Cynomolgus Monkey Liver Microsomes Research Articles

Deciphering the species differences in CES1A-mediated hydrolytic metabolism by using a bioluminescence substrate

Carboxylesterases 1A (CES1A) is a key enzyme responsible for the hydrolytic metabolism of a great deal of endogenous and exogenous substrates bearing ester- or amide-bond(s). This study aimed to decipher the species difference in CES1A-mediated hydrolytic metabolism by using a newly developed bioluminescence CES1A sensor (termed NLMe) as the probe substrate, while the liver microsomes from six different mammalian species (human, cynomolgus monkey, dog, minipig, rat and mouse) were used as the enzyme sources. Metabolite profiling demonstrated that all tested liver microsomes from various species could catalyze NLMe hydrolysis, but significant difference in hydrolytic rate was observed. Kinetic plots of NLMe hydrolysis in liver microsomes from different species showed that the inherent clearance rates (Clint) of NLMe in human liver microsomes (HLM), cynomolgus monkey liver microsomes (CyLM), and pig liver microsome (PLM) were comparable, while the Clint values of NLMe in dog liver microsomes (DLM), mouse liver microsomes (MLM), and rat liver microsomes (RLM) were relatively small. Moreover, chemical inhibition assays showed that NLMe hydrolysis in all tested liver microsomes could be competently inhibited by BNPP (a potent broad-spectrum inhibitor of CES), but CUA (a selective inhibitor of human CES1A) only inhibited NLMe hydrolysis in human liver microsomes and dog liver microsomes. In summary, the species differences in CES1A-catalyzed NLMe hydrolysis were carefully investigated from the views of the similarities in metabolite profile, hydrolytic kinetics and inhibitor response. All these findings provide new insights into the species differences in CES1A-mediated hydrolytic metabolism and suggest that it is necessary for the pharmacologists to choose appropriate animal models to replace humans for evaluating the in vivo effects of CES1A inhibitors.

Hydrolase activities of cynomolgus monkey liver microsomes and recombinant CES1, CES2, and AADAC

The cynomolgus monkey is a nonhuman primate that is often used for pharmacokinetic and toxicokinetic studies of new chemical entities. Species differences in drug metabolism are obstacles for the extrapolation of animal data to humans. This study aimed to characterize hydrolase activities for typical compounds by cynomolgus monkey liver microsomes and recombinant monkey carboxylesterases (CES1 and CES2) and arylacetamide deacetylase (AADAC) compared with the activities in humans. To estimate the contribution of each hydrolase, the ratios of the expression level of each hydrolase in the liver microsomes and recombinant systems were used. For almost all of the tested human CES1 substrates, hydrolase activities in cynomolgus monkey liver microsomes tended to be lower than those in human liver microsomes, and recombinant cynomolgus monkey CES1 showed catalytic activity, but not for all substrates. For human CES2 substrates, hydrolase activities in cynomolgus monkey liver were higher than those in human liver microsomes, and recombinant monkey CES2 was responsible for their hydrolysis. Among human AADAC substrates, phenacetin was mainly hydrolyzed by monkey AADAC, whereas indiplon and ketoconazole were hydrolyzed by AADAC and other unknown enzymes. Flutamide was hydrolyzed by monkey CES2, not by AADAC. Rifamycins were hardly hydrolyzed in monkey liver microsomes. In conclusion, this study characterized the hydrolase activities of cynomolgus monkeys compared with those in humans. The findings would be helpful for pharmacokinetic or toxicokinetic studies of new chemical entities whose main metabolic pathway is hydrolysis.

Differences in xanthotoxin metabolites in seven mammalian liver microsomes.

