Monkey Liver Microsomes Research Articles

In Vitro Characterization of Glucuronidation of Vanillin: Identification of Human UDP‐Glucuronosyltransferases and Species Differences

Vanillin is a food flavoring agent widely utilized in foods, beverages, drugs, and perfumes and has been demonstrated to exhibit multiple pharmacological activities. Given the importance of glucuronidation in the metabolism of vanillin, the UDP-glucuronosyltransferase conjugation pathway of vanillin was investigated in this study. Vanillin glucuronide was identified by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and a hydrolysis reaction catalyzed by β-glucuronidase. The kinetic study showed that vanillin glucuronidation by HLMs and HIMs followed Michaelis-Menten kinetics and the kinetic parameters were as follows: 134.9 ± 13.5 μM and 81.3 ± 11.3 μM for K(m) of HLMs and HIMs, 63.8 ± 2.0 nmol/min/mg pro and 13.4 ±2.0 nmol/min/mg pro for Vmax of HLMs and HIMs. All UDP-glucuronosyltransferase (UGT) isoforms except UGT1A4, 1A9, and 2B7 showed the capability to glucuronidate vanillin, and UGT1A6 exerted the higher V(max)/K(m) values than other UGT isoforms for the glucuronidation of vanillin when assuming expression of isoforms is similar in recombinant UGTs. Kinetic analysis using liver microsomes from six studied speices indicated that vanillin had highest affinity for the monkey liver microsomes enzyme (K(m) = 25.6 ± 3.2 μM) and the lowest affinity for the mice liver microsomes enzyme (K(m) = 149.1 ± 18.4 μM), and intrinsic clearance was in the following order: monkey > dog > minipig > mice > rat ~ human. These data collectively provided important information for understanding glucuronidation of vanillin.

In Vitro Bioactivation of a Selective Estrogen Receptor Modulator (2S,3R)-(+)-3-(3-Hydroxyphenyl)-2-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (I) in Liver Microsomes: Formation of Adenine Adducts

As part of our efforts to develop safer selective estrogen receptor modulators (SERMs), compound I {(2S,3R)-(+)-3-(3-hydroxyphenyl)-2-[4-(2-pyrrolidin-1-ylethoxy)-phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol} was previously identified as a lead for further development. Subsequent studies showed that compound I is genotoxic in both in vitro Chinese hamster ovary (CHO) cells and in vivo mouse studies. To better understand the possible mechanisms for the observed genetoxicity effects, in vitro incubations of I with liver microsomes of human, monkey, and mouse in the presence of adenine were performed, which led to the detection of five adenine adducts. The formation of these adducts was NADPH-dependent, suggesting the involvement of oxidative bioactivation catalyzed by cytochrome P450 enzymes. The mechanism for the formation of the major adenine adduct (A1) involves the formation of a reactive ring-opened para-quinone intermediate. The formation of four other adenine adducts may involve the formation of a reactive epoxide or ortho-quinone intermediate. Furthermore, incubations of compound I with human hepatocytes showed dose-dependent DNA damages in Comet assays. All of the above suggest that some reactive metabolites of compound I, formed through bioactivation mechanisms, have a potential to interact with DNA molecules in vitro and in vivo. This may be one of the causes of the genotoxicity observed preclinically both in vitro and in vivo. This case study demonstrated an approach using in vitro DNA trapping assays for assessing the genotoxicity potential of drug candidates.

Monkey liver cytochrome P450 2C19 is involved in R- and S-warfarin 7-hydroxylation

