Species Differences In Metabolism Research Articles

Acceleration of infectious disease drug discovery and development using a humanized model of drug metabolism

A key step in drug discovery, common to many disease areas, is preclinical demonstration of efficacy in a mouse model of disease. However, this demonstration and its translation to the clinic can be impeded by mouse-specific pathways of drug metabolism. Here, we show that a mouse line extensively humanized for the cytochrome P450 gene superfamily ("8HUM") can circumvent these problems. The pharmacokinetics, metabolite profiles, and magnitude of drug-drug interactions of a test set of approved medicines were in much closer alignment with clinical observations than in wild-type mice. Infection with Mycobacterium tuberculosis, Leishmania donovani, and Trypanosoma cruzi was well tolerated in 8HUM, permitting efficacy assessment. During such assessments, mouse-specific metabolic liabilities were bypassed while the impact of clinically relevant active metabolites and DDI on efficacy were well captured. Removal of species differences in metabolism by replacement of wild-type mice with 8HUM therefore reduces compound attrition while improving clinical translation, accelerating drug discovery.

Mass Balance of the IDO Inhibitor Navoximod (GDC-0919) in Rats and Dogs: Unexpected Cyanide Release from Imidazo[5,1-a]isoindole and Species Differences in Glucuronidation.

Navoximod (GDC-0919) is a small molecule inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1), developed to reduce T cell immunosuppression associated with cancer. This study describes the absorption, metabolism, and excretion (AME) of navoximod in rats and dogs following a single oral dose of [14C]-navoximod. An unexpected thiocyanate metabolite M1 and a chiral inversion metabolite M51 were captured as the major circulating metabolites in rats, accounting for 30% and 18% of 0-24 hr exposure, respectively. These two metabolites combined had much lower systemic exposure in dogs and humans (<6% and <1%). The novel cyanide release is proposed to occur via 4,5-epoxidation on the fused imidazole ring, leading to ring opening and rearrangement along with the release of cyanide. The decyanated metabolites were identified and confirmed by synthetic standards, which supported the proposed mechanism. In dogs, glucuronidation to M19 was the major clearance mechanism, representing 59% of the dose in the bile of bile duct-cannulated (BDC) dogs and 19% of the dose in the urine of intact dogs. Additionally, M19 also represented 52% of drug related exposure in circulation in dogs. In comparison, in humans, navoximod was mainly cleared through glucuronidation to M28 and excreted in urine (60% of the dose). The differences in the metabolism and elimination observed in vivo were qualitatively recapitulated in vitro with liver microsomes, suspended hepatocytes and co-cultured primary hepatocytes. The striking species differences in regioselective glucuronidation is likely explained by the species differences in UGT1A9, which was mainly responsible for M28 formation in humans. Significance Statement The results from this study demonstrated significant species differences in metabolism (especially glucuronidation) and elimination of navoximod among rats, dogs, and humans. The study also illustrated the mechanism of a novel cyanide release metabolism from the fused imidazo[5,1-a]isoindole ring. Such biotransformation should be kept in mind when working with imidazole containing new chemical entities (NCE) in drug discovery and development.

Preclinical safety assessment of JNJ-10450232 (NTM-006), a structural analog of acetaminophen, that does not cause hepatotoxicity at supratherapeutic doses

JNJ-10450232 (NTM-006) is a new molecular entity that is structurally related to acetaminophen. A comprehensive non-clinical safety program was conducted to support first-in-human and clinical efficacy studies based on preclinical data suggesting that the compound has comparable or enhanced antinociceptive and antipyretic efficacy without causing hepatotoxicity at supratherapeutic doses. No hepatic toxicity was noted in a mouse model sensitive to acetaminophen hepatotoxicity or in rats, dogs, and non-human primates in 28-day repeat dose toxicity studies at and above doses/exposures at which acetaminophen is known to cause hepatotoxicity. In the 28-day toxicity studies, all treatment-related findings were monitorable and reversible. Methemoglobinemia, which was observed in dogs and to a lesser extent in rats, is also observed with acetaminophen. This finding is considered not relevant to humans due to species differences in metabolism. Thyroid hypertrophy and hyperplasia were also observed in dogs and were shown to be a consequence of a species-specific UGT induction also demonstrated with increased thyroid hormone metabolism. Indirect bilirubin elevation was observed in rats as a result of UGT1A1 Inhibition. JNJ-10450232 (NTM-006) had no toxicologically relevant findings in safety pharmacology or genotoxicity studies. Together, these data supported progressing into safety and efficacy studies in humans.

