Seigo Sanoh

Prediction of in vivo hepatic clearance and half-life of drug candidates in human using chimeric mice with humanized liver

Accurate prediction of pharmacokinetics (PK) parameters in humans from animal data is difficult for various reasons, including species differences. However, chimeric mice with humanized liver (PXB mice; urokinase-type plasminogen activator/severe combined immunodeficiency mice repopulated with approximately 80% human hepatocytes) have been developed. The expression levels and metabolic activities of cytochrome P450 (P450) and non-P450 enzymes in the livers of PXB mice are similar to those in humans. In this study, we examined the predictability for human PK parameters from data obtained in PXB mice. Elimination of selected drugs involves multiple metabolic pathways mediated not only by P450 but also by non-P450 enzymes, such as UDP-glucuronosyltransferase, sulfotransferase, and aldehyde oxidase in liver. Direct comparison between in vitro intrinsic clearance (CLint,in vitro) in PXB mice hepatocytes and in vivo intrinsic clearance (CLint,in vivo) in humans, calculated based on a well stirred model, showed a moderate correlation (r2 = 0.475, p = 0.009). However, when CLint,in vivo values in humans and PXB mice were compared similarly, there was a good correlation (r2 = 0.754, p = 1.174 × 10−4). Elimination half-life (t1/2) after intravenous administration also showed a good correlation (r2 = 0.886, p = 1.506 × 10−4) between humans and PXB mice. The rank order of CL and t1/2 in human could be predicted at least, although it may not be possible to predict absolute values due to rather large prediction errors. Our results indicate that in vitro and in vivo experiments with PXB mice should be useful at least for semiquantitative prediction of the PK characteristics of candidate drugs in humans.

Significance of aldehyde oxidase during drug development: Effects on drug metabolism, pharmacokinetics, toxicity, and efficacy

Aldehyde oxidase contributes to drug metabolism and pharmacokinetics (PK), and a few clinical studies were discontinued because of aldehyde oxidase metabolism. Its AOX1, AOX3, AOX3L1, and AOX4 isoforms are expressed in mammals, and species differences in expression profiles reflect differences in drug metabolism and PK between animals and humans. Individual differences in aldehyde oxidase activity also influence drug metabolism in humans. Moreover, the reduced solubility of the aldehyde oxidase metabolites may induce drug toxicity. Because various drugs inhibit aldehyde oxidase, assessments of ensuing drug-drug interactions (DDI) are critical for drug optimization. Although drug metabolism, PK, safety, and DDI are important, drugs such as famciclovir and O6-benzylguanine that affect aldehyde oxidase activity in humans have been reported. Recently, various in vitro approaches have been developed to predict PK in humans. However, in vitro studies on aldehyde oxidase may be hampered because of its instability. In contrast, in vivo studies on chimeric mice with humanized livers have also been focused on to predict aldehyde oxidase-mediated metabolism. Additionally, the ratios of N1-methylnicotinamide to metabolites in urinary excretions may represent useful biomarkers of aldehyde oxidase activity in humans. Thus, assessing the contributions of aldehyde oxidase to drug metabolism in humans is necessary.

Metabolism of UV-filter benzophenone-3 by rat and human liver microsomes and its effect on endocrine-disrupting activity

Benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) is widely used as sunscreen for protection of human skin and hair from damage by ultraviolet (UV) radiation. In this study, we examined the metabolism of BP-3 by rat and human liver microsomes, and the estrogenic and anti-androgenic activities of the metabolites. When BP-3 was incubated with rat liver microsomes in the presence of NADPH, 2,4,5-trihydroxybenzophenone (2,4,5-triOH BP) and 3-hydroxylated BP-3 (3-OH BP-3) were newly identified as metabolites, together with previously detected metabolites 5-hydroxylated BP-3 (5-OH BP-3), a 4-desmethylated metabolite (2,4-diOH BP) and 2,3,4-trihydroxybenzophenone (2,3,4-triOH BP). In studies with recombinant rat cytochrome P450, 3-OH BP-3 and 2,4,5-triOH BP were mainly formed by CYP1A1. BP-3 was also metabolized by human liver microsomes and CYP isoforms. In estrogen reporter (ER) assays using estrogen-responsive CHO cells, 2,4-diOH BP exhibited stronger estrogenic activity, 2,3,4-triOH BP exhibited similar activity, and 5-OH BP-3, 2,4,5-triOH BP and 3-OH BP-3 showed lower activity as compared to BP-3. Structural requirements for activity were investigated in a series of 14 BP-3 derivatives. When BP-3 was incubated with liver microsomes from untreated rats or phenobarbital-, 3-methylcholanthrene-, or acetone-treated rats in the presence of NADPH, estrogenic activity was increased. However, liver microsomes from dexamethasone-treated rats showed decreased estrogenic activity due to formation of inactive 5-OH BP-3 and reduced formation of active 2,4-diOH BP. Anti-androgenic activity of BP-3 was decreased after incubation with liver microsomes.

