Richard J. Weaver

Cytochrome P450 specificities of alkoxyresorufin O-dealkylation in human and rat liver.

The O-dealkylations of ethoxyresorufin and pentoxyresorufin are widely used activity probes for measuring the cytochrome P450 forms, CYP1A1 and CYP2B1, respectively, and their induction by xenobiotics, but there is confusion in the literature about which P450 forms are detected in human and rat liver microsomes by these and homologous alkoxyresorufins. High performance liquid chromatographic analysis confirmed that O-dealkylation to resorufin was the sole or predominant route of metabolism for both short-chain and long-chain alkoxyresorufins and benzyloxyresorufin by rat liver microsomes. The purified 3-methylcholanthrene (3MC)-induced rat P450 forms, CYP1A1 and CYP1A2, and a possible variant form, CYP1A1*, showed substrate selectivities for propoxyresorufin, methoxyresorufin and ethoxyresorufin, respectively. Purified phenobarbitone (PB)-induced CYP2B1 was selective for benzyloxyresorufin and pentoxyresorufin. Purified constitutive CYP2C6 was much less active than CYP2B1 or the CYP1A forms but showed distinctive selectivity for benzyloxy-, propoxy- and butoxyresorufin. CYP1A2 and CYP2C6 metabolised n-propoxy- and n-butoxyresorufin much more rapidly (8-23-fold) than iso-propoxy- and iso-butoxyresorufin, whereas CYP1A1 and CYP2B1 showed only small differences (2-5-fold) between the n- and iso-homologues and CYP1A1* and CYP2B2 did not discriminate between them. The results show that ratios between different alkoxyresorufin O-dealkylation (AROD) activities can be more useful than absolute values of single activities for identifying P450 forms. Anti-P450 antibody and furafylline inhibition of rat liver microsomal AROD confirmed that ethoxyresorufin was a selective probe for CYP1A1 in 3MC-induced and isosafrole (ISF)-induced microsomes and that pentoxy- and benzyloxyresorufins both selectively measured CYP2B1 in PB-induced and ISF-induced microsomes. Ethoxyresorufin was not a selective probe for CYP1A in liver microsomes from untreated or PB-induced rats, however, where it was metabolised mainly by CYP2C6 and CYP2B1, respectively. Pentoxyresorufin and benzyloxyresorufin were metabolised by several different P450 forms in non-induced rat liver microsomes but mainly by the CYP1A subfamily in 3MC-induced microsomes and by CYP2B1 in PB- and ISF-induced microsomes. Although with purified rat P450s methoxyresorufin appeared not effectively to discriminate CYP1A2 from CYP1A1, CYP1A1* or CYP2C6, furafylline inhibition indicated that methoxyresorufin was a selective measure of CYP1A2 in uninduced and 3MC-induced rat liver microsomes but not in ISF- or PB-induced microsomes. In human liver microsomes, antibody inhibition and furafylline inhibition showed that ethoxyresorufin and methoxyresorufin were metabolised mainly by CYP1A2, whilst benzyloxyresorufin metabolism was due mainly to the CYP3A subfamily but also involved CYP1A2 and CYP2A6.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression and transcriptional regulation of ABC transporters and cytochromes P450 in hCMEC/D3 human cerebral microvascular endothelial cells

