UGT Enzymes Research Articles

Morphine Glucuronidation and Glucosidation Represent Complementary Metabolic Pathways That Are Both Catalyzed by UDP-Glucuronosyltransferase 2B7: Kinetic, Inhibition, and Molecular Modeling Studies

Morphine 3-β-D-glucuronide (M3G) and morphine 6-β-D-glucuronide (M6G) are the major metabolites of morphine in humans. More recently, morphine-3-β-d-glucoside (M-3-glucoside) was identified in the urine of patients treated with morphine. Kinetic and inhibition studies using human liver microsomes (HLM) and recombinant UGTs as enzyme sources along with molecular modeling were used here to characterize the relationship between morphine glucuronidation and glucosidation. The M3G to M6G intrinsic clearance (C(Lint)) ratio (∼5.5) from HLM supplemented with UDP-glucuronic acid (UDP-GlcUA) alone was consistent with the relative formation of these metabolites in humans. The mean C(Lint) values observed for M-3-glucoside by incubations of HLM with UDP-glucose (UDP-Glc) as cofactor were approximately twice those for M6G formation. However, although the M3G-to-M6G C(Lint) ratio remained close to 5.5 when human liver microsomal kinetic studies were performed in the presence of a 1:1 mixture of cofactors, the mean C(Lint) value for M-3-glucoside formation was less than that of M6G. Studies with UGT enzyme-selective inhibitors and recombinant UGT enzymes, along with effects of BSA on morphine glycosidation kinetics, were consistent with a major role of UGT2B7 in both morphine glucuronidation and glucosidation. Molecular modeling identified key amino acids involved in the binding of UDP-GlcUA and UDP-Glc to UGT2B7. Mutagenesis of these residues abolished morphine glucuronidation and glucosidation. Overall, the data indicate that morphine glucuronidation and glucosidation occur as complementary metabolic pathways catalyzed by a common enzyme (UGT2B7). Glucuronidation is the dominant metabolic pathway because the binding affinity of UDP-GlcUA to UGT2B7 is higher than that of UDP-Glc.

Targeted quantitative proteomics for the analysis of 14 UGT1As and -2Bs in human liver using NanoUPLC-MS/MS with selected reaction monitoring.

Targeted quantitative proteomics using heavy isotope dilution techniques is increasingly being utilized to quantify proteins, including UGT enzymes, in biological matrices. Here we present a multiplexed method using nanoLC-MS/MS and multiple reaction monitoring (MRM) to quantify 14 UGT1As and UGT2Bs in liver matrices. Where feasible, we employ two or more proteotypic peptides per protein, with only four proteins quantified with only one proteotypic peptide. We apply the method to analysis of a library of 60 human liver microsome (HLM) and matching S9 samples. Ten of the UGT isoforms could be detected in liver, and the expression of each was consistent with mRNA expression reported in the literature. UGT2B17 was unusual in that ∼30% of liver microsomes had no or little (<0.5 pmol/mg protein) content, consistent with a known common polymorphism. Liver S9 UGT concentrations were approximately 10-15% those of microsomes. The method was robust, precise, and reproducible and provides novel UGT expression data in human liver that will benefit rational approaches to evaluate metabolism in drug development.

Abstract 4552: Defining the metabolic pathways of 13-cis retinoic acid.

