The identification and relative contribution of human cytochrome P450 enzyme(s) involved in the metabolism of SCH 351125 were investigated. In human liver microsomes, O-deethylation was the major metabolic pathway, whereas aromatization of a piperidine ring to pyridine and the reduction of the N-oxide moiety were minor routes. Recombinant human CYP3A4 and CYP2C9 both exhibited catalytic activity with respect to the formation of rotameric O-deethylated metabolites (M12, M13), the metabolites resulting from aromatization (M22/M24) and N-oxide reduction (M31). Using the relative activity factor (RAF) approach, the relative contributions of CYP3A4 and CYP2C9 to M13 formation were estimated to be 76 and 24%, respectively. There was a high correlation (r > 0.96) between the rate of formation of M12 and M13 and 6β-hydroxylation of testosterone catalysed by CYP3A4/5. Ketoconazole (2 µM) and CYP3A4/5-specific inhibitory monoclonal antibody inhibited the formation of M12 and M13 from human liver microsomes by approximately 60 and 71%, respectively. The results demonstrate that the in vitro metabolism of SCH 351125 is mediated primarily via CYP3A4 and that CYP2C9 plays a minor role. Clinical study designs should encompass these enzymology data to address any potential drug interactions.

Ezetimibe [1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone] (Zetia; Schering-Plough, Kenilworth, NJ) is the first in a new class of cholesterol-lowering agents known as cholesterol absorption inhibitors. The objective of this study was to identify the isoform(s) of human liver and intestinal UDP-glucuronosyltransferase (UGT) enzymes responsible for the glucuronidation of ezetimibe. The main circulating metabolite of this drug in human plasma is SCH 60663, the phenolic glucuronide conjugate of ezetimibe. SCH 60663 [m/z = 584 Thompsons (Th)] is also the major in vitro metabolite formed by human liver microsomes supplemented with UDP glucuronic acid (UDPGA). In contrast to the liver, human jejunum microsomes supplemented with UDPGA converted ezetimibe to two glucuronides with the same mass (m/z = 584 Th) by liquid chromatography-mass spectrometry. One corresponds to the phenolic glucuronide (1-O-[4-trans-2S,3R)-1-(4-fluorophenyl)-4-oxo-3-[3(S)-hydroxy-3-(4-fluorophenyl)propyl]-2-azetidinyl]phenyl-beta-D-glucopyranuronic acid; SCH 60663) and the other was identified as the benzylic glucuronide of ezetimibe (1-O-[1(S)-(4-fluorophenyl)-3-[1-(4-fluorophenyl)-2(S)-(4-hydroxyphenyl)-4-oxo-3(R)-azetidinyl]propyl]-beta-D-glucopyranuronic acid; SCH 488128). Recombinant human UGT1A1, UGT1A3, and UGT2B15 all exhibited catalytic activity with respect to the formation of the phenolic glucuronide. However, UGT2B7 exclusively formed SCH 488128, a trace metabolite detected in dog and human plasma samples after oral administration of ezetimibe. In conclusion, the formation of SCH 60663 is mediated via UGT1A1, UGT1A3, and UGT2B15, and the formation SCH 488128 is mediated via UGT2B7.

Desloratadine is a non‐sedating antihistamine recently approved for the treatment of seasonal allergic rhinitis. The major metabolite of desloratadine in human plasma and urine is the glucuronide conjugate of 3‐hydroxydesloratadine. 3‐Hydroxydesloratadine‐glucuronide is also the major in vitro metabolite of 3‐hydroxydesloratadine formed by incubation of 3‐hydroxydesloratadine with human liver microsomes supplemented with uridine 5′‐diphosphate‐glucuronic acid (UDPGA). The metabolite structure was confirmed by LC‐MS and LC‐MS/MS. Out of ten recombinant human UDP‐glucuronosyltransferases (UGTs), UGT1A1, UGT1A3, UGT1A8 and UGT2B15 exhibited catalytic activity with respect to the formation of 3‐hydroxydesloratadine‐glucuronide. Inhibition studies with known inhibitors of UGT (diclofenac, flunitrazepam and bilirubin) confirmed the involvement of UGT1A1, UGT1A3 and UGT2B15 in the formation of 3‐hydroxydesloratadine‐glucuronide. The results from this study demonstrated that the in vitro formation of 3‐hydroxydesloratadine‐glucuronide from 3‐hydroxydesloratadine was mediated via UGT1A1, UGT1A3 and UGT2B15 in human liver. Copyright © 2004 John Wiley & Sons, Ltd.