Cynomolgus monkeys are widely used as primate models in preclinical studies. However, some differences are occasionally seen between monkeys and humans in the activities of cytochrome P450 enzymes. R- and S-warfarin are model substrates for stereoselective oxidation in humans. In this current research, the activities of monkey liver microsomes and 14 recombinantly expressed monkey cytochrome P450 enzymes were analyzed with respect to R- and S-warfarin 6- and 7-hydroxylation. Monkey liver microsomes efficiently mediated both R- and S-warfarin 7-hydroxylation, in contrast to human liver microsomes, which preferentially catalyzed S-warfarin 7-hydroxylation. R-Warfarin 7-hydroxylation activities in monkey liver microsomes were not inhibited by α-naphthoflavone or ketoconazole, and were roughly correlated with P450 2C19 levels and flurbiprofen 4-hydroxylation activities in microsomes from 20 monkey livers. In contrast, S-warfarin 7-hydroxylation activities were not correlated with the four marker drug oxidation activities used. Among the 14 recombinantly expressed monkey P450 enzymes tested, P450 2C19 had the highest activities for R- and S-warfarin 7-hydroxylations. Monkey P450 3A4 and 3A5 slowly mediated R- and S-warfarin 6-hydroxylations. Kinetic analysis revealed that monkey P450 2C19 had high Vmax and low Km values for R-warfarin 7-hydroxylation, comparable to those for monkey liver microsomes. Monkey P450 2C19 also mediated S-warfarin 7-hydroxylation with Vmax and Vmax/Km values comparable to those for recombinant human P450 2C9. R-warfarin could dock favorably into monkey P450 2C19 modeled. These results collectively suggest high activities for monkey liver P450 2C19 toward R- and S-warfarin 6- and 7-hydroxylation in contrast to the saturation kinetics of human P450 2C9-mediated S-warfarin 7-hydroxylation.

Stereoselective metabolism of tetrahydropalmatine enantiomers in rat liver microsomes

Tetrahydropalmatine (THP), with one chiral center, is an active alkaloid ingredient in Rhizoma Corydalis. The aim of the present paper is to study whether THP enantiomers are metabolized stereoselectively in rat, mouse, dog, and monkey liver microsomes, and then, to elucidate which Cytochrome P450 (CYP) isoforms are predominately responsible for the stereoselective metabolism of THP enantiomers in rat liver microsomes (RLM). The results demonstrated that (+)-THP was preferentially metabolized by liver microsomes from rats, mice, dogs, and monkeys, and the intrinsic clearance (Cl(int)) ratios of (+)-THP to (-)-THP were 2.66, 2.85, 4.24, and 1.67, respectively. Compared with the metabolism in untreated RLM, the metabolism of (-)-THP and (+)-THP was significantly increased in dexamethasone (Dex)-induced and β-naphthoflavone (β-NF)-induced RLM; meanwhile, the Cl(int) ratios of (+)-THP to (-)-THP in Dex-induced and β-NF-induced RLM were 5.74 and 0.81, respectively. Ketoconazole had stronger inhibitory effect on (+)-THP than (-)-THP, whereas fluvoxamine had stronger effect on (-)-THP in untreated and Dex-induced or β-NF-induced RLM. The results suggested that THP enantiomers were predominately metabolized by CYP3A1/2 and CYP1A2 in RLM, and CYP3A1/2 preferred to metabolize (+)-THP, whereas CYP1A2 preferred (-)-THP.

Characterization of intestinal and hepatic P450 enzymes in cynomolgus monkeys with typical substrates and inhibitors for human P450 enzymes

Cynomolgus monkeys are widely used to predict human pharmacokinetic and/or toxic profiles in the drug developmental stage. Characterization of cynomolgus monkey P450s such as the mRNA expression level, substrate specificity, and inhibitor selectivity were conducted to provide helpful information in designing monkey in vivo studies and monkey-to-human extrapolation.The expression levels of 12 monkey P450 mRNAs, which are considered to be important P450 subfamilies in drug metabolism, were investigated in the liver, small intestine (duodenum, jejunum, and ileum), and colon of individual monkeys.iIn vitro activities and intrinsic clearance values were determined in monkey intestinal and liver microsomes (MIM and MLM, respectively) using nine typical oxidative reactions for human P450s. Paclitaxel 6α-hydroxylation, diclofenac 4′-hydroxylation, and S-mephenytoin 4′-hydroxylation showed low activities in MIM and MLM.IC50 values of eight selective inhibitors of human P450s were determined in MIM and MLM. Inhibitory effects of furafylline and sulfaphenazole were weak in monkeys on phenacetin O-deethylation and diclofenac 4′-hydroxylation, respectively.These results show profiles of monkey P450s in both the intestine and liver in detail and contribute to a better understanding of the species difference in substrate specificity and inhibitor selectivity between cynomolgus monkeys and humans.

