Rory P. Remmel

Uridine diphosphoglucuronosyltransferase pharmacogenetics and cancer.

The uridine diphosphoglucuronosyltransferases (UGTs) belong to a superfamily of enzymes that catalyse the glucuronidation of numerous endobiotics and xenobiotics. Several human hepatic and extrahepatic UGT isozymes have been characterized with respect to their substrate specificity, tissue expression and gene structure. Genetic polymorphisms have been identified for almost all the UGT family members. A wide variety of anticancer drugs, dietary chemopreventives and carcinogens are known to be conjugated by members of both UGT1A and UGT2B subfamilies. This review examines in detail each UGT isozyme known to be associated with cancer and carcinogenesis. The cancer-related substrates for several UGTs are summarized, and the functionally relevant genetic polymorphisms of UGTs are reviewed. A number of genotype–phenotype association studies have been carried out to characterize the role of UGT pharmacogenetics in several types of cancer, and these examples are discussed here. In summary, this review focuses on the role of the human UGT genetic polymorphisms in carcinogenesis, chemoprevention and cancer risk.

Zidovudine triphosphate and lamivudine triphosphate concentration-response relationships in HIV-infected persons.

ObjectiveTo quantitate intracellular concentrations of zidovudine and lamivudine triphosphate and explore relationships with virologic and immunologic responses to antiretroviral therapy. DesignEight antiretroviral-naive, HIV-infected persons with CD4 T cell counts > 100 × 106 cells/l, and HIV RNA in plasma > 5000 copies/ml participating in a prospective, randomized, open-label study of standard dose versus concentration-controlled therapy with zidovudine, lamivudine, and indinavir. MethodsPeripheral blood mononuclear cells and plasma were collected frequently throughout the study for quantitation of intracellular zidovudine triphosphate and lamivudine triphosphate concentrations, and zidovudine and lamivudine concentrations in plasma. CD4 T cells and HIV RNA in plasma (Roche Amplicor Ultrasensitive Assay) were measured at baseline and every 4 weeks throughout the study. Relationships among intracellular and plasma concentrations, and CD4 T cells and HIV RNA in plasma were investigated with regression analyses. ResultsSignificant relationships were observed between the intracellular concentrations of zidovudine triphosphate and lamivudine triphosphate and the baseline level of CD4 cells. Lamivudine triphosphate concentrations were related in a linear manner to the apparent oral clearance of lamivudine from plasma. A direct linear relationship was found between the intracellular concentrations of zidovudine triphosphate and lamivudine triphosphate. The percent change in CD4 cells during therapy and the rate of decline in HIV RNA in plasma were related to the intracellular concentrations of zidovudine triphosphate and lamivudine triphosphate. ConclusionThese studies into the intracellular clinical pharmacology of nucleoside reverse transcriptase inhibitors illustrate potential clinical implications as determinants of therapeutic success. Moreover, these findings provide several leads and a strong impetus for future investigations with nucleoside reverse transcriptase inhibitors particularly when given in combination and sequentially.

Tissue-specific, Inducible, and Hormonal Control of the Human UDP-Glucuronosyltransferase-1 (UGT1) Locus

The human UDP-glucuronosyltransferase 1 (UGT1) locus spans nearly 200 kb on chromosome 2 and encodes nine UGT1A proteins that play a prominent role in drug and xenobiotic metabolism. Transgenic UGT1 (Tg-UGT1) mice have been created, and it has been demonstrated that tissue-specific and xenobiotic receptor control of the UGT1A genes is influenced through circulating humoral factors. In Tg-UGT1 mice, the UGT1A proteins are differentially expressed in the liver and gastrointestinal tract. Gene expression profiles confirmed that all of the UGT1A genes can be targeted for regulation by the pregnane X receptor activator pregnenolone-16α-carbonitrile (PCN) or the Ah receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition, the selective induction of glucuronidation activity toward lamotrigine, ethinyl estradiol, chenodeoxycholic acid, and lithocholic acid by either PCN or TCDD in small intestine from Tg-UGT1 mice corresponded to expression of the locus in this tissue. Induction of UGT1A1 by PCN and TCDD is believed to be highly dependent upon glucocorticoids, because submicromolar concentrations of dexamethasone actively promote PCN and TCDD induction of UGT1A1 in Tg-UGT1 primary hepatocytes. The role of hormonal control of the UGT1 locus was further verified in pregnant and nursing Tg-UGT1 mice. In maternal 14-day post-conception Tg-UGT1mice, liver UGT1A1, UGT1A4, and UGT1A6 were induced, with the levels returning to near normal by birth. However, maternal liver UGT1A4 and UGT1A6 were dramatically elevated and maintained after birth, indicating that these proteins may play a critical role in maternal metabolism during lactation. With expression of the UGT1 locus confirmed in a variety of mouse tissues, these results suggested that the Tg-UGT1 mice will be a useful model to examine the regulatory and functional properties of human glucuronidation.

