Monkey Liver Research Articles

In vitro metabolism of l-corydalmine, a potent analgesic drug, in human, cynomolgus monkey, beagle dog, rat and mouse liver microsomes

l-Corydalmine (l-CDL) was under development as an oral analgesic agent, exhibiting potent analgesic activity in preclinical models. The objective of this study was to compare metabolic profiles of l-CDL in liver microsomes from mouse, rat, monkey, dog and human. Six metabolites (M1–M6) were identified using LC-Q/TOF in liver microsomes from the five species. The metabolism of l-CDL included O-demethylation (M1–3) and hydroxylation (M4–6). The desmethyl metabolites were the major ones among the five species, which accounted for more than 84%. Data from chemical inhibition in human liver microsomes (HLM) and human recombinant CYP450s demonstrated that CYP2D6 exhibited strong catalytic activity towards M1 and M2 formations, while CYP2C9 and CYP2C19 also catalyzed M2 formation. Formations of M3 and hydroxyl metabolites (M4 and M5) were mainly catalyzed by CYP3A4. Further studies showed that M1 and M2 were main metabolites in HLM. The kinetics of M1 and M2 formations in HLM and recombinant CYP450s were also investigated. The results showed that M1 and M2 formations in HLM and recombinant CYP2D6 characterized biphasic kinetics, whereas sigmoid Vmax model was better used to fit M2 formation by recombinant CYP2C9 and CYP2C19. The contributions of CYP2D6 to M1 and M2 formations in HLM were estimated to be 75.3% and 50.7%, respectively. However, the contributions of CYP2C9 and CYP2C19 to M2 formation were only 5.0% and 4.1%, respectively. All these data indicated that M1 and M2 were main metabolites in HLM, and CYP2D6 was the primary enzyme responsible for their formations.

143. Durable Expression of TT-034 in Cynomolgus Monkey Hepatic and Cardiac Tissues without Long-Term Adverse Effects on Endogenous MicroRNA Levels

Background: The hepatitis C virus (HCV) is an attractive target for RNA interference (RNAi) based therapeutics because its genome consists of a single, positive stranded RNA and its replication occurs strictly within the cytoplasm. TT-034 is a DNA-directed RNAi (ddRNAi) based gene therapy product for the treatment of chronic HCV infection and is currently in a phase I/IIa clinical trial. TT-034 is comprised of a self-complementary recombinant adeno-associated virus (AAV) serotype 8 vector for transduction of hepatic tissues and is systemically administered as a single intravenous dose. Inside the hepatocyte, TT-034 uses the cell's own transcriptional machinery to continuously express three independent short hairpin RNAs (shRNAs) that simultaneously target the 5’ UTR and NS5B regions of the HCV genome. Results: Following a single administration of up to 3.75E12 vg/kg of TT-034, biodistribution analyses demonstrated that greater than 90% of the quantified vector is found in the Cynomolgus monkey liver tissues. In situ hybridization analyses demonstrated almost 100% transduction of the hepatocytes when dosed at 1.25E12 vg/kg. Furthermore, qPCR data revealed that shRNA levels persisted for the duration of the 180-day experiment, demonstrating the durability of expression following a single injection. We examined the impact of continuous TT-034 expression on endogenous miRNAs in liver and cardiac tissues because previous reports have suggested that high expression of shRNA may cause global dysregulation of endogenous microRNA (miRNA) levels within cells. Non-human primates were dosed with saline control or either 1.00E11, 1.00E12, 1.00E13 vg/kg of TT-034. Expression profiles of mature miRNAs were generated from liver biopsies taken 15 days after TT-034 administration. Additionally, samples from liver and heart tissues were collected 60 and 180 days after administration. Approximately 260 different miRNAs were reliably detected in each sample type and used for the analyses. The day 15 liver samples showed significant differences in the expression levels of 27 miRNAs. When analyzing all 260 miRNAs, the day 60 and day 180 liver samples showed no significant statistical differences from the control group at any dose. In the heart tissues, no significant statistical differences between the TT-034 treated group and the control group were noted at any timepoint. Conclusions: These data suggest that a single, intravenous infusion of TT-034 results in almost complete transduction of Cynomolgus monkey hepatocytes. Furthermore, TT-034 results in durable expression of three anti-HCV shRNAs. These doses of TT-034 do not cause long-term perturbation of endogenous miRNA levels.