Different Metabolites of Human Hepatotoxic Pyrazolopyrimidine Derivative 5‐n‐Butyl‐Pyrazolo[1,5‐a]Pyrimidine Produced by Human, Rat and Monkey Cytochrome P450 1A2 and Liver Microsomes

Different Metabolites of Human Hepatotoxic Pyrazolopyrimidine Derivative 5‐<i>n</i>‐Butyl‐Pyrazolo[1,5‐<i>a</i>]Pyrimidine Produced by Human, Rat and Monkey Cytochrome P450 1A2 and Liver Microsomes

In Vitro Glucuronidation of the Antibacterial Triclocarban and Its Oxidative Metabolites

Triclocarban (3,4,4'-trichlorocarbanilide; TCC) is widely used as an antibacterial in bar soaps. During use of these soaps, a significant portion of TCC is absorbed by humans. For the elimination from the body, glucuronidation plays a key role in both biliary and renal clearance. To investigate this metabolic pathway, we performed microsomal incubations of TCC and its hydroxylated metabolites 2'-OH-TCC, 3'-OH-TCC, and 6-OH-TCC. Using a new liquid chromatography-UV-mass spectrometry method, we could show a rapid glucuronidation for all OH-TCCs by the uridine-5'-diphosphate-glucuronosyltransferases (UGT) present in liver microsomes of humans (HLM), cynomolgus monkeys (CLM), rats (RLM), and mice (MLM). Among the tested human UGT isoforms, UGT1A7, UGT1A8, and UGT1A9 showed the highest activity for the conjugation of hydroxylated TCC metabolites followed by UGT1A1, UGT1A3, and UGT1A10. Due to this broad pattern of active UGTs, OH-TCCs can be efficiently glucuronidated in various tissues, as shown for microsomes from human kidney (HKM) and intestine (HIM). The major renal metabolites in humans, TCC-N-glucuronide and TCC-N'-glucuronide, were formed at very low conversion rates (<1%) by microsomal incubations. Low amounts of N-glucuronides were generated by HLM, HIM, and HKM, as well as by MLM and CLM, but not by RLM, according to the observed species specificity of this metabolic pathway. Among the human UGT isoforms, only UGT1A9 had activity for the N-glucuronidation of TCC. These results present an anomaly where in vivo the predominant urinary metabolites of TCC are N and N'-glucuronides, but these compounds are slowly produced in vitro.

Comparative metabolism of sildenafil in liver microsomes of different species by using LC/MS-based multivariate analysis

Sildenafil metabolism in liver microsomes obtained from different species was studied in vitro and compared using liquid chromatography–mass spectrometry (LC/MS) and multivariate statistical analysis. Sildenafil (1, 5, and 25 μM) was incubated with rat, mouse, dog, monkey, and human liver microsomes along with NADPH, and the reaction mixtures were analyzed by LC/MS to obtain species-specific metabolic profiles of sildenafil. A total of 12 metabolites were detected and their peak area ratio values were used as variables for multivariate analyses to evaluate the interspecies differences in sildenafil metabolism. Principal components analysis of the metabolic profiles showed that the mouse samples were generally clustered closer to the human samples on the principal component score plot. Similarity index (SI) indicated that sildenafil metabolism in mice, compared to the other animals, was highly analogous (SI = 0.764 at 25 μM) to that in humans. These results suggest that LC/MS-based multivariate analytical approaches are useful for the evaluation of interspecies differences in the metabolism of xenobiotics.

Significant Species Difference in Amide Hydrolysis of GDC-0834, a Novel Potent and Selective Bruton's Tyrosine Kinase Inhibitor