We investigated the expression of genes encoding ABC transporters, cytochromes P450 (CYPs) and some transcription factors in the hCMEC/D3 immortalized human cerebral microvascular endothelial cell line, a promising in vitro model of the human BBB, and we compared these expressions to a non-brain endothelial cell line (HUVEC) and freshly human brain microvessels. qRT-PCR showed that the MDR1, BCRP, MRP1, MRP3, MRP4 and MRP5 genes were expressed and that the main CYP gene was CYP2U1 in hCMEC/D3. The pattern of ABC and CYPs gene expression in hCMEC/D3 differed from HUVEC which did not express MDR1. Moreover, expression of P-gp and BCRP was lower in hCMEC/D3 than in human brain microvessels but remain functional as shown by rhodamine 123 efflux assay. The gene encoding the aryl hydrocarbon receptor (AhR), a transcription factor that regulates the expression of some ABC and CYPs was highly expressed in hCMEC/D3 and HUVEC, while the pregnane-X-receptor (PXR) and the constitutive androstane receptor (CAR) were barely detected. We investigated the function of the AhR-mediated regulatory pathway in hCMEC/D3 by treating them with the AhR agonist TCDD. The expressions of two AhR-target genes, CYP1A1 and CYP1B1, were increased 26-fold and 28-fold. But the expressions of ABC transporter genes were not significantly altered. We have thus determined the pattern of expression of the genes encoding ABC transporters, CYPs and three transcription factors in hCMEC/D3 and shown that the AhR pathway might afford an original functional transport and metabolic pattern in cerebral endothelial cells that is different from other peripheral endothelial cells.

Challenges and approaches for the development of safer immunomodulatory biologics.

Immunomodulatory biologics, which render their therapeutic effects by modulating or harnessing immune responses, have proven their therapeutic utility in several complex conditions including cancer and autoimmune diseases. However, unwanted adverse reactions — including serious infections, malignancy, cytokine release syndrome, anaphylaxis and hypersensitivity as well as immunogenicity — pose a challenge to the development of new (and safer) immunomodulatory biologics. In this article, we assess the safety issues associated with immunomodulatory biologics and discuss the current approaches for predicting and mitigating adverse reactions associated with their use. We also outline how these approaches can inform the development of safer immunomodulatory biologics.

The Expression of Cytochrome P-450, Epoxide Hydrolase, and Glutathione S- Transferase in Hepatocellular Carcinoma

Background. Cytochrome P‐450, epoxide hydrolase, and glutathione S‐transferase (GST) all play a key role in the metabolism of chemical carcinogens, mutagens, and various anti‐cancer drugs. All these functionally associated enzymes might be involved in both the development of hepatocellular carcinoma and in determining the anti‐cancer drug sensitivity of such tumors.

Cisplatin nephrotoxicity is mediated by gamma glutamyltranspeptidase, not via a C-S lyase governed biotransformation pathway.

Cisplatin exhibits dose-limiting nephrotoxicity in rodents and man. This study investigates the mechanism of cisplatin nephrotoxicity in vivo and in an in vitro model system. Nephrotoxicity was induced in rats (6 mg/kg cisplatin i.p.) and mice (10 mg/kg cisplatin i.p.). Cisplatin administration significantly elevated blood urea nitrogen (BUN) and serum creatinine in male Sprague Dawley rats day 5 post-treatment (BUN Delta+28+/-5 micromol/ml; serum creatinine Delta+108+/-4 nmol/ml, P<0.05) and in male C57BL6 mice day 4 post-treatment (BUN Delta+21+/-4 micromol/ml; serum creatinine Delta+81+/-5 nmol/ml, P<0.05). Nephrotoxicity was confirmed by histological analysis that revealed significant damage to the proximal tubules of cisplatin- versus saline vehicle-treated animals. Inhibition of gamma glutamyltranspeptidase prevented cisplatin nephrotoxicity in Sprague Dawley rats (day 5 BUN Delta+1+/-2 micromol/ml; serum creatinine Delta+8+/-4 nmol/ml) and C57BL6 mice (day 4 BUN Delta+1+/-0.8 micromol/ml; serum creatinine Delta-1+/-2 nmol/ml), but not cellular toxicity in rat proximal tubular (RPT) or human proximal tubular (HPT) cultures. Inhibition of aminopeptidase N (AP-N) or renal dipeptidase (RDP) in male Sprague Dawley rats, or in RPT and HPT cell cultures, did not reduce cisplatin toxicity. In contrast to published findings inhibition of C-S lyase did not prevent the nephrotoxicity of cisplatin in vivo or cellular toxicity in vitro. These data demonstrate that the biotransformation enzymes AP-N, RDP and C-S lyase are not implicated in the metabolism of cisplatin to a nephrotoxic metabolite as has been previously hypothesised. Instead, our data demonstrate that gamma glutamyltranspeptidase is a key enzyme involved in mediating cisplatin nephrotoxicity, which potentially acts to cleave cisplatin-GSH conjugates to a toxic metabolite.

