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Aims Carbamazepine can cause hypersensitivity reactions in ~10% of patients. An immunogenic effect can be produced by the electrophilic 10,11-epoxide metabolite but not by carbamazepine. Hypothetically, certain single nucleotide polymorphisms might increase the formation of immunogenic metabolites, leading ultimately to hypersensitivity reactions. This study explores the role of clinical and genetic factors in the pharmacokinetics (PK) of carbamazepine and 3 metabolites known to be chemically reactive or formed through reactive intermediates. Methods A combination of rich and sparse PK samples were collected from healthy volunteers and epilepsy patients. All subjects were genotyped for 20 single nucleotide polymorphisms in 11 genes known to be involved in the metabolism or transport of carbamazepine and carbamazepine 10,11-epoxide. Nonlinear mixed effects modelling was used to build a population-PK model. Results In total, 248 observations were collected from 80 subjects. A 1-compartment PK model with first-order absorption and elimination best described the parent carbamazepine data, with a total clearance of 1.96 L/h, central distribution volume of 164 L and absorption rate constant of 0.45 h-1 . Total daily dose and coadministration of phenytoin were significant covariates for total clearance of carbamazepine. EPHX1-416G/G genotype was a significant covariate for the clearance of carbamazepine 10,11-epoxide. Conclusion Our data indicate that carbamazepine clearance was affected by total dose and phenytoin coadministration, but not by genetic factors, while carbamazepine 10,11-epoxide clearance was affected by a variant in the microsomal epoxide hydrolase gene. A much larger sample size would be required to fully evaluate the role of genetic variation in carbamazepine pharmacokinetics, and thereby predisposition to carbamazepine hypersensitivity.

Hypersensitivity reactions occur frequently in patients upon treatment with sulfamethoxazole (SMX). These adverse effects have been attributed to nitroso sulfamethoxazole (SMX-NO), the reactive product formed from auto-oxidation of the metabolite SMX hydroxylamine. The ability of SMX-NO to prime naïve T-cells in vitro and also activate T-cells derived from hypersensitive patients has illustrated that T-cell activation may occur through the binding of SMX-NO to proteins or through the direct modification of MHC-bound peptides. SMX-NO has been shown to modify cysteine residues in glutathione, designer peptides, and proteins in vitro; however, the presence of these adducts have not yet been characterized in vivo. In this study a parallel in vitro and in vivo analysis of SMX-NO adducts was conducted using mass spectrometry. In addition to the known cysteine adducts, multiple SMX-NO-derived haptenic structures were found on lysine and tyrosine residues of human serum albumin (HSA) in vitro. On lysine residues two haptenic structures were identified including an arylazoalkane adduct and a Schiff base adduct. Interestingly, these adducts are labile to heat and susceptible to hydrolysis as shown by the presence of allysine. Furthermore, SMX-modified HSA adducts were detected in patients on long-term SMX therapy illustrated by the presence of an arylazoalkane adduct derived from a proposed carboxylic acid metabolite of SMX-NO. The presence of these adducts could provide an explanation for the immunogenicity of SMX and the strong responses to SMX-NO observed in T-cell culture assays. Also, the degradation of these adducts to allysine could lead to a stress-related innate immune response required for T-cell activation.

Dapsone (DDS) causes hypersensitivity reactions in 0.5-3.6% of patients. Although clinical diagnosis is indicative of a hypersensitivity reaction, studies have not been performed to define whether dapsone or a metabolite activates specific T-cells. Thus, the aims of this study were to explore the immunogenicity DDS and nitroso DDS (DDS-NO) using peripheral blood mononuclear cells from healthy donors and splenocytes from mice and generate human T-cell clones to characterize mechanisms of T-cell activation. DDS-NO was synthesized from DDS-hydroxylamine and shown to bind to the thiol group of glutathione and human and mouse albumin through sulfonamide and N-hydroxyl sulphonamide adducts. Naïve T-cell priming to DDS and DDS-NO was successful in three human donors. DDS-specific CD4+ T-cell clones were stimulated to proliferate in response to drug via a MHC class II restricted direct binding interaction. Cross reactivity with DDS-NO, DDS-analogues, and sulfonamides was not observed. DDS-NO clones were CD4+ and CD8+, MHC class II and I restricted, respectively, and activated via a pathway dependent on covalent binding and antigen processing. DDS and DDS-NO-specific clones secreted a mixture of Th1 and Th2 cytokines, but not granzyme-B. Splenocytes from mice immunized with DDS-NO were stimulated to proliferate in vitro with the nitroso metabolite, but not DDS. In contrast, immunization with DDS did not activate T-cells. These data show that DDS- and DDS-NO-specific T-cell responses are readily detectable.

The transcription factor NRF2, governed by its repressor KEAP1, protects cells against oxidative stress. There is interest in modelling the NRF2 response to improve the prediction of clinical toxicities such as drug-induced liver injury (DILI). However, very little is known about the makeup of the NRF2 transcriptional network and its response to chemical perturbation in primary human hepatocytes (PHH), which are often used as a translational model for investigating DILI. Here, microarray analysis identified 108 transcripts (including several putative novel NRF2-regulated genes) that were both downregulated by siRNA targeting NRF2 and upregulated by siRNA targeting KEAP1 in PHH. Applying weighted gene co-expression network analysis (WGCNA) to transcriptomic data from the Open TG-GATES toxicogenomics repository (representing PHH exposed to 158 compounds) revealed four co-expressed gene sets or ‘modules’ enriched for these and other NRF2-associated genes. By classifying the 158 TG-GATES compounds based on published evidence, and employing the four modules as network perturbation metrics, we found that the activation of NRF2 is a very good indicator of the intrinsic biochemical reactivity of a compound (i.e. its propensity to cause direct chemical stress), with relatively high sensitivity, specificity, accuracy and positive/negative predictive values. We also found that NRF2 activation has lower sensitivity for the prediction of clinical DILI risk, although relatively high specificity and positive predictive values indicate that false positive detection rates are likely to be low in this setting. Underpinned by our comprehensive analysis, activation of the NRF2 network is one of several mechanism-based components that can be incorporated into holistic systems toxicology models to improve mechanistic understanding and preclinical prediction of DILI in man. Electronic supplementary material The online version of this article (10.1007/s00204-018-2354-1) contains supplementary material, which is available to authorized users.

Carbamazepine (CBZ) is an effective antiepileptic drug that has been associated with hypersensitivity reactions. The pathogenesis of those reactions is incompletely understood but is postulated to involve a complex interplay between the drug's metabolism, genetic variation in human leukocyte antigens and adverse activation of the immune system. Multiple T-cell activation mechanisms have been hypothesised, including activation by drug-peptide conjugates derived from proteins haptenated by reactive metabolites. However, definitive evidence of the drug-protein adducts in patients has been lacking. In this study, mass spectrometry was used to characterize protein modifications by microsomally generated metabolites of CBZ and in patients taking CBZ therapy. CBZ 10,11-epoxide (CBZE), a major electrophilic plasma metabolite of CBZ, formed adducts with glutathione-S-transferase pi (GSTP; Cys47) and human serum albumin (HSA; His146 and His338, but not Cys34) in vitro, via notably divergent side-chain selectivity. Both proteins were adducted at the same residues by undefined monoxygenated metabolites ([O]CBZ) when they were incubated with human liver microsomes, NADPH and CBZ. There was also evidence for formation of a CBZ adduct at His146 and His338 of HSA derived via dehydration from an intermediate arene oxide adduct. Glutathione trapping of reactive metabolites confirmed microsomal production of CBZE, and indicated simultaneous production of arene oxides. In 15 patients prescribed CBZ therapy, [O]CBZ-modified HSA (His146) was detected in all the subjects. The relative amount of adduct was moderately positively correlated with plasma concentrations of CBZ (r2 = 0.44, p = 0.002) and CBZE (r2 = 0.35, p = 0.006). Our results have provided the first chemical evidence for microsomal production of [O]CBZ species that are able to escape the microsomal domain to react covalently with soluble proteins. This study has also demonstrated the presence of circulating [O]CBZ-modified HSA in patients without hypersensitivity reactions who were receiving standard CBZ therapy. The implications of those circulating adducts for susceptibility to CBZ hypersensitivity merits further immunological investigation in hypersensitive patients.