Abstract Introduction: Isotretinoin (13-cRA) induces differentiation of human neuroblastoma cells. Althoughhigh-dose, pulse 13-cis-RA treatment significantly improves overall survival of high-risk neuroblastoma patients, most recently together with ch14.18 antibody + cytokines, many children develop recurrent disease during or after 13-cis-RA treatment. Pharmacokinetic data from clinical trials showed a large variation in the levels of 13-cRA or a metabolite 4-oxo-13-cis-retinoic acid (4-oxo-13-cRA) with the metabolite being present up to 7-fold higher than 13-cRA in the plasma at steady-state (Br J Cancer 96:424-31, 2007). We sought to define the enzymes involved in metabolism of 13-cis-RA using liver microsomes. Methods: Phase I metabolism was assessed using pooled human liver (HLM) and intestinal (HIM) microsomes as well as supersomes expressing the recombinant human CYP isoenzymes (2B6, 2C9*1, 2C9*3, 3A5, 3A4, 2C19 and 2C8) for phase I metabolism. We tested inhibitors of CYP enzymes: fluconazole (50μM) (CYP2C9) and ketoconazole (50μM) (CYP3A4). For UGT (phase II) metabolism, we utilized UGT1A1, UGT1A3, UGT1A7, UGT1A9 and UGT1A8 isoenzymes. The generation of metabolites was analyzed by high performance liquid chromatography (HPLC). Results: We identified 4-oxo-13-cRA, monohydroxy-13-cRA, monohydroxy-4-oxo-13-cRA, and 13-cRA glucuronide and 4-oxo-13-cRA-glucuronide as metabolites in the plasma of a 13-cRA treated patient, of which 4-oxo-13-cRA was by far the most abundant. The enzyme kinetic studies revealed that the formation of 4-oxo-13-cRA by HLM (velocity: v=1.67) was much greater compared with HIM (v=0.08) at 100 μM 13-cRA. The formation of 4-oxo-13-cRA from 13-cRA by CYP3A4 isoenzyme was the highest, with CYP3A4 generating more than twice the 4-oxo-13ciRA compared with other isoenzymes tested at a given range of concentrations. In HLM, the CYP3A4 inhibitor ketoconazole (50μM) caused an 83% ± 0.0009 reduction in the formation of 4-oxo-13-cRA relative to no inhibitor controls, supporting that CYP3A4 is a key enzyme in the generation of 4-oxo-13-cRA. Other CYP isoenzymes (2B6, 2C9*1, 2C9*3, 3A5, 2C19 and 2C8) generated minimal amounts of 4-oxo-13-cRA. Glucuronidation of 13-cRA was &amp;gt;5 fold higher by UGT1A3 as compared to other UGT isoenzymes, while 4-oxo-13-cRA was glucuronized at similar rates by multiple isoenzymes of UGT (UGT1A1, UGT1A3, UGT1A7, UGT1A9, UGT1A8). Conclusions: CYP3A4 and UGT1A3 are the major enzymes metabolizing 13-cRA while the major metabolite, 4-oxo-13-cRA is a substrate for a broader spectrum of UGT enzymes. Future studies on the association between enzymatic polymorphisms and pharmacokinetics of 13-cRA may inform strategies to optimize dosing of 13-cRA in high-risk neuroblastoma patients. Citation Format: Poonam Sonawane, Hardeep Singh, C Patrick Reynolds, Min H. Kang. Defining the metabolic pathways of 13-cis retinoic acid. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4552. doi:10.1158/1538-7445.AM2013-4552

Effects of amino acid substitutions at positions 33 and 37 on UDP-glucuronosyltransferase 1A9 (UGT1A9) activity and substrate selectivity

UGT1A9 contributes to the glucuronidation of numerous drugs and xenobiotics. There is evidence to suggest that the Met33Thr substitution, as occurs in the polymorphic variant UGT1A9*3, variably affects xenobiotic glucuronidation. The equivalent position in UGT1A4 is also known to influence enzyme activity, whilst an N-terminal domain histidine (His37 in UGT1A9) is believed to function as the catalytic base in most UGT enzymes. To elucidate the roles of key amino acids and characterise structure–function relationships, we determined the effects of amino acid substitutions at positions 33 and 37 of UGT1A9 on the kinetics of 4-methylumbelliferone (4-MU), mycophenolic acid (MPA), propofol (PRO), sulfinpyrazone (SFZ), frusemide (FSM), (S)-naproxen (NAP) and retigabine (RTB) glucuronidation, compounds that undergo glucuronidation at either a phenolic (4-MU, MPA, PRO), carboxylate (FSM, NAP), acidic carbon (SFZ) or amine (RTB) function. Substitution of Met33 with Val, Ile, Thr, and Gln, as occur in UGT1A1, UGT1A3, UGT1A4 and UGT1A6 respectively, variably affected kinetics and catalytic efficiency. Whilst Km values were generally higher and Vmax and CLint values were generally lower than for wild-type UGT1A9 with most substrate-mutant pairs, the pattern and the magnitude of the changes in each parameter differed substantially. Moreover, exceptions occurred; CLint values for MPA and FSM glucuronidation by the position-33 mutants were the same as or higher than that of UGT1A9. Mutation of His37 abolished activity towards all substrates, except RTB N-glucuronidation. The data confirm the importance of single amino acids for UGT enzyme activity and substrate selectivity, and support a pivotal role for residue-33 in facilitating substrate binding to UGT1A9.

