Clozapine-Induced Cytotoxicity in Freshly Isolated Hepatocyte Suspensions

Sophie Regan1, Daniel Antoine1, Dominic Williams1, Gerry Kenna2, Kevin Park1
1University of Liverpool, Liverpool, United Kingdom, 2AstraZeneca, Cheshire, United Kingdom

Idiosyncratic drug-induced liver injury is a major clinical problem, posing an increasing challenge in the area of drug development (Boersterli et al 2007). A number of marketed drugs have been implicated in drug-induced liver injury, despite pre-clinical screenings. Idiosyncratic reactions are infrequent but can be severe (Uetrecht 2007). Clozapine (CLZ), an antipsychotic agent, is associated with idiosyncratic hepatotoxicity. Severe cases of clozapine-induced hepatotoxicity are rare (0.06%) whilst the asymptomatic elevation in liver enzymes is more frequent (37%) Markowitz et al 1997). Reactive metabolite formation has previously been reported, agranulocytosis (Williams et al 1997). We aim to assess the role of drug bioactivation in CLZ-induced cytotoxicity in fresh hepatocyte suspensions.

Hepatocytes were isolated from male CD1 mice (20-25g) and male Wistar rats (125-150g) by a two-step in situ collagenase perfusion method. Fresh hepatocyte suspensions were incubated for 6 hours with clozapine (CLZ) at a concentration range (0-1000μM). Cytotoxicity was assessed through trypan blue exclusion and MTS reduction. Drug bioactivation was assessed through the incubation with a non-selective P450 inhibitor, 1-amino-benzotriazole (ABT 1mM). Further consideration of the reactive metabolite involvement in cytotoxicity was examined through a nucleophilic trapping agent, glutathione ethyl ester (GSH-EE 1mM). Total glutathione content and irreversible binding were assessed in parallel with concentration and time-dependent cytotoxic assessment.

CLZ was cytotoxic in both mouse and rat hepatocyte suspensions with concentrations above 500μM yielding 100% mortality in cells from both species, conveying no species selectivity. Cytotoxicity was both dose and time dependent. CLZ became irreversibly bound to hepatocyte protein in both mouse (CLZ 300μM 6H, 55.27 pmol equiv/mg protein (SE ± 6.73) and rat hepatocyte suspensions (CLZ 300μM 6hrs, 79.58 pmol equiv/mg of protein (SE ± 15.5), which was dose and time dependent. Evidence of glutathione depletion was seen in both mouse and rat hepatocyte suspensions. The role of drug bioactivation in CLZ-induced cytotoxicity was assessed through a pre-incubation with ABT. This resulted in no protection against CLZ-induced cytotoxicity in mouse hepatocytes (Viability: CLZ 58.5% (SE ± 5.25); CLZ +ABT 55.7% (SE ±13.05) or rat hepatocytes (Viability: CLZ 60.1% (SE ± 6.25); CLZ +ABT 50.8% (SE ± 8.32). Instead, an increase in CLZ-induced cytotoxicity was evident. However, in contrast ABT did result in a decrease in irreversible binding of CLZ in both mouse and rat hepatocyte suspensions although it was only significant in the rat. GSH-EE gave no protection against CLZ-induced cytotoxicity in hepatocyte suspensions. It can be concluded from this that drug bioactivation does not play a role in CLZ-induced cytotoxicity in rodent hepatocyte suspensions. Idiosyncratic CLZ-induced hepatotoxicity may therefore be attributed to the parent drug rather than a cytotoxic metabolite. However, further research is required.

Boersterli, U.A, Lim, P.L, 2007, Toxicol Appl Pharmacol; 220 (1):92-107.
Markowitz, J.S., Grinberg, R., Jackson, C., 1997, J Clin Psychopharmacol; 17: 70-71.
Uetrecht, J., 2007 Annu Rev Pharmacol Toxicol; 47: 513-39.
Williams, D.P.,Pirmohamed, M., Naisbitt, D.J., Maggs, J.L. and Park, B.K., 1997; J Pharmacol Exp Ther; 283: 1375-82.