Sarinj Fattah

Sandwich-cultured hepatocytes: utility for in vitro exploration of hepatobiliary drug disposition and drug-induced hepatotoxicity

Introduction: The sandwich-cultured hepatocyte (SCH) model has become an invaluable in vitro tool for studying hepatic drug transport, metabolism, biliary excretion and toxicity. The relevant expression of many hepatocyte-specific functions together with the in vivo-like morphology favor SCHs over other preclinical models for evaluating hepatobiliary drug disposition and drug-induced hepatotoxicity. Areas covered: In this review, the authors highlight recommended procedures required for reproducibly culturing hepatocytes in sandwich configuration. It also provides an overview of the SCH model characteristics as a function of culture time. Lastly, the article presents a summary of the most prominent applications of the SCH model, including hepatic drug clearance prediction, drug–drug interaction potential and drug-induced hepatotoxicity. Expert opinion: When human (cryopreserved) hepatocytes are used to establish sandwich cultures, the model appears particularly valuable to quantitatively investigate clinically relevant mechanisms related to in vivo hepatobiliary drug disposition and hepatotoxicity. Nonetheless, the SCH model would largely benefit from better insight into the fundamental cell signaling mechanisms that are critical for long-term in vitro maintenance of the hepatocytic phenotype. Studies systematically exploring improved cell culture conditions (e.g., co-cultures or extracellular matrix modifications), as well as in vitro work identifying key transcription factors involved in hepatocyte differentiation are currently emerging.

Sodium fluorescein is a probe substrate for hepatic drug transport mediated by OATP1B1 and OATP1B3

The aim of this study was to characterize the in vitro hepatic uptake kinetics of sodium fluorescein (NaFluo) and identify the transporters involved. NaFluo exhibited saturable uptake kinetics in suspended rat and human hepatocytes as reflected by K(m) values of 22.5 and 14.1 µM, and V(max) values of 98.3 and 5.8 pmol/(million cells • min), respectively. Coincubation with known inhibitors (e.g. rifampicin) of organic anion transporting polypeptide (OATP/Oatp; SLCO gene family) significantly decreased NaFluo uptake in hepatocytes. In contrast, neither inhibitors/substrates of the organic cation transporter or organic anion transporter family nor depletion of extracellular sodium resulted in significant inhibition of NaFluo uptake. To explore the contribution of individual uptake transporters, NaFluo uptake was determined in Chinese hamster ovary cells transfected with OATP1B1, OATP1B3, and OATP2B1. Transporter-mediated uptake of NaFluo was observed in OATP1B1- and OATP1B3-transfected cells (K(m) = 4.2 and 10.9 µM; V(max) = 30.9 and 135 [pmol/(mg protein • min)], respectively). NaFluo can be used as a probe substrate to study Oatp/OATP1B-mediated drug interactions in fluorescence-based in vitro transport assays of rat and human liver. Labeling of drugs or bile salts with a fluorescein moiety can be expected to result in fluorescent conjugates with substantially altered hepatic uptake characteristics as compared with the unconjugated compounds.

Age-dependent Activity of the Uptake Transporters Ntcp and Oatp1b2 in Male Rat Hepatocytes: From Birth till Adulthood

Recognition of the role of hepatic drug transporters in elimination of xenobiotics continues to grow. Hepatic uptake transporters, such as hepatic isoforms of the organic anion-transporting polypeptide (Oatp) family as well as the bile acid transporter Na+-taurocholate cotransporting polypeptide (Ntcp) have been studied extensively both at the mRNA and protein expression levels in adults. However, in pediatric/juvenile populations, there continues to be a knowledge gap about the functional activity of these transporters. Therefore, the aim of this study was to examine the functional maturation of Ntcp and Oatp isoforms as major hepatic transporters. Hepatocytes were freshly isolated from rats aged between birth and 8 weeks. Transporter activities were assessed by measuring the initial uptake rates of known substrates: taurocholate (TCA) for Ntcp and sodium fluorescein (NaFluo) for Oatp. Relative to adult values, uptake clearance of TCA in hepatocytes from rats aged 0, 1, 2, 3, and 4 weeks reached 19, 43, 22, 46, and 63%, respectively. In contrast, Oatp-mediated NaFluo uptake showed a considerably slower developmental pattern: uptake clearance of NaFluo in hepatocytes from rats aged 0, 1, 2, 3, 4, and 6 weeks were 24, 20, 19, 8, 19, and 64%, respectively. Maturation of NaFluo uptake activity correlated with the previously reported ontogeny of Oatp1b2 mRNA expression, confirming the role of Oatp1b2 for NaFluo uptake in rat liver. The outcome of this project will help in understanding and predicting age-dependent drug exposure in juvenile animals and will eventually support safe and more effective drug therapies for children.

Hepatobiliary and intestinal elimination of darunavir in an integrated preclinical rat model

Abstract 1. Although valuable in vitro models exist to study drug elimination from the systemic circulation, more integrated models may improve mechanistic insight in a biorelevant setting. 2. This study aimed to explore (1) intestinal and biliary excretion of the HIV protease inhibitor darunavir and its impact on systemic disposition and (2) to evaluate to what extent findings in an in situ excretion model in rat can be captured by individual in vitro models. 3. Contemporary in vitro models were applied to study intestinal and hepatobiliary disposition of darunavir and data were compared with findings in the in situ excretion model. 4. Both in situ and in vitro experiments demonstrated significant metabolism of darunavir, which could be strongly inhibited by the P450 inhibitor 1-aminobenzotriazole. Using the P-gp inhibitor zosuquidar, P-gp mediated excretion of darunavir from blood towards gastrointestinal lumen was evidenced and this was confirmed by transport studies in Caco-2 cells. Moreover, involvement of P-gp in the biliary excretion of darunavir was also demonstrated in situ. 5. In general, in situ findings corresponded well with in vitro data. The in situ excretion model offers the possibility to gain mechanistic insight in intestinal and hepatobiliary excretion processes and, at the same time, evaluate their impact on the systemic disposition of a compound.

Effect of Age on The Hepatocellularity Number for Wistar rats

Recently there has been a substantial increase in the number of juvenile animal toxicity studies that are conducted to support pediatric drug safety evaluation. Adequate design of juvenile toxicity studies in rats, for instance with respect to dose levels per age group, requires an understanding of age-dependent pharmacokinetics. In vitro–in vivo extrapolation (IVIVE) and physiologically based pharmacokinetic (PBPK) modeling can help to anticipate age-dependent drug exposure in juvenile toxicity studies provided age-dependent profiles for animal physiology and scaling factors are available. For instance, when hepatocytes are used to predict hepatic drug clearance, the hepatocellularity (number of hepatocytes per gram liver, HPGL) is required as one of the scaling factors. Although HPGL is known for adult rats, information on the influence of age on HPGL is missing. The present work profiles the hepatocellularity number in male Wistar rats as a function of age. Using the NADPH–cytochrome P450 reductase (NCR) activity method, the mean HPGL for the adult rat (8 weeks) was 104 × 106 cells/gram liver (relative standard deviation 17%). This value was calculated as a ratio between NRC activities in liver homogenates and suspended hepatocytes. The HPGL values were significantly higher (p <0.001) for rat pups until 3 weeks of age compared with adults. Our results revealed that the HPGL value showed a rapid decrease after 3 weeks (end of weaning), essentially reaching adult values by 4 weeks. This age-dependent HPGL profile will be instrumental for hepatic drug clearance prediction when designing juvenile toxicity studies in Wistar rats.