A Comparison with Pyronaridine and Related Antimalarial Drugs

Life-threatening agranulocytosis and hepatotoxicity during prophylactic administration of amodiaquine have led to its withdrawal. Agranulocytosis is t...
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Chem. Res. Toxicol. 1998, 11, 1586-1595

Metabolism-Dependent Neutrophil Cytotoxicity of Amodiaquine: A Comparison with Pyronaridine and Related Antimalarial Drugs Dean J. Naisbitt,† Dominic P. Williams,† Paul M. O’Neill,† James L. Maggs,† David J. Willock,‡ Munir Pirmohamed,† and B. Kevin Park*,† Department of Pharmacology and Therapeutics, The University of Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K., and Department of Chemistry, University of Wales, P.O. Box 912, Cardiff CF1 3TB, U.K. Received June 19, 1998

Life-threatening agranulocytosis and hepatotoxicity during prophylactic administration of amodiaquine have led to its withdrawal. Agranulocytosis is thought to involve bioactivation to a protein-reactive quinoneimine metabolite. The toxicity of amodiaquine and the lack of cheap drugs have prompted a search for alternative antimalarial agents. The aim of this study was to determine the metabolism and neutrophil toxicity of amodiaquine, pyronaridine, and other related antimalarial agents. Horseradish peroxidase and hydrogen peroxide were used to activate drugs to their respective quinoneimine metabolites. Metabolites were trapped as stable glutathione conjugates, prior to analysis by LC/MS. Amodiaquine was metabolized to a polar metabolite (m/z 661), identified as a glutathione adduct. Tebuquine was converted to two polar metabolites. The principal metabolite (m/z 686) was derived from glutathione conjugation and side chain elimination, while the minor metabolite gave a protonated molecule (m/z 496). Only parent ions were identified when chloroquine, cycloquine, or pyronaridine was incubated with the activating system and glutathione. Calculation of the heat of formation of the drugs, however, demonstrated that amodiaquine, tebuquine, cycloquine, and pyronaridine readily undergo oxidation to their quinoneimine. None of the antimalarial compounds depleted the level of intracellular glutathione (1-300 µM) when incubated with neutrophils alone. Additionally, with the exception of tebuquine, no cytotoxicity below 100 µM was observed. In the presence of the full activating system, however, all compounds except chloroquine resulted in depletion of the level of glutathione and were cytotoxic. Pretreating the cells with glutathione and other antioxidants inhibited metabolism-dependent cytotoxicity. In summary, our data show that amodiaquine and related antimalarials containing a p-aminophenol moiety undergo bioactivation in vitro to chemically reactive and cytotoxic intermediates. In particular, pyronaridine, which is currently being investigated in humans, was metabolized to a compound which was toxic to neutrophils. Thus, the possibility that it will cause agranulocytosis in clinical practice cannot be excluded, and will require careful monitoring.

Introduction Malaria kills between 1 and 2 million people every year, most of them African children (1). There are few effective antimalarial drugs; most of Africa still relies on chloroquine (CQ)1 because of its low cost, widespread availability, and good oral tolerance. CQ, however, can no longer be relied upon for the treatment of Plasmodium falciparum malaria because of the spread of resistant parasites throughout Africa and most of the developing world (2). In Asia, Oceania, and South America, resistance has been overcome primarily by using mefloquine * To whom correspondence should be addressed: Department of Pharmacology and Therapeutics, The University of Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K. Telephone: (+44) 0151 794 5559. Fax: (+44) 0151 794 5540. E-mail: [email protected]. † The University of Liverpool. ‡ University of Wales. 1 Abbreviations: ACN, acetonitrile; AQ, amodiaquine; ASC, ascorbic acid; CQ, chloroquine; CYC, cycloquine; DMSO, dimethyl sulfoxide; GSH, glutathione; HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; HRP, horseradish peroxidase; MPO, myeloperoxidase; NAC, N-acetylcysteine; PYRO, pyronaridine; TEB, tebuquine.