Rat hepatocyte spheroids formed by rocked technique maintain differentiated hepatocyte gene expression and function

The culture of primary hepatocytes as spheroids creates an efficient three‐dimensional tissue construct for hepatic studies in vitro. Spheroids possess structural polarity and functional bile canaliculi with normal differentiated function. Thus, hepatocyte spheroids have been proposed as the cell source in a variety of diagnostic, discovery, and therapeutic applications, such as a bioartificial liver. Using a novel rocking technique to induce spheroid formation, kinetics of spheroid formation, cell‐cell adhesion, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. Evidence was provided that the formation of spheroids occurred faster and with fewer nonadherent hepatocytes in rocked suspension culture compared to a traditional rotational system. Hepatocyte spheroids in rocked culture showed stable expression of more than 80% of 242 liver‐related genes including those of albumin synthesis, urea cycle, phase I and II metabolic enzymes, and clotting factors. Biochemical activity of rocked spheroid hepatocytes was superior to monolayer culture of hepatocytes on tissue culture plastic and collagen. Conclusion: Spheroid formation by rocker technique was more rapid and more efficient than by rotational technique. Rocker‐formed spheroids appear suitable for application in a bioartificial liver or as an in vitro liver tissue construct. (HEPATOLOGY 2009.)

Rapid, Large-Scale Formation of Porcine Hepatocyte Spheroids in a Novel Spheroid Reservoir Bioartificial Liver

We have developed a novel bioreactor based on the observation that isolated porcine hepatocytes rapidly and spontaneously aggregate into spheroids under oscillation conditions. The purpose of this study was to characterize the influence of oscillation frequency (0.125 Hz, 0.25 Hz), cell density (1‐10 × 106 cells/mL), and storage condition (fresh, cryopreserved) of porcine hepatocytes on the kinetics of spheroid formation. The viability and metabolic performance of spheroid hepatocytes was also compared to monolayer culture. We observed that both fresh and cryopreserved porcine hepatocytes began formation of spheroids spontaneously at the onset of oscillation culture. Spheroid size was directly related to cell density and time in culture, though inversely related to oscillatory frequency. Spheroid formation by fresh porcine hepatocytes was associated with decreased cell death (lactate dehydrogenase release, 1.3 ± 1.0 vs. 3.1 ± 0.7 U/mL, P < 0.05) and increased metabolic performance (albumin production, 14.7 ± 3.3 vs. 4.6 ± 1.4 fg/c/h, P < 0.0001; ureagenesis from ammonia, 267 ± 63 vs. 92 ± 13 μmol/L/h, P < 0.001) compared with monolayer culture. In conclusion, based on the favorable properties of rapid spheroid formation, increased hepatocellular function, and ease of scale‐up, the spheroid reservoir bioreactor warrants further investigation as a bioartificial liver for support of liver failure. (Liver Transpl 2005;11:901–910.)

Expression of the human UGT1 locus in transgenic mice by 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY-14643) and implications on drug metabolism through peroxisome proliferator-activated receptor α activation

The UDP-glucuronosyltransferase (UGT) 1A genes in humans have been shown to be differentially regulated in a tissue-specific fashion. Transgenic mice carrying the human UGT1 locus (Tg-UGT1) were recently created, demonstrating that expression of the nine UGT1A genes closely resembles the patterns of expression observed in human tissues. In the present study, UGT1A1, UGT1A3, UGT1A4, and UGT1A6 have been identified as targets of the peroxisome proliferator-activated receptor (PPAR) α in human hepatocytes and Tg-UGT1 mice. Oral administration of the PPARα agonist 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (pirinixic acid, WY-14643) to Tg-UGT1 mice led to induction of these proteins in either the liver, gastrointestinal tract, or kidney. The levels of induced UGT1A3 gene transcripts in liver and UGT1A4 protein in small intestine correlated with induced lamotrigine glucuronidation activity in these tissues. With UGT1A3 previously identified as the major human enzyme involved in human C24-glucuronidation of lithocholic acid (LCA), the dramatic induction of liver UGT1A3 RNA in Tg-UGT1 mice was consistent with the formation of LCA-24G in plasma. Furthermore, PPAR-responsive elements (PPREs) were identified flanking the UGT1A1, UGT1A3, and UGT1A6 genes by a combination of site-directed mutagenesis, specific binding to PPARα and retinoic acid X receptor α, and functional response of the concatenated PPREs in HepG2 cells overexpressing PPARα. In conclusion, these results suggest that oral fibrate treatment in humans will induce the UGT1A family of proteins in the gastrointestinal tract and liver, influencing bile acid glucuronidation and first-pass metabolism of other drugs that are taken concurrently with hypolipidemic therapy.