Background: Mevalonic acid (MVA), as a product of 3-hydroxy-3-methylglutaryl coenzyme A reductase, represents a potential multipurpose biomarker in health and disease. A translational urinary MVA quantification method was developed, validated and used to demonstrate the diurnal variation of urinary MVA excretion in rats and healthy children. Methods: Urinary MVA was converted to mevalonolactone at pH 2, extracted with ethyl acetate and quantified by reversed-phase liquid chromatography–tandem mass spectrometry. Results: The assay had a dynamic range of 0.0156–10 µg/ml with precision

Dispiro-1,2,4,5-tetraoxanes and 1,2,4-trioxolanes represent attractive classes of synthetic antimalarial peroxides due to their structural simplicity, good stability, and impressive antimalarial activity. We investigated the reactivity of a series of potent amide functionalized tetraoxanes with Fe(II)gluconate, FeSO(4), FeSO(4)/TEMPO, FeSO(4)/phosphatidylcholine, and heme to gain knowledge of their potential mechanism of bioactivation and to compare the results with the corresponding 1,2,4-trioxolanes. Spin-trapping experiments demonstrate that Fe(II)-mediated peroxide activation of tetraoxanes produces primary and secondary C-radical intermediates. Reaction of tetraoxanes and trioxolanes with phosphatidylcholine, a predominant unsaturated lipid present in the parasite digestive vacuole membrane, under Fenton reaction conditions showed that both endoperoxides share a common reactivity in terms of phospholipid oxidation that differs with that of artemisinin. Significantly, when tetraoxanes undergo bioactivation in the presence of heme, only the secondary C-centered radical is observed, which smoothly produces regioisomeric drug derived-heme adducts. The ability of these tetraoxanes to alkylate the porphyrin ring was also confirmed with Fe(II)TPP and Mn(II)TPP, and docking studies were performed to rationalize the regioselectivity observed in the alkylation process. The efficient process of heme alkylation and extensive lipid peroxidation observed here may play a role in the mechanism of action of these two important classes of synthetic endoperoxide antimalarial.

The artemisinin compounds are the frontline drugs for the treatment of drug-resistant malaria. They are selectively cytotoxic to mammalian cancer cell lines and have been implicated as neurotoxic and embryotoxic in animal studies. The endoperoxide functional group is both the pharmacophore and toxicophore, but the proposed chemical mechanisms and targets of cytotoxicity remain unclear. In this study we have used cell models and quantitative drug metabolite analysis to define the role of the mitochondrion and cellular heme in the chemical and molecular mechanisms of cell death induced by artemisinin compounds. HeLa ρ(0) cells, which are devoid of a functioning electron transport chain, were used to demonstrate that actively respiring mitochondria play an essential role in endoperoxide-induced cytotoxicity (artesunate IC(50) values, 48 h: HeLa cells, 6 ± 3 μM; and HeLa ρ(0) cells, 34 ± 5 μM) via the generation of reactive oxygen species and the induction of mitochondrial dysfunction and apoptosis but do not have any role in the reductive activation of the endoperoxide to cytotoxic carbon-centered radicals. However, using chemical modulators of heme synthesis (succinylacetone and protoporphyrin IX) and cellular iron content (holotransferrin), we have demonstrated definitively that free or protein-bound heme is responsible for intracellular activation of the endoperoxide group and that this is the chemical basis of cytotoxicity (IC(50) value and biomarker of bioactivation levels, respectively: 10β-(p-fluorophenoxy)dihydroartemisinin alone, 0.36 ± 0.20 μM and 11 ± 5%; and with succinylacetone, >100 μM and 2 ± 5%).

Exposure to sulfamethoxazole (SMX) is associated with T-cell-mediated hypersensitivity reactions in human patients. T-cells can be stimulated by the putative metabolite nitroso SMX, which binds irreversibly to protein. The hydroxylamine and nitroso derivatives of three arylamine benzenesulfonamides, namely, sulfamethozaxole, sulfadiazine, and sulfapyridine, were synthesized, and their T-cell stimulatory capacity in the mouse was explored. Nitroso derivatives were synthesized by a three-step procedure involving the formation of nitro and hydroxylamine sulfonamide intermediates. For immune activation, female Balb-c strain mice were administered nitroso sulfonamides four times weekly for 2 weeks. After 14 days, isolated splenocytes were incubated with the parent compounds, hydroxylamine metabolites, and nitroso derivatives to measure antigen-specific proliferation. To explore the requirement of irreversible protein binding for spleen cell activation, splenocytes were incubated with nitroso derivatives in the presence or absence of glutathione. Splenocytes from nitroso sulfonamide-sensitized mice proliferated and secreted interleukin (IL)-2, IL-4, IL-5, and granulocyte monocyte colony-stimulating factor following stimulation with nitroso derivatives but not the parent compounds. Splenocytes from sensitized mice were also stimulated to proliferate with hydroxylamine and nitroso derivatives of the structurally related sulfonamides. The addition of glutathione inhibited the nitroso-specific T-cell response. Hydroxylamine metabolites were unstable in aqueous solution: Spontaneous transformation yielded appreciable amounts of nitroso and azoxy compounds as well as the parent compounds within 0.1 h. T-cell cross-reactivity with nitroso sulfonamides provides a mechanistic explanation as to why structurally related arylamine benzenesulfonamides are contraindicated in hypersensitive patients.

Exposure to the skin sensitizer p-phenylenediamine (PPD) is associated with allergic contact dermatitis; however, the ability of PPD to modify protein has not been fully investigated. The aims of this study were to characterize the reactions of PPD and the structurally related chemical 2,5-dimethyl-1,4-benzoquinonediamine with model nucleophiles, a synthetic peptide (DS3) containing each of the naturally occurring amino acids and His-tagged glutathione-S-transferase pi (GSTP), and to explore the effect of dimethyl substitution on PPD-specific T-cell responses using lymphocytes from allergic patients. The reductive soft nucleophiles N-acetyl cysteine and glutathione prevented PPD self-conjugation reactions and Bandrowski's base formation, but no adducts were detected. N-Acetyl lysine, a hard nucleophile, did not alter the rate of PPD degradation or form PPD adducts. With PPD and 2,5-dimethyl-1,4-benzoquinonediamine, only cysteine was targeted in the DS3 peptide. PPD and 2,5-dimethyl-1,4-benzoquinonediamine were also found to selectively modify the reactive Cys 47 residue of GSTP, which has a pK(a) of 3.5-4.2 and therefore exists in a largely protonated form. Glutathione formed mixed disulfides with the DS3 peptide, reducing levels of PPD binding. Lymphocytes from PPD allergic patients proliferated in the presence of PPD but not with 2,5-dimethyl-1,4-benzoquinonediamine. These results reveal that PPD and 2,5-dimethyl-1,4-benzoquinonediamine bind selectively to specific cysteine residues in peptides and proteins. Lymphocytes from PPD allergic patients were capable of discriminating between the different haptenic structures, suggesting that the hapten, but not the peptide moiety associated with MHC, is an important determinant for T-cell recognition.