ATP-dependent transport of statins by human and rat MRP2/Mrp2.

Multidrug resistance associated protein-2, MRP2 (human), Mrp2 (rat) are an efflux transporter, responsible for the transport of numerous endogenous and xenobiotic compounds including taurocholate, methotrexate and carboxydichlorofluorescein (CDF). The present study aims to characterise transport of statins by human and rat MRP2/Mrp2 using membrane and vesicle preparations. All statins tested (simvastatin, pravastatin, pitavastatin, fluvastatin, atorvastatin, lovastatin and rosuvastatin) stimulated vanadate-sensitive ATPase activity in membranes expressing human or rat MRP2/Mrp2, suggesting that all statins are substrates of human and rat MRP2/Mrp2. The substrate affinity (Km) of all statins for MRP2/Mrp2 was comparable and no correlation between lipophilicity (logD7.0) and Km was seen. All statins also inhibited uptake of the fluorescent Mrp2 substrate, CDF (1μM) into vesicles expressing human or rat MRP2/Mrp2 with similar IC50 values. Fitting of the inhibitory data to the hill slope equation, gave hill coefficients (h) of greater than one, suggesting that transport involved more than one binding site for inhibitors of MPR2 and Mrp2. We conclude that statins were transported by both human and rat MRP2/Mrp2 with similar affinity. Statins were also shown to compete with other substrates for transport by MRP2/Mrp2 and that this transport involved more than one binding site on the Mrp2/MRP2 protein.

Cytochrome P450 2C9 is responsible for hydroxylation of the naphthoquinone antimalarial drug 58C80 in human liver

2-(4-t-Butylcyclohexyl)-3-hydroxy-1,4-naphthoquinone (58C80) is an experimental naphthoquinone antimalarial drug which undergoes extensive alky hydroxylation in man. By means of purification, N-terminal amino acid sequencing and inhibition by antibodies and sulfaphenazole, we have identified the form of cytochrome P450 primarily responsible for 58C80 hydroxylation in human liver, P450hB20-27, to be a member of the P450 2C9 subfamily. P450hB20-27 is a low-spin haemoprotein with molecular mass 54 kDa. 58C80 hydroxylation in human liver microsomes was dependent on either NADPH or NADH, with the activity supported by NADH being 35% of that supported by NADPH. With purified P450hB20-27 cytochrome b5 stimulated the NADH-dependent activity 8-fold but inhibited the NADPH-dependent activity by 30%. 58C80 is a novel substrate structure for human P450 2C and these results significantly broaden the range of drugs which have been directly shown (i.e. using a purified enzyme as opposed to expressed cDNA) to be metabolized by human P450 2C forms that are incontrovertibly expressed in human liver in vivo.

Comparative Proteomic Characterization of 4 Human Liver-Derived Single Cell Culture Models Reveals Significant Variation in the Capacity for Drug Disposition, Bioactivation, and Detoxication

In vitro preclinical models for the assessment of drug-induced liver injury (DILI) are usually based on cryopreserved primary human hepatocytes (cPHH) or human hepatic tumor-derived cell lines; however, it is unclear how well such cell models reflect the normal function of liver cells. The physiological, pharmacological, and toxicological phenotyping of available cell-based systems is necessary in order to decide the testing purpose for which they are fit. We have therefore undertaken a global proteomic analysis of 3 human-derived hepatic cell lines (HepG2, Upcyte, and HepaRG) in comparison with cPHH with a focus on drug metabolizing enzymes and transport proteins (DMETs), as well as Nrf2-regulated proteins. In total, 4946 proteins were identified, of which 2722 proteins were common across all cell models, including 128 DMETs. Approximately 90% reduction in expression of cytochromes P450 was observed in HepG2 and Upcyte cells, and approximately 60% in HepaRG cells relative to cPHH. Drug transporter expression was also lower compared with cPHH with the exception of MRP3 and P-gp (MDR1) which appeared to be significantly expressed in HepaRG cells. In contrast, a high proportion of Nrf2-regulated proteins were more highly expressed in the cell lines compared with cPHH. The proteomic database derived here will provide a rational basis for the context-specific selection of the most appropriate ‘hepatocyte-like’ cell for the evaluation of particular cellular functions associated with DILI and, at the same time, assist in the construction of a testing paradigm which takes into account the in vivo disposition of a new drug.