Use of biomarker data and metabolite relative potencies to support derivation of noncancer reference values based on the reproductive and developmental toxicity effects of 1,3-butadiene

Subchronic and chronic reference values (RfVs) were derived for 1,3-butadiene (BD) based upon its ability to cause reproductive and developmental effects observed in laboratory mice and rats. Metabolism has been well-established as an important determinant of the toxicity of BD. A major challenge to human health risk assessment is presented by large quantitative species differences in the metabolism of BD, differences that should be accounted for when the rodent toxicity responses are extrapolated to humans. The methods of Fred et al. (2008)/Motwani and Törnqvist (2014) were extended and applied here to the noncancer risk assessment of using data-derived extrapolation factors to account for species differences in metabolism, as well as differences in cytotoxic potency of three BD metabolites. This approach made use of biomarker data (hemoglobin adducts) to quantify species differences in the internal doses of BD metabolites experienced in mice, rats and humans. Using these methods, the dose-response relationships in mice and rats exhibit improved concordance, and result in subchronic and chronic inhalation reference values of 29 and 10 ppm, respectively, for BD. Confidence in these reference values is considered high, based on high confidence in the key studies, medium-to-high confidence in the toxicity database, high confidence in the estimates of internal dose, and high confidence in the dose-response modeling.

Use of Biomarker Data and Relative Potencies of Mutagenic Metabolites to Support Derivation of Cancer Unit Risk Values for 1,3-Butadiene from Rodent Tumor Data.

Unit Risk (UR) values were derived for 1,3-butadiene (BD) based upon its ability to cause tumors in laboratory mice and rats. Metabolism has been established as the significant molecular initiating event of BD’s carcinogenicity. The large quantitative species differences in the metabolism of BD and potency of critical BD epoxide metabolites must be accounted for when rodent toxicity responses are extrapolated to humans. Previously published methods were extended and applied to cancer risk assessments to account for species differences in metabolism, as well as differences in mutagenic potency of BD metabolites within the context of data-derived adjustment factors (DDEFs). This approach made use of biomarker data (hemoglobin adducts) to quantify species differences in the internal doses of BD metabolites experienced in mice, rats, and humans. Using these methods, the dose–response relationships in mice and rats exhibit improved concordance, and result in upper bound UR values ranging from 2.1 × 10−5 to 1.2 × 10−3 ppm−1 for BD. Confidence in these UR values was considered high based on high confidence in the key studies, medium-to-high confidence in the toxicity database, high confidence in the estimates of internal dose, and high confidence in the dose–response modeling.

Species-dependent hepatic and intestinal metabolism of selective estrogen receptor degrader LSZ102 by sulfation and glucuronidation

1. LSZ102 is an orally bioavailable selective estrogen receptor degrader in clinical development for the treatment of breast cancer. Preclinical studies showed efficacy in xenograft models on oral dosing. However, oral bioavailability was relatively low in several preclinical species (7-33%), and was associated with first-pass metabolism, particularly intestinal first-pass. 2. To investigate metabolism and first-pass effects, metabolites were analysed in human plasma samples after oral dosing of LSZ102 to patients, rat plasma samples after oral dosing of [14C]LSZ102, and in vitro incubations of [14C]LSZ102 with human and rat hepatocytes and intestinal S9 fractions. The kinetics of human sulfotransferase enzymes potentially involved in metabolism of LSZ102 were characterized. 3. Sulfate metabolites were found to be the major components in human plasma, as well as in human hepatocytes and intestinal S9 fractions. Contrastingly, glucuronidation was predominant in rat plasma, hepatocytes and intestinal S9. LSZ102 was found to be metabolized by several human sulfotransferases expressed in liver and intestine. The combined metabolism data in rat and human provide supporting evidence for an extensive intestinal first-pass metabolism effect via sulfation in human but glucuronidation in rat. 4. As LSZ102 is metabolized by a number of different sulfotransferases, drug-drug interactions resulting from the inhibition of one sulfotransferase are unlikely. 5. Despite the observed species difference in metabolism, the major human metabolites of LSZ102, sulfate M5, glucuronide M4, and secondary glucuronide/sulfate metabolite M12, have no or weak pharmacological activity and are not considered a toxicity risk as they are phase II conjugative metabolites.