Sulfamethoxazole (SMX) induces immunoallergic reactions that are thought to be a result of intracellular protein haptenation by its nitroso metabolite (SMX-NO mass, 267 amu). SMX-NO reacts with protein thiols in vitro, but the conjugates have not been defined chemically. The reactions of SMX-NO with glutathione (GSH), a synthetic peptide (DS3), and two model proteins, human GSH S-transferase pi (GSTP) and serum albumin (HSA), were investigated by mass spectrometry. SMX-NO formed a semimercaptal (N-hydroxysulfenamide) conjugate with GSH that rearranged rapidly (1-5 min) to a sulfinamide. Reaction of SMX-NO with DS3 also yielded a sulfinamide adduct (mass increment, 267 amu) on the cysteine residue. GSTP was exclusively modified at the reactive Cys47 by SMX-NO and exhibited mass increments of 267, 283, and 299 amu, indicative of sulfinamide, N-hydroxysulfinamide, and N-hydroxysulfonamide adducts, respectively. HSA was modified at Cys34, forming only the N-hydroxysulfinamide adduct. HSA modification by SMX-NO under these conditions was confirmed with ELISA and immunoblotting with an antisulfonamide antibody. It is proposed that cysteine-linked N-hydroxysulfinamide and N-hydroxysulfonamide adducts of SMX are formed via the reaction of SMX-NO with cysteinyl sulfoxy acids. Evidence for a multistep assembly of model sulfonamide epitopes on GSH and polypeptides via hydrolyzable intermediates is also presented. In summary, novel, complex, and metastable haptenic structures have been identified on proteins exposed in vitro to the nitroso metabolite of SMX.

On the basis of a mechanistic understanding of the toxicity of the 4-aminoquinoline amodiaquine (1b), three series of amodiaquine analogues have been prepared where the 4-aminophenol "metabolic alert" has been modified by replacement of the 4'-hydroxy group with a hydrogen, fluorine, or chlorine atom. Following antimalarial assessment and studies on mechanism of action, two candidates were selected for detailed ADME studies and in vitro and in vivo toxicological assessment. 4'-Fluoro-N-tert-butylamodiaquine (2k) was subsequently identified as a candidate for further development studies based on potent activity versus chloroquine-sensitive and resistant parasites, moderate to excellent oral bioavailability, low toxicity in in vitro studies, and an acceptable safety profile.

N-tert-Butyl isoquine (4) (GSK369796) is a 4-aminoquinoline drug candidate selected and developed as part of a public-private partnership between academics at Liverpool, MMV, and GSK pharmaceuticals. This molecule was rationally designed based on chemical, toxicological, pharmacokinetic, and pharmacodynamic considerations and was selected based on excellent activity against Plasmodium falciparum in vitro and rodent malaria parasites in vivo. The optimized chemistry delivered this novel synthetic quinoline in a two-step procedure from cheap and readily available starting materials. The molecule has a full industry standard preclinical development program allowing first into humans to proceed. Employing chloroquine (1) and amodiaquine (2) as comparator molecules in the preclinical plan, the first preclinical dossier of pharmacokinetic, toxicity, and safety pharmacology has also been established for the 4-aminoquinoline antimalarial class. These studies have revealed preclinical liabilities that have never translated into the human experience. This has resulted in the availability of critical information to other drug development teams interested in developing antimalarials within this class.

Methapyrilene (MP), a 2-thiophene H(1)-receptor antagonist, is a model toxicant in the genomic and proteomic analyses of hepatotoxicity. In rats, it causes an unusual periportal necrosis that is hypothetically attributed to chemically reactive and cytotoxic metabolites. We have characterized the bioactivation of MP by hepatic microsomes and primary rat hepatocytes, and we established a possible causal linkage with cytotoxicity. Methapyrilene tritiated at C-2 of the diaminoethane moiety ([3H]MP) was metabolized via an NADPH-dependent pathway to intermediates that combined irreversibly with microsomes (rat > mouse approximately human). This binding was attenuated by the cytochrome P450 (P450) inhibitor 1-aminobenzotriazole and thiols but not by trapping agents for iminium ions and aldehydes. Reactive intermediates were trapped as thioether adducts of monooxygenated MP. Mass spectrometric and hydrogen/deuterium exchange analysis of the glutathione adduct produced by rat liver microsomes indicated that the metabolite was most probably a thioether of MP S-oxide substituted in the thiophene ring. The glutathione adduct was formed by rat hepatocytes and eliminated in bile by rats administered [3H]MP intravenously. MP produced concentration- and time-dependent cytotoxicity, depleted glutathione, and underwent irreversible binding to the hepatocytes before a significant increase in cell damage was observed. P450 inhibitors reduced turnover of the drug, production of the glutathione adduct, irreversible binding, and cytotoxicity but inhibited glutathione depletion selectively. MP underwent lesser turnover and bioactivation in mouse hepatocytes and was not cytotoxic. Analogs with phenyl and p-methoxyphenyl rings were much less hepatocytotoxic than MP. Hepatotoxicity in rats was diminished by predosing with 1-aminobenzotriazole. For the first time, a thiophene ring substituent is identified as a bioactivation-dependent toxicophore in hepatocytes.

Acyl glucuronides are key metabolites for many carboxylic acid-containing drugs, notably those of the non-steroidal anti-inflammatory class. In the processes of drug safety assessment and new drug development, it is essential that acyl glucuronides, if formed in vivo, should be made conveniently available for bioevaluation. We recently showed that selective acylation of allyl glucuronate is a promising method for the synthesis of these metabolites in good yield and with excellent β-anomeric selectivity. We now give fuller details of the allyl ester method and further report that benzyl glucuronate performs at least equally well in the acylation step, offering the advantage of very mild deprotection by catalytic transfer (or conventional) hydrogenation. Depending on the compatibility of other functional groups, as discussed below, this will be the method of choice for many acyl glucuronide syntheses. The value of the method is demonstrated in particular by the synthesis of several acyl glucuronides that are known metabolites of important drugs.

Artemisinin and its derivatives are currently recommended as first-line antimalarials in regions where Plasmodium falciparum is resistant to traditional drugs. The cytotoxic activity of these endoperoxides toward rapidly dividing human carcinoma cells and cell lines has been reported, and it is hypothesized that activation of the endoperoxide bridge by an iron(II) species, to form C-centered radicals, is essential for cytotoxicity. The studies described here have utilized artemisinin derivatives, dihydroartemisinin, 10beta-(p-bromophenoxy)dihydroartemisinin, and 10beta-(p-fluorophenoxy)dihydroartemisinin, to determine the chemistry of endoperoxide bridge activation to reactive intermediates responsible for initiating cell death and to elucidate the molecular mechanism of cell death. These studies have demonstrated the selective cytotoxic activity of the endoperoxides toward leukemia cell lines (HL-60 and Jurkat) over quiescent peripheral blood mononuclear cells. Deoxy-10beta-(p-fluorophenoxy)dihydroartemisinin, which lacks the endoperoxide bridge, was 50- and 130-fold less active in HL-60 and Jurkat cells, respectively, confirming the importance of this functional group for cytotoxicity. We have shown that chemical activation is responsible for cytotoxicity by using liquid chromatography-mass spectrometry analysis to monitor endoperoxide activation by measurement of a stable rearrangement product of endoperoxide-derived radicals, which was formed in sensitive HL-60 cells but not in insensitive peripheral blood mononuclear cells. In HL-60 cells the endoperoxides induce caspase-dependent apoptotic cell death characterized by concentration- and time-dependent mitochondrial membrane depolarization, activation of caspases-3 and -7, sub-G(0)/G(1) DNA formation, and attenuation by benzyloxycarbonyl-VAD-fluoromethyl ketone, a caspase inhibitor. Overall, these results indicate that endoperoxide-induced cell death is a consequence of activation of the endoperoxide bridge to radical species, which triggers caspase-dependent apoptosis.