Extracorporeal application of a gel-entrapment, bioartificial liver: Demonstration of drug metabolism and other biochemical functions

Metabolic activity of a gel-entrapment, hollow fiber, bioartificial liver was evaluated in vitro and during extracorporeal hemoperfusion in an anhepatic rabbit model. The bioartificial liver contained either 100 million rat hepatocytes (n = 12), fibroblasts (n = 3), or no cells (n = 7) during hemoperfusion of anhepatic rabbits. Eight other anhepatic rabbits were studied without hemoperfusion as anhepatic controls, and three sham rabbits served as normal controls. Albumin production rates (mean ± SEM) were similar during in vitro (17.0 ± 2.8 μg/h) and extracorporeal (18.0 ± 4.0 μg/h) application of the hepatocyte bioartificial liver. Exogenous glucose requirements were reduced (p < 0.01) and euglycemia was prolonged (p < 0.001) in anhepatic rabbits treated with the hepatocyte bioartificial liver. The maximum rate of glucose production by the hepatocyte bioartificial liver ranged from 50-80 μg/h. Plasma concentrations of aromatic amino acids, proline, alanine, and ammonia were normalized in anhepatic rabbits during hepatocyte hemoperfusion. Gel-entrapped hepatocytes in the bioartificial liver performed sulfation and glucuronidation of 4-methylumbelliferone. P450 activity was demonstrated during both in vitro and extracorporeal application of the BAL device by the formation of 3-hydroxy-lidocaine, the major metabolite of lidocaine biotransformation by gel-entrapped rat hepatocytes. In summary, a gel-entrapment, bioartificial liver performed multiple hepatocyte-specific functions without adverse side effects during extracorporeal application in an anhepatic, small animal model. With its potential for short term support of acute liver failure, scale-up of the current bioartificial liver device is indicated for further investigations in large animal, preclinical trials.

Extended liver-specific functions of porcine hepatocyte spheroids entrapped in collagen gel.

The potential use of porcine hepatocytes in a bioartificial liver device requires large quantities of viable and highly active cells. To facilitate the scaling up of the system, liver specific activities of hepatocytes should be maximized. One way of enhancing the specific activities is to cultivate hepatocytes as multicellular spheroids. Freshly isolated porcine hepatocytes form spheroids when cultivated in suspended cultures. These spheroids exhibit higher activities for a number of liver specific functions compared to hepatocytes cultivated as monolayers. However, these activities decreased in a few days in culture. Entrappment of spheroids in collagen gel sustained their metabolic activities at a stable level over 21 days. Production of albumin and urea by spheroid hepatocytes entrapped in collagen gels were 2 to 3 times higher than those by freshly isolated single cells. P-450 activity was demonstrated by metabolism of lidocaine to its main metabolite, monoethylglycinexylidide. Phase II drug metabolism was demonstrated by glucuronidation of 4-methylumbelliferone. This work shows that porcine hepatocyte spheroids entrapped in collagen maintain differentiated functions for an extended time period. Such hepatocyte spheroid entrappment system may facilitate the development of a bioartificial liver support device.

Concentration-controlled zidovudine therapy

Heterogeneity in the response to antiretroviral therapy has been attributed to pharmacologic, immunologic, and virologic differences between patients. Currently available antiretroviral agents used for the treatment of human immunodeficiency virus (HIV) infection in adults are administered in standard fixed doses. The active moiety of nucleoside anti‐HIV drugs is the intracellular anabolite. Therefore the heterogeneity in response to nucleoside agents may arise as a result of pharmacologic variability at both the systemic and cellular level.

Effect of Aging on Glucuronidation of Valproic Acid in Human Liver Microsomes and the Role of UDP-Glucuronosyltransferase UGT1A4, UGT1A8, and UGT1A10

Valproic acid (VPA) is a widely used anticonvulsant that is also approved for mood disorders, bipolar depression, and migraine. In vivo, valproate is metabolized oxidatively by cytochromes P450 and β-oxidation, as well as conjugatively via glucuronidation. The acyl glucuronide conjugate (valproate-glucuronide or VPAG) is the major urinary metabolite (30–50% of the dose). It has been hypothesized that glucuronidation of antiepileptic drugs is spared over age, despite a known decrease in liver mass. The formation rates of VPAG in a bank of elderly (65 years onward) human liver microsomes (HLMs) were measured by liquid chromatography/tandem mass spectrometry and compared with those in a younger (2–56 years) HLM bank. In vitro kinetic studies with recombinant UDP-glucuronosyltransferases (UGTs) were completed. A 5- to 8-fold variation for the formation of VPAG was observed within the microsomal bank obtained from elderly and younger donors. VPAG formation ranged from 6.0 to 53.4 nmol/min/mg protein at 1 mM substrate concentration (n = 36). The average velocities at 0.25, 0.5, and 1 mM VPA were 7.0, 13.4, and 25.4 nmol/min/mg protein, respectively, in the elderly HLM bank. Rates of VPAG formation were not significantly different in the HLM bank obtained from younger subjects. Intrinsic clearances (Vmax/Km) for several cloned, expressed UGTs were determined. UGT1A4, UGT1A8, and UGT1A10 also were found to catalyze the formation of VPAG in vitro. This is the first reported activity of these UGTs toward VPA glucuronidation. UGT2B7 had the highest intrinsic clearance, whereas UGT1A1 demonstrated no activity. In conclusion, our investigation revealed no differences in VPAG formation in younger versus elderly HMLs and revealed three other UGTs that form VPAG in vitro.