Abstract 10979: Liver-Humanized Mice Provide a New Platform to Study Human Cardiometabolic Diseases

Introduction: Conventional mouse models do not fully recapitulate human cardiometabolic diseases (CMD), e.g. , atherosclerosis and nonalcoholic steatohepatitis (NASH), due to species differences in metabolism. Liver-humanized mice (LHM), i.e. , mice repopulated with human hepatocytes, have been shown to be an improved translatable model to study human hepatic and lipoprotein metabolism. Hypothesis: As dietary challenge is often used to induce CMD in animal models, we sought to characterize lipoprotein and lipid metabolism in LHM challenged by different diets. Methods: Immune-deficient FRGN mice receiving hepatocytes from different human donors (LHM) or NOD mouse (liver-murinized mice, LMM) were fed a high-fat/high-sucrose diet (HFHSD) for 8 weeks or a high-fat/high-fructose/high-cholesterol diet (NASH-diet) for 8, 12, 16 or 20 weeks. Plasma lipids and lipoproteins were quantified after size-exclusion chromatography separation or by routine clinical assays. Cholesterol in the descending aorta was analyzed by GC-MS. Histologic analysis of hearts or livers was done using hematoxylin/eosin or Oil Red O staining, and anti-mouse CD68-antibody was used to detect murine macrophages. Results: Compared with LMM, LHM fed either HFHSD or NASH-diet accumulated plasma cholesterol and triglycerides within LDL and triglyceride-rich particles (VLDL and remnants). However, only the NASH-diet induced a notable hyperlipidemia and a substantial increase in cholesteryl esters within the aorta. NASH-diet also determined a clear accumulation of neutral lipids and CD68-positive cells in the aortic valves of LHM. Similarly, hepatocyte ballooning, liver steatosis and fibrosis were also induced by the NASH-diet. All these pathologic changes occurred during diet challenge at different time points. Conclusions: LHM developed severe hyperlipidemia, atherosclerosis and NASH when fed a NASH-diet, but not on HFHSD. The high quantity of cholesterol in the NASH-diet likely compensates for the high utilization of cholesterol of LHM due to their known increase in bile acid synthesis, prompting dyslipidemia and CMD. LHM fed a NASH-diet is an improved model for human CMD and for the development of therapeutic strategies rapidly translatable to the human condition.

Species Differences in Metabolism of Soluble Epoxide Hydrolase Inhibitor, EC1728, Highlight the Importance of Clinically Relevant Screening Mechanisms in Drug Development.

There are few novel therapeutic options available for companion animals, and medications rely heavily on repurposed drugs developed for other species. Considering the diversity of species and breeds in companion animal medicine, comprehensive PK exposures in the companion animal patient is often lacking. The purpose of this paper was to assess the pharmacokinetics after oral and intravenous dosing in domesticated animal species (dogs, cats, and horses) of a novel soluble epoxide hydrolase inhibitor, EC1728, being developed for the treatment of pain in animals. Results: Intravenous and oral administration revealed that bioavailability was similar for dogs, and horses (42 and 50% F) but lower in mice and cats (34 and 8%, respectively). Additionally, clearance was similar between cats and mice, but >2× faster in cats vs. dogs and horses. Efficacy with EC1728 has been demonstrated in mice, dogs, and horses, and despite the rapid clearance of EC1728 in cats, analgesic efficacy was demonstrated in an acute pain model after intravenous but not oral dosing. Conclusion: These results demonstrate that exposures across species can vary, and investigation of therapeutic exposures in target species is needed to provide adequate care that addresses efficacy and avoids toxicity.