▪ Abstract The importance of reactive metabolites in the pathogenesis of drug-induced toxicity has been a focus of research interest since pioneering investigations in the 1950s revealed the link between toxic metabolites and chemical carcinogenesis. There is now a great deal of evidence that shows that reactive metabolites are formed from drugs known to cause hepatotoxicity, but how these toxic species initiate and propagate tissue damage is still poorly understood. This review summarizes the evidence for reactive metabolite formation from hepatotoxic drugs, such as acetaminophen, tamoxifen, diclofenac, and troglitazone, and the current hypotheses of how this leads to liver injury. Several hepatic proteins can be modified by reactive metabolites, but this in general equates poorly with the extent of toxicity. Much more important may be the identification of the critical proteins modified by these toxic species and how this alters their function. It is also important to note that the toxicity of reactive metabolites may be mediated by noncovalent binding mechanisms, which may also have profound effects on normal liver physiology. Technological developments in the wake of the genomic revolution now provide unprecedented power to characterize and quantify covalent modification of individual target proteins and their functional consequences; such information should dramatically improve our understanding of drug-induced hepatotoxic reactions.

SB-209247 [(E)-3-[6-[[(2,6-dichlorophenyl)-thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenoic acid], an anti-inflammatory leukotriene B4 receptor antagonist, was associated in beagle dogs but not male rats with an inflammatory hepatopathy. It also produced a concentration-dependent (10-1000 microM) but equal leakage of enzymes from dog and rat precision-cut liver slices. The hepatic metabolism of SB-209247 was investigated with reference to the formation of reactive acyl glucuronides. [14C]SB-209247 (100 micromol/kg) administered i.v. to anesthetized male rats was eliminated by biliary excretion of the acyl glucuronides of the drug and its sulfoxide. After 5 h, 1.03 +/- 0.14% (mean +/- S.E.M., n = 4) of the dose was bound irreversibly to liver tissue. The sulfoxide glucuronide underwent pH-dependent rearrangement in bile more rapidly than did the SB-209247 conjugate. [14C]SB-209247 was metabolized by sulfoxidation and glucuronidation in rat and dog hepatocytes, and approximately 1 to 2% of [14C]SB-209247 (100 microM) became irreversibly bound to cellular material. [14C]SB-209247 sulfoxide and glucuronide were the only metabolites produced by dog, rat, and human liver microsomes in the presence of NADPH and UDP-glucuronic acid (UDPGA), respectively. V(max) values for [14C]SB-209247 glucuronidation by dog, rat, and human microsomes were 2.6 +/- 0.1, 1.2 +/- 0.1, and 0.4 +/- 0.0 nmol/min/mg protein, respectively. Hepatic microsomes from all three species catalyzed UDPGA-dependent but not NADPH-dependent irreversible binding of [14C]SB-209247 (100-250 microM) to microsomal protein. Although a reactive acyl glucuronide was formed by microsomes from every species, the binding did not differ between species. Therefore, neither the acute cellular injury nor glucuronidation-driven irreversible protein binding in vitro is predictive of the drug-induced hepatopathy.

In humans, metabolism of the commonly used nonsteroidal antiinflammatory drug diclofenac 1 yields principally the 4'-hydroxy 2, 5-hydroxy 3, and acyl glucuronide 4 metabolites. All three metabolites have been implicated in rare idiosyncratic adverse reactions associated with this widely used drug. Therefore, for mechanistic toxicological studies of 1, substantial quantities of 2-4 are required and their syntheses and characterization are described here. Key steps were a convenient two-step preparation of aniline 5 from phenol, efficient and selective 6-iodination of amide 18, and high-yielding Ullmann couplings to generate diarylamines 11 and 21. The acyl glucuronide 4 was obtained by Mitsunobu reaction of 1 (free acid) with allyl glucuronate 23 followed by Pd(0) deprotection, using a modification of a published procedure. We report full characterization of 4 and note that this important metabolite has been made available pure and in quantity for the first time. We report also the metabolic fates of the synthetic metabolites: 2 and 3 were glucuronidated in rats, but only 3 formed glutathione adducts in vivo and by enzymatic synthesis via a quinoneimine intermediate. A previously undescribed glutathione adduct of 3 was obtained by enzymatic synthesis. Compound 4 formed an imine-linked protein conjugate as evinced by sodium cyanoborohydride trapping.

Drug hypersensitivity remains a major concern, as it causes high morbidity and mortality. Understanding the mechanistic basis of drug hypersensitivity is complicated by the multiple risk factors implicated. This study utilized sulfamethoxazole (SMX) as a model drug to (1) relate SMX metabolism in antigen presenting cells (APCs) to the activation of T-cells and (2) characterize covalent adducts of SMX and myeloperoxidase, which might represent antigenic determinants for T-cells. The SMX metabolite nitroso-SMX (SMX-NO) was found to bind irreversibly to APCs. Time- and concentration-dependent drug-protein adducts were also detected when APCs were cultured with SMX. Metabolic activation of SMX was significantly reduced by the oxygenase/peroxidase inhibitor methimazole. Similarly, SMX-NO-specific T-cells were activated by APCs pulsed with SMX, and the response was inhibited by pretreatment with methimazole or glutaraldehyde, which blocks antigen processing. Western blotting, real-time polymerase chain reaction (RT-PCR), and mass spectrometry analyses suggested the presence of low concentrations of myeloperoxidase in APCs. RT-PCR revealed mRNA expression for flavin-containing monooxygenases (FMO1-5), thyroid peroxidase, and lactoperoxidase, but the corresponding proteins were not detected. Mass spectrometric characterization of SMX-NO-modified myeloperoxidase revealed the formation of N-hydroxysulfinamide adducts on Cys309 and Cys398. These data show that SMX's metabolism in APCs generates antigenic determinants for T-cells. Peptides derived from SMX-NO-modified myeloperoxidase may represent one form of functional antigen.

Covalent protein modifications by electrophilic acyl glucuronide (AG) metabolites are hypothetical causes of hypersensitivity reactions associated with certain carboxylate drugs. The complex rearrangements and reactivities of drug AG have been defined in great detail, and protein adducts of carboxylate drugs, such as diclofenac, have been found in liver and plasma of experimental animals and humans. However, in the absence of definitive molecular characterization, and specifically, identification of signature glycation conjugates retaining the glucuronyl and carboxyl residues, it cannot be assumed any of these adducts is derived uniquely or even fractionally from AG metabolites. We have therefore undertaken targeted mass spectrometric analyses of human serum albumin (HSA) isolated from diclofenac patients to characterize drug-: derived structures and, thereby, for the first time, have deconstructed conclusively the pathways of adduct formation from a drug AG and its isomeric rearrangement products in vivo. These analyses were informed by a thorough understanding of the reactions of HSA with diclofenac AG in vitro. HSA from six patients without drug-: related hypersensitivities had either a single drug-: derived adduct or one of five combinations of 2-8 adducts from among seven diclofenac N-acylations and three AG glycations on seven of the protein's 59 lysines. Only acylations were found in every patient. We present evidence that HSA modifications by diclofenac in vivo are complicated and variable, that at least a fraction of these modifications are derived from the drug's AG metabolite, and that albumin adduction is not inevitably a causation of hypersensitivity to carboxylate drugs or a coincidental association.

A two-step-approach to title compounds (IV) based on N-arylation of indole derivatives (I), followed by acid-catalyzed rearrangement is reported.

Abacavir (ABC), a nucleoside-analogue reverse transcriptase inhibitor, is associated with severe hypersensitivity reactions that are thought to involve the activation of CD8+ T cells in a HLA-B*57:01-restricted manner. Recent studies have claimed that noncovalent interactions of ABC with HLA-B*57:01 are responsible for the immunological reactions associated with ABC. However, the formation of hemoglobin-ABC aldehyde (ABCA) adducts in patients exposed to ABC suggests that protein conjugation might represent a pathway for antigen formation. To further characterize protein conjugation reactions, we used mass spectrometric methods to define ABCA modifications in patients receiving ABC therapy. ABCA formed a novel intramolecular cross-linking adduct on human serum albumin (HSA) in patients and in vitro via Michael addition, followed by nucleophilic adduction of the aldehyde with a neighboring protein nucleophile. Adducts were detected on Lys159, Lys190, His146, and Cys34 residues in the subdomain IB of HSA. Only a cysteine adduct and a putative cross-linking adduct were detected on glutathione S-transferase Pi (GSTP). These findings reveal that ABC forms novel types of antigens in all patients taking the drug. It is therefore vital that the immunological consequences of such pathways of haptenation are explored in the in vitro models that have been used by various groups to define new mechanisms of drug hypersensitivity exemplified by ABC.