Effect of a Natural Inulin-Containing Product, “Kikuimo Extract” on Intestinal Microbes and Gene Expression in the Liver and Adipose Tissue of Menopausal Monkeys

Inulin is a soluble and indigestible fiber derived from natural plants such as Jerusalem artichoke (Helianthus tuberosus), “Kikuimo”. In the current study, a nutrigenomics approach was utilized to evaluate the in vivo function of “Kikuimo Extract” (KE) in ovariectomized cynomolgus macaque, a post-menopausal non-human primate model. KE was administered orally before feeding, for 3 months for the following examinations: 1) the effect of KE on intestinal microbes was examined by quantitative analyses of the intestinal bacteria using real-time PCR with DNA extracted from monkey feces; 2) the effect of KE on gene expression was investigated by real-time RT-PCR using RNA extracted from both the liver and adipose tissue of the monkeys. KE administration modulated menopause-mediated altered microbes to increase Lactobacilli, Veillonella, and Bacteroides in all monkeys. KE administration regulated the altered expression of functional genes, SCAP, LDLR, and LXRA (lipid metabolism); GLUT-4 (glucose transport); CYP1A1 and CYP1A2 (drug metabolism); and CYP-17-2 and CYP-19-2 (E2 synthesis) in the menopausal monkeys. In menopausal monkeys, KE showed potent prebiotic effect on beneficial microflora and regulating effect on altered expression of functional genes associated with metabolism and E2 production. Thus, KE appears to be a practical functional food that alleviates the altered conditions of intestinal microbes and gene expression in the liver and adipose tissue in a menopausal state.

Alterations in levels and ratios of n–3 and n–6 polyunsaturated fatty acids in the temporal cortex and liver of vervet monkeys from birth to early adulthood

Deficiencies in omega-3 (n–3) long chain polyunsaturated fatty acids (LC-PUFAs) and increases in the ratio of omega-6 (n–6) to n–3 LC-PUFAs in brain tissues and blood components have been associated with psychiatric and developmental disorders. Most studies have focused on n–3 LC-PUFA accumulation in the brain from birth until 2years of age, well before the symptomatic onset of such disorders. The current study addresses changes that occur in childhood and adolescence. Postmortem brain (cortical gray matter, inferior temporal lobe; n=50) and liver (n=60) from vervet monkeys fed a uniform diet from birth through young adulthood were collected from archived tissues. Lipids were extracted and fatty acid levels determined. There was a marked reduction in the ratio of n–6 LC-PUFAs, arachidonic acid (ARA) and adrenic acid (ADR), relative to the n–3 LC-PUFA, docosahexaenoic acid (DHA), in temporal cortex lipids from birth to puberty and then a more gradual decrease though adulthood. This decrease in ratio resulted from a 3-fold accumulation of DHA levels while concentrations of ARA remained constant. Early childhood through adolescence appears to be a critical period for DHA accretion in the cortex of vervet monkeys and may represent a vulnerable stage where lack of dietary n–3 LC-PUFAs impacts development in humans.

Targeting TMPRSS6 Using Antisense Technology for the Treatment of Beta-Thalassemia