Xanthotoxin, abundantly occurring in fruits, vegetables, grapefruit juice and oils, is widely used in medicine for the treatment of psoriasis and vitiligo. Xanthotoxin possesses the ability to inhibit mechanism-based cytochrome P450 (CYP450)-mediated activities in rats and mice. Furthermore, it time-dependently obstructs a number of CYP450-mediated functions in humans. CYP450 enzymes are most abundant in the liver and induce metabolic activation of numerous xenobiotic compounds. The present study aimed to identify the similarities and differences in xanthotoxin metabolism in liver microsomes of 7 mammalian species, including human liver microsomes (HLM), Rhesus monkey liver microsomes (RMLM), Cynomolgus monkey liver microsomes (CMLM), Sprague Dawley rat liver microsomes (RLM), mouse liver microsomes (MLM), Dunkin Hartley guinea pig liver microsomes (PLM) and Beagle dog liver microsomes (DLM). Ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometric analysis was used to determine the metabolites. A total of 3 metabolites were detected in RMLM, CMLM and RLM. Furthermore, two metabolites were observed in MLM, HLM, PLM and DLM. By analyzing the type and quantity of metabolites, the metabolism of xanthotoxin in MLM was indicated to be most similar to that in HLM. The metabolic transformations of xanthotoxin in the liver microsomes of the 7 species were analyzed in further detail. On the whole, the results of the present study provide a deeper understanding of the metabolic patterns of xanthotoxin in liver microsomes of different species, which may prove to be advantageous regarding the metabolic mechanisms of action of xanthotoxin. Further insight into drug metabolism with respect to different species will also aid in the selection of appropriate animal models for further research.

A Novel RAD51 Inhibitor, CYT01B, Shows Anti-Cancer Activity in Preclinical Models of Aid Expressing Lymphomas

Activation Induced Cytidine Deaminase (AID) is a DNA directed cytidine deaminase that is normally only expressed in activated B-cells to promote somatic hypermutations and immunoglobulin class switching. Unlike site-specific recombinases (e.g. RAG1/2), AID lacks target site specificity and can generate DNA damage at widespread locations throughout the genome. In cancer cells, AID expression promotes high levels of DNA replication stress, and results in dependency upon the homologous recombination factor RAD51. We have shown previously that a novel RAD51 inhibitor, CYT01B, functions by disrupting RAD51 focus formation which reduces the nuclear concentration of RAD51 and promotes RAD51 protein degradation. Here we present the in vitro and in vivo pharmacological characterization of CYT01B. We first analyzed the permeability and stability of the compound using Caco-2 and liver microsome assays. CYT01B shows low efflux and is stable with low intrinsic clearance rates (< 30 ml/min/mg protein) in mouse, rat, dog, and human, but not in cynomolgus monkey liver microsomes. In vivo, CYT01B showed oral bioavailability that correlated well with Caco-2 permeability (36.9% in monkeys up to 86.5% in rats). Additionally, CYT01B exhibited a minimum half-life of 4 hours in all species tested (mouse, rat, dog, and monkey). We then examined off target liabilities by performing kinome inhibition and Panlabs safety panel screens. CYT01B showed negligible (<50%) inhibition across 371 kinases tested, while only two targets showed greater than 50% inhibition in the Panlabs panel. CYT01B displayed activity in several human-to-mouse cell line xenograft models. In a systemic model of chronic lymphoblastic leukemia (CLL), the tumor burden in the bone marrow of CYT01B-treated mice was reduced by approximately 30%. In two different subcutaneous engraftment models of either a Burkitt's lymphoma cell line (Daudi) or an acute lymphoblastic leukemia cell line (CCRF-SB), mice treated with CYT01B showed tumor growth inhibition of 49% and 88%, respectively. Lastly, CYT01B was well tolerated in rat 7-day dose range finding studies. Rats were treated with 20, 80, and 240 mg/kg once per day by oral gavage. There were no observed changes in hematology or clinical chemistry, and no observed histopathological toxicities. Taken together, these data demonstrate that CYT01B is a cell permeable, metabolically stable, and orally bioavailable small molecule, with anti-cancer activity in multiple preclinical lymphoid cancer models. Overall, this provides the basis for continued preclinical development of an AID/RAD51 synthetic lethal therapeutic paradigm that may be applicable to various hematologic malignancies. DisclosuresPatrick:Cyteir Therapeutics: Consultancy. Bedard:Cyteir Therapeutics: Consultancy. Vacca:Cyteir Therapeutics: Consultancy. McComas:Cyteir Therapeutics: Consultancy. Castro:Cyteir Therapeutics: Consultancy. Day:Cyteir Therapeutics: Employment. Mills:Cyteir Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Identification of cytochrome P450 isoforms involved in the metabolism of Syl930, a selective S1PR1 agonist acting as a potential therapeutic agent for autoimmune encephalitis