Simultaneous Determination of Sulfation and Glucuronidation of Flavones in FVB Mouse Intestine in Vitro and in Vivo

Glucuronidation and sulfation are the two major phase II metabolic pathways for flavones, natural compounds that hold great potential for improving human health. We investigated the positional preference for sulfation and glucuronidation of seven structurally similar flavones in vitro and in situ. An FVB mouse intestinal perfusion model was used in addition to three small intestine S9 fractions catalyzing sulfation only (Sult enzymes), glucuronidation only (Ugt enzymes) or both (Sult and Ugt enzymes). In both the single and co-reaction S9 systems, flavones containing 7-OH groups were conjugated only at 7-OH despite the presence of other hydroxyl groups, and 7-OH glucuronidation was faster than sulfation (P <0.05). The sulfation rate was enhanced in the Sult-Ugt co-reaction system, while glucuronidation was usually unchanged by the presence of Sult. In the intestinal perfusate, sulfation patterns were the same in the small intestine and colon, and the excretion rate of 7-O-sulfate was the fastest or second fastest. The excretion of 7-O-glucuronidates was faster in small intestine (P < 0.05) than in colon. The S9-mediated sulfation rates of the different flavones were significantly correlated with the excretion rates of the same flavones from perfused intestine. In conclusion, flavone glucuronidation and sulfation rates were sensitive to minor changes in molecular structure. In intestinal S9 fractions, both Ugts and Sults preferentially catalyzed reactions at 7-OH. The sulfation rate was significantly enhanced by simultaneous glucuronidation, but glucuronidation was unaltered by sulfation. Sulfation rates in mouse S9 fractions correlated with sulfation rates in perfused intestine.

Phenylbutyl Isoselenocyanate Modulates Phase I and II Enzymes and Inhibits 4-(Methylnitrosamino)-1-(3-Pyridyl)- 1-Butanone–Induced DNA Adducts in Mice

Lung cancer remains one of the most preventable forms of cancer with about 90% of cases attributed to cigarette smoking. Over the years, the development of chemopreventive agents that could inhibit, delay, or reverse the lung carcinogenesis process has been an active field of research, however, without much attainment. Through extensive structure-activity relationship studies, we recently identified a novel agent phenylbutyl isoselenocyanate (ISC-4), designed on the basis of naturally occurring isothiocyanates well known for their lung cancer prevention properties, as a potential chemopreventive agent. In this study, we used A/J mice to evaluate the lung cancer chemopreventive potential of ISC-4. A single intragastric dose of 1.25 μmol ISC-4 resulted in a time-dependent increase of selenium levels in serum, liver, and lung, suggesting that ISC-4 is orally bioavailable, a key requirement for a chemopreventive agent. This dose also resulted in a time-dependent inhibition of microsomal cytochrome P450 (Cyp450) activity and delayed increases in phase II UDP-glucuronyl transferase (Ugt) and glutathione-S-transferase (Gst) activity. ISC-4 was able to induce mRNA expression of Cyp, Ugt, and Gst enzyme isoforms in liver, but in lung, it inhibited Cyp isoforms while inducing Ugt and Gst isoforms. In addition, ISC-4 effectively inhibited methyl-DNA adduct formation in mice fed diet supplemented with ISC-4 for two weeks and then treated with the tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. These results suggest that ISC-4 is a strong candidate for development as a chemopreventive agent.

N-vinylpyrrolidone dimer, a novel formulation excipient, causes hepatic and thyroid hypertrophy through the induction of hepatic microsomal enzymes in rats

N-vinylpyrrolidone dimer (VPD) is a novel experimental formulation excipient intended for preclinical toxicology studies. In a previous 4-week toxicity study, VPD induced dose-dependent hepatocellular and thyroid gland hypertrophy in Sprague-Dawley (SD) rats. The objectives of the current investigation were to define the underlying molecular mechanisms of these changes. Two separate studies were conducted using male SD rats, daily doses of 300, 1000 or 3000 mg/kg of VPD, and a positive control (phenobarbital at 75 mg/kg/day): (1) a 28-day study to monitor thyroid hormone levels after 7 and 28 days of dosing; (2) a 5-day study to evaluate hepatic and thyroid gland transcriptomic changes, as well as hepatic UGT activity levels. At VPD dosages of 300 mg/kg/day and higher, 2-fold increases of serum thyroid stimulating hormone (TSH) levels were observed in male SD rats after 28 days of dosing, while serum thyroxine (T4) and triiodothyronine (T3) levels were unchanged. Liver UGT enzyme activity levels were increased in VPD-treated rats after 5 days. In addition, in the 5-day study, VPD caused increased hepatic mRNA levels of a panel of drug metabolizing enzymes (DMEs) and transporters, including Cyp3a1, Cyp2b1, Ugt 2b1, and Abcc3. Similar patterns of induction were observed in primary rat hepatocytes exposed to VPD. Transcriptomic changes in the thyroid gland were identified for genes involved in thyroid hormone biosynthesis and in the FAK, PTEN, and Wnt/β-catenin signaling pathways. Collectively, these data indicate that VPD acts as an inducer of hepatic DMEs in SD rats and that this likely leads to enhanced peripheral metabolism of T3/T4, resulting in a feedback response characterized by increased serum TSH levels, and thyroid gland hypertrophy and hyperplasia.