Weight of evidence approach using a TK gene mutation assay with human TK6 cells for follow-up of positive results in Ames tests: a collaborative study by MMS/JEMS

BackgroundConflicting results between bacterial mutagenicity tests (the Ames test) and mammalian carcinogenicity tests might be due to species differences in metabolism, genome structure, and DNA repair systems. Mutagenicity assays using human cells are thought to be an advantage as follow-up studies for positive results in Ames tests. In this collaborative study, a thymidine kinase gene mutation study (TK6 assay) using human lymphoblastoid TK6 cells, established in OECD TG490, was used to examine 10 chemicals that have conflicting results in mutagenicity studies (a positive Ames test and a negative result in rodent carcinogenicity studies).ResultsTwo of 10 test substances were negative in the overall judgment (20% effective as a follow-up test). Three of these eight positive substances were negative after the short-term treatment and positive after the 24 h treatment, despite identical treatment conditions without S9. A toxicoproteomic analysis of TK6 cells treated with 4-nitroanthranilic acid was thus used to aid the interpretation of the test results. This analysis using differentially expressed proteins after the 24 h treatment indicated that in vitro specific oxidative stress is involved in false positive response in the TK6 assay.ConclusionsThe usefulness of the TK6 assay, by current methods that have not been combined with new technologies such as proteomics, was found to be limited as a follow-up test, although it still may help to reduce some false positive results (20%) in Ames tests. Thus, the combination analysis with toxicoproteomics may be useful for interpreting false positive results raised by 24 h specific reactions in the assay, resulting in the more reduction (> 20%) of false positives in Ames test.

Characterization of the metabolites of trans-resveratrol-3-O-glucoside in monkeys and dogs

In this study, a liquid chromatography-tandem multi-stage mass spectrometry (LC/MSn) method was established to characterize the metabolites of TRG in monkeys and dogs. A total of seven metabolites of TRG besides the prototype were found, which were identified as TR (M1), TRN (M2), trans-resveratrol-4'-O-glucuronide (M2'), trans-resveratrol-3-O-glucoside-4'-O-glucuronide (M3), trans-resveratrol-3-O-glucoside-5-O-glucuronide (M3'), trans-resveratrol-3-sulfate (M4) and trans-resveratrol-4'-sulfate (M4'). Additionally, the metabolic pathways of TRG in monkeys and dogs were proposed. There were also species differences of metabolism of TRG between monkeys and dogs.

Integrated Array Chip for High-Throughput Screening of Species Differences in Metabolism.

Species differences in metabolism may produce failure prediction of drug efficacy/toxicity in humans. Integration of metabolic competence and cellular effect assays in vitro can provide insight into the species differences in metabolism; however, a co-culture platform with features of high throughput, operational simplicity, low sample consumption, and independent layouts is required for potential usage in industrial test settings. Herein, we developed an integrated array chip (IAC) to evaluate the species differences in metabolism through metabolism-induced anticancer bioactivity as a case. The IAC consisted of two functional parts: a micropillar chip for immobilization of liver microsomes and a microwell chip for three-dimensional (3D) tumor cell culture. First, optimized parameters of the micropillar chip for microsomal encapsulation were obtained by cross-shaped protrusions and a 2.5 μL volume of 3D agarose spots. Next, we examined factors influencing metabolism-induced anticancer bioactivity. Feasibility of the IAC was validated by four model prodrugs using image-based bioactivity detection and mass spectrometry (MS)-based metabolite analysis. Finally, a species-specific IAC was used for selection of animal species that best resembles metabolism-induced drug response to humans at throughputs. Overall, the IAC provides a promising co-culture platform for identifying species differences in metabolism and selection of animal models to accelerate drug discovery.

S11.4 - Management of mist in a case of multiple metabolic species differences

S11.4 - Management of mist in a case of multiple metabolic species differences

In vitro Drug Metabolism Investigation of 7-Ethoxycoumarin in Human, Monkey, Dog and Rat Hepatocytes by High Resolution LC-MS/MS.