Adverse drug reactions remain a major clinical problem and impairment to safe drug development. The CPMP provides guidelines for preclinical studies. Safety margins for target-organ toxicity must be calculated. It is necessary to be aware of species differences in pharmacodynamic effects and in routes and rates of metabolism. The relevance of the test system to the intended human use must be defined. It is important to learn lessons from past experience with drugs that have caused serious toxicity in man. Idiosyncratic toxicity is only detected late in drug development. The pathophysiology of such reactions is complex and poorly understood. In man, genetics and disease may be predisposing factors. Therefore, it is not surprising that conventional animal models fail to identify drugs which cause idiosyncratic toxicity. At present, we have to depend on the identification of structural alerts in drugs and metabolites to screen out the potential for toxicity. Recent advances in molecular toxicology allow the investigator to explore biochemical stress, in both in vitro and in vivo models, at doses of drugs and chemicals that do not cause overt toxicity. It is, thus, possible to address physiological, pharmacological and toxicological aspects of adverse reactions. Ultimately, it may be possible to relate cellular changes in these test systems to serious toxicities seen in man (anaphylaxis, hepatotoxicity, blood dyscrasias and severe skin reactions) and, thus, provide more physiologically relevant assays for the prediction of human drug toxicity.

The mechanisms by which acute administration of methapyrilene, an H1-receptor antihistamine causes periportal necrosis to rats are unknown. This study investigated the role of the hepato-biliary system in methapyrilene hepatotoxicity following daily administration of 150 mg/kg per day over 3 consecutive days. Biliary metabolites of methapyrilene were tentatively identified. In male Han Wistar rats administration of methapyrilene significantly increased hepatic reduced glutathione (GSH) to 140% of control levels 24 h following the last dose. There were no significant changes in the activities of glutathione-related enzymes, glutathione peroxidase (GPx) and reductase (GSH), glutathione S-transferase (GST), and gamma-glutamyl cysteine synthetase (γ-GCS) over 3 days of methapyrilene administration. Methapyrilene treatment resulted in no significant increase in excretion of biliary oxidized glutathione (GSSG), a sensitive marker of oxidative stress in vivo, following the third dose. [3H]Methapyrilene-derived radioactivity was detected in bile, to a greater extent than in feces, indicating that methapyrilene and/or metabolites underwent enterohepatic recirculation. Cannulation and exteriorization of the bile duct (to interrupt enterohepatic recirculation) afforded some protection against the hepatotoxicity, assessed by clinical chemistry and histopathology. Liquid chromatography-mass spectrometry (LC-MS) analysis of bile indicated the presence of unmetabolized methapyrilene, methapyrilene O-glucuronide and desmethyl methapyrilene O-glucuronide. These data demonstrate that acute methapyrilene hepatotoxicity in vivo is not a consequence of GSH depletion, or oxidative stress, but that enterohepatic recirculation of biliary metabolites may be important. Progressive exposure to non-oxidizing, reactive metabolic intermediates may be responsible for hepatotoxicity.

Lamotrigine [3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine] is an antiepileptic drug associated with hypersensitivity reactions which are thought to be an immunological response to metabolically generated drug-protein adducts. The o-dichlorophenyl moiety is a potential site for bioactivation of the drug to an arene oxide. The metabolites of [(14)C]lamotrigine (78 micromol/kg, iv) in adult male Wistar rats were characterized with particular reference to thioether derivatives of an epoxide intermediate. Biliary recovery of radioactivity from anesthetized and cannulated animals was 7.3 +/- 3.0% (mean +/- SD, n = 4) of the dose over 4 h; 5.5 +/- 0.5% was recovered in bladder urine after 4 h. Bile contained [(14)C]lamotrigine (1.4 +/- 0.3%), a glutathione adduct of [(14)C]dihydrohydroxylamotrigine (1.8 +/- 0.3%), i.e., an adduct of an arene oxide, and the glutathione (1.5 +/- 0.7%), cysteinylglycine (1.9 +/- 0.5%), and N-acetylcysteine (0.4 +/- 0.2%) adducts of [(14)C]lamotrigine. Formation of the thioether metabolites was partially blocked by the cytochrome P450 inhibitor, ketoconazole. Urine contained [(14)C]lamotrigine (4.5 +/- 0.5%) and [(14)C]lamotrigine N-oxide (0.9 +/- 0.2%). The radiolabeled material in skin (15.6 +/- 1.4%) was almost entirely [(14)C]lamotrigine. Isolated rat hepatocytes achieved a low rate of turnover to the glutathione adduct and N-oxide. However, neither rat nor human liver microsomes catalyzed NADPH-dependent irreversible binding. Lamotrigine can be bioactivated to an arene oxide by rat hepatocytes in the absence of a major competing pathway such as N-glucuronidation. Inhibition of N-glucuronidation has been associated with an increased risk of skin reactions in patients.

The metabolic formation of a-hydroxytamoxifen, a reactive metabolite of tamoxifen in rat liver, was characterized and quantified in vitro (hepatic microsomal incubations) and in vivo (bile-duct cannulated animals). This minor metabolite was identified by chromatographic and mass spectral comparisons with the authentic compound. The rates of formation of alpha-hydroxytamoxifen in incubations (30 min) of tamoxifen (25 microM) with liver microsomal preparations from women (pool of six), female CD1 mice or female Sprague-Dawley rats, as quantified by liquid chromatography-mass spectrometry (LC-MS), were 1.15+/-0.03, 0.30+/-0.05 and 2.70+/-0.35 pmol/min/mg protein, respectively. Selective inhibition of microsomal P450 indicated that alpha-hydroxylation was catalysed predominantly by CYP3A in humans. Bile-duct cannulated and anaesthetized female rats and mice given [14C]tamoxifen (43 micromol/kg, i.v.) excreted, respectively, 24 and 21% of the administered radioactivity in bile over 5 and 3.5 h. The major radiolabelled biliary metabolite in rats, characterized by LC-MS after enzymic hydrolysis of conjugates, was the glucuronide of 4-hydroxytamoxifen (10% of dose) and only 0.1% of the dose was recovered as alpha-hydroxytamoxifen. After administration of alpha-hydroxytamoxifen (43 micromol/kg, i.v.) to rats, only 1.19% of the administered compound was recovered from a glucuronide metabolite in bile, indicating a possible 0.84% alpha-hydroxylation of tamoxifen in vivo. There was, however, no indication of the presence in bile of either O-sulphonate or glutathione conjugates derived from alpha-hydroxytamoxifen. This study shows for the first time that alpha-hydroxytamoxifen can be glucuronylated in rat liver. Whereas sulphonation results in electrophilic genotoxic intermediates, glucuronidation may represent a means of detoxifying alpha-hydroxytamoxifen.