Homodimerization of UDP-glucuronosyltransferase 2B7 (UGT2B7) and identification of a putative dimerization domain by protein homology modeling

Although homodimerization of UGT1A proteins is well established, direct evidence for dimerization of UGT2B7, which is arguably the most important enzyme involved in human drug glucuronidation, is currently lacking. This study characterized UGT2B7 homodimerization by co-immunopreciptation and generated a UGT2B7 homology model that identified the dimerization domain. It was demonstrated that co-expressed, solubilized UGT2B7 proteins differentially tagged with hemagglutinin (UGT2B7-HA) and c-MYC (UGT2B7-cMYC) co-immunoprecipitated as active homodimers that catalyzed 4-methylumbelliferone glucuronidation. Substrate binding affinities (assessed as S50 values) of the tagged and co-expressed tagged proteins were essentially identical to that of native UGT2B7. Co-association was not observed in a ‘mixed’ UGT2B7-HA and UGT2B7-cMYC protein preparation. Generation of a UGT2B7 homology model established from plant and human templates was achieved using SYBYLX1.2 with all residues energy minimized using the Tripos Force Field. The UGT2B7 model allowed elucidation of a putative protein dimerization domain within the B′–C loop of each UGT2B7 monomer. The eighteen amino acid dimerization domain is present in all UGT2B enzymes and comprises a proposed dimerization signature motif (FPPSYVPVVMS). Stabilization of the dimer interface is maintained by the formation of two salt bridges, aromatic π–π stacking interactions, two S-aromatic (face) interactions, and the presence of ‘proline brackets’. The homology model further provides important insights into structure–function relationships of this enzyme and the mechanism responsible for the atypical glucuronidation kinetics for substrates of UGT2B7 and other human UGT enzymes.

New approach in the synthesis of M6G

Morphine-6-glucuronide (M6G) is the primarily active metabolite of morphine, produced endogenously by the UGT enzyme, which displays analgesic activity. For this reason, M6G is a promising drug candidate for the treatment of severe pain. We described herein a new, efficient and scalable synthesis of M6G using dimorphinic derivatives. This preparation of M6G could be performed on multigram scale with good yield and high purity.

Repaglinide‐gemfibrozil drug interaction: inhibition of repaglinide glucuronidation as a potential additional contributing mechanism

To further explore the mechanism underlying the interaction between repaglinide and gemfibrozil, alone or in combination with itraconazole. Repaglinide metabolism was assessed in vitro (human liver subcellular fractions, fresh human hepatocytes, and recombinant enzymes) and the resulting incubates were analyzed, by liquid chromatography-mass spectrometry (LC-MS) and radioactivity counting, to identify and quantify the different metabolites therein. Chemical inhibitors, in addition to a trapping agent, were also employed to elucidate the importance of each metabolic pathway. Finally, a panel of human liver microsomes (genotyped for UGT1A1*28 allele status) was used to determine the importance of UGT1A1 in the direct glucuronidation of repaglinide. The results of the present study demonstrate that repaglinide can undergo direct glucuronidation, a pathway that can possibly contribute to the interaction with gemfibrozil. For example, [³H]-repaglinide formed glucuronide and oxidative metabolites (M2 and M4) when incubated with primary human hepatocytes. Gemfibrozil effectively inhibited (∼78%) both glucuronide and M4 formation, but had a minor effect on M2 formation. Concomitantly, the overall turnover of repaglinide was also inhibited (∼80%), and was completely abolished when gemfibrozil was co-incubated with itraconazole. These observations are in qualitative agreement with the in vivo findings. UGT1A1 plays a significant role in the glucuronidation of repaglinide. In addition, gemfibrozil and its glucuronide inhibit repaglinide glucuronidation and the inhibition by gemfibrozil glucuronide is time-dependent. Inhibition of UGT enzymes, especially UGT1A1, by gemfibrozil and its glucuronide is an additional mechanism to consider when rationalizing the interaction between repaglinide and gemfibrozil.