Tributyltin-induced endoplasmic reticulum stress and its Ca2 +-mediated mechanism

Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls post-translational modification and intracellular Ca(2+) signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca(2+) homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that 700nM TBT induced ER stress markers such as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2α. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca(2+) depletion, and to test this idea, we examined the effect of TBT on intracellular Ca(2+) concentration using fura-2 AM, a Ca(2+) fluorescent probe. TBT increased intracellular Ca(2+) concentration in a TBT-concentration-dependent manner, and Ca(2+) increase in 700nM TBT was mainly blocked by 50μM dantrolene, a ryanodine receptor antagonist (about 70% inhibition). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca(2+) concentration by releasing Ca(2+) from ER, thereby causing ER stress.

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.

Prediction of Human Metabolism of FK3453 by Aldehyde Oxidase Using Chimeric Mice Transplanted with Human or Rat Hepatocytes

During drug development, it is important to predict the activities of multiple metabolic enzymes, not only cytochrome P450 (P450) but also non-P450 enzymes, such as conjugative enzymes and aldehyde oxidase (AO). In this study, we focused on prediction of AO-mediated human metabolism and pharmacokinetics (PK) of 6-(2-amino-4-phenylpyrimidine-5-yl)-2-isopropylpyridazin-3(2H)-one (FK3453) (Astellas Pharma Inc.), the development of which was suspended due to extremely low exposure in human, despite good oral bioavailability in rat and dog. We examined species difference in oxidative metabolism of the aminopyrimidine moiety of FK3453, catalyzed by AO, using human-chimeric mice with humanized liver (h-PXB mice) and rat-chimeric mice (r-PXB mice) transplanted with rat hepatocytes. AO activity of h-PXB mouse hepatocytes was higher than that of r-PXB mouse hepatocytes. Moreover, higher concentrations of human-specific AO-generated FK3453 metabolite A-M were detected in urine and feces after administration of FK3453 to h-PXB mice versus r-PXB mice. The total clearance of h-PXB mice was 2-fold higher than that of r-PXB mice. These results agreed reasonably well with the metabolism and PK profiles of FK3453 in human and rat. Our results indicated that h-PXB mice should be helpful for predicting the metabolic profile of drugs in humans, and the use of both h-PXB and r-PXB mice should be helpful for evaluation of species differences of AO metabolic activity.

Chimeric mice transplanted with human hepatocytes as a model for prediction of human drug metabolism and pharmacokinetics

Preclinical studies in animal models are used routinely during drug development, but species differences of pharmacokinetics (PK) between animals and humans have to be taken into account in interpreting the results. Human hepatocytes are also widely used to examine metabolic activities mediated by cytochrome P450 (P450) and other enzymes, but such in vitro metabolic studies also have limitations. Recently, chimeric mice with humanized liver (h-chimeric mice), generated by transplantation of human donor hepatocytes, have been developed as a model for the prediction of metabolism and PK in humans, using both in vitro and in vivo approaches. The expression of human-specific metabolic enzymes and metabolic activities was confirmed in humanized liver of h-chimeric mice with high replacement ratios, and several reports indicate that the profiles of P450 and non-P450 metabolism in these mice adequately reflect those in humans. Further, the combined use of h-chimeric mice and r-chimeric mice, in which endogenous hepatocytes are replaced with rat hepatocytes, is a promising approach for evaluation of species differences in drug metabolism. Recent work has shown that data obtained in h-chimeric mice enable the semi-quantitative prediction of not only metabolites, but also PK parameters, such as hepatic clearance, of drug candidates in humans, although some limitations remain because of differences in the metabolic activities, hepatic blood flow and liver structure between humans and mice. In addition, fresh h-hepatocytes can be isolated reproducibly from h-chimeric mice for metabolic studies.