Antisense technology is a powerful drug discovery approach for identifying oligonucleotide analogs that can specifically modify RNA expression through multiple mechanisms including RNase H-mediated degradation of RNA and modulation of RNA splicing. We have successfully applied this technology towards targeting a number of transcripts in a wide-range of therapeutic areas. Beta-thalassemia, one of the most common genetic disorders worldwide, is characterized by reductions in beta-globin and ineffective erythropoiesis. This in turn leads to suppression of hepcidin, a peptide hormone that serves as the master regulator of iron homeostasis. Inappropriately low levels of hepcidin trigger increased dietary iron absorption resulting in iron overload, which is the major cause of morbidity and mortality in beta-thalassemia patients. TMPRSS6 is a transmembrane serine protease mainly produced by hepatocytes that negatively regulates hepcidin expression. Previous mouse and human genetic data from multiple groups suggest that lowering TMPRSS6 expression could up-regulate hepcidin and ameliorate many of the disease symptoms associated with β-thalassemia.We identified potent antisense oligonucleotides (ASOs) against mouse TMPRSS6. Downregulation of TMPRSS6 with ASO treatment resulted in dose-dependent hepcidin upregulation and reduction in serum iron and transferrin saturation in normal mice. In a mouse model of beta-thalassemia (th3/+ mice), which effectively recapitulates beta-thalassemia intermedia in humans, TMPRSS6 reduction resulted in induction of hepcidin and dramatic reductions of serum transferrin saturation (from 55-63% in control group down to 20-26% in treatment group). Liver iron concentration (LIC) was also greatly reduced (40-50%). Moreover, anemia endpoints were significantly improved with ASO treatment, including increases in red blood cells (~30-40%), hemoglobin (~2 g/dl), and hematocrit (~20%); reduction of splenomegaly; decreases in serum erythropoietin levels; improved erythroid maturation as indicated by a strong reduction in reticulocyte number and a normalized proportion between the pool of erythroblasts and enucleated erythroid cells.Encouraged by the strong pharmacology of TMPRSS6 suppression in animal models, we initiated an effort to identify a human TMPRSS6 clinical candidate with a liver-targeted delivery strategy. Over 2000 ASOs were screened in cell lines and the most active compounds were evaluated in rodent tolerability studies. A human TMPRSS6 transgenic mouse model was established enabling evaluation of ASO activity toward human TMPRSS6 transcript in vivo. Furthermore, lead compounds were tested in a 3-month study in normal monkeys. With repeated dosing, TMPRSS6 mRNA levels in monkey liver were reduced by >90%, accompanied by time-dependent reductions of serum iron (from ~100-120ug/dl to <40ug/dl), transferrin saturation (from ~30-35% to <10%), and hemoglobin. These compounds were well tolerated in rodents and in monkeys. Collectively, our data demonstrate that TMPRSS6 ASO could be an effective therapeutic for patients with beta-thalassemia and related disorders. A Phase 1 clinical trial is planned to initiate in 2016. DisclosuresGuo:Isis Pharmaceuticals: Employment, Other: Shareholder. Aghajan:Isis Pharmaceuticals: Employment, Other: Shareholder. Booten:Isis Pharmaceuticals: Employment, Other: Shareholder. Monia:Isis Pharmaceuticals: Employment, Other: Shareholder.

Investigating the effect of autoinduction in cynomolgus monkeys of a novel anticancer MDM2 antagonist, idasanutlin, and relevance to humans

1. Idasanutlin (RG7388) is a potent p53-MDM2 antagonist currently in clinical development for treatment of cancer. The purpose of the present studies was to investigate the cause of marked decrease in plasma exposure after repeated oral administration of RG7388 in monkeys and whether the autoinduction observed in monkeys is relevant to humans.2. In monkey liver and intestinal microsomes collected after repeated oral administration of RG7388 to monkeys, significantly increased activities of homologue CYP3A8 were observed (ex vivo). Investigation using a physiologically based pharmacokinetic (PBPK) model suggested that the loss of exposure was primarily due to induction of metabolism in the gut of monkeys.3. Studies in monkey and human primary hepatocytes showed that CYP3A induction by RG7388 only occurred in monkey hepatocytes but not in human hepatocytes, which suggests the observed CYP3A induction is monkey specific.4. The human PK data obtained from the first cohorts confirmed the lack of relevant induction as predicted by the human hepatocytes and the PBPK modelling based on no induction in humans.

Metabolism of dictamnine in liver microsomes from mouse, rat, dog, monkey, and human

Dictamnine, a furoquinoline alkaloid isolated from the root bark of Dictamnus dasycarpus Turcz. (Rutaceae), is reported to have a wide range of pharmacological activities. In this study, the in vitro metabolic profiles of dictamnine in mouse, rat, dog, monkey, and human liver microsomes were investigated and compared. Dictamnine was incubated with liver microsomes in the presence of an NADPH-regenerating system, resulting in the formation of eight metabolites (M1–M8). M1 is an O-desmethyl metabolite. M5 and M6 are formed by a mono-hydroxylation of the benzene ring of dictamnine. M8 was tentatively identified as an N-oxide metabolite. The predominant metabolic pathway of dictamnine occurs through the epoxidation of the 2,3-olefinic to yield a 2,3-epoxide metabolite (M7), followed by the ring of the epoxide opening to give M4. Likewise, cleavage of the furan ring forms M2 and M3. Slight differences were observed in the in vitro metabolic profiles of dictamnine among the five species tested. A chemical inhibition study with a broad and five specific CYP450 inhibitors revealed that most of the dictamnine metabolites in liver microsomes are mediated by CYP450, with CYP3A4 as the predominant enzyme involved in the formation of M7, the major metabolite. These findings provide vital information to better understand the metabolic processes of dictamnine among various species.