and derivatives of artemisinin which are unfortunately too expensive for widespread use in Africa. Amodiaquine (AQ) is a 4-aminoquinoline antimalarial agent synthesized in the late 1940s by Burckhalter et al. (3). It contains a 7-chloroquinoline-substituted system connected to a phenolic-butyldiamino side chain. In the 1980s, investigators demonstrated that AQ was effective against both CQ-resistant and -sensitive isolates of P. falciparum (4). This observation resulted in an increase in its use; however, life-threatening agranulocytosis and hepatotoxicity in about 1 in 2000 patients during prophylactic administration led to its withdrawal (5-7). Despite its toxicity, AQ is still used for the treatment of acute malaria if the risk of infection outweighs the potential for drug toxicity (8). The mechanism of AQ-induced agranulocytosis remains unclear, but both direct stem cell toxicity and immune-mediated mechanisms have been implicated (9, 10). AQ undergoes extensive bioactivation to an electrophilic quinoneimine metabolite in vivo in rats (11) and in vitro by both hepatic microsomes (12) and phorbol

10.1021/tx980148k CCC: $15.00 © 1998 American Chemical Society Published on Web 11/26/1998

Metabolism-Dependent Antimalarial Cytotoxicity

Figure 1. Chemical structures of antimalarial drugs.

ester-stimulated neutrophils (13). Subsequent oxidative stress or conjugation to cysteinyl sulfydryl groups on proteins is likely to be involved in the induction of toxicity by either cytotoxic or immunological mechanisms (14, 15). However, the factors determining individual susceptibility are unknown. The toxicity of AQ, the inability to prevent its development, and the lack of a cheap replacement for the treatment of CQ-resistant P. falciparum have prompted a search for alternative antimalarial agents. One promising candidate is pyronaridine (PYRO), a compound showing a favorable efficacy profile (16, 17), and to date, little toxicity has been observed in clinical trials carried out with it (18). In a recent study, we compared AQ to other antimalarials, including cycloquine (CYC), bispyroquine, and PYRO, to elucidate the chemical features involved in drug accumulation, bioactivation, and cellular function (19). This study demonstrated that bis-mannich antimalarials, including CYC and PYRO, appeared to have an advantage over mono-mannichs with respect to the avoidance of bioactivation and excessive lysosomal accumulation. These data suggested that if PYRO were to be administered for malaria prophylaxis, it may be less likely than AQ to cause life-threatening agranulocytosis. However, Winstanley (20) expressed concern over the structural similarities of AQ and PYRO, and thus the potential for PYRO to cause idiosyncratic toxicity. Consequently, the aim of this study was to investigate the metabolism and cytotoxicity of AQ, PYRO, and structurally related antimalarial agents toward peripheral blood neutrophils (Figure 1). An in vitro model comprised of horseradish peroxidase (HRP) and hydrogen peroxide was used to generate the same metabolites as neutrophils (21, 22). Furthermore, the ability of reactive metabolite scavengers to inhibit drug-induced cytotoxicity was assessed.

Experimental Procedures Warning: AQ quinoneimine is a potent sensitizing agent and should be handled with care. Chemicals. AQ, ASC, bromobimane, CQ, gentamycin, GSH, HRP (type VI), human serum albumin, NAC, N-ethylmorpholine, trichloroacetic acid, and trypan blue were all obtained from Sigma Chemical Co. (Poole, U.K.). Hydrogen peroxide was obtained from Merck Ltd. (Dorest, U.K.). Tebuquine (TEB) was a gift from Parke Davis (Ann Arbor, MI), and PYRO was a gift

Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1587 from D. C. Warhurst (London School of Hygiene and Tropical Medicine, London). CYC was synthesized using the method of Barlin and Tan (23), and AQ quinoneimine was synthesized by the method of Harrison et al. (11). The purity of all compounds ranged from 95 to 100%, as assessed by HPLC and NMR spectroscopy. Monopoly resolving medium (Ficoll Hypaque, density of 1.114 g/mL) and Lymphoprep (1.077 g/mL) were obtained from ICN Biomedicals (Bucks, U.K.) and Nycomed (Birmingham, U.K.), respectively. All solvents were HPLC grade and were purchased from Fisher Scientific plc (Loughborough, U.K.). Isolation of Human Peripheral Blood Cells. Neutrophils and lymphocytes were isolated from the venous blood of 10 healthy volunteers (ages ranging from 21 to 40 years). To prevent contamination by microorganisms, buffers were filter sterilized prior to use with a 0.22 µM pore size disposable membrane filter (Millipore), and all work was carried out in a class II biohazard cabinet with a vertical laminar air flow (Gelaire BSB 4A, Flow Laboratories). The blood was layered onto a dual-density gradient of Lymphoprep (4 mL) and Monopoly resolving medium (8 mL). After centrifugation (750g for 45 min), the bands of neutrophils and lymphocytes were removed from the resolving medium using a sterile Pasteur pipet and washed with PBS [8 g/L sodium chloride, 1.15 g/L disodium hydrogen orthophosphate, 0.2 g/L potassium chloride, and 0.04 g/L potassium dihydrogen orthophosphate (pH 7.4)]. Red blood cells were removed by relaying the cells on Monopoly resolving medium (5 mL) and centrifuging them (750g) for an additional 5 min. The cells were washed again and resuspended in HEPES buffer [3.58 g/L HEPES, 7.31 g/L sodium chloride, 0.45 g/L potassium chloride, 0.24 g/L magnesium chloride, 0.16 g/L sodium dihydrogen orthophosphate, 0.15 g/L calcium chloride, and 1.8 g/L glucose (pH 7.4)]. The procedure yielded a final preparation of cells that was >99% neutrophils and >97% lymphocytes as assessed by Wright’s staining. The viability of both cell types as determined by trypan blue dye exclusion was >97%. Determination of the Depletion of Cellular Glutathione Concentrations by Antimalarial Drugs. AQ has previously been shown to deplete the level of intracellular GSH from neutrophils in the presence of phorbol 12-myristate 13-acetate, a cell-activating factor (13, 19). To determine whether AQ and chemically related antimalarial drugs cause peroxidase-dependent GSH depletion, the fluorescent probe, bromobimane, which reacts with intracellular GSH, was used (24). Initial experiments were designed to evaluate the role of individual components of the peroxidase-activating system on AQ-induced GSH depletion. GSH (3 µM) was incubated with AQ (30 µM), AQ/HRP (20 units), AQ/hydrogen peroxide (10 µM), and AQ/HRP (20 units)/hydrogen peroxide (10 µM) in HEPES buffer at 37 °C for 20 min. The total incubation volume was 1 mL. Bromobimane (3 mM) was added to the incubations, which were left in the dark at 37 °C for a further 5 min. Aliquots (50 µL) were analyzed by fluorescence HPLC, as described previously (19, 25). A cell concentration of 0.5 × 106 neutrophils/mL was used to demonstrate intracellular GSH depletion. The cells were incubated with AQ, AQ quinoneimine, CQ, TEB, CYC, and PYRO (1-100 µM) in HEPES buffer at 37 °C for 1 h in the presence and absence of HRP (20 units) and hydrogen peroxide (10 µM) as described previously (26). GSH levels were determined using the protocol described above. The compounds were dissolved in DMSO (1% v/v), which did not deplete the level of GSH by itself. Determination of the Direct and Metabolism-Dependent Cytotoxicity of the Antimalarial Drugs. The direct and peroxidase-dependent cytotoxicities of AQ, AQ quinoneimine, and structurally related antimalarial drugs (CQ, TEB, CYC, and PYRO, 1-100 µM) were determined by incubating either neutrophils (1 × 106/incubation) or lymphocytes (1 × 106/ incubation) in 15 mL plastic conical tubes in a shaking water bath for 2 h at 37 °C. All drugs were added in DMSO, which as a 1% (v/v) solution was not toxic to the cells. The cells were

