Cytochrome P450-dependent toxic effects of primaquine on human erythrocytes

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Abstract

Primaquine, an 8-aminoquinoline, is the drug of choice for radical cure of relapsing malaria. Use of primaquine is limited due to its hemotoxicity, particularly in populations with glucose-6-phosphate dehydrogenase deficiency [G6PD(−)]. Biotransformation appears to be central to the anti-infective and hematological toxicities of primaquine, but the mechanisms are still not well understood. Metabolic studies with primaquine have been hampered due to the reactive nature of potential hemotoxic metabolites. An in vitro metabolism-linked hemotoxicity assay has been developed. Co-incubation of the drug with normal or G6PD(−) erythrocytes, microsomes or recombinant cytochrome P450 (CYP) isoforms has allowed in situ generation of potential hemotoxic metabolite(s), which interact with the erythrocytes to generate hemotoxicity. Methemoglobin formation, real-time generation of reactive oxygen intermediates (ROIs) and depletion of reactive thiols were monitored as multiple biochemical end points for hemotoxicity. Primaquine alone did not produce any hemotoxicity, while a robust increase was observed in methemoglobin formation and generation of ROIs by primaquine in the presence of human or mouse liver microsomes. Multiple CYP isoforms (CYP2E1, CYP2B6, CYP1A2, CYP2D6 and CYP3A4) variably contributed to the hemotoxicity of primaquine. This was further confirmed by significant inhibition of primaquine hemotoxicity by the selective CYP inhibitors, namely thiotepa (CYP2B6), fluoxetine (CYP2D6) and troleandomycin (CYP3A4). Primaquine caused similar methemoglobin formation in G6PD(−) and normal human erythrocytes. However, G6PD(−) erythrocytes suffered higher oxidative stress and depletion of thiols than normal erythrocytes due to primaquine toxicity. The results provide significant insights regarding CYP isoforms contributing to hemotoxicity and may be useful in controlling toxicity of primaquine to increase its therapeutic utility.

Introduction

Primaquine, an 8-aminoquinoline, is the only drug available for radical cure of relapsing malaria caused due to infections with Plasmodium vivax and Plasmodium ovale (Baird and Hoffman, 2006). The utility of primaquine has also been established for prophylaxis of malaria (Hill et al., 2006) and treatment of other protozoan infections, as well as Pneumocystis pneumonia, (Tekwani and Walker, 2006). However, primaquine has been long known to cause dose-limiting side effects such as hemolytic anemia and methemoglobinemia, especially in the individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency (Taylor and White, 2004). Oxidative metabolites, rather than the parent drug, are primarily responsible for toxic hemolytic effects of primaquine (Fletcher et al., 1988, Bolchoz et al., 2001, Bowman et al., 2004). Redox reaction of the metabolites with hemoglobin and molecular oxygen yields methemoglobin and reactive oxygen intermediates (ROIs), which subsequently lead to hemolysis (Fletcher et al., 1988). Majority of the drug is metabolized to carboxyprimaquine through a non-CYP-mediated mechanism (Frischer et al., 1991). Only a small fraction of the drug is believed to undergo metabolism through hepatic cytochrome P450 (CYP), and account for the hemotoxic response to the drug. Biotransformation mechanisms appear to be central to both anti-infective and hematological toxicities of 8-aminoquinolines, but are still not well understood (Brueckner et al., 2001). Hemoglobin is believed to be the first protein to be damaged when the erythrocytes undergo oxidative damage by oxidant drugs, which subsequently may lead to hemolysis (Hall et al., 1986). Methemoglobin is produced by oxidation of the iron from ferrous (Fe+ 2) to ferric (Fe+ 3) form, which can not carry oxygen, leading to hypoxemia and cyanosis (Percy et al., 2005). Methemoglobin is normally maintained at ∼ 1–2% of total hemoglobin by the protective diaphorase systems. When the exogenous oxidative stress overwhelms the cellular protective NADH diaphorase system, the auxiliary NADPH diaphorase function also helps to reduce the oxidative stress (Bradberry, 2003). However, due to deficiency of NADPH in G6PD(−) individuals, the oxidation of hemoglobin leads to accumulation of methemoglobin, oxidative stress and increased hemolytic response (Jollow and McMillan, 2001). The ultimate mechanism leading to hemolysis by primaquine is still ambiguous, as each oxidant may trigger specific biochemical changes leading to faster clearance of erythrocytes from the physiological system (Sivilotti, 2004). A few studies conducted with putative primaquine metabolites have shown that hemolytic effects of 6-methoxy-8-hydroxylaminoquinoline (MAQ-NOH) result from peroxidative damage to the lipids of the red cell membrane (Bolchoz et al., 2001, McMillan et al., 2005). However, with 5-hydroxyprimaquine (5-HPQ), another putative phenolic metabolite of primaquine, generation of reactive oxygen intermediates was not associated with the onset of lipid peroxidation or an alteration in phosphatidylserine asymmetry (Bowman et al., 2004). Instead, 5-HPQ induced oxidative injury to the erythrocytes' cytoskeleton occurs primarily due to the formation of disulfide-linked hemoglobin adducts with cytoskeletal proteins. This leads to an accelerated phagocytosis and faster clearance of the damaged erythrocytes by the host macrophages (Bowman et al., 2005).