(R)-N-(3-(6-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide (GDC-0834) is a potent and selective inhibitor of Bruton's tyrosine kinase (BTK), investigated as a potential treatment for rheumatoid arthritis. In vitro metabolite identification studies in hepatocytes revealed predominant formation of an inactive metabolite (M1) via amide hydrolysis in human. The formation of M1 appeared to be NADPH-independent in human liver microsomes. M1 was found in only minor to moderate quantities in plasma from preclinical species dosed with GDC-0834. Human clearance predictions using various methodologies resulted in estimates ranging from low to high. In addition, GDC-0834 exhibited low clearance in PXB chimeric mice with humanized liver. Uncertainty in human pharmacokinetic prediction and high interest in a BTK inhibitor for clinical evaluation prompted an investigational new drug strategy, in which GDC-0834 was rapidly advanced to a single-dose human clinical trial. GDC-0834 plasma concentrations in humans were below the limit of quantitation (<1 ng/ml) in most samples from the cohorts dosed orally at 35 and 105 mg. In contrast, substantial plasma concentrations of M1 were observed. In human plasma and urine, only M1 and its sequential metabolites were identified. The formation kinetics of M1 was evaluated in rat, dog, monkey, and human liver microsomes in the absence of NADPH. The maximum rate of M1 formation (V(max)) was substantially higher in human compared with that in other species. In contrast, the Michaelis-Menten constant (K(m)) was comparable among species. Intrinsic clearance (V(max)/K(m)) of GDC-0834 from M1 formation in human was 23- to 169-fold higher than observed in rat, dog, and monkey.

Abstract 3233: Comparative in vitro and in vivo metabolism of MPC-3100, an oral HSP90 inhibitor, in rat, dog, monkey and human

Abstract MPC-3100, an 8, 9-disubstituted purine, is an orally bioavailable HSP90 inhibitor currently in Phase 1 clinical development. The objectives of these studies were to compare the metabolism of MPC-3100 in preclinical species to select the species most appropriate for toxicological testing and to identify the major phase I and II metabolites formed both in vitro and in vivo in rats, dogs, monkeys and humans. MPC-3100 was incubated with liver microsomes from rats, dogs, monkeys, and humans. In addition, urine, feces, and bile were collected from rats dosed with MPC-3100 intravenously (5 mg/kg) or orally (50 mg/kg), and urine was collected from dogs (2 mg/kg) and cynomolgus monkeys (2.5 mg/kg) dosed intravenously. Metabolites were identified by liquid chromatography electrospray-ionization mass spectrometry. Quantitative analysis was performed with an AB Sciex 4000 Q-trap and qualitative analysis was conducted on a high resolution Agilent Q-TOF 6520 mass spectrometer. Six authentic standards were synthesized and used to confirm structural identity. In human liver microsomes, four distinct peaks were observed following chromatographic analysis. Three of these were conclusively identified using synthetic standards, accurate mass, and chromatographic retention time. The most abundant metabolite in all species was the catechol. In human, monkey, and dog liver microsomes, the next most abundant metabolite was formed by oxidation of the 2-hydroxypropan-1-one moiety to propane-1, 2-dione. A third metabolite present in all incubations was the de-amidated product of MPC-3100. It was formed in microsomes in the absence of NADPH suggesting that its formation was due to pH-mediated hydrolysis. A fourth metabolite formed by the addition of oxygen (+16 Da) within the methylenedioxyphenyl ring was assigned based solely upon its product ion spectrum. Following intravenous administration of MPC-3100 to rats, fourteen metabolites were observed in the feces; whereas, only 6 metabolites were observed in urine. No glucuronides were found in either the urine or feces. Less than 1% of the dose was recovered in rat urine; whereas, up to 40% of the dose was recovered as MPC-3100 and metabolites in feces over a 24 hour period. As many as 25 different metabolites were observed in the bile based upon differences in their retention time and molecular weight. Most of the metabolites in the bile resulted from either glucuronidation or sulfation, some of which were conclusively identified with authentic standards. Rat, dog, and monkey liver microsomes all produced the four major metabolites formed in human liver microsomes. Three of these metabolites formed in human microsomes were conclusively identified by comparison to authentic synthetic standards. Although MPC-3100 and several metabolites were found in rat, dog and monkey urine, the primary route of elimination of MPC-3100 was through biliary excretion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3233. doi:10.1158/1538-7445.AM2011-3233

P.5.010 The behavioural and neurochemical effects of NGF dipeptide fragment GK-2 in MPTP-lesioned C57BL mice