S 101–200 101. IDENTIFICATION OF HUMAN UDP-GLUCURONOSYLTRANSFERASE ENZYME(S) RESPONSIBLE FOR THE GLUCURONIDATION OF 3-HYDROXYDESLORATADINE Anima Ghosal*, Yuan Yuan, Neil Hapangama, Ai Duen (Iris) Su, Narciso Alvarez, Swapan Chowdhury, Kevin B. Alton, James E. Patrick, and Shmuel Zbaida Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, Kenilworth, New Jersey Desloratadine (Clarinex) is a nonsedating antihistamine recently approved for the treatment of seasonal allergic rhinitis. The main circulating metabolite of desloratadine in human plasma and urine is SCH 354202, the glucuronide conjugate of its metabolite 3-hydroxy-desloratadine (SCH 45581). SCH 354202 is also the major in vitro metabolite of SCH 45581 formed by human liver microsomes supplemented with uridine 50-diphosphate-glucuronic acid (UDPGA). The metabolite structure was confirmed by LC-MS and LC-MS/MS. Recombinant human UGT1A1, UGT1A3, and UGT2B15 exhibited catalytic activity with respect to the formation of SCH 354202. Inhibition studies with known inhibitors of UGT (diclofenac, flunitrazepam and bilirubin) confirmed the involvement of UGT1A1, UGT1A3, and UGT2B15 in the formation of SCH 354202. The results from this study demonstrated that the in vitro formation of SCH 45581-glucuronide from SCH 45581 was mediated via UGT1A1, UGT1A3, and UGT2B15. 102. COMPARISON OF QUANTITATIVE AND QUALITATIVE ESTIMATION OF METABOLITES Ping Wang*, Daksha Desai-Krieger, Thanh Duong, and Wu-Nan Wu Johnson & Johnson Pharmaceutical R&D, Spring House, PA 19477 Analysts are often under pressure to give their best estimation of the metabolite level in the biological matrices while analytical standards are not readily available in the early stages of drug development. The estimations are usually based on the comparison of ion intensity of parent drug and metabolites. Although the estimation can provide some valuable information, the discrepancy between the estimation and the true level could be dramatic due to the structural differences. In this presentation, we compare the quantitative results of a new chemical entity (NCE) in human plasma using an analytical standard and the semi-quantitative results obtained without standards. Direct plasma injection into an on-line extraction system coupled with LC/MS/MS was used to carry out the analysis. This NCE contains a carbamate structure with an aliphatic hydroxyl group (-OH). The major metabolite observed is a glucuronide conjugate. The initial semi-quantitative studies showed that in human plasma samples 72% of parent drug was metabolized to glucuronide conjugate while 19% remain intact. However, when a reference standard became available and was used for quantitation, the results showed that glucuronide conjugate is only about 10% of parent in human plasma. The discrepancy can be explained since glucuronide conjugates have much higher ionization efficiency in the negative mode than a compound with a single aliphatic OH group. The results show that extra precautions should be exercised during the process of semi-quantitative studies. When analytical standards are not available, an analog with similar structure may be used for estimation to obtain more meaningful data. Abstracts 51 ©2003 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc. MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016 103. EFFECT OF BILIRUBIN ON THE METABOLISM OF CODEINE, MORPHINE, AND ESTRADIOL IN C57/BL MICE Swati Nagar* and Cheryl L. Zimmerman* Department of Pharmaceutics, University of Minnesota, Minneapolis, MN 55455 This study examined the hypothesis that increased bilirubin is one possible reason for the observed differences in opioid metabolism in sickle cell transgenic mice. Male age-matched