Functional Consequences of Active Hepatic Uptake on Cytochrome P450 Inhibition in Rat and Human Hepatocytes

Abstract

ABSTRACT:A series of cytochrome P450 (P450) inhibition experiments were conducted with four hepatic uptake substrates (AZ3, AZ25, atorvastatin, and pitavastatin) using hepatocytes and recombinant P450s. The uptake was shown to be temperature-dependent and was inhibited by estrone sulfate, signifying an active component. At the lowest concentrations tested, the inhibitors concentrated up to 1000-fold in rat hepatocytes, but demonstrated only 5-fold greater P450 inhibition relative to recombinant rat P450s, indicating high intracellular binding. Inhibitor accumulation was considerably lower in human hepatocytes and an increase in inhibitory potency relative to recombinant human P450s was not obvious. This study highlights several technical and conceptual issues in the study of P450 inhibition mediated by compounds actively transported across the basolateral hepatocyte membrane. Primarily, the incubation medium concentration once the inhibitor has fully accumulated into the hepatocytes rather than the starting medium concentration, along with the extent of intracellular binding, must be considered as a foundation for in vitro-in vivo extrapolations. Additionally, it is suggested that if the K m value for the active uptake process is close to the P450 inhibition K i , hepatocytes may be used only to establish the free drug accumulation ratio at a clinically relevant drug concentration, and this information, along with the (recombinant P450) K i value, may be used to simulate the likely impact of active hepatic uptake on P450 inhibition in vivo.

Comparative Use of Isolated Hepatocytes and Hepatic Microsomes for Cytochrome P450 Inhibition Studies: Transporter-Enzyme Interplay

“…The IC 50 curves reported by Grime et al (2008) were somewhat flat, contrasting with the traditional sigmoidal shape, and the reasons for this are open to question. These authors discussed the scenario in which the affinity constant for the transporter may be of a value similar to the K i and showed by simulation that this can result in a relatively flat IC 50 profile.…”

“…For these compounds we demonstrated good concordance between K i values (range 0.05-30 M) in microsomes and hepatocytes despite the differing importance of hepatic accumulation observed for these compounds (Kp values ranging from 4 to 6000). In contrast, Grime et al, (2008) demonstrated a 5-fold difference in inhibition between recombinant rat P450s and isolated rat hepatocytes for four lipophilic carboxylic acids including atorvastatin and pitavastatin. As with the present study there was a disconnect between the change in the K i value between these systems and the extent of hepatic accumulation (approximately 1000-fold).…”

In Vitro Inhibition of the Bile Salt Export Pump Correlates with Risk of Cholestatic Drug-Induced Liver Injury in Humans

“…Although our studies have not addressed this issue directly, our data suggest that a useful operational threshold could be as high as 300 M. This is higher than the C max, u values of all but a few of the tested drugs, even if dmd.aspetjournals.org adjustment is made for higher compound concentrations typically observed at the hepatic inlet compared with peripheral blood (Ito et al, 2002). It may be relevant that many drugs accumulate within hepatocytes at concentrations that are much higher than extracellular concentrations (Grime et al, 2008). In addition, whereas the calculated potency values were IC 50 values, the fraction of transport inhibition required to cause functional alterations in vivo that contribute to DILI progression after long-term dosing are unknown and could be much lower (e.g., IC 10 values).…”

The Impact of Solute Carrier (SLC) Drug Uptake Transporter Loss in Human and Rat Cryopreserved Hepatocytes on Clearance Predictions

“…ABBREVIATIONS: ACN, acetonitrile; AZ, AstraZeneca; C blood /C plasma , blood-to-plasma concentration ratio; CL, clearance; CL int , intrinsic clearance; DMSO, dimethylsulfoxide; FCS, fetal calf serum; f hepatic , hepatically cleared fraction; fu, fraction unbound, free fraction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; h, as prefix, human; HRP, horseradish peroxidase; IVIVE, in vitro-in vivo extrapolation; KHL, KrebsHenseleit buffer; LOQ, limit of quantification; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MCT1, monocarboxylate transporter 1; mPGES-1, microsomal prostaglandin E synthase-1; MS, mass spectrometry; NTCP, Na active uptake of a compound can influence both its metabolism and its P450 inhibitory effect (Grime et al, 2008;Kusuhara and Sugiyama, 2009;Watanabe et al, 2009;Brown et al, 2010). The International Transporter Consortium has recently published guidelines for the application of drug uptake transporter kinetics in pharmacokinetics and strongly encourages screening for interactions with the major drug uptake transporters during drug development (in the liver: OAT2, OATP1B1, OATP1B3, OCT1, and OCT3) (Giacomini et al, 2010;Zamek-Gliszczynski et al, 2013).…”