Converting Skin Fibroblasts into Hepatic-like Cells by Transient Programming.

Transplantation of hepatocytes is a promising therapy for end-stage liver disease, but the availability of functional cells currently precludes its clinical application. We now report a simple transient reprogramming approach to convert fibroblasts into hepatic-like cells. Human skin fibroblasts were treated with fish egg extracts to become the transiently remodeled cells (TRCs). After infected with retroviral EGFP, they were directly injected into the fetal monkey liver, where they underwent in situ differentiation in the hepatic niche. The hepatic-like cells were functional as shown by the synthesis of hepatic markers in vivo, including albumin, cytokeratin-18, and hepatic serum antigen. Similarly, when implanted in the mouse liver, the TRCs were differentiated into hepatic-like cells that synthesize albumin and CK18 and became completely integrated into the liver parenchyma. The potency of TRCs was mechanistically related to the activation of several signal pathways, which reactivate endogenous genes related to cell potency. This study demonstrates the feasibility of a simple and inexpensive epigenetic remodeling approach to convert human fibroblasts into therapeutic hepatic-like cells for the treatment of end-stage liver disease.

The different metabolism of morusin in various species and its potent inhibition against UDP-glucuronosyltransferase (UGT) and cytochrome p450 (CYP450) enzymes

1. The aim of this study was to investigate the inhibitory effect of morusin on Glucuronosyltransferase (UGT) isoforms and cytochrome P450 enzymes (CYP450s). We also investigated the metabolism of morusin in human, rat, dog, monkey, and minipig liver microsomes.2. 100 μM of morusin exhibited strong inhibition on all UGTs and CYP450s. The half inhibition concentration (IC50) values for CYP3A4, CYP1A2, CYP2C9, CYP2E1, UGT1A6, UGT1A7, and UGT1A8 were 2.13, 1.27, 3.18, 9.28, 4.23, 0.98, and 3.00 μM, and the inhibition kinetic parameters (Ki) were 1.34, 1.16, 2.98, 6.23, 4.09, 0.62, and 2.11 μM, respectively.3. Metabolism of morusin exhibited significant species differences. The quantities of M1 from minipig, monkey, dog, and rat were 7.8, 11.9, 2.0, and 6.3-fold of human levels. The Km values in HLMs, RLMs, MLMs, DLMs, and PLMs were 7.84, 22.77, 14.32, 9.13, and 22.83 μM, and Vmax for these species were 0.09, 1.23, 1.43, 0.15, and 0.75 nmol/min/mg, respectively. CLint (intrinsic clearance) values (Vmax/Km) for morusin obeyed the following order: monkey > rat > minipig > dog > human. CLH (hepatic clearance) values for humans, dogs, and rats were calculated to be 8.28, 17.38, and 35.12 mL/min/kg body weight, respectively.4. This study provided vital information to understand the inhibitory potential and metabolic behavior of morusin among various species.

Rosuvastatin Liver Partitioning in Cynomolgus Monkeys: Measurement In Vivo and Prediction Using In Vitro Monkey Hepatocyte Uptake