Background: Recently, it has been an increasing concern on the bioactivation and adverse re-actions associated with consumption of herbal and nature products. 7-Ethoxycoumarin is one of coumarin family compounds, but little information is available regarding its potential reactive metabolites.Method: 7-ethoxylcoumarin was incubated individually with human, monkey, dog and rat hepatocytes for 2 hr, metabolites were detected, identified and characterized using high resolution liquid chromagraphy – tandem mass spectrometry.Results: Twenty-eight metabolites (M1 - M28) were detected and identified. O-deethylation, glucuronida-tion, sulfation, oxygenation, oxidative ring-opening, hydrogenation, glutathionation, dehydrogenation, cysteination, glucosidation, methylation, and hydrolysis were observed. At least sixteen metabolites not reported previously, were newly identified. M1 (O-deethylation, mono-oxygenation and glucuronidation), M3 (O-deethylation and glucuronidation), M5 (hydrolysis and mono-oxygenation), M14 (O-deethylation), M16 (hydrolysis), M22 (oxidative ring-opening and oxygenation) and M27 (mono-oxygenation) exhibited high mass spectrometric responses in human hepatocytes. M3, M5, M8, M13 (mono-oxygenation), M14, M16, M18 (O-deethylation and sulfation), M22 and M27 exhibited high mass spectrometric responses in monkey hepatocytes. M14, M16, M18, M20 (glutathionation and dehy-drogenation) and M27 exhibited high mass spectrometric responses in dog hepatocytes. M1 (O-deethylation, mono-oxygenation and glucuronidation), M3, M5, M13, M14, M16, M17 (cysteination), M18, M20, and M22 exhibited high mass spectrometric responses in rat hepatocytes.Conclusion: Most of new metabolites via oxidative ring-opening and glutathionation were identified. Species differences in metabolism of 7-ethoxylcoumarin in hepatocytes were observed. The analysis of metabolites suggests that 7-ethoxylcoumarin may undergo 3,4-epoxidation responsible for formation of glutathione and its derived cysteine conjugates, carboxylic acid and its glucuronides, glucosides and sul-fate.

Embryo-fetal development studies with the dietary supplement vinpocetine in the rat and rabbit.

Dietary supplement and natural product use is increasing within the United States, resulting in growing concern for exposure in vulnerable populations, including young adults and women of child-bearing potential. Vinpocetine is a semisynthetic derivative of the Vinca minor extract, vincamine. Human exposure to vinpocetine occurs through its use as a dietary supplement for its purported nootropic and neuroprotective effects. To investigate the effects of vinpocetine on embryo-fetal development, groups of 25 pregnant Sprague-Dawley rats and 8 pregnant New Zealand White rabbits were orally administered 0, 5, 20, or 60 mg vinpocetine/kg and 0, 25, 75, 150, or 300 mg/kg daily from gestational day (GD) 6-20 and GD 7-28, respectively. Pregnant rats dosed with vinpocetine demonstrated dose-dependent increases in postimplantation loss, higher frequency of early and total resorptions, lower fetal body weights, and fewer live fetuses following administration of 60 mg/kg, in the absence of maternal toxicity. Additionally, the rat fetuses displayed dose-dependent increases in the incidences of ventricular septum defects and full supernumerary thoracolumbar ribs. Similarly, albeit at higher doses than the rats, pregnant rabbits administered vinpocetine displayed an increase in postimplantation loss and fewer live fetuses (300 mg/kg), in addition to significantly lower fetal body weights (≥75 mg/kg). In conclusion, vinpocetine exposure resulted in similar effects on embryo-fetal development in the rat and rabbit. The species differences in sensitivity and magnitude of response is likely attributable to a species difference in metabolism. Taken together, these data suggest a potential hazard for pregnant women who may be taking vinpocetine.

Pooled human liver preparations, HepaRG, or HepG2 cell lines for metabolism studies of new psychoactive substances? A study using MDMA, MDBD, butylone, MDPPP, MDPV, MDPB, 5-MAPB, and 5-API as examples

Metabolism studies play an important role in clinical and forensic toxicology. Because of potential species differences in metabolism, human samples are best suitable for elucidating metabolism. However, in the case of new psychoactive substances (NPS), human samples of controlled studies are not available. Primary human hepatocytes have been described as gold standard for in vitro metabolism studies, but there are some disadvantages such as high costs, limited availability, and variability of metabolic enzymes. Therefore, the aim of our study was to investigate and compare the metabolism of six methylenedioxy derivatives (MDMA, MDBD, butylone, MDPPP, MDPV, MDPB) and two bioisosteric analogues (5-MAPB, 5-API) using pooled human liver microsomes (pHLM) combined with cytosol (pHLC) or pooled human liver S9 fraction (pS9) all after addition of co-substrates for six phase I and II reactions. In addition, HepaRG and HepG2 cell lines were used. Results of the different in vitro tools were compared to each other, to corresponding published data, and to metabolites identified in human urine after consumption of MDMA, MDPV, or 5-MAPB. Incubations with pHLM plus pHLC showed similar results as pS9. A more cost efficient model for prediction of targets for toxicological screening procedures in human urine should be identified. As expected, the incubations with HepaRG provided better results than those with HepG2 concerning number and signal abundance of the metabolites. Due to easy handling without special equipment, incubations with pooled liver preparations should be the most suitable alternative to find targets for toxicological screening procedures for methylenedioxy derivatives and bioisosteric analogues.