C57BL mice were treated with two intra-peritoneal doses of 20mg/kg bilirubin or vehicle 48 and 24 h before euthanasia. Livers were harvested and liver microsomes were prepared by differential centrifugation and used for in vitro incubations. The substrates used were codeine, morphine, and estradiol, and the formation of their respective metabolites, morphine, morphine 3-glucuronide (M3G), and estradiol 3and 17-glucuronides (E3G and E17G) were quantitated with HPLC analysis. Data were fit with the Michaelis-Menten or Hill equations. For the formation of morphine from codeine, microsomes from bilirubin-treated mice had a markedly higher Vmax than the vehicle group (0.25 0.01 vs. 0.19 0.01 nmol/min/mg protein; estimate SE). There was no difference in M3G formation. The formation of E3G as well as E17G had lower Vmax values in the bilirubin group. E17G formation also showed a lower Km estimate. These studies suggest that higher than normal bilirubin levels in sickle cell anemia may be responsible for altered metabolism of some substrates, depending upon the enzyme system involved. 104. EVALUATION OF 5-HTOL AND OTHER ENDOGENOUS SEROTONIN ANALOGUES AS UGT1A6 SUBSTRATES IN HUMAN LIVER MICROSOMES Soundararajan Krishnaswamy*, Qin Hao, and Michael H. Court Comparative and Molecular Pharmacogenetics Laboratory, Department of Pharmacology and Experimental Therapeutics, Tufts University, Boston, MA 02111, USA We have previously shown serotonin to be a specific in vitro substrate for human UGT1A6 isoform. Here, we studied the role of UGT1A6 in the glucuronidation of endogenous structural analogues of serotonin (5-hydroxytryptophol [5HTOL], N-acetylserotonin and 6-hydroxymelatonin) using recombinant UGT isoforms and human liver microsomes. Out of 10 expressed UGTs tested, only UGT1A6 and UGT1A9 catalyzed the glucuronidation of 5-HTOL, although activity with UGT1A6 was over 10 times that of UGT1A9. UGT1A6 and UGT1A9 also had vastly different Km values of 135 and 3674mM, respectively. Km values for human liver microsomes (156, 141, and 134mM) were most similar to that of UGT1A6. 5-HTOL glucuronidation by human liver microsomes (n1⁄4 55) measured at 100mM substrate concentration varied over six fold and correlated well with serotonin glucuronidation (Rs1⁄4 0.832; n1⁄4 55) and immunoreactive UGT1A6 protein (Rs1⁄4 0.848; n1⁄4 52). 5-Hydroxytryptophol also competitively inhibited an index activity of for UGT1A6 (serotonin glucuronidation). N-Acetylserotonin was glucuronidated highest by UGT1A6, although other UGT isomers, mainly UGT1A9 and UGT1A10, also showed significant catalysis. 6-Hydroxymelatonin (6-hydroxyl-N-acetylserotonin) was not glucuronidated by UGT1A6 to any extent. In conclusion, UGT1A6 appears to play a major role in the glucuronidation of 5-HTOL and N-acetylserotonin, but not 6-hydroxymelatonin. (Supported by NIH grant R01-GM-61834.) 52 Abstracts ©2003 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc. MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016 105. THE EFFECT OF ALAMETHICIN ON HEPATIC MICROSOMAL METABOLIC STABILITY MEASUREMENTS Song Lin*, Wan Y. Feng, Emil Samara, and Peter Haroldsen Chiron Corporation, Emeryville, CA 94608 Hepatic microsomes have been commonly used in drug discovery to screen for metabolic stability of new chemical entities. Although liver microsomes contain hepatic uridine diphosphate glucuronosyltransferases, glucuronidation is typically not available by conventional metabolic stability assays. In recent years, reports have shown that alamethicin was capable of facilitating glucuronidation in vitro when used with microsomes. Nevertheless, the impact of such coupled experimentation on metabolic stability screening in drug discovery has not