Unbound plasma concentrations may not reflect those in target tissues, and there is a need for methods to predict tissue partitioning. Here, we investigate the unbound liver partitioning (Kpu,u) of rosuvastatin, a substrate of hepatic organic anion transporting peptides, in cynomolgus monkeys and compare it with that determined using hepatocytes in vitro. Rosuvastatin (3 mg/kg) was administered orally to monkeys and plasma and liver (by ultrasound-guided biopsy) collected over time. Uptake into monkey hepatocytes was evaluated up to steady state. Binding in monkey plasma, liver, and hepatocytes was determined using equilibrium dialysis. Mean in vivo Kpu,u was 118 after correcting total liver partitioning by plasma and liver binding. In vitro uptake data were analyzed by compartmental modeling to determine active uptake clearance, passive diffusion, the intracellular unbound fraction, and Kpu,u. In vitro Kpu,u underpredicted that in vivo, resulting in the need for an empirical in vitro to in vivo scaling factor of 10. Adjusting model parameters using hypothetical scaling factors for transporter expression and surface area or assuming no effect of protein binding on active transport increased partitioning values by 1.1-, 6-, and 9-fold, respectively. In conclusion, in vivo rosuvastatin unbound liver partitioning in monkeys was underpredicted using hepatocytes in vitro. Modeling approaches that allow integrating corrections from passive diffusion or protein binding on active uptake could improve the estimation of in vivo intracellular partitioning of this organic anion transporting peptide substrate. A similar assessment of other active hepatic transport mechanisms could confirm and determine the extent to which limited accumulation in isolated hepatocytes needs to be considered in drug development.

Raloxifene glucuronidation in liver and intestinal microsomes of humans and monkeys: contribution of UGT1A1, UGT1A8 and UGT1A9

1. Raloxifene is an antiestrogen that has been marketed for the treatment of osteoporosis, and is metabolized into 6- and 4′-glucuronides by UDP-glucuronosyltransferase (UGT) enzymes. In this study, the in vitro glucuronidation of raloxifene in humans and monkeys was examined using liver and intestinal microsomes and recombinant UGT enzymes (UGT1A1, UGT1A8 and UGT1A9).2. Although the Km and CLint values for the 6-glucuronidation of liver and intestinal microsomes were similar between humans and monkeys, and species differences in Vmax values (liver microsomes, humans > monkeys; intestinal microsomes, humans < monkeys) were observed, no significant differences were noted in the Km or S50, Vmax and CLint or CLmax values for the 4′-glucuronidation of liver and intestinal microsomes between humans and monkeys.3. The activities of 6-glucuronidation in recombinant UGT enzymes were UGT1A1 > UGT1A8 >UGT1A9 for humans, and UGT1A8 > UGT1A1 > UGT1A9 for monkeys. The activities of 4′-glucuronidation were UGT1A8 > UGT1A1 > UGT1A9 in humans and monkeys.4. These results demonstrated that the profiles for the hepatic and intestinal glucuronidation of raloxifene by microsomes were moderately different between humans and monkeys.

Comprehensive Evaluation for Substrate Selectivity of Cynomolgus Monkey Cytochrome P450 2C9, a New Efavirenz Oxidase.

Cynomolgus monkeys are widely used as primate models in preclinical studies, because of their evolutionary closeness to humans. In humans, the cytochrome P450 (P450) 2C enzymes are important drug-metabolizing enzymes and highly expressed in livers. The CYP2C enzymes, including CYP2C9, are also expressed abundantly in cynomolgus monkey liver and metabolize some endogenous and exogenous substances like testosterone, S-mephenytoin, and diclofenac. However, comprehensive evaluation regarding substrate specificity of monkey CYP2C9 has not been conducted. In the present study, 89 commercially available drugs were examined to find potential monkey CYP2C9 substrates. Among the compounds screened, 20 drugs were metabolized by monkey CYP2C9 at a relatively high rates. Seventeen of these compounds were substrates or inhibitors of human CYP2C9 or CYP2C19, whereas three drugs were not, indicating that substrate specificity of monkey CYP2C9 resembled those of human CYP2C9 or CYP2C19, with some differences in substrate specificities. Although efavirenz is known as a marker substrate for human CYP2B6, efavirenz was not oxidized by CYP2B6 but by CYP2C9 in monkeys. Liquid chromatography-mass spectrometry analysis revealed that monkey CYP2C9 and human CYP2B6 formed the same mono- and di-oxidized metabolites of efavirenz at 8 and 14 positions. These results suggest that the efavirenz 8-oxidation could be one of the selective markers for cynomolgus monkey CYP2C9 among the major three CYP2C enzymes tested. Therefore, monkey CYP2C9 has the possibility of contributing to limited specific differences in drug oxidative metabolism between cynomolgus monkeys and humans.