Meeting the Vitamin A Requirement: The Efficacy and Importance ofβ-Carotene in Animal Species

Vitamin A is essential for life in all vertebrate animals. Vitamin A requirement can be met from dietary preformed vitamin A or provitamin A carotenoids, the most important of which is β-carotene. The metabolism of β-carotene, including its intestinal absorption, accumulation in tissues, and conversion to vitamin A, varies widely across animal species and determines the role that β-carotene plays in meeting vitamin A requirement. This review begins with a brief discussion of vitamin A, with an emphasis on species differences in metabolism. A more detailed discussion of β-carotene follows, with a focus on factors impacting bioavailability and its conversion to vitamin A. Finally, the literature on how animals utilize β-carotene is reviewed individually for several species and classes of animals. We conclude that β-carotene conversion to vitamin A is variable and dependent on a number of factors, which are important to consider in the formulation and assessment of diets. Omnivores and herbivores are more efficient at converting β-carotene to vitamin A than carnivores. Absorption and accumulation of β-carotene in tissues vary with species and are poorly understood. More comparative and mechanistic studies are required in this area to improve the understanding of β-carotene metabolism.

Species differences in metabolism of ripasudil (K-115) are attributed to aldehyde oxidase

1. We examined the metabolism of ripasudil (K-115), a selective and potent Rho-associated coiled coil-containing protein kinase (ROCK) inhibitor, by in vitro and in vivo studies.2. First, we identified metabolites and metabolic enzymes involved in ripasudil metabolism. Species differences were observed in metabolic clearance and profiles of metabolites in liver S9 fraction and hepatocytes. In addition, ripasudil was metabolised in humans and monkey S9 without nicotinamide adenine dinucleotide phosphate (NADPH). Studies using specific inhibitors and human recombinant enzyme systems showed that M1 (main metabolite in humans) formation is mediated by aldehyde oxidase (AO).3. Therefore, we developed ripasudil as an ophthalmic agent. First, we compared the pharmacokinetic profiles of ripasudil in humans and rats. The results indicated rapid disappearance of ripasudil from the circulation after instillation in humans and its level remained relatively high only in M1. In contrast, we found six metabolites from M1 to M6 in plasma after oral administration to rats.4. Analysis of enzyme kinetics using S9 showed that the formation of M1 is the major metabolic pathway of ripasudil in humans even though CYP3A4/3A5 and CYP2C8/3A4/3A5 were associated with the formation of M2 and M4, respectively. In conclusion, AO causes differences in ripasudil metabolism between species.

Species differences in metabolism of EPZ015666, an oxetane-containing protein arginine methyltransferase-5 (PRMT5) inhibitor

1. Metabolite profiling and identification studies were conducted to understand the cross-species differences in the metabolic clearance of EPZ015666, a first-in-class protein arginine methyltransferase-5 (PRMT5) inhibitor, with anti-proliferative effects in preclinical models of Mantle Cell Lymphoma. EPZ015666 exhibited low clearance in human, mouse and rat liver microsomes, in part by introduction of a 3-substituted oxetane ring on the molecule. In contrast, a higher clearance was observed in dog liver microsomes (DLM) that translated to a higher in vivo clearance in dog compared with rodent.2. Structure elucidation via high resolution, accurate mass LC-MSn revealed that the prominent metabolites of EPZ015666 were present in hepatocytes from all species, with the highest turnover rate in dogs. M1 and M2 resulted from oxidative oxetane ring scission, whereas M3 resulted from loss of the oxetane ring via an N-dealkylation reaction.3. The formation of M1 and M2 in DLM was significantly abrogated in the presence of the specific CYP2D inhibitor, quinidine, and to a lesser extent by the CYP3A inhibitor, ketoconazole, corroborating data from human recombinant isozymes.4. Our data indicate a marked species difference in the metabolism of the PRMT5 inhibitor EPZ015666, with oxetane ring scission the predominant metabolic pathway in dog mediated largely by CYP2D.