Trabectedin (YONDELIS®) is a potent anticancer agent which was recently approved in Europe for the treatment of soft tissue sarcoma. The drug is currently also in clinical development for the treatment of ovarian carcinoma. In vitro experiments were conducted to investigate the hepatic metabolism of [14C]trabectedin in Cynomolgus monkey and human liver subcellular fractions. The biotransformation of trabectedin was qualitatively similar in 12,000 × g supernatants of both species, and all human metabolites were also produced by the monkey. The trabectedin metabolites were identified by QTOF mass spectrometry, and HPLC co-chromatography with reference compounds. Trabectedin was metabolized via different biotransformation pathways. Most of the metabolic conversions occurred at the trabectedin A domain including mono-oxidation and di-oxidation, carboxylic acid formation with and without additional oxidation, and demethylation either without (N-demethylation to ET-729) or with additional mono-, di- or tri-oxidation. Another metabolite resulted from O-demethylation at the trabectedin C subunit, and in addition, aliphatic ring opening of the methylene dioxybridge at the B domain was detected. Overall, demethylation and oxidation played a major role in phase I metabolism of the drug. Human cDNA expressed CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, 3A4 and 3A5 in E. coli membranes, but not CYP1B1, 2C19, and 4A11 were able to metabolize [14C]trabectedin. Experiments with chemical inhibitors and CYP inhibitory antibodies indicated that, at therapeutic levels, CYP3A4 is the main human CYP isoform involved in trabectedin's hepatic metabolism. In monkey and human liver microsomes, trabectedin was not substantially metabolized by glucuronidation.

Metabolism of the c-Fos/Activator Protein-1 Inhibitor T-5224 by Multiple Human UDP-Glucuronosyltransferase Isoforms

We developed 3-{5-[4-(cyclopentyloxy)-2-hydroxybenzoyl]-2-[(3-hydroxy-1,2-benzisoxazol-6-yl)methoxy]phenyl} propionic acid (T-5224) as a novel inhibitor of the c-Fos/activator protein-1 for rheumatoid arthritis therapy. We predicted the metabolism of T-5224 in humans by using human liver microsomes (HLM), human intestinal microsomes (HIM), recombinant human cytochrome P450 (P450), and UDP-glucuronosyltransferases (UGTs). T-5224 was converted to its acyl O-glucuronide (G2) by UGT1A1 and UGT1A3 and to its hydroxyl O-glucuronide (G3) by several UGTs, but it was not metabolized by the P450s. A comparison of the intrinsic clearances (CL(int)) between HLM and HIM suggested that the glucuronidation of T-5224 occurs predominantly in the liver. UGT1A1 showed a higher k(cat)/K(m) value than UGT1A3 for G2 formation, but a lower k(cat)/K(m) value than UGT1A3 for G3 formation. A high correlation was observed between G2 formation activity and UGT1A1-specific activity (β-estradiol 3-glucuronidation) in seven individual HLM. A high correlation was also observed between G2 formation activity and UGT1A1 content in the HLM. These results strongly suggest that UGT1A1 is responsible for G2 formation in human liver. In contrast, no such correlation was observed with G3 formation, suggesting that multiple UGT isoforms, including UGT1A1 and UGT1A3, are involved in G3 formation. G2 is also observed in rat and monkey liver microsomes as a major metabolite of T-5224, suggesting that G2 is not a human-specific metabolite. In this study, we obtained useful information on the metabolism of T-5224 for its clinical use.

Biotransformation of a Novel Antimitotic Agent, I-387, by Mouse, Rat, Dog, Monkey, and Human Liver Microsomes and In Vivo Pharmacokinetics in Mice

3-(1H-Indol-2-yl)phenyl)(3,4,5-trimethoxyphenyl)methanone (I-387) is a novel indole compound with antitubulin action and potent antitumor activity in various preclinical models. I-387 avoids drug resistance mediated by P-glycoprotein and showed less neurotoxicity than vinca alkaloids during in vivo studies. We examined the pharmacokinetics and metabolism of I-387 in mice as a component of our preclinical development of this compound and continued interest in structure-activity relationships for antitubulin agents. After a 1 mg/kg intravenous dose, noncompartmental pharmacokinetic analysis in plasma showed that clearance (CL), volume of distribution at steady state (Vd(ss)), and terminal half-life (t(1/2)) of I-387 were 27 ml per min/kg, 5.3 l/kg, and 7 h, respectively. In the in vitro metabolic stability study, half-lives of I-387 were between 10 and 54 min by mouse, rat, dog, monkey, and human liver microsomes in the presence of NADPH, demonstrating interspecies variability. I-387 was most stable in rat liver microsomes and degraded quickly in monkey liver microsomes. Liquid chromatography-tandem mass spectrometry was used to identify phase I metabolites. Hydroxylation, reduction of a ketone group, and O-demethylation were the major metabolites formed by the liver microsomes of the five species. The carbonyl group of I-387 was reduced and identified as the most labile site in human liver microsomes. The results of these drug metabolism and pharmacokinetic studies provide the foundation for future structural modification of this pharmacophore to improve stability of drugs with potent anticancer effects in cancer patients.