Endogenous neurotoxic dopamine derivative covalently binds to Parkinson's disease‐associated ubiquitin C‐terminal hydrolase L1 and alters its structure and function

Parkinsons disease (PD) is a common neurodegenerative disease, but its pathogenesis remains elusive. A mutation in ubiquitin C‐terminal hydrolase L1 (UCH‐L1) is responsible for a form of genetic PD which strongly resembles the idiopathic PD. We previously showed that 1‐(3′,4′‐dihydroxybenzyl)‐1,2,3,4‐tetrahydroisoquinoline (3′,4′DHBnTIQ) is an endogenous parkinsonism‐inducing dopamine derivative. Here, we investigated the interaction between 3′,4′DHBnTIQ and UCH‐L1 and its possible role in the pathogenesis of idiopathic PD. Our results indicate that 3′,4′DHBnTIQ binds to UCH‐L1 specifically at Cys152 in vitro. In addition, 3′,4′DHBnTIQ treatment increased the amount of UCH‐L1 in the insoluble fraction of SH‐SY5Y cells and inhibited its hydrolase activity to 60%, reducing the level of ubiquitin in the soluble fraction of SH‐SY5Y cells. Catechol‐modified UCH‐L1 as well as insoluble UCH‐L1 were detected in the midbrain of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐treated PD model mice. Structurally as well as functionally altered UCH‐L1 have been detected in the brains of patients with idiopathic PD. We suggest that conjugation of UCH‐L1 by neurotoxic endogenous compounds such as 3′,4′DHBnTIQ might play a key role in onset and progression of idiopathic PD.

Estrogenic activity of an environmental pollutant, 2-nitrofluorene, after metabolic activation by rat liver microsomes.

In this study, the metabolic activation of 2-nitrofluorene (NF) to estrogenic compounds was examined. NF was negative in estrogen reporter assays using estrogen-responsive yeast and human breast cancer cell line MCF-7. However, the compound exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH. Minor estrogenic activity was observed when liver microsomes of untreated or phenobarbital-treated rats were used instead of those from 3-methylcholanthrene-treated rats. When the compound was incubated with the liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH, 7-hydroxy-2-nitrofluorene (7-OH-NF) was formed as a major metabolite. However, little of the metabolite was formed by liver microsomes of untreated or phenobarbital-treated rats. Rat recombinant cytochrome P450 1A1 exhibited a significant oxidase activity toward NF, affording 7-OH-NF. Liver microsomes of phenobarbital-treated rats also enhanced oxidase activity toward NF. In this case, 9-hydroxy-2-nitrofluorene was formed. 7-OH-NF exhibited a significant estrogenic activity, while the activity of 9-hydroxy-2-nitrofluorene was much lower. These results suggest that the estrogenic activity of NF was due to formation of the 7-hydroxylated metabolite by liver microsomes.

Predictability of plasma concentration–time curves in humans using single-species allometric scaling of chimeric mice with humanized liver

Abstract 1. We used chimeric mice (PXB mice®), which were repopulated with human hepatocytes, to evaluate their predictabilities of human pharmacokinetics. 2. The relationships of total clearance (CLt) and the volume of distribution at steady state (Vdss) between that predicted from single-species allometric scaling (SSS) of PXB mice and the observed human values indicated good correlations for various drugs metabolized by cytochrome P450s (CYPs) and non-CYPs. 3. We examined the Dedrick plot with which the plasma concentration–time curves can exhibit superimposability using SSS of PXB mice for CLt and Vdss. The predicted plasma concentration–time curves using the complex Dedrick plot from PXB mice were generally superimposed with the observed human data. 4. However, the predicted curve of diazepam was not superimposable with the observed profile. Residual mouse hepatocytes in the livers of PXB mice may affect predictability of CLt of diazepam because significant discrepancy of in vitro intrinsic clearance in PXB mouse liver microsomes consisted of low and high replacement of human hepatocytes were observed. 5. The complex Dedrick plot with SSS from PXB mice is useful for predicting the plasma concentration–time curve in drug discovery, although there are some limitations.