Modulation of the Human Glucuronosyltransferase UGT1A Pathway by Splice Isoform Polypeptides Is Mediated through Protein-Protein Interactions

This study investigated the molecular mechanisms underlying the regulatory effect of the newly discovered 45-kDa enzymatically inactive UGT1A spliced polypeptides, named isoform i2, upon UGT1A-mediated glucuronidation. Initially, using an inducible system that mimics the relative abundance of isoforms 1 and 2 of UGT1A1 in human tissues, the rates of formation of glucuronides were significantly reduced. We then used a heterologous system constitutively expressing both isoforms i1 and i2 for an in-depth investigation of the presence of spliced i2 on glucuronidation kinetics. UGT1A1, UGT1A7, and UGT1A8 were selected as candidates for these studies. In all cases, co-expression of i1 and i2 in HEK293 cells leads to a significant reduction of the velocity of the glucuronidation reaction without affecting the affinity (K(m) (app)) for all substrates tested and the K(m) for the co-substrate, UDP-glucuronic acid. The data are consistent with a dominant-negative model of inhibition but do not sustain with an UGT1A_i2-mediated inhibition by competitive binding for substrate or the co-substrate. In contrast, the data from the co-immunoprecipitation experiments are indicative of the existence of a mixture homo-oligomeric (i1-i1 or i2-i2) and hetero-oligomeric (i1-i2) complexes in which the i2-i2 and i1-i2 subunits would be inactive. Thus, protein-protein interactions are likely responsible for the inhibition of active UGT1A_i1 by i2 spliced polypeptides. This new regulatory mechanism may alternatively modulate cellular response to endo/xeno stimulus.

An in vitro approach to estimate putative inhibition of buprenorphine and norbuprenorphine glucuronidation

An in vitro inhibition study was performed to investigate potential drug-drug interactions on glucuronidation of buprenorphine (BUP) and norbuprenorphine (NBUP), which represents the major elimination pathway of the drug using cDNA-expressed uridine 5'-diphosphate glucuronosyltransferases (UGTs) and human liver microsomes (HLMs). Following identification of major UGT enzymes for BUP and NBUP glucuronidation, substrates were incubated with drugs (amitriptyline, nortriptyline, lamotrigine, oxazepam, and temazepam), which are extensively cleared by glucuronidation as well as are often used during maintenance treatment. To evaluate the inhibitory potential, the half maximal inhibitor concentration (IC(50)), the inhibition constant (K (i)), and the inhibitor concentration (K (I)) that yield half the maximum rate of inactivation and the enzyme inactivation rate constant (k (inact)) were determined, if appropriate. Amitriptyline and temazepam are inhibitors of NBUP glucuronidation (UGT1A3, HLMs), whereas BUP glucuronidation was affected by amitriptyline (HLMs), oxazepam, and temazepam (UGT2B7). Additionally, BUP inhibits NBUP glucuronidation (UGT1A1, 1A3, HLMs) and vice versa (UGT1A3). A decrease in the metabolic clearance of NBUP may increase the risk of adverse effects such as respiratory depression. Further investigations are needed to evaluate whether inhibition of BUP and NBUP glucuronidation contributes to adverse events.

Identification of Human UGT2B7 as the Major Isoform Involved in theO-Glucuronidation of Chloramphenicol

Chloramphenicol (CP), a broad spectrum antibiotic, is eliminated in humans by glucuronidation. The primary UGT enzymes responsible for CP O-glucuronidation remain unidentified. We have previously identified the 3-O-CP (major) and 1-O-CP (minor) glucuronides by beta-glucuronidase hydrolysis, liquid chromatography-tandem mass spectrometry, and 1D/2D H NMR. Reaction phenotyping for the glucuronidation of CP with 12 expressed human liver UGT isoforms has identified UGT2B7 as having the highest activity for 3-O- and 1-O-CP glucuronidation with minor contributions from UGT1A6 and UGT1A9. The kinetics of CP 3-O-glucuronidation by pooled human liver microsomes (HLMs) exhibited biphasic Michaelis-Menten kinetics with the apparent high-affinity K(m1) and low-affinity K(m2) values of 46.0 and 1027 microM, whereas expressed UGT2B7 exhibited Michaelis-Menten kinetics with the apparent K(m) value of 109.1 microM. The formation of 1-O-CP glucuronide by pooled HLM and expressed UGT2B7 exhibited substrate inhibition kinetics with apparent K(m) values of 408.2 and 115.0 microM, respectively. Azidothymidine (AZT) and hyodeoxycholic acid (substrates of UGT2B7) inhibited 3-O- and 1-O-CP glucuronidation in pooled HLMs. In 10 donor HLM preparations, both CP 3-O- and CP 1-O-glucuronidation showed a significant correlation with AZT glucuronidation (UGT2B7) (r(s) = 0.85 and r(s) = 0.83, respectively) at 30 microM CP, whereas no significant correlation was observed between CP 3-O-glucuronidation and serotonin glucuronidation (UGT1A6) or propofol glucuronidation (UGT1A9) at this CP concentration. These results suggest that UGT2B7 is the primary human hepatic UDP-glucuronosyltransferase isoform catalyzing 3-O- and 1-O-CP glucuronidation with minor contributions from UGT1A6 and UGT1A9.