Preclinical Characterization of BI 201335, a C-Terminal Carboxylic Acid Inhibitor of the Hepatitis C Virus NS3-NS4A Protease

BI 201335 is a hepatitis C virus (HCV) NS3-NS4A (NS3 coexpressed with NS4A) protease inhibitor that has been shown to have potent clinical antiviral activity. It is a highly optimized noncovalent competitive inhibitor of full-length NS3-NS4A proteases of HCV genotypes 1a and 1b with K(i) values of 2.6 and 2.0 nM, respectively. K(i) values of 2 to 230 nM were measured against the NS3-NS4A proteases of HCV genotypes 2 to 6, whereas it was a very weak inhibitor of cathepsin B and showed no measurable inhibition of human leukocyte elastase. BI 201335 was also shown to be a potent inhibitor of HCV RNA replication in vitro with 50% effective concentrations (EC(50)s) of 6.5 and 3.1 nM obtained in genotype 1a and 1b replicon assays. Combinations of BI 201335 with either interferon or ribavirin had additive effects in replicon assays. BI 201335 had good permeability in Caco-2 cell assays and high metabolic stability after incubation with human, rat, monkey, and dog liver microsomes. Its good absorption, distribution, metabolism, and excretion (ADME) profile in vitro, as well as in rat, monkey, and dog, predicted good pharmacokinetics (PK) in humans. Furthermore, drug levels were significantly higher in rat liver than in plasma, suggesting that distribution to the target organ may be especially favorable. BI 201335 is a highly potent and selective NS3-NS4A protease inhibitor with good in vitro and animal ADME properties, consistent with its good human PK profile, and shows great promise as a treatment for HCV infection.

Lack of Appreciable Species Differences in Nonspecific Microsomal Binding

Species differences in microsomal binding were evaluated for 43 drug molecules in human, monkey, dog and rat liver microsomes, using a fixed concentration of microsomal protein. The dataset included 32 named drugs and 11 proprietary compounds encompassing a broad spectrum of physicochemical properties (11 acids, 24 bases, 8 neutral, c log D -1 to 7, MW 200 to 700 and free fraction <0.001 to 1). Free fractions (f(u,mic)) in monkey, dog, rat and human microsomes were highly correlated, with linear regression correlation coefficients greater than 0.97. The average fold-difference in f(u,mic) between monkey, dog, or rat, and human was 1.6-, 1.3-, and 1.5-fold, respectively. Species differences in f(u,mic) were also assessed for a range of microsomal protein concentrations (0.2-2 mg/mL) for midazolam, clomipramine, astemizole, and tamoxifen, drugs with low to high microsomal binding. The mean fold species-difference in f(u,mic) for midazolam, clomipramine, astemizole, and tamoxifen was 1.1-, 1.2-, 1.3-, and 2.0-fold, respectively, and was independent of normalized microsomal protein concentration. For a fixed concentration of microsomal protein, greater than 76% and 90% of drugs examined in this study had preclinical species f(u,mic) within 1.5- and 2-fold, respectively, of experimentally measured human values.

Functional characterization of human and cynomolgus monkey UDP-glucuronosyltransferase 1A1 enzymes