Peroxides represent a new chemical class of antimalarial drugs (Figure 1). The first generation compounds are all derivatives of artemisinin, the active principle of a Chinese herbal remedy [1]. Artemisinin, or qinghaosu (green plant extract), is extracted from Artemesia annua, a plant which grows widely in South China. The first generation compounds currently in use are derivatives of the lactol, dihydroartemisinin, which is a major metabolite for each of the derivatives, and may be the most important chemical species for antimalarial activity in vivo. The promise of these compounds lies in their effectiveness against chloroquine-resistant parasites and their rapid action against malaria. Moreover, they have been used for the treatment of malaria in three million patients with few reports of clinically significant toxicity [2]. Figure 1 Chemical structures of ‘First’ and ‘Second’ generation peroxides. The fenozan, tetroxane and tricyclic trioxanes are totally synthetic compounds prepared from readily available starting materials. The first generation DHA ... The drawback with artemisinin and related compounds is that they suffer from poor bioavailability: the relative bioavailability of oral artemisinin (vs intramuscular) and intramuscular artemether (vs oral) and the bioavailability of artesunate are all within a range of 15–30% [3–5]. This may be one of the reasons for the relatively high rate of recrudescence associated with peroxide monotherapy. Their complex chemical structures are not essential for pharmacological activity and there has, therefore, been a drive to design and synthesize simpler endoperoxides which have greater in vivo potency. To date, more than 1000 new endoperoxides belonging to several chemical classes have been prepared. A number of ‘second generation’ peroxides have been developed, including arteflene (Figure 1). This is a synthetic peroxide developed from yingzhaosu A, the complex active constituent of the Chinese plant Artabotrys uncinatus, after it was shown to have antimalarial activity [6]. It has been tested in clinical trials in Africa. The ‘first generation’ artemisinin derivatives, as described above, are chemically simple esters and ethers of the parent lactol, dihydroartemisinin (Figure 1) [7, 8]. These compounds are generally more potent than artemisinin both in vitro and in vivo, and provide a wider choice of formulations which are more easily administered to patients in the field. Artemether and arteether are more oil soluble than artemisinin and are currently undergoing clinical trials under WHO sponsorship [8]. Other first generation derivatives include sodium artesunate, the DHA β-O-succinate half ester sodium salt, and sodium artelinate, the corresponding DHA β-O-p-carboxybenzyl ether sodium salt. Both of these compounds, which are water soluble, are currently under investigation as intravenous treatments for severe malarial infections [8]. Given the obvious pharmacokinetic limitations of the ‘first generation’ analogues and the realization that the peroxide bridge is the essential pharmacophore for biological activity, medicinal chemists have developed a range of second generation derivatives. Representative compounds include the bicyclic fenozan type derivatives, synthesized by Jefford et al. [9], the tetroxanes synthesized by Vennerstrom [10] and tricyclic simplified derivatives prepared by Posner and coworkers [11]. The discovery that several of the first generation artemisinin derivatives are metabolised extensively to DHA has prompted other workers to prepare C-10-carba deoxy derivatives (for a review see Meshnick et al. [1]). These compounds cannot be metabolised to DHA, which undergoes fairly rapid clearance [8], and should therefore have longer half-lives in vivo. The initial focus of the medicinal chemist is clearly directed towards the discovery of simpler (hence often less expensive) and more potent compounds, which provides not only lead compounds for therapeutic use, but also pharmacological tools to define the mechanism of action of this class of agent and the essential pharmacophore. A QSAR study of antimalarial activity indicates that docking between an active trioxane and the receptor, haem, could be the crucial step preliminary to drug action [12]. Drug safety is an important consideration which has to be evaluated in concert with efficacy at the earliest possible stage in drug development. It is therefore essential to define the potential hazards associated with second or third generation peroxides that present greater and more prolonged systemic exposure and design appropriate test systems for screening for drug toxicity. The specific questions which need to be addressed during further chemical development of peroxide antimalarials are as follows. Will such chemical modifications lead to loss of the high degree of selective therapeutic potency noted for first generation peroxide antimalarials? Will chemical modification result in greater exposure of mammalian systems to hazards posed by the peroxide group such as the neurotoxicity observed in rodents given first generation peroxides? The purpose of this review is to address each of these questions from chemical and clinical perspectives.

1. The clearance of dihydroartemisinin (DHA) in control and malaria-infected (MI) rats was investigated using the isolated perfused rat liver (IPRL) model and hepatic microsomal studies. 2. In the recirculating IPRL, clearance of DHA was reduced from a mean (s.d.) of 8.2+/-1.8 ml min(-1) in controls (n=8) to 6.0+/-1.0 ml min(-1) in MI (n=8; P

Ozonolysis of R-carvone and in situ trapping with primary alcohols ROH (R= Me, Et, Bu, Pent, Oct) produces hydroperoxy ketals (5a-e) as a 1:1 mixture of diastereomers. Cyclisation of these intermediates with catalytic sodium methoxide in methanol produces the corresponding endoperoxide derivatives (6a-6e). The pentyl and octyl endoperoxide derivatives demonstrate reasonable antimalarial potency in vitro against the HB3 strain of Plasmodium falciparum . A mechanism for antimalarial action involving the formation of a C-centred radical is proposed.

Dexamethasone (DEX) has previously been shown to be extensively metabolised to 6-hydroxylated and side-chain cleaved metabolites in human liver in vitro. CYP3A4 is responsible for 6alpha- and 6beta-hydroxylation of DEX and CYP17 is thought to mediate side-chain cleavage to generate 9alphafluoro-androsta-1,4-diene-11beta-hydroxy-16alpha-methyl-3,17-dione (9alphaF-A). Although 9alphaF-A has not previously been isolated as a metabolite in its unhydroxylated form in human liver incubations, it is formed as an intermediate metabolite, which is subsequently rapidly hydroxylated to OH-9alphaF-A. A main part of this study has been to conclusively show that DEX undergoes extensive side-chain cleavage to form 9alphaF-A in human kidney fractions, which is in contrast to profiles obtained for DEX metabolism in parallel human liver microsomal incubations where 6-hydroxylation is the predominant pathway. Furthermore, molecular models of CYP3A4 and CYP17 (17,20 lyase) have been used to model the enzyme fits of DEX. From these modelling studies it has been shown that DEX complements both putative enzyme active sites in orientations likely to lead to the formation of the metabolites identified in vitro. We have also been able to rationalise the preferential formation of the 6betaOH-DEX isomer.

Arteflene 4 reacts with FeCl 2 .4H 2 O in acetonitrile or with hemin / N-acetylcysteine (heme Fe(II)) in acetonitrile to produce the diol 5 and the enone 6 . Treatment of arteflene with Zn AcOH , a model of NADH dehydrogenase, results in the formation of the diol 5 in 80% yield. The formation of the enone 6 indicates that arteflene fragments to a non-stabilised carbon-centred radical. This radical intermediate is proposed to mediate the potent antimalarial activity of 4 .

1. Cytochrome P450-mediated bioactivation of sulphamethoxazole to a hydroxylamine has been implicated in the hypersensitivity reactions associated with co-trimoxazole administration. Inhibiting the formation of the hydroxylamine may be one method of preventing the high frequency of toxicity which is observed in HIV-infected patients. Therefore, in this study, we have investigated the ability of fluconazole and ketoconazole, known cytochrome P450 inhibitors, to inhibit the formation of sulphamethoxazole hydroxylamine. 2. Ten healthy male volunteers were given co-trimoxazole (800 mg sulphamethoxazole and 160 mg trimethoprim) alone or 1 h after either fluconazole (150 mg) or ketoconazole (200 mg) in a randomized fashion with a washout period of at least 1 week between each phase. Urine was collected for 24 h, and sulphamethoxazole and its metabolites were quantified by electrospray LC-MS. 3. Ketoconazole had no effect on the urinary recovery of sulphamethoxazole or any of its metabolites. In contrast, fluconazole significantly (P < 0.001) inhibited the formation of sulphamethoxazole hydroxylamine by 50.0 +/- 15.1%. Fluconazole also inhibited the oxidation of sulphamethoxazole to the 5-methylhydroxy and 5-methylhydroxy acetate metabolites by 69.9 +/- 15.8% and 64.0 +/- 12.0%, respectively, but had no effect on the amount of sulphamethoxazole, N4-acetyl sulphamethoxazole, or sulphamethoxazole N1-glucuronide excreted in urine. 4. The potential clinical benefit of using fluconazole to prevent hypersensitivity to co-trimoxazole in patients with AIDS needs to be assessed in a prospective study using both metabolite formation and the clinical occurrence of adverse reactions as end-points.