Abstract C133: Effect of casopitant, a novel neurokinin-1 receptor antagonist, on the pharmacokinetics (PK) of the oral contraceptive pill (OCP)

Abstract Background: Clinical studies suggest that casopitant, a novel neurokinin-1 receptor antagonist in development for prevention of postoperative and chemotherapy-induced nausea and vomiting (PONV and CINV, respectively), is a mild to moderate inhibitor of CYP3A4. However, in cultured human hepatocytes, induction of CYP3A4 and UGT enzymes also has been observed. Since casopitant is intended for use in women receiving moderately emetogenic chemotherapy for breast cancer, the current studies evaluated the pharmacokinetics (PK) of a combined monophasic oral contraceptive pill (OCP) coadministered with repeat-dose casopitant. Methods: Healthy female subjects of non-childbearing or of childbearing potential taking OCP were enrolled. Study 1 (N=11) was a randomized, double-blind, placebo-controlled, repeat-dose, crossover study. OCP containing 30 µg ethinylestradiol (ETE) and 150 µg levonorgestrel (LEV) was administered for 2 complete menstrual cycles (21 days + 7-day washout), plus oral placebo or 60 mg casopitant on days 1–14 of each cycle. Study 2 (N=61) was a single-blind, placebo-controlled, repeat-dose, single-sequence study. OCP was administered for 2 complete cycles as above with oral casopitant (30 mg or 120 mg) or placebo administered for cycles 1 and 2, respectively. Casopitant or placebo was administered for 28 consecutive days, beginning 7 days before OCP administration. The geometric least squares mean ratios and 90% confidence intervals (CI) for ETE and LEV AUC and Cmax were calculated to compare OCP + casopitant vs OCP + placebo. Results: In study 1, ETE AUC(0−) and Cmax were decreased on average by 16% and 17%, respectively, when the OCP was coadministered with 60 mg casopitant vs placebo. Geometric mean LEV AUC(0−) increased 4%, and Cmax decreased 17%. In study 2, for ETE, OCP + casopitant decreased AUC(0−) on average by 15% and 40%, and Cmax by 24% and 32% with 30 mg and 120 mg, respectively. For LEV, the largest mean change in exposure was a 13% decrease in Cmax with 120 mg casopitant given daily for 14 days. Coadministration of casopitant with the combined OCP was well tolerated in both studies. Conclusions: Continuous coadministration of casopitant for 14 or 28 days with a combined monophasic OCP resulted in only a small change in LEV exposure, but a dose-dependent reduction in ETE exposure that may reduce the efficacy of hormonal contraceptives. The effect of a single oral dose of casopitant on hormonal contraceptives has not been studied. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C133.

UGT1A1 genotyping: a predictor of irinotecan-associated side effects and drug efficacy?

Irinotecan [Camptosar (CPT-11), Pfizer Pharmaceuticals, New York, USA] is one of the most effective chemotherapeutic agents in the treatment of metastatic colorectal cancer. In vivo, the prodrug CPT-11 is biotransformed by carboxylesterase into its active metabolite SN-38. SN-38 is inactivated by uridine disphosphate glucuronosyl transferase 1 (UGT1A1) into the inactive compound SN-38G, which is excreted with the bile.This review concentrates on a critical evaluation of UGT1A1 gene polymorphism as a predictor of toxicity and treatment efficacy in patients who received irinotecan for metastatic colorectal cancer. Irinotecan is explained with its main toxicities as well as the underlying mechanisms. The enzyme UGT1A1 is shown in the context of other metabolic pathways and different UGT enzymes involved. We will review in detail the controversy of the current literature with regard to the significance of identifying patients carrying the homozygous genotype UGT1A1 28. Racial differences concerning UGT enzymes have to be considered when discussing a pragmatic approach to determine gene polymorphisms as a predictor of treatment efficacy and outcome in patients receiving irinotecan-based chemotherapy. Dose dependency of toxicity and the clinical relevance of various UGT1 enzymes and single nucleotide polymorphisms in different alternative metabolic pathways are clarified to put UGT1A1 genotyping in a broad context with additional and competing strategies of patient-tailored therapy.