Aims UDP-glucuronosyltransferase 1A1 (UGT1A1) plays important roles in the glucuronidation of various drugs and endogenous substances. Cynomolgus monkeys are regarded as experimental animals closer to humans in studies on safety evaluation and biotransformation for drug development. In this study, the similarities and differences in the enzymatic properties of UGT1A1 between humans and cynomolgus monkeys were precisely identified. Main methods Human and cynomolgus monkey UGT1A1s (humUGT1A1 and monUGT1A1, respectively) were cloned, and the corresponding proteins were heterologously expressed in insect cells. The enzymatic properties of UGT1A1 proteins were characterized by kinetic analysis of 7-hydroxy-4-trifluoromethylcoumarin (7-HFC), estradiol at 3-hydroxy position (E-3OH) and 7-ethyl-10-hydroxycamptothecin (SN-38) glucuronidation. Key findings There were no significant differences in the levels of kinetic parameters for 7-HFC, E-3OH and SN-38 glucuronidation between humans and cynomolgus monkeys in both enzyme sources of liver microsomes and recombinant UGT1A1s. 7-HFC and E-3OH glucuronidation by human liver microsomes exhibited biphasic and sigmoidal kinetics, respectively, whereas the kinetics by cynomolgus monkey liver microsomes fitted the typical Michaelis–Menten model. SN-38 glucuronidation by human and cynomolgus monkey liver microsomes exhibited autoactivation kinetics. In recombinant UGT1A1 enzymes expressed in insect cells, the kinetics of 7-HFC, E-3OH and SN-38 glucuronidation fitted the substrate inhibition (7-HFC glucuronidation) or Hill equation (E-3OH and SN-38 glucuronidation), and each glucuronidation showed the same kinetic profile between humans and cynomolgus monkeys. Significance These findings suggest that the enzymatic properties of human and cynomolgus monkey UGT1A1 enzymes are very similar.

Abstract 5783: In vitro ADME properties of ARQ 621: A specific Eg5 inhibitor

Abstract ARQ 621 is a potent and selective allosteric inhibitor of Eg5, a microtubule-based ATPase motor protein involved in cell division. Eg5 inhibition is recognized as a potential therapeutic strategy in cancer, supported by the observation that over-expression of Eg5 causes genomic instability and tumor formation in mice. ARQ 621 is currently being tested in a Phase I clinical trial in cancer patients. Prior to its entry into the clinic, the in vitro ADME properties of ARQ 621 were studied. Data from these studies showed that ARQ 621 had a t1/2 of 53 min in human liver microsomes. The t1/2 of ARQ 621 in male and female mouse, rat, dog and monkey liver microsomes was similar to that of human liver microsomes with t1/2 values of 43, 53, 56, 53, 47, 44, 36, and 32 minutes, respectively. Consistent with the microsomal stability data, in vitro metabolic studies conducted with individual human CYP P450 isoforms indicated that ARQ 621 was relatively stable with t1/2 values all greater than 27 min. IC50 values of ARQ 621 were measured for CYP 1A2, 2C9, 2D6, 3A4, 2C19, and 2C8 and determined to be &amp;gt;20, &amp;gt;20, &amp;gt;20, 4.1, 4.0, and 15 µM, respectively. ARQ 621 was found to modestly induce 1A2 but not 2A6 or 3A4. Data from in vitro Caco 2 studies demonstrated that ARQ 621 has poor GI absorption potential with a Papp value of 0.69 × 10−6 cm/s. Additionally, Caco 2 bi-directional experiments suggested that ARQ 621 may be a P-glycoprotein substrate with an efflux ratio of 45. These data are consistent with rat in vivo oral bioavailability studies in which the bioavailability of ARQ 621 was determined to be approximately 9%. Hence, ARQ 621 was subsequently developed for IV administration only. Protein binding studies conducted in human plasma showed that ARQ 621 is highly bound (∼96.4-99.2%) to plasma proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5783.

Pharmacokinetics and interspecies scaling of a novel VEGF receptor inhibitor, SU5416.

The pharmacokinetics and allometric relationships of SU5416, a novel small anti-angiogenesis agent, were studied. The pharmacokinetics of SU5416 were examined in mice, rats, dogs, and cancer patients. The in-vitro intrinsic clearance (CLint) was estimated from the in-vitro metabolism study in mouse, rat, dog, monkey and human liver microsomes. The parameters of interest were correlated across species as a function of bodyweight using an allometric approach. The steady-state volume of distribution (Vd(ss)), plasma clearance (CLs), and CLint of SU5416 were well correlated across species. The exponent of the allometric relationship (b) of the corresponding parameters was 0.92, 0.80 and 0.66, respectively. The elimination half-life (t1/2) was consistent across species and independent of bodyweight. The prediction of CLs, Vd(ss), CLint, and t1/2 in humans using the data from mouse, rat, and dog, and monkey (for CLint) was reasonably good (within 4-fold of the observed values). However, an improved prediction (within 2-fold of the observed values) of the corresponding parameters in humans was obtained when extrapolation from only the rodent data was performed, suggesting that the rodent data are sufficient for the scale-up of SU5416 pharmacokinetic parameters in humans. Using allometry, it was possible to achieve reasonable predictions of the pharmacokinetic parameters of SU5416 in cancer patients with various solid tumours.