In Vitro Drug Metabolism of Green Tea Catechins in Human, Monkey, Dog, Rat and Mouse Hepatocytes

The metabolic fate of green tea catechins [(-)-epicatechin ((-)-EC), (-)-epicatechin-3-gallate (ECG) (-)- epigallocatechin (EGC) and (-)-epigallocatechin-3-gallate (EGCG)] in cryopreserved human, monkey, dog, rat and mouse hepatocytes was studied. Methylation, glucuronidation, sulfation and isomerization pathways of (-)-EC in all five species were found. Methylation, glucuronidation, sulfation, hydrolysis, isomerization and glucosidation pathways of ECG were found. Species differences in metabolism of (-)-EC or ECG were observed. Surprisingly, no metabolites of EGC or EGCG were detected, but chemical oxidation and polymerization were observed under these experimental conditions. It appeared that enzymatic reactions and chemical reactions were differentiated by an additional hydroxyl group on the B-ring between (-)-EC/ECG and EGC/EGCG. For (-)-EC, thirty-five metabolites including isomerized (M6. M10 and M25), glucuronidated (M3, M5 and M11), sulfated (M7, M15, M16, M18, M20, M23, M26), methylated (M2, M9, M12, M17, M19, M21, M27, M30, M32), glucuronated/methylated (M4, M8, M13, M14), sulfated/methylated (M22, M24, M28, M29, M31, M33, M34, M35) and diglucuronidate (M1), were detected and characterized. M11, M18, M19 and M23 were major metabolites in human hepatocytes; M11, M26 and M31 were major metabolites in monkey hepatocytes; M10, M20, M22, M26 and M31 were major metabolites in dog hepatocytes; M5, M6 and M10 were major metabolites in rat hepatocytes; and M5, M6 and M13 were major metabolites in mouse hepatocytes. For ECG, twelve metabolites including isomerized (M1), hydrolyzed (M3), glucosidated (M2), glucuronidated (M4 and M6), sulfated (M9, M11 and M12), methylated (M7), sulfated/glucuronidated/methylated (M8 and M10) and diglucuronidated (M5), were detected and characterized. M4, M11 and M12 were major metabolites in human hepatocytes; M11 and M12 were major metabolites in monkey hepatocytes; M3 and M11 were major metabolites in dog hepatocytes; M4, M6 and M11 were major metabolites in rat hepatocytes, and M3 was a major metabolite in mouse hepatocytes. The experimental results have demonstrated that fate of catechins in in vitro hepatocytes depends on metabolism and chemical stability. In certain experimental conditions, the chemical reaction may become a dominant pathway. Keywords: Catechins, metabolism, humans, monkeys, dogs, rats, mice, hepatocytes

Predictability of Metabolism of Ibuprofen and Naproxen Using Chimeric Mice with Human Hepatocytes

Prediction of human drug metabolism is important for drug development. Recently, the number of new drug candidates metabolized by not only cytochrome P450 (P450) but also non-P450 has been increasing. It is necessary to consider species differences in drug metabolism between humans and experimental animals. We examined species differences of drug metabolism, especially between humans and rats, for ibuprofen and (S)-naproxen as nonsteroidal anti-inflammatory drugs, which are metabolized by P450 and UDP-glucuronosyltransferase, sulfotransferase, and amino acid N-acyltransferase for taurine conjugation in liver, using human chimeric mice (h-PXB mice) repopulated with human hepatocytes and rat chimeric mice (r-PXB mice) transplanted with rat hepatocytes. We performed the direct comparison of excretory metabolites in urine between h-PXB mice and reported data for humans as well as between r-PXB mice and rats after administration of ibuprofen and (S)-naproxen. Good agreement for urinary metabolites (percentage of dose) was observed not only between humans and h-PXB mice but also between rats and r-PXB mice. Therefore, the metabolic profiles in humans and rats reflected those in h-PXB mice and r-PXB mice. Our results indicated that h-PXB mice should be helpful for predicting the quantitative metabolic profiles of drugs mediated by P450 and non-P450 in liver, and r-PXB mice should be helpful for evaluation of species differences in these metabolic enzymes.