP3A4 and CYP2D6 showed the highest specific activity. Enzyme kinetical characterization of these P450s showed comparable intrinsic clearance of bioactivation, implicating that CYP3A4 would be more important because of its higher hepatic expression, compared with CYP2D6. Inhibition experiments using pooled human liver microsomes confirmed the major role of CYP3A4 in hepatic bioactivation of clozapine. By studying bioactivation of clozapine in human liver microsomes from 100 different individuals, an 8-fold variability in bioactivation activity was observed. In two individuals bioactivation activity exceeded N-demethylation and N-oxidation activity. Quinidine did not show significant inhibition of bioactivation in any of these liver fractions, suggesting that CYP2D6 polymorphism is not an important factor in determining susceptibility to hepatotoxicity of clozapine. Therefore, interindividual differences and drug-drug interactions at the level of CYP3A4 might be factors determining exposure of hepatic tissue to reactive clozapine metabolites.

Mass Spectrometric Characterization of Protein Adducts of Multiple P450-Dependent Reactive Intermediates of Diclofenac to Human Glutathione-S-transferase P1-1

Use of the nonsteroidal anti-inflammatory drug diclofenac (DF) is associated with serious idiosyncratic hepatotoxicity. Covalent binding of reactive intermediates of DF to proteins is considered to initiate the process leading to this severe side-effect. The aim of this study was to characterize the nature of covalent protein modifications by reactive metabolites of DF which result from bioactivation by cytochrome P450. DF and its major monohydroxylated metabolites 4-hydroxydiclofenac (4-OH-DF) and 5-hydroxydiclofenac (5-OH-DF) were bioactivated using a highly active P450 BM3 mutant (CYP102A1M11H) in the presence of the model target protein human glutathione-S-transferase P1-1 (hGST P1-1). Protein-adducts were subsequently identified by LC-MS/MS analysis of tryptic digests of hGST P1-1. In total, 10 different peptide adducts were observed which result from modifications of Cys-47 and Cys-14 of hGST P1-1. The majority of the protein thiol modifications appeared to be derived from 5-OH-DF, which produced seven different peptide adducts with mass increments of 289.0, 309.0, and 339.0 Da. Remarkably, no peptide adducts were observed upon the bioactivation of 4-OH-DF. Incubations of P450 BM3 with DF also showed the peptide adducts derived from 5-OH-DF and peptide adducts that are not derived from quinone imine. A peptide adduct with a mass increment of 249.0 Da most likely results from the o-imine methide formed by oxidative decarboxylation of DF. In addition, a peptide adduct was observed with a mass increment of 259.0 Da, which corresponds to the substitution of one of the chlorine atoms of DF by protein thiol. A corresponding GSH-conjugate with a similar mass increment was only observed if incubations of DF with P450 and GSH were supplemented by human GST P1-1. The results of this study not only confirm the importance of 5-OH-DF in covalent protein-binding but also suggest that the nature of protein adduction is not necessarily reflected by chemical conjugation with GSH.

Human NAD(P)H:quinone Oxidoreductase 1 (NQO1)-Mediated Inactivation of Reactive Quinoneimine Metabolites of Diclofenac and Mefenamic Acid