Identification of activating enzymes of a novel FBPase inhibitor prodrug, CS-917.

CS-917 (MB06322) is a selective small compound inhibitor of fructose 1,6-bisphosphatase (FBPase), which is expected to be a novel drug for the treatment of type 2 diabetes by inhibiting gluconeogenesis. CS-917 is a bisamidate prodrug and activation of CS-917 requires a two-step enzyme catalyzed reaction. The first-step enzyme, esterase, catalyzes the conversion of CS-917 into the intermediate form (R-134450) and the second-step enzyme, phosphoramidase, catalyzes the conversion of R-134450 into the active form (R-125338). In this study, we biochemically purified the CS-917 esterase activity in monkey small intestine and liver. We identified cathepsin A (CTSA) and elastase 3B (ELA3B) as CS-917 esterases in the small intestine by mass spectrometry, whereas we found CTSA and carboxylesterase 1 (CES1) in monkey liver. We also purified R-134450 phosphoramidase activity in monkey liver and identified sphingomyelin phosphodiesterase, acid-like 3A (SMPADL3A), as an R-134450 phosphoramidase, which has not been reported to have any enzyme activity. Recombinant human CTSA, ELA3B, and CES1 showed CS-917 esterase activity and recombinant human SMPDL3A showed R-134450 phosphoramidase activity, which confirmed the identification of those enzymes. Identification of metabolic enzymes responsible for the activation process is the requisite first step to understanding the activation process, pharmacodynamics and pharmacokinetics of CS-917 at the molecular level. This is the first identification of a phosphoramidase other than histidine triad nucleotide-binding protein (HINT) family enzymes and SMPDL3A might generally contribute to activation of the other bisamidate prodrugs.

Metabolism of Opicapone, a Novel COMT Inhibitor, in the Rat: Characterization of In Vitro Sulfation

In this study, opicapone, a novel catechol‐O‐methyltransferase (COMT) inhibitor, metabolism was evaluated in the rat and the sulfotransferases (SULTs) responsible for opicapone sulfation were characterized. Opicapone 3‐O‐sulfate was present in monkey, dog, rat and human liver cellular fractions and represented the major metabolite in the rat. Regarding human kidney, liver and small intestine cellular fractions, the later displayed the highest activity towards opicapone sulfation. A systematic analysis of seven recombinant human SULTs, showed that SULT1A1*1 and SULT1A1*2 exhibited the highest catalytic activity for opicapone sulfation. The kinetic analysis of opicapone sulfation revealed that the affinity for the conjugation of opicapone by intestinal S9 fraction was 20‐fold higher than that for the liver S9 fraction. Similar apparent Km values were obtained for SULT1A1 and human liver S9. Opicapone sulfation by human S9 fractions and SULT1A1 isoform was inhibited in a concentration manner by quercetin and 2,6‐dichloro‐4‐nitrophenol, highly selective inhibitors of SULT1A1. Dopamine, the major endogenous substrate of SULT1A3, also inhibited opicapone sulfation, but acetaminophen had no effect. The enhanced catalytic activity for opicapone sulfation and the high level of expression of SULT1A1 in liver together with the similar Km values obtained for SULT1A1 and for human liver S9 fraction, suggest that SULT1A1 may play a major role in opicapone sulfation, though other SULTs, such as SULT1A3, may also be involved in opicapone sulfation, namely at the intestinal level.

Guanfu base A, an antiarrhythmic alkaloid of Aconitum coreanum, Is a CYP2D6 inhibitor of human, monkey, and dog isoforms.