The reactive and unstable nature of potential hemotoxic aminophenolic metabolites have hampered the studies on biotransformation mechanisms leading to toxicities caused by 8-aminoquinolines (Strother et al., 1981). Metabolism through microsomal CYPs has been assumed to be involved in toxicity of primaquine. CYP ranks first in phase I enzymes in terms of the catalytic versatility and the wide range of xenobiotics it metabolizes (Johnson, 2008). The intensity and duration of drug action is governed by these microsomal enzymes which are involved either in detoxification or activation of drugs to reactive intermediates. An in vitro metabolism-linked hemotoxicity assay has been developed based on the earlier microsomal incubation test reported by Bloom et al. (1983). This assay allows in situ formation of potential toxic metabolites to generate toxicity in the co-incubated erythrocytes. The previous assay described by Bloom et al. (1983) involved the use of phenobarbital induced rat liver microsomes and evaluation of glutathione depletion in normal rat erythrocytes by the potential hemolytic drugs. Significant variations occur regarding CYP contents and substrate selectivity among animal and human liver microsomes and erythrocytes. Bashan et al. (1988) failed to reproduce the observations by Bloom et al. (1983), while attempting to predict hemolytic potential of drugs in G6PD-deficient blood. The in vitro metabolism-linked assay described here utilizes human liver microsomes or recombinant human CYPs and human erythrocytes. Multiple endpoints for hemolytic toxicity were monitored, namely formation of methemoglobin, generation of oxidative stress (reactive oxygen intermediates-ROS) and depletion of reactive thiols. The evaluation of multiple endpoints for hemotoxicity should provide a better correlation for prediction of hemotoxic potential of the test drugs. The assay was also employed to determine potential contribution of individual CYP isoforms for hemotoxicity of primaquine.

Section snippets

Reagents

The pooled human (mixed sexes) and male ICR/CD-1 mouse liver microsomes were procured from In Vitro Technologies (Celsis) (Chicago, IL USA) (Table S1 and S2—Supplementary data). The recombinant cDNA-expressed human CYPs (BD Gentest Supersomes™) were procured from BD Biosciences (San Jose, CA USA). The ROS probe 2,7-dichlorofluorescein diacetate (DCFDA), and the reactive thiol probe monobromobimane were obtained from Molecular Probes/Invitrogen Labeling & Detection, Eugene, OR, USA.

In vitro methemoglobin toxicity

Treatment of human erythrocytes in vitro with either primaquine or the microsomes alone did not cause significant increase in formation of methemoglobin. A robust increase in the formation of methemoglobin was noticed when the erythrocytes were treated with primaquine in the presence of pooled human or mouse liver microsomes (Fig. 1). The primaquine induced methemoglobin formation was almost 2 fold higher in the presence of mouse liver microsomes as compared to that observed with pooled human

Discussion

Despite more than five decades of clinical use of primaquine, the biotransformation mechanisms, which appear to be central to anti-infective and hematological toxicities of the drug, have still remained elusive (Brueckner et al., 2001). The reactive nature of the potential hemotoxic metabolites poses major challenges. The limited evidence available suggesting a role for CYP-mediated metabolism in generating potential hemotoxic metabolites (Strother et al., 1981, Link et al., 1985). However, the

Acknowledgments

This work has received financial support from Medicines for Malaria Venture (MMV), Geneva and the US Army Medical Research and Materiel Command (W81XWH-07-2-0095). The National Center for Natural Products Research is also supported by the United States Department of Agriculture-Agricultural Research Services (USDA-ARS) through a cooperative scientific agreement.

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