1588 Chem. Res. Toxicol., Vol. 11, No. 12, 1998 incubated in the presence or absence of HRP (20 units) and hydrogen peroxide (10 µM). Some incubations also contained the antioxidants GSH (1 mM), NAC (1 mM), and ASC (1 mM). Reactions were initiated with hydrogen peroxide, and after 2 h, the tubes were centrifuged (650g for 10 min) to pellet the cells. The supernatant was discarded, and the cells were resuspended in 1 mL of drug-free HEPES buffer containing human serum albumin (5 mg/mL) and gentamycin (50 µg/mL). Samples were then placed in an incubator at 37 °C. Cell viability was assessed after 16 h by trypan blue dye exclusion (0.2% w/v, trypan blue), as reported previously (27). Metabolism of the Antimalarial Drugs by Horseradish Peroxidase and Hydrogen Peroxide. Drugs (AQ, CYC, PYRO, and TEB; 100 µM in 1% v/v DMSO) were incubated at 37 °C for 2 h in the presence or absence of HRP (20 units), hydrogen peroxide (10 µM), and GSH (1 mM), as described previously (21, 28, 29). The reaction was initiated by the addition of hydrogen peroxide, and GSH was added within 30 s. After 2 h, the reaction was terminated by the addition of ethanol (1 mL). The solutions were evaporated to dryness under a stream of nitrogen and stored at -20 °C for no longer than 3 days prior to analysis by HPLC and LC/MS. Analysis of Metabolites by HPLC. Incubations containing drug, HRP, hydrogen peroxide, and GSH were reconstituted in an ethanol/water mixture (1:1; 500 µL) prior to HPLC analysis. A gradient HPLC system consisting of a Spectra-physics pump (Spectra-physics, San Jose, CA) and a Spectrasystem UV100 wavelength detector (254 nm; Hemel Hempstead, Herts, U.K.) was used for all the studies. GSH conjugates were eluted from either a µBondapak C18 (AQ and TEB; flow rate of 1 mL/min) or Columbus column (CYC and PYRO; flow rate of 0.75 mL/ min) (HPLC Technology, Macclesfield, U.K.) with a mobile phase consisting of acetonitrile (ACN) and 20 mM ammonium formate (pH 2.75). Gradient conditions varied slightly with each drug: AQ, 5 to 20% ACN over the course of 18 min, followed by 20% ACN for 8 min, with re-equilibration at 5% for 5 min; TEB, 10 to 40% ACN over the course of 25 min, followed by 40% ACN for 20 min, with re-equilibration at 10% for 5 min; CYC, 5 to 20% ACN over the course of 35 min, with re-equilibration at 5% for 5 min; and PYRO, 5 to 12% ACN over the course of 12 min, followed by 12% ACN for 18 min, with re-equilibration at 5% for 5 min. Analysis of Metabolites by LC/MS. Triplicate incubations containing drug, HRP, hydrogen peroxide, and GSH were reconstituted in ethanol/water (1:1; 100 µL) and combined to provide the mass required for mass spectrometry. Samples were eluted using the HPLC gradients described above. The mobile phase was delivered by two Jasco PU980 pumps (Jasco Corporation, Tokyo, Japan) via an HG-980-30 mixing module. The eluate was passed through a Jasco UV-975 absorbance detector (254 nm), a Valco tee-union splitter (Phase Separations, Deeside, Clwydd, U.K.), and a fused silica capillary (75 µM i.d., 85 cm long) to the electrospray probe and interphase of a Quattro II tandem quadrupole mass spectrometer (Micromass Ltd., Manchester, U.K.). Full-scan spectra were acquired over the range of m/z 50-800, and the extent of fragmentation was modulated via the cone voltage. The data were processed by MassLynx 2.1 software. Determination of the Reaction Enthalpy for the Oxidation of the Antimalarial Drugs. The presence of a hydroxyl group on the phenyl ring of AQ introduces the possibility of enzyme-catalyzed oxidation (12-14). In this study, we have used semiempirical quantum chemistry calculations of the heat of formation for the drugs (AQ, CYC, PYRO, and TEB) and the corresponding quinoneimine to estimate the enthalpy change on oxidation. Each molecule was constructed using the Insight II 4.0.0 package (MSI, San Diego, CA). Geometry optimization was carried out using the MOPAC program (30) with the PM3 Hamiltonian (31) and using the eigenvector follower algorithm (32). In our recent study, we found that AQ and all the other compounds discussed here form an internal hydrogen bond between the hydroxyl hydrogen and the nitrogen of the N-

Naisbitt et al.