Caco-2 cell monolayers as a tool to study simultaneous phase II metabolism and metabolite efflux of indomethacin, paracetamol and 1-naphthol

The human intestinal cell line, Caco-2, was used to study compounds – indomethacin, paracetamol and 1-naphthol – that undergo intestinal phase II metabolism followed by apical and/or basolateral efflux of the metabolites and/or parent compounds. The interplay was studied during permeability experiments across fully differentiated Caco-2 cell monolayers. The parent compounds and their glucuronide and/or sulfate metabolites were detected by LC–MS/MS. Conjugation of the model compounds and effluxes of their metabolites were observed. The efflux of indomethacin glucuronide was apical, but complementary basolateral efflux was observed at the highest indomethacin concentration (500 μM), probably due to apical saturation. Paracetamol glucuronide was not formed in these experiments, but apical and basolateral effluxes of paracetamol sulfate were observed. A typical bell-shaped inhibition curve was observed for the formation of 1-naphthol glucuronides, indicating substrate or product inhibition of the UGT enzyme(s) at higher 1-naphthol concentrations (200 μM and 500 μM). Based on these results, the fully differentiated Caco-2 cell monolayers can be applied as a platform for qualitative in vitro studies, where phase II metabolism and efflux activities are ongoing simultaneously.

Aldosterone glucuronidation by human liver and kidney microsomes and recombinant UDP‐glucuronosyltransferases: Inhibition by NSAIDs

To characterize: i) the kinetics of aldosterone (ALDO) 18beta-glucuronidation using human liver and human kidney microsomes and identify the human UGT enzyme(s) responsible for ALDO 18beta-glucuronidation and ii) the inhibition of ALDO 18beta-glucuronidation by non-selective NSAIDs. Using HPLC and LC-MS methods, ALDO 18beta-glucuronidation was characterized using human liver (n= 6), human kidney microsomes (n= 5) and recombinant human UGT 1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, 2B17 and 2B28 as the enzyme sources. Inhibition of ALDO 18beta-glucuronidation was investigated using alclofenac, cicloprofen, diclofenac, diflunisal, fenoprofen, R- and S-ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, S-naproxen, pirprofen and tiaprofenic acid. A rank order of inhibition (IC(50)) was established and the mechanism of inhibition investigated using diclofenac, S-ibuprofen, indomethacin, mefenamic acid and S-naproxen. ALDO 18beta-glucuronidation by hepatic and renal microsomes exhibited Michaelis-Menten kinetics. Mean (+/-SD) K(m), V(max) and CL(int) values for HLM and HKCM were 509 +/- 137 and 367 +/- 170 microm, 1075 +/- 429 and 1110 +/- 522 pmol min(-1) mg(-1), and 2.36 +/- 1.12 and 3.91 +/- 2.35 microl min(-1) mg(-1), respectively. Of the UGT proteins, only UGT1A10 and UGT2B7 converted ALDO to its 18beta-glucuronide. All NSAIDs investigated inhibited ALDO 18beta-G formation by HLM, HKCM and UGT2B7. The rank order of inhibition (IC(50)) of renal and hepatic ALDO 18beta-glucuronidation followed the general trend: fenamates > diclofenac > arylpropionates. A NSAID-ALDO interaction in vivo may result in elevated intra-renal concentrations of ALDO that may contribute to the adverse renal effects of NSAIDs and their effects on antihypertensive drug response.

Metabolism and transport pathways of the blood stable camptothecin AR-67 (7-t-butyldimethylsilyl-10-hydroxycamptothecin)

2553 Background: AR-67 is a 3rd generation camptothecin analog with superior stability of its active lactone form (&gt;85%). This stability has been credited in part to the -R10 hydroxyl group (-OH); a group also present on the two clinically approved analogs, topotecan and irinotecan. As with SN-38, the active metabolite of irinotecan, the -OH of AR-67 may be a site for glucuronidation by a UDP glucuronosyltransferase. Moreover, many camptothecins are substrates for the efflux transporter BCRP (ABCG2) and the anionic carboxylate may be a substrate for the liver uptake transporter OATP1B1 (SLCO1B1). Pharmacogenetic studies have established that significant portions of the population have polymorphisms in these genes, which lead to altered pharmacokinetics and toxicity profiles of their substrates. This report describes in-vitro metabolism and transport studies with AR-67 as well as identification of metabolites in plasma and urine from patients enrolled in a phase I study. Methods: AR-67 and its metabolites were detected using chromatography methods with fluorescence or MS/MS detection. In vitro metabolism studies were done with UGT and CYP450 enzyme isoforms expressed in Sf9 cells. In vivo AR-67 metabolic products were identified in clinical plasma and urine specimens. Collection of clinical specimens has been described (control #09-AB-30336-ASCOAM). AR-67 uptake and efflux studies were performed in cells transfected with ABCG2 or SLCO1B1. Results: AR-67 glucuronidation was demonstrated to occur in vitro, primarily by UGT1A8. AR-67 was also found to be a substrate of CYP3A4 and CYP3A5. Only limited glucuronide and oxidation metabolite peaks were observed in patient samples at the highest concentrations. Diminished accumulation of AR-67 lactone was observed in BCRP expressing Saos-2 cells and significant uptake of AR-67 carboxylate was observed in OATP1B1 expressing cells. Conclusions: Unlike SN-38, AR-67 was not found to be a major substrate of UGT1A1, but was rapidly processed by UGT1A8, an isoform that is highly expressed in the GI tract. These data may explain the absence of diarrhea during this phase I study. The pharmacologic significance of ABCG2 and SLCO1B1 polymorphisms should be explored further in the context of population studies. [Table: see text]