Syl930 is a novel sphingosine-1-phosphate receptor subtype 1 (S1PR1) agonist for the treatment of autoimmune encephalitis with promising receptor selectivity and little risk of bradycardia. Syl930 could be reversibly converted to its phosphorylated metabolite, acting as the active form to provide therapeutic effects, but eliminated principally in the form of oxidative metabolites. The aim of the present study was to identify the cytochrome P450 isoforms (CYPs) responsible for the oxidative metabolism of Syl930. Considerable production of hydroxylated metabolite (Syl930-M1) was found in both rat blood and tissue homogenates in vivo and in vitro. Moreover, another hydroxylated metabolite, Syl930-M2, was detected in human, beagle dog and cynomolgus monkey liver microsomes with significant differences in the Km, Vmax and CLint of the metabolites among species. CYP1A1, CYP2J2, CYP4F2 and CYP3A4 were identified to be the major CYPs mediated in the hydroxylation of Syl930 by using 14 recombinant human CYPs, selective chemical inhibitors and monoclonal antibodies against CYPs. The multiple CYPs mediated oxidation was believed to be one of the reasons for the relatively short elimination half-life of Syl930.

Regioselective glucuronidation of andrographolide and its major derivatives: metabolite identification, isozyme contribution, and species differences.

Andrographolide (AND) and two of its derivatives, deoxyandrographolide (DEO) and dehydroandrographolide (DEH), are widely used in clinical practice as anti-inflammatory agents. However, UDP-glucuronosyltransferase (UGT)-mediated phase II metabolism of these compounds is not fully understood. In this study, glucuronidation of AND, DEO, and DEH was characterized using liver microsomes and recombinant UGT enzymes. We isolated six glucuronides and identified them using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. We also systematically analyzed various kinetic parameters (K m, V max, and CLint) for glucuronidation of AND, DEO, and DEH. Among 12 commercially available UGT enzymes, UGT1A3, 1A4, 2B4, and 2B7 exhibited metabolic activities toward AND, DEO, and DEH. Further, UGT2B7 made the greatest contribution to glucuronidation of all three anti-inflammatory agents. Regioselective glucuronidation showed considerable species differences. 19-O-Glucuronides were present in liver microsomes from all species except rats. 3-O-Glucuronides were produced by pig and cynomolgus monkey liver microsomes for all compounds, and 3-O-glucuronide of DEH was detected in mouse and rat liver microsomes (RLM). Variations in K m values were 48.6-fold (1.93-93.6 μM) and 49.5-fold (2.01-99.1 μM) for 19-O-glucuronide and 3-O-glucuronide formation, respectively. Total intrinsic clearances (CLint) for 3-O- and 19-O-glucuronidation varied 4.8-fold (22.7-110 μL min(-1) mg(-1)), 10.6-fold (94.2-991 μL min(-1) mg(-1)), and 8.3-fold (122-1,010 μL min(-1) mg(-1)), for AND, DEH, and DEO, respectively. Our results indicate that UGT2B7 is the major UGT enzyme involved in the metabolism of AND, DEO, and DEH. Metabolic pathways in the glucuronidation of AND, DEO, and DEH showed considerable species differences.

Comparison of P450 Enzymes Between Cynomolgus Monkeys and Humans: P450 Identities, Protein Contents, Kinetic Parameters, and Potential for Inhibitory Profiles

Cynomolgus monkeys are used to predict human pharmacokinetic and/or toxic profiles in the drug developmental stage. Cynomolgus P450s exhibit a high degree of identity (more than 90%) in both cDNA and amino acid sequences with corresponding human P450s. CYP3A protein predominantly exists in cynomolgus monkey liver microsomes, followed by CYP2A, CYP2C, CYP2B6, CYP2E1, and CYP2D. There are many similarities of metabolic properties in cytochrome P450s between cynomolgus monkeys and humans, but the species differences between cynomolgus monkey and human P450s are clearly present in substrate specificity and inhibitor selectivity. Diclofenac 4'-hydroxylation (DFOH) in monkey liver and intestinal microsomes shows much lower activities compared with those in human liver and intestinal microsomes. Sulfaphenazole strongly inhibits DFOH in human liver microsomes, but does not effectively inhibit DFOH in monkey liver and intestinal microsomes. Cynomolgus CYP2C19 exhibits higher activity for DFOH than cynomolgus CYP2C9 although this reaction is a marker reaction of human CYP2C9. On the other hand, cynomolgus CYP2C76 orthologue is not expressed in humans and shows 70-72% identity in amino acid sequences of human CYP2C subfamilies. Cynomolgus CYP2C76 metabolizes non-CYP2C substrates, 7-ethoxyresorufin (human CYP1A substrate) and bufuralol (human CYP2D6 substrate). In addition, cynomolgus CYP3A4 and CYP3A5 also exhibits wider substrate selectivity toward human CYP2D6 and CYP2E1 substrates. These enzymes may be responsible for species difference in drug metabolism between cynomolgus monkeys and humans. The comparative data presented here can be helpful for designing in vivo metabolic assays using cynomolgus monkeys in terms of substrate specificity and inhibitor selectivity.