Amodiaquine, a 4-aminoquinoline antimalarial, has been associated with hepatitis and agranulocytosis in humans. Drug hypersensitivity reactions, especially agranulocytosis, have been attributed to reactive intermediates generated by the oxidants discharged from stimulated polymorphonuclear leucocytes (PMN). The metabolism of amodiaquine to both stable and chemically reactive metabolites by human PMN has been investigated in vitro. Incubation of [14C]-amodiaquine with PMN resulted in irreversible binding of radiolabel to protein and depletion of intracellular reduced glutathione, which were enhanced by phorbol myristate acetate (PMA), a PMN activator. Two metabolites were identified: the C-5′ glutathione adduct of amodiaquine, derived from both endogenous and exogenous glutathione, and 4-amino-7-chloroquinoline, which was presumed to be formed by hydrolysis of amodiaquine quinoneimine. Desethylamodiaquine, the major plasma metabolite of amodiaquine in humans, also underwent bioactivation to a chemically reactive species in the presence of PMA-stimulated PMN. Substitution of the 4′-hydroxyl group in amodiaquine with fluorine significantly reduced irreversible binding to protein and abolished depletion of intracellular glutathione in the presence of PMA. These findings indicate that the bioactivation of amodiaquine by PMN is associated with the formation of a quinoneimine intermediate. Such a reactive metabolite, if produced in PMN or bone marrow in vivo, may be responsible for the drug's myelotoxicity.

The metabolic fates of 2-chloro-, 2-bromo-, 4-bromo- and 2-iodo-17α-ethynyloestradiol (EE2) in rats were determined. 6,7-3H-labelled analogues (0.1–2.0 μmol/kg) were administered i.v. to anaesthetized animals. The metabolites of all four compounds were rapidly and extensively excreted in bile (79–93% of the dose over 6 h). Unlike EE2 and 2-fluoro-EE2 (2-FEE2), neither 2-chloro(Cl)-(2.0 μmol/kg),2-bromo(Br)-(0.1 μmol/kg), nor 2-iodo(I)-EE2-(0.1 μmol/kg) underwent C-2 hydroxylation in female rats; 2-BrEE2 was similarly refractory in male rats; 4-BrEE2(0.1 μmol/kg), in females, was subject to approx. 2-fold greater C-2 hydroxylation than 2-FEE2 but this equalled only approx. 60% of that undergone by EE2. All three of the C-2 halogenated derivatives were substantially excreted unchanged except for conjugation. 2-ClEE2 alone was C-4 hydroxylated to an appreciable extent. The oxidative metabolism of 2- and 4-BrEE2 in rats was sexually differentiated: 2-BrEE2 yielded an alkyl hydroxylated metabolite and a two-component dihydroxylated fraction in the ratio 1:0.09 and 1:0.76 in males and females, respectively; 4-BrEE2 underwent C-2 and alicyclic (C-15) hydroxylation in the ratio 1:4.8 and 1:0.07 in males and females, respectively. 2-ClEE2 formed much less alkyl monohydroxylated metabolite (C-16 hydroxylated for 2-Cl- and 2-IEE2) than did either 2-BrEE2 or 2-IEE2. The observed structure-metabolism relationships are discussed.

Efficient procedures for the synthesis of benzo-fluorinated dibenz[b,f]azepines (iminostilbenes) from fluorinated isatins or indoles using a number of ring-expansion reactions are described. A range of mono- and difluorinated analogues is accessible, and the syntheses can deliver gram quantities of the final products, which are precursors of fluoro analogues of the important anticonvulsant carbamazepine.

The oxidative and reductive biotransformations of 9 alpha-fluorocortisol (fluorocortisol) by human liver microsomes and cytosol have been characterized. 9 alpha-Fluorination greatly simplified cortisol metabolism in microsomes: dehydrogenation of the 11 beta-hydroxyl group and A-ring (4-ene-5 beta and 3 alpha-keto) reduction, the principle pathways, were completely blocked. Fluorocortisol was essentially metabolized by the remaining pathways, 20 beta-reduction and 6 beta-hydroxylation. In cytosol, 20 beta-reduction replaced the A-ring reduction of cortisol as the sole biotransformation. The major structure-metabolism relationships of fluorocortisol in man, i.e. complete and extensive inhibition of 11 beta-dehydrogenation and 4-ene-5 beta-reduction, respectively, were attributed to hepatic enzyme systems. Their mechanistic basis is discussed with reference to the electronic and conformational changes induced by 9 alpha-fluorination.

Acyl glucuronidation is the major metabolic conjugation reaction of most carboxylic acid drugs in mammals. The physiological consequences of this biotransformation have been investigated incompletely but include effects on drug metabolism, protein binding, distribution and clearance that impact upon pharmacological and toxicological outcomes. In marked contrast, the exceptional but widely disparate chemical reactivity of acyl glucuronides has attracted far greater attention. Specifically, the complex transacylation and glycation reactions with proteins have provoked much inconclusive debate over the safety of drugs metabolised to acyl glucuronides. It has been hypothesised that these covalent modifications could initiate idiosyncratic adverse drug reactions. However, despite a large body of in vitro data on the reactions of acyl glucuronides with protein, evidence for adduct formation from acyl glucuronides in vivo is limited and potentially ambiguous. The causal connection of protein adduction to adverse drug reactions remains uncertain. This review has assessed the intrinsic reactivity, metabolic stability and pharmacokinetic properties of acyl glucuronides in the context of physiological, pharmacological and toxicological perspectives. Although numerous experiments have characterised the reactions of acyl glucuronides with proteins, these might be attenuated substantially in vivo by rapid clearance of the conjugates. Consequently, to delineate a relationship between acyl glucuronide formation and toxicological phenomena, detailed pharmacokinetic analysis of systemic exposure to the acyl glucuronide should be undertaken adjacent to determining protein adduct concentrations in vivo. Further investigation is required to ascertain whether acyl glucuronide clearance is sufficient to prevent covalent modification of endogenous proteins and consequentially a potential immunological response.

Nevirapine (NVP), an antiretroviral drug, is associated with idiosyncratic hepatotoxicity and skin reactions. Metabolic pathways of haptenation and immunotoxicity mechanisms have been proposed. NVP is metabolized by liver microsomes to a reactive intermediate that binds irreversibly to protein and forms a GSH adduct. However, no reactive metabolite of NVP, trapped as stable thioether conjugates, has hitherto been identified in vivo. This study has defined the metabolism of NVP with respect to reactive intermediate formation in patients and a rat model of NVP-induced skin reactions. An integrated NMR and mass spectrometry approach has been developed to discover and quantify stable urinary metabolite biomarkers indicative of NVP bioactivation in patients. Two isomeric NVP mercapturates were identified in the urine of HIV-positive patients undergoing standard antiretroviral chemotherapy. The same conjugates were found in rat bile and urine. The mercapturates were isolated from rat bile and characterized definitively by NMR as thioethers substituted at the C-3 and exocyclic C-12 positions of the methylpyrido ring of NVP. It is proposed that NVP undergoes bioactivation to arene oxide and quinone methide intermediates. The purified major mercapturate was quantified by NMR and used to calibrate a mass spectrometric assay of the corresponding metabolite in patient urine. This is the first evidence for metabolic activation of NVP in humans, and only the second minimum estimate in patients of bioactivation of a widely prescribed drug associated with idiosyncratic toxicities. The method can be used as a template for comparative estimations of bioactivation of any drug in patients.