In vitro and in vivo glucuronidation of midazolam in humans

Midazolam (MDZ) is a benzodiazepine used as a CYP3A4 probe in clinical and in vitro studies. A glucuronide metabolite of MDZ has been identified in vitro in human liver microsome (HLM) incubations. The primary aim of this study was to understand the in vivo relevance of this pathway. An authentic standard of N-glucuronide was generated from microsomal incubations and isolated using solid-phase extraction. The structure was confirmed using proton nuclear magnetic resonance (NMR) and (1)H-(13)C long range correlation experiments. The metabolite was quantified in vivo in human urine samples. Enzyme kinetic behaviour of the pathway was investigated in HLM and recombinant UGT (rUGT) enzymes. Additionally, preliminary experiments were performed with 1'-OH midazolam (1'-OH MDZ) and 4-OH-midazolam (4-OH MDZ) to investigate N-glucuronidation. NMR data confirmed conjugation of midazolam N-glucuronide (MDZG) standard to be on the alpha-nitrogen of the imidazole ring. In vivo, MDZG in the urine accounted for 1-2% of the administered dose. In vitro incubations confirmed UGT1A4 as the enzyme of interest. The pathway exhibited atypical kinetics and a substrate inhibitory cooperative binding model was applied to determine K(m) (46 microM, 64 microM), V(max) (445 pmol min(-1) mg(-1), 427 pmol min(-1) mg(-1)) and K(i) (58 microM, 79 microM) in HLM and rUGT1A4, respectively. From incubations with HLM and rUGT enzymes, N-glucuronidation of 1'-OH MDZ and 4-OH MDZ is also inferred. A more complete picture of MDZ metabolism and the enzymes involved has been elucidated. Direct N-glucuronidation of MDZ occurs in vivo. Pharmacokinetic modelling using Simcyp illustrates an increased role for UGT1A4 under CYP3A inhibited conditions.

Mechanism of trifluralin-induced thyroid tumors in rats

Trifluralin, an herbicide, has been reported to cause a significant increase in thyroid follicular cell tumors in male Fischer 344 rats. This study was designed to determine the mechanism of thyroid hyperactivity after trifluralin exposure. A group of 15 male Fischer 344 rats were exposed to trifluralin-fortified (6500 ppm) diet for 2 weeks. The time weighted average daily intake of trifluralin was 441 ± 77 mg/kg/day. Ten rats of the group were sacrificed and the sera analyzed for T3, T4, and TSH levels. The livers were also analyzed for selected T4-specific UGT gene expression and total UGT enzyme activity. In the trifluralin treated rats, the serum T3 and T4 levels decreased by 17% and 90%, respectively and TSH increased by 37% more than the control rats. Trifluralin-induced total hepatic UGT enzymes (2.4-fold) and mRNA expression of selected hepatic UGT isozymes (UGT1A1, 1.4-fold; UGT1A6, 6.4-fold; UGT2B1, 3.7-fold). For the remaining 5 rats in the group, bile was collected for 2 h and analyzed for free and conjugated T3 and T4. The total amount of T4 in bile more than doubled in trifluralin treated rats. Trifluralin treatment increased bile flow, caused a 3.2-fold increase in biliary elimination of conjugated T4 and 63% increase in conjugated T3. Based on these data, the decrease in total serum T3 and T4 levels in the trifluralin treated rats was due to enhanced peripheral metabolism and an increase in bile flow that results in a compensatory increase in TSH synthesis and secretion. The increased levels of TSH with chronic exposure to trifluralin would exert a continuous stimulation of the thyroid gland leading to cellular hypertrophy and proliferation predisposing to the development of follicular cell tumors in rats.