been fully realized. In the present study, we have compared metabolic stability studies with or without alamethicin for a set of structurally diverse compounds. The in vitro half lives for some phenols, N-hydroxy and acylhydroxy containing compounds, where glucuronidation may occur without prior phase I oxidations, differed by as much as 60 fold (i.e., phenolphthalein). However, for compounds whose major pathway was through CYP450 mediated reactions prior to phase II conjugation (i.e., midazolam), inclusion of alamethicin in the incubations did not significantly change the in vitro half lives and intrinsic clearance under the conditions set for discovery metabolic stability screening. These findings demonstrated that the ability to predict in vivo metabolic stability could be improved with further characterization for phase II metabolic pathways for those candidates selected after conventional metabolic stability screening. Furthermore, alamethicin may ideally be used for classes of compounds with apriori potential glucuronidation liabilities. 106. IN VITRO GLUCURONIDATION OF MYCOPHENOLIC ACID BY HUMAN UGTs AND THE IMPACT OF KNOWN POLYMORPHISMS OF UGT1A8, UGT1A9, AND UGT2B7 Olivier Bernard and Chantal Guillemette* Canada Research Chair in Pharmacogenomics, Pharmacogenomics Laboratory, CHUL Research Center, Faculty of Pharmacy of Laval University, Quebec, Canada Mycophenolic acid (MPA) is a standard immunosuppressive used after solid organ transplant. MPA is metabolized in the liver to the pharmacologically inactive 7-O-mycophenolic acid glucuronide (MPAG) and to an acyl glucuronide (AcMPAG)which has pharmacologic activity. The identity of specific UGT enzymes involved in the formation of both metabolites is still not defined. Moreover, the formation of MPAG is subject to an important variability amongst individuals which as yet to be explained. The objectives of our study were (i) to determine the specific UGTs responsible for the formation of MPAG and AcMPAG and (ii) to assess the impact of common polymorphic UGTs on MPA metabolism. Data on the catalytic efficiencies (Vmax/Km) performedwith 16 recombinantUGTs revealed thatUGT1A9>UGT1A8 aremajor in the conjugation of MPAG while UGT2B7 is specifically involved in the formation of AcMPAG. Using U

Cytochrome P450 (CYP) substrates that yield fluorescent metabolites were used for rapid screening of drug metabolism activities of 13 recombinant human cytochromes P450, human liver microsomes and human hepatocytes. Reproducible results were obtained using a fluorescent plate reader (CytoFluor) more expediently than those generated using conventional HPLC methods. Typically, results for 96 samples were obtained with the plate reader in less than 10 min as opposed to 15–35 min/sample required by conventional HPLC. The fluorescent substrates used to measure CYP activities were as follows: 3‐cyano‐7‐ethoxycoumarin (CEC) for CYP1A1, CYP1A2, CYP2C9 and CYP2C19; 7‐ethoxyresorufin (7‐ER) for CYP1A1, CYP1A2 and CYP1B1; 3‐[2‐( N , N ‐diethyl‐ N ‐methylammonium)ethyl]‐7‐methoxy‐4‐methylcoumarin (AMMC) for CYP2D6; dibenzylfluorescein (DBF) for CYP3A4, CYP3A5 and CYP2C8; 7‐methoxy‐4‐trifluoromethylcoumarin (7‐MFC) for CYP2E1, CYP2B6 and CYP2C18; and coumarin for CYP2A6. The chemical inhibition and correlation data indicated that the following substrates can be used as specific functional probes for individual cytochrome P450 present in human liver microsomes: coumarin for CYP2A6 ( r =0.82), AMMC for CYP2D6 ( r =0.83) and DBF for CYP3A4 ( r =0.92). The fluorescent plate reader was found to be useful for the rapid assessment of CYP activities (positive control) in both intact cells and subcellular fractions. Copyright © 2003 John Wiley & Sons, Ltd.