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.

Stereoselective Glucuronidation of Propranolol in Human and Cynomolgus Monkey Liver Microsomes: Role of Human Hepatic UDP-Glucuronosyltransferase Isoforms, UGT1A9, UGT2B4 and UGT2B7

The stereoselective glucuronidation of propranolol (PL) in human and cynomolgus monkey liver microsomes, and the roles of human hepatic UDP-glucuronosyltransferase (UGT) isofoms involved in the enantiomeric glucuronidation of PL using recombinant UGT enzymes were investigated. In Michaelis-Menten plots, R- and S-PL glucuronidation by human liver microsomes showed sigmoidal kinetics whereas the kinetics of enantiomeric PL glucuronidation by cynomolgus monkey liver microsomes was monophasic. The K_m, V_max and CL_int values of cynomolgus monkey liver microsomes were generally higher than the S₅₀, V_max and CL_max values of human liver microsomes in R- and S-PL glucuronidation. The glucuronidation of R- and S-PL was catalyzed by at least 3 UGT isoforms: UGT1A9, UGT2B4 and UGT2B7. Michaelis-Menten plots for R- and S-PL glucuronidation by UGT1A9 were monophasic, whereas the kinetics of UGT2B7 showed sigmoidal curves. Enantiomeric R-PL glucuronidation by UGT2B4 showed sigmoidal kinetics, whereas S-PL glucuronidation displayed monophasic kinetics. UGT1A9 showed remarkable stereoselectivity in V_max and CL_int values of R-PL < S-PL. These findings demonstrate that the profiles of enantiomeric PL glucuronidation in human and cynomolgus monkey liver microsomes are largely different and suggest that the human hepatic UGT isofoms UGT1A9, UGT2B4 and UGT2B7 play distinctive roles in enantiomeric PL glucuronidation.

Functional characterization of human and cynomolgus monkey UDP-glucuronosyltransferase 1A6 enzymes

UDP-glucuronosyltransferase 1A6 (UGT1A6) is a major isoform in the human liver that glucuronidates numerous drugs, environmental chemicals and endogenous substrates. In this study, human and cynomolgus monkey UGT1A6 cDNAs (humUGT1A6 and monUGT1A6, respectively) were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate UGT1A6s. The enzymatic properties of UGT1A6 proteins were characterized by the kinetic analysis of serotonin (5-hydroxytryptamine, 5-HT) and 4-methylumbelliferone (4-MU) glucuronidation. humUGT1A6 and monUGT1A6 showed 96% identity in their nucleotide and amino acid sequences. Immunoblotting analysis using an antibody raised against human UGT1A6 showed that protein staining intensities were different between human and cynomolgus monkey UGT1A6 enzymes in microsomal fractions from livers and yeast cells, although both enzymes were detectable. The apparent K(m) value (15 mM) for 5-HT glucuronidation of cynomolgus monkey liver microsomes was significantly higher than that (8.6mM) of human liver microsomes, whereas V(max) values were lower in cynomolgus monkeys (2.8 nmol/min/mg protein) than in humans (8.6 nmol/min/mg protein). No significant species difference was observed in K(m) (approximately 90 microM) or V(max) (approximately 25 nmol/min/mg protein) values for liver microsomal 4-MU glucuronidation. In yeast cell microsomes, K(m) values (approximately 6mM) for 5-HT glucuronidation by recombinant UGT1A6s were similar, while a V(max) value (0.1nmol/min/mg protein) of monUGT1A6 was significantly lower than that (0.7 nmol/min/mg protein) of humUGT1A6. In 4-MU glucuronidation, both K(m) (210 microM) and V(max) (3.5 nmol/min/mg protein) values of monUGT1A6 were significantly higher than those of humUGT1A6 (K(m), 110 microM; V(max), 1.5nmol/min/mg protein). These findings suggest that the enzymatic properties of UGT1A6 were extensively different between humans and cynomolgus monkeys, although humUGT1A6 and monUGT1A6 showed high homology at the amino acid level. The information gained in this study should help with in vivo extrapolation and to assess the toxicity of xenobiotics.