NAD(P)Hnquinone oxidoreductase 1 (NQO1) is an enzyme capable of reducing a broad range of chemically reactive quinones and quinoneimines (QIs) and can be strongly upregulated by Nrf2/Keap1-mediated stress responses. Several commonly used drugs implicated in adverse drug reactions (ADRs) are known to form reactive QI metabolites upon bioactivation by P450, such as acetaminophen (APAP), diclofenac (DF), and mefenamic acid (MFA). In the present study, the reductive activity of human NQO1 toward the QI metabolites derived from APAP and hydroxy-metabolites of DF and MFA was studied, using purified bacterial P450 BM3 (CYP102A1) mutant M11 as a bioactivation system. The NQO1-catalyzed reduction of the QI metabolites was quantified relative to spontaneous glutathione (GSH) conjugation. Addition of NQO1 to the incubations strongly reduced the formation of all corresponding GSH conjugates, and this activity could be prevented by dicoumarol, a selective NQO1 inhibitor. The GSH conjugation was strongly increased by adding human GSTP1-1 in a wide range of GSH concentrations. Still, NQO1 could effectively compete with the GST catalyzed GSH conjugation by reducing the QIs. In conclusion, we identified the QI metabolites of the 4- and 5-hydroxy-metabolites of DF and MFA as novel substrates for human NQO1. NQO1-mediated reduction proves to be an effective pathway to detoxify these QI metabolites in addition to GSH conjugation. Genetically determined deficiency of NQO1 therefore might be a risk factor for ADRs induced by reactive QI drug metabolites.

Effect of Human Glutathione S-Transferases on Glutathione-Dependent Inactivation of Cytochrome P450-Dependent Reactive Intermediates of Diclofenac

Idiosyncratic adverse drug reactions due to the anti-inflammatory drug diclofenac have been proposed to be caused by the generation of reactive acyl glucuronides and oxidative metabolites. For the oxidative metabolism of diclofenac by cytochromes P450 at least five different reactive intermediates have been proposed previously based on structural identification of their corresponding GSH-conjugates. In the present study, the ability of four human glutathione S-transferases (hGSTs) to catalyze the GSH-conjugation of the different reactive intermediates formed by P450s was investigated. Addition of pooled human liver cytosol and recombinant hGSTA1-1, hGSTM1-1, and hGSTP1-1 to incubations of diclofenac with human liver microsomes or purified CYP102A1M11 L437N as a model system significantly increased total GSH-conjugation. The strongest increase of total GSH-conjugation was observed by adding hGSTP1-1, whereas hGSTM1-1 and hGSTA1-1 showed lower activity. Addition of hGSTT1-1 only showed a minor effect. When considering the effects of hGSTs on GSH-conjugation of the different quinoneimines of diclofenac, it was found that hGSTP1-1 showed the highest activity in GSH-conjugation of the quinoneimine derived from 5-hydroxydiclofenac (5-OH-DF). hGSTM1-1 showed the highest activity in inactivation of the quinoneimine derived from 4-hydroxydiclofenac (4-OH-DF). Separate incubations with 5-OH-DF and 4-OH-DF as substrates confirmed these results. hGSTs also catalyzed GSH-conjugation of the o-iminemethide formed by oxidative decarboxylation of diclofenac as well as the substitution of one of the chlorine atoms of DF by GSH. hGSTP1-1 showed the highest activity for the formation of these minor GSH-conjugates. These results suggest that hGSTs may play an important role in the inactivation of DF quinoneimines and its minor reactive intermediates especially in stress conditions when tissue levels of GSH are decreased.

Effect of human glutathione S-transferase hGSTP1-1 polymorphism on the detoxification of reactive metabolites of clozapine, diclofenac and acetaminophen

Recent association studies suggest that genetically determined deficiencies in GSTs might be a risk factor for idiosyncratic adverse drug reactions resulting from the formation of reactive drug metabolites. hGSTP1-1 is polymorphic in the human population with a number of single nucleotide polymorphisms that yield an amino acid change in the encoded protein. Three allelic variants of hGSTP1-1 containing an Ile105Val or Ala114Val substitution, or a combination of both, have been the most widely studied and showed different activity when compared to wild-type hGSTP1-1*A (Ile105/Ala114). In the present study, we studied the ability of these allelic variants to catalyze the GSH conjugation of reactive metabolites of acetaminophen, clozapine, and diclofenac formed by bioactivation in in vitro incubations by human liver microsomes and drug metabolizing P450 BM3 mutants. The results show that effects of the change of amino acid at residue 105 and 114 on conjugation reactions were substrate dependent. A single substitution at residue 105 affects the ability to catalyze GSH conjugation, while when both residue 105 and 114 were substituted the effect was additionally enhanced. Single mutation at position 114 did not show a significant effect. The different hGSTP1-1 mutants showed slightly altered regioselectivities in formation of individual GSH conjugates of clozapine which suggests that the binding orientation of the reactive nitrenium ion of clozapine is affected by the mutations. For diclofenac, a significant decrease in activity in GSH-conjugation of diclofenac 1,4-quinone imine was observed for variants hGSTP1-1*B (Val105/Ala114) and hGSTP1-1*C (Val105/Val114). However, since the differences in total GSH conjugation activity catalyzed by these allelic variants were not higher than 30%, differences in inactivation of reactive intermediates by hGSTP1-1 are not likely to be a major factor in determining interindividual difference in susceptibility to adverse drug reactions induced by the drugs studied.

Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs.

Cytochrome P450 BM3 mutants are promising biocatalysts for the production of drug metabolites. In the present study, the ability of cytochrome P450 BM3 mutants to produce oxidative metabolites of structurally related NSAIDs meclofenamic acid, mefenamic acid and tolfenamic acid was investigated. A library of engineered P450 BM3 mutants was screened with meclofenamic acid (1) to identify catalytically active and selective mutants. Three mono-hydroxylated metabolites were identified for 1. The hydroxylated products were confirmed by NMR analysis to be 3-OH-methyl-meclofenamic acid (1a), 5-OH-meclofenamic acid (1b) and 4-OH-meclofenamic acid (1c) which are human relevant metabolites. P450 BM3 variants containing V87I and V87F mutation showed high selectivity for benzylic and aromatic hydroxylation of 1 respectively. The applicability of these mutants to selectively hydroxylate structurally similar drugs such as mefenamic acid (2) and tolfenamic acid (3) was also investigated. The tested variants showed high total turnover numbers in the order of 4000-6000 and can be used as biocatalysts for preparative scale synthesis. Both 1 and 2 could undergo benzylic and aromatic hydroxylation by the P450 BM3 mutants, whereas 3 was hydroxylated only on aromatic rings. The P450 BM3 variant M11 V87F hydroxylated the aromatic ring at 4 position of all three drugs tested with high regioselectivity. Reference metabolites produced by P450 BM3 mutants allowed the characterisation of activity and regioselectivity of metabolism of all three NSAIDs by thirteen recombinant human P450s. In conclusion, engineered P450 BM3 mutants that are capable of benzylic or aromatic hydroxylation of fenamic acid containing NSAIDs, with high selectivity and turnover numbers have been identified. This shows their potential use as a greener alternative for the generation of drug metabolites.

Inter-donor variability of phase I/phase II metabolism of three reference drugs in cryopreserved primary human hepatocytes in suspension and monolayer.

Cytochrome P450s (CYPs), UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) are the most important enzymes for metabolic clearance. Characterization of phase I and phase II metabolism of a given drug in cellular models is therefore important for an adequate interpretation of the role of drug metabolism in toxicity. We investigated phase I (CYP) and phase II (UGT and SULT) metabolism of three drugs related to drug-induced liver injury (DILI), namely acetaminophen (APAP), diclofenac (DF) and tolcapone (TC), in cryopreserved primary human hepatocytes from 5 donors in suspension and monolayer. The general phase II substrate 7-hydroxycoumarin (7-HC) was included for comparison. Our results show that the decrease in CYP, UGT and SULT activity after plating is substrate dependent. As a consequence the phase I/phase II metabolism ratio is significantly affected, with a shift in monolayer towards phase I metabolism for TC and towards phase II metabolism for APAP and DF. Inter-donor variability in drug metabolism is significant, especially in sulfation of 7-HC or APAP. As CYP, UGT and SULT metabolism may lead to bioactivation and/or detoxification of drugs, a changed ratio in phase I/phase II metabolism may have important consequences for metabolism-related toxicity.