Stem cell-derived models to improve mechanistic understanding and prediction of human drug-induced liver injury.

Current preclinical drug testing does not predict some forms of adverse drug reactions in humans. Efforts at improving predictability of drug‐induced tissue injury in humans include using stem cell technology to generate human cells for screening for adverse effects of drugs in humans. The advent of induced pluripotent stem cells means that it may ultimately be possible to develop personalized toxicology to determine interindividual susceptibility to adverse drug reactions. However, the complexity of idiosyncratic drug‐induced liver injury means that no current single‐cell model, whether of primary liver tissue origin, from liver cell lines, or derived from stem cells, adequately emulates what is believed to occur during human drug‐induced liver injury. Nevertheless, a single‐cell model of a human hepatocyte which emulates key features of a hepatocyte is likely to be valuable in assessing potential chemical risk; furthermore, understanding how to generate a relevant hepatocyte will also be critical to efforts to build complex multicellular models of the liver. Currently, hepatocyte‐like cells differentiated from stem cells still fall short of recapitulating the full mature hepatocellular phenotype. Therefore, we convened a number of experts from the areas of preclinical and clinical hepatotoxicity and safety assessment, from industry, academia, and regulatory bodies, to specifically explore the application of stem cells in hepatotoxicity safety assessment and to make recommendations for the way forward. In this short review, we particularly discuss the importance of benchmarking stem cell–derived hepatocyte‐like cells to their terminally differentiated human counterparts using defined phenotyping, to make sure the cells are relevant and comparable between labs, and outline why this process is essential before the cells are introduced into chemical safety assessment. (Hepatology 2017;65:710‐721).

PPAR agonists reduce steatosis in oleic acid-overloaded HepaRG cells

UNLABELLED Although non-alcoholic fatty liver disease (NAFLD) is currently the most common form of chronic liver disease there is no pharmacological agent approved for its treatment. Since peroxisome proliferator-activated receptors (PPARs) are closely associated with hepatic lipid metabolism, they seem to play important roles in NAFLD. However, the effects of PPAR agonists on steatosis that is a common pathology associated with NAFLD, remain largely controversial. In this study, the effects of various PPAR agonists, i.e. fenofibrate, bezafibrate, troglitazone, rosiglitazone, muraglitazar and tesaglitazar on oleic acid-induced steatotic HepaRG cells were investigated after a single 24-hour or 2-week repeat treatment. Lipid vesicles stained by Oil-Red O and triglycerides accumulation caused by oleic acid overload, were decreased, by up to 50%, while fatty acid oxidation was induced after 2-week co-treatment with PPAR agonists. The greatest effects on reduction of steatosis were obtained with the dual PPARα/γ agonist muraglitazar. Such improvement of steatosis was associated with up-regulation of genes related to fatty acid oxidation activity and down-regulation of many genes involved in lipogenesis. Moreover, modulation of expression of some nuclear receptor genes, such as FXR, LXRα and CAR, which are potent actors in the control of lipogenesis, was observed and might explain repression of de novo lipogenesis. CONCLUSION Altogether, our in vitro data on steatotic HepaRG cells treated with PPAR agonists correlated well with clinical investigations, bringing a proof of concept that drug-induced reversal of steatosis in human can be evaluated in in vitro before conducting long-term and costly in vivo studies in animals and patients.