Posaconazole (Noxafil, SCH 56592), an orally available broad-spectrum triazole antifungal, is currently in phase III clinical studies for treating serious opportunistic fungal infections. The major in vitro metabolite of posaconazole formed by human liver microsomes supplemented with uridine 5'-diphosphate-glucuronic acid was a glucuronide of posaconazole (m/z877). Screening of 10 cDNA-expressed recombinant human UDP-glucuronosyltransferase (UGT) enzymes showed that only UGT1A4 exhibited catalytic activity with respect to the formation of the glucuronide of posaconazole. The formation of glucuronide by human liver microsomes and UGT1A4 was inhibited by bilirubin, a known inhibitor of UGT1A4. There was a high correlation (r =0.90) between the rate of formation of glucuronide, determined in 10 human liver microsomal samples, and trifluoperazine glucuronidation catalyzed by UGT1A4. These results confirmed that the formation of major posaconazole-glucuronide produced from human liver microsomes was mediated via UGT1A4.

Ezetimibe [SCH 58235; 1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone], a selective cholesterol absorption inhibitor, is being developed for the treatment of primary hypercholesterolemia. The absorption, metabolism, and excretion of ezetimibe were characterized in eight healthy male volunteers in this single-center, single-dose, open-label study. Subjects received a single oral 20-mg dose of [14C]ezetimibe (approximately 100 microCi) with 200 ml of noncarbonated water after a 10-h fast. Concentrations of radioactivity and/or ezetimibe (conjugated and unconjugated) were determined in plasma, urine, and fecal samples. Ezetimibe was rapidly absorbed and extensively conjugated following oral administration. The main circulating metabolite in plasma was SCH 60663 [1-O-[4-[trans-(2S,3R)-1-(4-fluorophenyl)-4-oxo-3-[3(S)-hydroxy-3-(4-fluorophenyl)propyl]-2-azetidinyl]phenyl]-beta-D-glucuronic acid], the glucuronide conjugate of ezetimibe. Plasma concentration-time profiles of unconjugated and conjugated drug exhibited multiple peaks, indicating enterohepatic recycling. Approximately 78 and 11% of the administered [14C]ezetimibe dose were excreted in feces and urine, respectively, by 240 h after drug administration. Total recovery of radioactivity averaged 89% of the administered dose. The main excreted metabolite was the glucuronide conjugate of ezetimibe. The primary metabolite in urine (0- to72-h composite) was also the glucuronide conjugate (about 9% of the administered dose). Significant amounts (69% of the dose) of ezetimibe were present in the feces, presumably as a result of SCH 60663 hydrolysis and/or unabsorbed drug. No adverse events were reported in this study. A single 20-mg capsule of [(14)C]ezetimibe was safe and well tolerated after oral administration. The pharmacokinetics of ezetimibe are consistent with extensive glucuronidation and enterohepatic recirculation. The primary metabolic pathway for ezetimibe is by glucuronidation of the 4-hydroxyphenyl group.