In Vitro Profiling and Mass Balance of the Anti-Cancer Agent Laromustine [14C]-VNP40101M by Rat, Dog, Monkey and Human Liver Microsomes

Laromustine (VNP40101M; 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino) carbonylhydrazine) is a novel sulfonylhydrazine alkylating agent. For the first time In vitro profiling and mass balance of [14C] VNP40101M in rat, dog, monkey and human liver microsomes was investigated. Also, the role of human cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) enzymes in the conversion of [14C] VNP40101M by NADPH-fortified human liver microsomes was determined. In this study, [14C]-VNP40101M was converted to five radioactive components (C-1, C-2, C-3, C-4 and C-7) after 60 min of incubation with dog, monkey and human liver microsomes. With the exception of C-3, the same components were detected with rat liver microsomes. In the presence of NADPH, after 60 min of incubation, the loss of substrate for rat, dog, monkey and human was 63, 82, 76 and 64%, respectively and mass balance ranged from 91.0 – 99.3%. In the absence of NADPH, after 60 min of incubation with [14C] VNP40101M (100 µM), the loss of substrate for rat, dog, monkey and human liver microsomes was 59, 53, 61 and 59%, respectively and mass balance ranged from 100.6 – 116.4%. The profiles of metabolites were similar. The relative abundance of individual metabolites was not species dependent. The formation of C-7 was not observed in zero cofactor (no NADPH) or zero-protein samples, suggesting that its formation was enzymatic. The formation of C-1, C-2, C-3, and C-4 increased with respect to incubation time. Using a panel of CYP enzymes including rCYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4, it was shown that C-7 formation was catalyzed by CYP2B6 and CYP3A4/5. The results of this study suggest that (1) P450 plays a role in C-7 formation but plays little or no role in the conversion of [14C] VNP40101M to C-1 through C-4, and (2) the relative abundance of individual degradation/metabolite products were not species dependent. These findings provide a comprehensive understanding of the metabolism of this new agent.

Comparison of Cytochrome P450 3A Enzymes in Cynomolgus Monkeys and Humans

Drug metabolizing activities of cytochromes P450 (P450s, or CYPs) 3A4 and 3A5 in liver microsomes from the cynomolgus monkey [Macaca fascicularis (mf)] were investigated and compared with those of human P450 3A enzymes. Low activities for dealkylation of ethoxyresorufin and pentoxyresorufin were seen in recombinant monkey mfCYP3A4 and mfCYP3A5 and in recombinant human CYP3A4 and CYP3A5 expressed in bacterial membranes. Hydroxylation activities of mfCYP3A4 and mfCYP3A5 toward coumarin, paclitaxel, diclofenac, flurbiprofen, and S-mephenytoin were below detectable levels, as was also true for CYP3A4 and CYP3A5. Monkey mfCYP3A5 and mfCYP3A4 were highly active in bufuralol 1'-hydroxylation. mfCYP3A5 was efficient at dextromethorphan O-demethylation, although human CYP3A5 was unable to catalyze this reaction. Apparent bufuralol 1'-hydroxylation and dextromethorphan O-demethylation activities of monkey liver microsomes were higher than those of human liver microsomes, possibly because of contributions of mfCYP3A5 to these P450 2D-dependent drug oxidations. mfCYP3A5 and CYP3A5 catalyzed midazolam 1'-hydroxylation at a low substrate concentration more efficiently than the corresponding CYP3A4. mfCYP3A5 had higher testosterone 6beta-hydroxylase activity than mfCYP3A4, but the reverse relationship was observed in oxidation of nifedipine and hydroxylation of dexamethasone. These results demonstrate that monkey P450 3A enzymes have similar substrate selectivity to that of human P450 3A enzymes, but exhibit wider substrate selectivity toward P450 2D substrates.