Guanfu base A (GFA) is a novel heterocyclic antiarrhythmic drug isolated from Aconitum coreanum (Lèvl.) rapaics and is currently in a phase IV clinical trial in China. However, no study has investigated the influence of GFA on cytochrome P450 (P450) drug metabolism. We characterized the potency and specificity of GFA CYP2D inhibition based on dextromethorphan O-demethylation, a CYP2D6 probe substrate of activity in human, mouse, rat, dog, and monkey liver microsomes. In addition, (+)-bufuralol 1'-hydroxylation was used as a CYP2D6 probe for the recombinant form (rCYP2D6), 2D1 (rCYP2D1), and 2D2 (rCYP2D2) activities. Results show that GFA is a potent noncompetitive inhibitor of CYP2D6, with inhibition constant Ki = 1.20 ± 0.33 μM in human liver microsomes (HLMs) and Ki = 0.37 ± 0.16 μM for the human recombinant form (rCYP2D6). GFA is also a potent competitive inhibitor of CYP2D in monkey (Ki = 0.38 ± 0.12 μM) and dog (Ki = 2.4 ± 1.3 μM) microsomes. However, GFA has no inhibitory activity on mouse or rat CYP2Ds. GFA did not exhibit any inhibition activity on human recombinant CYP1A2, 2A6, 2C8, 2C19, 3A4, or 3A5, but showed slight inhibition of 2B6 and 2E1. Preincubation of HLMs and rCYP2D6 resulted in the inactivation of the enzyme, which was attenuated by GFA or quinidine. Beagle dogs treated intravenously with dextromethorphan (2 mg/ml) after pretreatment with GFA injection showed reduced CYP2D metabolic activity, with the Cmax of dextrorphan being one-third that of the saline-treated group and area under the plasma concentration-time curve half that of the saline-treated group. This study suggests that GFA is a specific CYP2D6 inhibitor that might play a role in CYP2D6 medicated drug-drug interaction.

Species differences and substrate specificity of CYP3A heteroactivation by efavirenz

1. The purpose of this study was to clarify species differences in the heteroactivation of CYP3A substrates by efavirenz, which is known from clinical studies to activate midazolam 1′-hydroxylation, and to assess the feasibility of an animal model.2. In monkey and human liver microsomes, efavirenz activated CYP3A-mediated midazolam 1′-hydroxylation, but had no effect in rat liver microsomes. The activating effect of efavirenz was also observed with recombinant human CYP3A4 and CYP3A5. Midazolam 4-hydroxylation, testosterone 6β-hydroxylation and the oxidation of nifedipine were not activated by efavirenz in any of the microsomes.3. In an in vivo study using monkeys, the AUC ratio of midazolam/1′-hydroxymidazolam was reduced from 0.85 to 0.30 by efavirenz treatment, which was comparable to that obtained in clinical studies. However, the AUC changes of midazolam caused by efavirenz were smaller than those observed in clinical results, therefore the effect of efavirenz on monkeys was not completely consistent with that seen in humans.4. In conclusion, this is the first report that efavirenz specifically activates midazolam 1′-hydroxylation only in monkey and human liver microsomes, revealing marked species differences and high substrate specificity in the heteroactivation. A further study is required to clarify whether this in vitro result reflects the in vivo situation.

Age-related changes of hepatic clearances of cytochrome P450 probes, midazolam and R-/S-warfarin in combination with caffeine, omeprazole and metoprolol in cynomolgus monkeys using in vitro–in vivo correlation

1. Pharmacokinetics of human cytochrome P450 probes (caffeine, racemic warfarin, omeprazole, metoprolol and midazolam) were investigated after single intravenous and oral administrations at doses of 0.20 and 1.0 mg kg−1, respectively, in combination to three young (3-year-old) and three aged (16-year-old) cynomolgus monkeys.2. The plasma concentrations of caffeine and R-/S-warfarin decreased slowly in a monophasic manner, but those of omeprazole, metoprolol and midazolam decreased rapidly, in a similar manner to those as reported for pharmacokinetics in humans.3. The mean maximum concentrations of R- and S-warfarin (4.6 and 3.7 µg/mL, respectively) in aged monkeys after oral administration were significantly higher than those in young monkeys (3.3 and 2.7 µg/mL). The mean clearance (CL) values of midazolam in aged monkeys (9.5 mL/min/kg) were significantly lower than those in young monkeys (13 mL/min/kg).4. Individual intrinsic CL values for omeprazole (r = 0.29) and metoprolol (r = 0.30) of individual monkey livers were inversely correlated with their ages significantly (p < 0.05) in liver microsomes prepared from 55 cynomolgus monkeys.5. These results suggest that cynomolgus monkeys could be a good model for humans, especially with particular characteristics in reduced CLs of some human P450 substrates by aging.