Adenoviral vectors are being actively investigated for their potential utility in gene therapy. SCH 58500, a replication-deficient adenoviral vector, carries the normal p53 tumor suppressor gene, which is frequently mutated or absent in several human cancers. To assess the potential toxicity associated with adenoviral use, Yorkshire pigs were dosed by intravenous, intrahepatic, or local routes (subcutaneous and intradermal) to support a variety of potential clinical indications. Porcine cells were shown to support replication of wild-type human adenovirus. The nonlethal and asymptomatic dose in pigs following dosing via the intrahepatic route was greater than 3 x 10(8) plaque-forming units (pfu)/kg (2.2 x 10(11) particles/kg), but less than 2.1 x 10(9) pfu/kg (1.5 x 10(12) particles/kg). By the intravenous route it was 1 x 10(8) pfu/kg, and by the ip route it was greater than or equal to 3 x 10(8) pfu/kg. In a multicycle intraperitoneal study in pigs, the high dose of 3 x 10(8) pfu/kg caused an increased antibody and/or an inflammatory response. By the intravenous route, plaque-forming units were present in most pigs at 5 min postdose, but only in a few at 10 min postdose. No expression was found in gonadal tissue approximately 3 weeks after a single intravenous injection of 3 x 10(8) pfu/kg. At high intrahepatic doses (about 1.5 x 10(12) particles/kg), acute cardiovascular and hemodynamic effects were found, which in subsequent studies were also present at high doses by intravenous administration. Based on these findings, careful evaluation of hemodynamic parameters in patients receiving systemic doses of SCH 58500 is warranted.

A rapid HPLC method was developed for quantification of unbound evernimicin in human plasma. Protein-free samples prepared by ultrafiltration were injected directly onto a polymeric reversed-phase column and the eluent monitored at 302 nm. Evernimicin that eluted within 3.5 min was well resolved from endogenous components. Linearity was established between peak height and evernimicin concentration from 25 to 2500 ng/ml. Assay precision (C.V.) was within 5% while bias was no greater than 3%. This method has been used for the ex vivo assessment of evernimicin protein binding in human plasma from safety and tolerance as well as liver dysfunction and renal insufficiency studies.

SCH 58500 is a replication-defective recombinant adenoviral vector containing the cloned human wild-type (normal) tumor suppressor gene p53. SCH 58500 is in trials to evaluate potential clinical utility. A series of toxicology studies in rats and mice were conducted via multiple routes of exposure to support these programs. The nonlethal and asymptomatic dose in rats following a 14-day observation period was equal to 7.5 x 10(7) plaque-forming units (pfu)/kg (5.6 x 10(10) particles/kg) by intravenous or intraperitoneal route and was similar by the ip route, following 4 weeks of dosing. The high dose of 1.5 x 10(9) pfu/kg (1.1 x 10(12) particles/kg) was lethal by the i.v. route and inflammatory to the peritoneal cavity by the ip route. SCH 58500 was rapidly cleared from the systemic circulation in rats (serum t(1/2) of 7 to 9 min) following iv administration. Administration by other routes resulted in no (sc) or delayed (ip) serum levels. Since most rats in the i.v. rat study died within 24 h postdose, another study to evaluate potential mechanisms of toxicity in rats was designed in which rats were killed at intervals following a single i.v. dosing. A single high i.v. dose of SCH 58500 (1.1 x 10(12) pfu/kg) was associated with lethargy, soft feces, a ruffled-hair coat, and death within 1 h postdose. Potential mechanisms of toxicity appeared to include a mild coagulopathy and/or vasculopathy, resulting in consumption of platelets and clotting factors, leakage or loss of intravascular fluid, hemoconcentration, electrolyte and/or fluid shifts, a moderate stress and/or inflammatory response, and a mild, direct or indirect toxic effect on liver and/or kidney tissue. These findings suggest a multifocal cause for acute lethality following i.v. dosing in rats.

ABSTRACT The in vitro uptake of [ 14 C]evernimicin ([ 14 C]SCH 27899) by primary cultures of rat alveolar macrophages and hepatocytes was determined. Both cell populations exhibited linear rates of uptake. However, the initial rate of drug uptake by alveolar macrophages was about threefold higher than that by hepatocytes. These findings demonstrate that [ 14 C]evernimicin is taken up by rat alveolar macrophages, supporting the likelihood that the drug is able to reach sites of infection.