James L. Maggs

Evidence for the Involvement of Carbon-centered Radicals in the Induction of Apoptotic Cell Death by Artemisinin Compounds *

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, 10β-(p-bromophenoxy)dihydroartemisinin, and 10β-(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-10β-(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-G0/G1 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.

The Role of Heme and the Mitochondrion in the Chemical and Molecular Mechanisms of Mammalian Cell Death Induced by the Artemisinin Antimalarials

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 ρ0 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 IC50 values, 48 h: HeLa cells, 6 ± 3 μm; and HeLa ρ0 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 (IC50 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%).

Acyl glucuronides: the good, the bad and the ugly.

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.

Role of Reactive Metabolites in Drug-Induced Hepatotoxicity

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.

Role of hepatic metabolism in the bioactivation and detoxication of amodiaquine

1. The hepatic metabolism of the antimalarial drug amodiaquine was investigated in order to gain further insight into the postulated metabolic causation of the hepatotoxicity, which restricts the use of the drug. After intraportal (i.p.) administration (54 mumol/kg) to the anaesthetized rat, the drug was excreted in bile (23 +/- 3% dose over 5 h; mean +/- SD, n = 6) primarily as thioether conjugates. 2. After i.p. administration, 20% of the dose was excreted into urine over 24 h as parent compound and products of N-dealkylation and oxidative deamination. Desethylamodiaquine accumulated in liver, but was not a substrate for bioactivation as measured by biliary elimination of a glutathione adduct. 3. Prior administration of ketoconazole, an inhibitor of P450, reduced biliary excretion by 50% and effected a corresponding decrease in the amount of drug irreversibly bound to liver proteins. This indicated a role for P450 in the bioactivation of amodiaquine to a reactive metabolite that conjugates with glutathione and protein. 4. De-ethylation and irreversible binding were observed in vitro using male rat liver microsomes, and were again inhibited by ketoconazole. However, no such binding was observed with human (six individuals) hepatic microsomes despite extensive turnover of amodiaquine to desethylamodiaquine. 5. Amodiaquine quinoneimine underwent rapid reduction in the presence of either human or rat liver microsomes. Therefore in vitro studies may underestimate the bioactivation of amodiaquine in vivo. These data indicate that the extent of protein adduct formation in the liver will depend on the relative rates of oxidation of amodiaquine and reduction of its quinoneimine. This in turn may be a predisposing factor in the idiosyncratic hepatotoxicity associated with amodiaquine. 6. Substitution of a fluorine for the phenolic hydroxyl group in amodiaquine blocked bioactivation of the drug in vivo. Insertion of an N-hydroxyethyl function enabled partial clearance of amodiaquine and its deshydroxyfluoro analogue via O-glucuronidation and altered the balance between phase I oxidation and direct phase II conjugation of amodiaquine.

The biliary and urinary metabolites of [3H]17α-ethynylestradiol in women

1. The metabolism of 17α-ethynyl[6,7-3H]estradiol (3H-EE2) (50 μg) given orally was studied in two groups of women: (a) six subjects from whom duodenal bile samples were obtained after 4h by endoscopic aspiration; (b) two subjects with bile-duct (T-tube) drainage.2. The first group eliminated 16.6 ± 7.8% (mean ± S.D.) of the dose in urine over 72 h, the second group 28.6% and 27.5%. Biliary excretion by the latter was 41.9% and 28.3% of the dose, respectively, during the first 24 h after dosing.3. The metabolites excreted in bile and urine were largely polar conjugates: 1–12% of the 3H was ether extractable. Approx. 70–90% of urinary and biliary 3H was extractable following β-glucuronidase-arylsulphohydrolase hydrolysis. Both β-glucuronidase and arylsulphates were excreted.4. Unchanged 3H-EE2 was the principal 3H-labelled component of the glucuronide and arylsulphate fractions of bile, and it was a major component of urinary fractions. 2-Hydroxy-EE2 and 2-methoxy-EE2 were identified as conjugated biliary ...

Immunogenicity of Amodiaquine in the Rat

Amodiaquine is an antimalarial drug that has been associated with adverse reactions which may be immune mediated. Specific IgG anti-amodiaquine antibodies were detected after administration of the drug to rats (269 mumols/kg for 4 days), using an enzyme-linked immunosorbent assay employing amodiaquine conjugated to metallothionein as an antigen. A positive immune response was observed regardless of the route of administration, but the magnitude of the response in terms of antibody titre was in the order intraperitoneal administration greater than intramuscular administration greater than oral administration. Hapten inhibition experiments with structurally related drugs defined the specificity of the antibody which appears to recognize a conjugate of amodiaquine quinone imine and cysteine residues present in protein. Amodiaquine was converted to a protein-reactive species by activated human polymorphonuclear leucocytes in vitro, and this may provide a mechanism for immunogen formation in vivo. A humoral immune response was observed with doses of amodiaquine that did not produce either direct hepatotoxicity or leucopenia. Thus an animal model has been developed with which to investigate the toxicological consequences of amodiaquine immunogenicity.

Candidate selection and preclinical evaluation of N-tert-butyl isoquine (GSK369796), an affordable and effective 4-aminoquinoline antimalarial for the 21st century.

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.

Multiple adduction reactions of nitroso sulfamethoxazole with cysteinyl residues of peptides and proteins: implications for hapten formation.

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.

Direct evidence for the formation of diastereoisomeric benzylpenicilloyl haptens from benzylpenicillin and benzylpenicillenic acid in patients

Covalent binding to proteins to form neoantigens is thought to be central to the pathogenesis of penicillin hypersensitivity reactions. We have undertaken detailed mass spectrometric studies to define the mechanism and protein chemistry of hapten formation from benzylpenicillin (BP) and its rearrangement product, benzylpenicillenic acid (PA). Mass spectrometric analysis of human serum albumin exposed to BP and PA in vitro revealed that at low concentrations (drug protein molar ratio 0.001:1) and during short time incubations BP and PA selectively target different residues, Lys199 and Lys525, respectively. Molecular modeling showed that the selectivity was a function of noncovalent interaction before covalent modification. With increased exposure to higher concentrations of BP and PA, multiple epitopes were detected on albumin, demonstrating that the multiplicity of hapten formation is a function of time and concentration. More importantly, we have demonstrated direct evidence that PA is a hapten accounting for the diastereoisomeric BP antigen formation in albumin isolated from the blood of patients receiving penicillin. Furthermore, PA was found to be more potent than BP with respect to stimulation of T cells from patients with penicillin hypersensitivity, illustrating the functional relevance of diastereoisomeric hapten formation.