Species-related difference between limonin and obacunone among five liver microsomes and zebrafish using ultra-high-performance liquid chromatography coupled with a LTQ-Orbitrap mass spectrometer.

Limonin and obacunone are two major limonoids distributed in the Rutaceae and Meliaceae families. Their defined anti-tumor activity is closely connected with the furan ring and the multi-carbonyls in their structures. In vivo and in vitro biotransformations may influence their structures and further change their effects. The metabolic profiles of limonin and obacunone have not been studied previously. In order to clarify their in vivo and in vitro metabolism, a comparative investigation of their metabolic pathways in five different species of liver microsomes and zebrafish was carried out. In the present study, ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC/HRMS) and related electrospray ionization (ESI) tandem mass spectrometric (MS/MS) dissociation of limonin and obacunone were applied for the analysis. Each metabolite was identified by its accurate mass data. Human liver microsomes (HLMs), monkey liver microsomes (MLMs), dog liver microsomes (DLMs), rat liver microsomes (RLMs), mice liver microsomes (XLMs) and zebrafish were included in the biotransformations. One phase I metabolite of limonin (M1-1) and two phase I metabolites of obacunone (M2-1, M2-2) were identified by accurate mass measurement and MS/MS fragmentation behaviors. A reduction reaction was regarded as the major metabolic pathway of limonoids in liver microsomes. The reduction reaction site of M1-1 and M2-1 was at the C-16 carbonyl, while for M2-2 it was at C-7. M1-1 was the major and unique metabolite of limonin and the metabolic rate of limonin varied from 11.5% to 17.8% in liver microsomes (LMs). M2-2 was the main metabolite of obacunone in LMs and zebrafish. M1-1 and M2-1 were only detected in LMs while M2-2 was found in both LMs and zebrafish incubation systems. The metabolic rate of obacunone varied from 2.5% to 19.1% and the content of M2-2 was about five times higher than that of M2-1. The ESI-HR-MS/MS fragmentation behaviors of limonin and obacunone were investigated for the first time. A qualitative and semi-quantitative method was developed for the in vivo and in vitro metabolic analysis of limonin and obacunone. The results demonstrated that the metabolic processes of limonin and obacunone were different between LMs and zebrafish. However, both of these two parent compounds presented similar metabolic processes in five species of LMs. This was caused by the metabolic difference between mammals and fish or because limonin probably cannot be absorbed in zebrafish.

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.

Cynomolgus monkey as a surrogate for human aldehyde oxidase metabolism of the EGFR inhibitor BIBX1382.

BIBX1382 was an epidermal growth factor receptor inhibitor under clinical investigation for treatment of cancer. This candidate possessed an attractive preclinical absorption, distribution, metabolism, and excretion profile, yet failed in clinical studies due in part to poor oral exposure, resulting from extensive metabolism by aldehyde oxidase (AO). In vitro metabolism studies were performed in liver cytosol and cryopreserved hepatocytes from multiple species. In addition, a pharmacokinetic study was performed in cynomolgus monkey for comparison with the reported human pharmacokinetics of BIBX1382. Estimated hepatic clearance of BIBX1382 in rhesus (42 ml/min per kg) and cynomolgus monkey (43 ml/min per kg) liver cytosol was comparable to human (≥93% of liver blood flow). Metabolite identification after incubation of BIBX1382 in liver cytosol fortified with the AO inhibitor raloxifene confirmed that AO is involved in the formation of the predominant metabolite (BIBU1476, M1) in cynomolgus monkey. After intravenous and oral administration of BIBX1382 to cynomolgus monkeys, high plasma clearance (118 ml/min per kg) and low oral exposure (C(max) = 12.7 nM and 6% oral bioavailability) was observed, with the exposure of M1 exceeding BIBX1382 after oral dosing. This pharmacokinetic profile compared favorably with the human clinical data of BIBX1382 (plasma clearance 25-55 ml/min per kg and 5% oral bioavailability). Thus, it appears that cynomolgus monkey represents a suitable surrogate for the observed human AO metabolism of BIBX1382. To circumvent clinical failures due to uncharacterized metabolism by AO, in vitro studies in the appropriate subcellular fraction, followed by pharmacokinetic and toxicokinetic studies in the appropriately characterized surrogate species should be conducted for substrates of AO.