Figure 2. GSH depletion by AQ (30 µM) after 20 min in the presence and absence of HRP (20 units) and hydrogen peroxide (H2O2, 10 µM). GSH levels were measured using bromobimane, by fluorescence HPLC. Each bar represents the mean ( SD of three separate experiments (all incubations were performed in triplicate). Statistical analysis was performed by comparing the ability of AQ to deplete the GSH level (in the presence and absence of HRP and hydrogen peroxide) with that of solvent alone (P < 0.05 for columns marked with an asterisk). (diethylamino) group (33). Accordingly, each molecule was optimized with this hydrogen bond in place, and then the hydroxyl group was rotated by 180° to obtain a second structure without the internal hydrogen bond. In all cases, the structures with the internal hydrogen bond were found to be significantly more stable than those in which it was absent. The calculation of the enthalpy of reaction for each molecule requires the heat of formation of the quinoneimine and a hydrogen molecule. These were geometry optimized in the same manner as above, with the quinoneimine structure being generated by removing the hydrogens from each compound and reoptimizing the structure. Statistical Analysis. The results are presented as means ( SD. Statistical analysis was performed by the Mann-Whitney test, accepting a P of 80% of the 3 Personal communication from L. M. Werbel and H. Chung, Walter Reed Army Institute of Research, Washington, DC.

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Figure 8. Typical absorbance (UV at 254 nm) and selected ion chromatograms and mass spectra of TEB (m/z 466) and its glutathionyl conjugate (m/z 686), when TEB (100 µM) was incubated with GSH (1 mM) in the presence of HRP (20 units) and hydrogen peroxide (10 µM). Ions were protonated molecules.

Scheme 1. Possible Mechanism of Tebuquine Bioactivation and GSH Conjugation

GSH was depleted from cells in the presence of the full activating system. LC/MS analysis of incubations containing peroxidase and GSH revealed distinct differences in the reactions of mono- and bis-mannich substituted compounds. GSH depletion by monosubstituted compounds such as AQ was primarily a result of conjugate formation; however, as no conjugates were formed with either CYC or PYRO, GSH depletion was likely to have been due to the oxidation of GSH to GSH disulfide. The mechanism of neutrophil toxicity associated with mono- and bis-mannich antimalarial agents correlates closely with work carried out in the late 1980s on acetaminophen and a series of 3,5-dimethylated ana-

logues (40-42). Acetaminophen is widely used as an analgesic and does not cause toxicity except when taken in overdose. The hepatocellular necrosis observed with acetaminophen overdosage has been linked to bioactivation of the parent compound to N-acetyl-p-benzoquinoneimine (42). Although the precise mechanism of the liver toxicity is still widely debated, it has been shown that the quinoneimine metabolite causes depletion of intracellular thiol stores, can bind covalently to protein thiol groups, and induce oxidative stress (43-45). It was expected that the 3,5-dimethylated analogues of acetaminophen would not bind to thiols and thus would not cause toxicity. However, 3,5-dimethylacetaminophen was found to be as hepatotoxic as acetaminophen in rats and mice (46). No GSH conjugates were identified, and therefore, the mechanism of 3,5-dimethylacetaminophen cytotoxicity was attributed to oxidative stress and protein thiol oxidation. A recent report by Weis et al. (47) confirmed these initial findings, and furthermore, the authors demonstrated that the cytotoxicity of 3,5-dimethylacetaminophen involved oxidative modification of essential cysteinyl proteins. It is important to note that AQ in addition to causing agranulocytosis also causes hepatotoxicity. Bioactivation to the quinoneimine metabolite by liver P450 enzymes has been demonstrated (12). Clearly, acetaminophen does not lead to agranulocytosis, and this may be due to two main reasons: (a) it is rapidly metabolized in the liver, and (b) it does not undergo accumulation within neutrophils, i.e., it is not lysosomotropic. The property of lysosomotropism is exhibited by most antimalarial agents, and apart from affecting neutrophil function (19), it may also play an important part in the pathogenesis of the agranulocytosis by allowing a high concentration of the drug and its reactive metabolite to be present within the target tissue.

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In summary, we have shown that AQ and related antimalarials containing a 4-aminophenol moiety can undergo bioactivation in vitro to a chemically reactive intermediate(s). In our system, PYRO was also metabolized to a compound which was toxic to neutrophils, and thus, the possibility of it causing white cell toxicity in clinical practice cannot be excluded, and will require careful monitoring.

Acknowledgment. TEB and PYRO were gifts kindly donated by Parke Davis (Ann Arbor, MI), and D. C. Warhurst (London School of Hygiene and Tropical Medicine, London), respectively. We also thank the Wellcome Trust (D.J.N.) and Novartis Pharmaceuticals (D.P.W.) for funding Ph.D. studentships. B.K.P. is a Wellcome Principal Fellow.

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