The measurement of urinary 6 beta-hydroxycortisol (6 beta-OHF) has been widely used as a non-invasive clinical test to detect cytochrome P450 induction. Although only a minor biotransformation, 6 beta-OHF formation represents a sensitive target for many P450-inducing drugs and environmental chemicals in man. There is good evidence that an isozyme of the P450IIIA subfamily is predominantly responsible for 6 beta-hydroxylase activity and therefore it has been suggested that urinary 6 beta-OHF is a marker of the induction of P450IIIA. The basis of the present study was that in order to realistically assign to 6 beta-OHF the status of a P450IIIA marker we should characterize all the metabolites of cortisol produced by human liver and assess inter-liver variability. Incubations at 37 degrees C for 2 h contained [3H]cortisol (0.1 microCi, 1 or 50 microM), MgCl2 (10 mM), microsomal or cytosolic protein (3 mg), an NADPH-regenerating system and 1/15 M phosphate buffer (pH 7.4) to give a final volume of 0.5 ml. Extraction with ethyl acetate (2 x 2 ml) was followed by radiometric HPLC analysis. Metabolites were identified by co-chromatography with authentic standards and mass spectrometry (electron impact and chemical ionization). All the microsomal incubations (n = 6 livers) produced 6 alpha-hydroxycortisol (6 alpha-OHF), 6 beta-OHF, 20 beta-dihydroxycortisol, 20 beta-dihydroxycortisone, cortisone, and 3 alpha, 5 beta-tetrahydrocortisone (3 alpha, 5 beta-THE), while five produced 6 beta-hydroxycortisone and four produced 3 alpha, 5 beta-tetrahydrocortisol (3 alpha, 5 beta-THF). The cytosolic incubations gave a much simpler metabolic profile, with 3 alpha, 5 beta-THF the major metabolite and 3 alpha, 5 beta-THE a minor metabolite. There was considerable inter-individual variability in metabolite profiles from microsomal incubations. 6 beta-OHF varied from 2.8 to 31.7%. Major metabolites were cortisone and 3 alpha, 5 beta-THE. Inter-liver variability was less for cytosolic incubations, the major metabolite always being 3 alpha, 5 beta-THF. In conclusion we have rigorously identified the hepatic metabolites of cortisol formed in vitro. The highly complex and variable hepatic metabolism of cortisol clearly limits the use of urinary 6 beta-OHF excretion as a marker of baseline P450IIIA activity in man.

Metabolic activation to catechols and their oxidation products is variously considered to contribute to the genotoxic, cytotoxic, transforming and tumour-promoting activities of exogenous steroidal oestrogens. 2-Fluoro-17α-ethynyloestradiol (2-FEE 2 ) was synthesized as a prototype of pharmacologically active derivatives of 17β-oestradiol which are resistant to metabolic activation in vivo . It possessed high affinity for the rat uterine oestrogen receptor and was oestrogenic in rats. Biliary metabolites of [6,7- 3 H]2-FEE 2 (0.73 μmol/kg, 157 μg/kg, i.v.) in female rats were characterized: 87% of the radiolabel was excreted, principally as 2-FEE 2 glucuronide, over 6hr. Although 2-nuoro-17β-oestradiol is not metabolized to C-2 oxygenated products in vivo , 2-FEE 2 underwent rapid and appreciable oxidative defluorination. 2-Hydroxy-17α-ethynyloestradiol and 2-methoxy-17α-ethynyl-oestradiol represented, respectively, 8% and 13% of the dose. Fluorination nevertheless restricted C-2 oxygenation to ca . 28% of that which 17α-ethynyloestradiol undergoes in female rats. C-4 oxygenation of 2-FEE 2 , resulting in catechol formation, occurred but to a lesser extent ( ca . 12% of dose). None of the major and identified minor biliary metabolites was a product of metabolic activation at the ethynyl function. A mechanistic rationalization of the long range enhancement by 17α-ethynylation of oxidative defluorination at C-2 is presented.

2-Fluoro-[6,7- 3 H]17β-oestradiol ([ 3 H]2-FE 2 ) and 4-fluoro-[6, 7- 3 H]17β-oestradiol([ 3 H]4-FE 2 ) were synthesized by the fluorination and reduction of [ 3 H]oestrone and purified by HPLC. [ 3 H]2-FE 2 and [ 3 H]4-FE 2 (72.5 μg/kg; 0.25 μmol/kg) were administered i.v. to anaesthetized female and male Wistar rats (N = 4) with biliary cannulae. Bile was collected for 6 hr. Female rats administered [ 3 H]2-FE 2 excreted 85% of the dose into bile over 6 hr whilst male rats excreted 77%. After the administration of [ 3 H]4-FE 2 , female and male rats excreted 72 and 83% of dose into bile over 6 hr, respectively. The biliary metabolites were glucuronides in all cases. The principal metabolite of [ 3 H]2-FE 2 liberated from biliary conjugates by β-glucuronidase was 2-fluoroestrone in both female rats (64% of dose) and male rats (57%). No 2-hydroxylated, i.e. oxidatively defluorinated, metabolites were detected in either sex. In contrast, 2-hydroxylation of [ 3 H]4-FE 2 did occur, but only in female rats: 2-hydroxy-4-fluoro-oestrone (22%) and 2-methoxy-4-fluoroestrone (17%) were identified as biliary aglycones. However, the major metabolite was 4-fluoroestrone (4FE 1 ]; 38%). In male rats, 4-FE 1 and 4-fluoro d -ring-oxygenated products were the principal biliary aglycones. The differences in metabolism between the two fluoro analogues and oestradiol are discussed with particular reference to the possible involvement of 2- and 4-hydroxy (catechol) oestrogens in oestrogen toxicity.

Drugs are generally converted to biologically inactive forms and eliminated from the body, principally by hepatic metabolism. However, certain drugs undergo biotransformation to metabolites that can interfere with cellular functions through their intrinsic chemical reactivity towards glutathione, leading to thiol depletion, and functionally critical macromolecules, resulting in reversible modification, irreversible adduct formation, and irreversible loss of activity. There is now a great deal of evidence which shows that reactive metabolites are formed from drugs known to cause hepatotoxicity, such as acetaminophen, tamoxifen, isoniazid, and amodiaquine. The main theme of this article is to review the evidence for chemically reactive metabolites being initiating factors for the multiple downstream biological events culminating in toxicity. The major objectives are to understand those idiosyncratic hepatotoxicities thought to be caused by chemically reactive metabolites and to define the role of toxic metabolites.

C-2 and C4 fluorinated and brominated derivatives of 17α-ethynyloestradiol have been efficiently prepared via the corresponding halo-oestrones

p-Phenylenediamine (PPD) exposure is associated with T-cell mediated contact dermatitis. T-cells from allergic patients proliferate following exposure to PPD and the oxido-conjugation product Bandrowski's base (BB). Both compounds are classified as sensitizers in the local lymph node assay; however, because of their instability the nature of the antigenic determinant remains ill-defined. The aim of this study was to explore the immunogenic potential of PPD and BB in mice. Spleen cell proliferation and cytokine secretion was measured ex vivo following antigen recall with soluble PPD or BB and either irradiated or glutaraldehyde fixed, antigen pulsed dendritic cells from syngeneic mice. Glutathione was added to certain incubations. LC-MS analysis and solvent extraction were used to monitor the fate of [(14)C]BB in culture and the extent of BB binding, respectively. Spleen cells from BB exposed, but not PPD- or vehicle-exposed, mice proliferated when stimulated with BB. Proliferating cells secreted high levels of IFN-gamma, GM-CSF and IL-2. Stimulation with PPD instigated low levels of proliferation. Irradiated, but not fixed, dendritic cells pulsed with BB stimulated proliferation signifying a classical hapten mechanism involving irreversible BB binding to protein and processing. BB bound preferentially to serum protein when incubated together with cells and serum. Degradation of BB in the presence of glutathione was associated with a stronger stimulation of specific T-cells at higher BB concentrations. These data demonstrate that BB is a potent immunogen in the mouse.