The usefulness of determining plasma and tissue concentrations of phosphorodiamidate morpholino oligonucleotides to estimate their efficacy in Duchenne muscular dystrophy patients.

Currently four kinds of phosphorodiamidate morpholino oligomers (PMOs) such as viltolarsen have been approved for the treatment of Duchenne muscular dystrophy (DMD); however, it is unclear whether human efficacy can be estimated using plasma concentrations. This study summarizes the tissue distribution of viltolarsen in mice and cynomolgus monkeys and evaluates the relationship between exposure and efficacy based on exon skipping. In the tissue distribution studies, all muscles in DMD model mice showed higher concentrations of viltolarsen than those in wild-type mice and cynomolgus monkeys, and the concentrations in skeletal muscle were correlated with the exon-skipping efficiency in mice and cynomolgus monkeys. In addition, a highly sensitive bioanalytical method using liquid chromatography with tandem mass spectrometry shows promise for determining plasma concentrations up to a later time point, and the tissue (muscle)/plasma concentration ratio (Kp) in DMD model mice was shown to be useful for predicting changes in pharmacodynamic (PD) markers in humans. Our results suggest that pharmacokinetic (PK)/PD analysis can be conducted by using the human PK profile or Kp values and skipping efficiency in DMD model mice. This information will be useful for the efficient and effective development of PMOs as therapeutic agents. Significance Statement We compare that plasma and tissue concentrations with the efficiency of exon skipping for viltolarsen as an example phosphorodiamidate morpholino oligomers in skeletal and cardiac muscle of mice and cynomolgus monkeys for pharmacokinetics/pharmacodynamics (PK/PD) analysis. Our results suggest that PK/PD analysis can be conducted by using the human PK profile or Kp values and skipping efficiency in DMD model mice.

Prediction of Human Pharmacokinetics of Phosphorodiamidate Morpholino Oligonucleotides in Duchenne Muscular Dystrophy Patients Using Viltolarsen.

Several modified antisense oligonucleotides (ASOs) have recently been approved for clinical use. Some are phosphorodiamidate morpholino oligomers (PMOs), which, unlike other nucleic acids, are not negatively charged. Thus, PMOs differ from other ASOs in their pharmacokinetic (PK) properties. Drugs with a PMO backbone have been administered to Duchenne muscular dystrophy pediatric patients; however, appropriate methodologies are not currently available to predict their human PK from nonclinical data. In this study, we used viltolarsen as a representative PMO to investigate the applicability of the allometric scaling approach to human PK prediction. We first summarized the nonclinical and clinical PK data for viltolarsen as showing high total clearance, low serum protein binding, metabolic resistance, and urinary excretion as the unchanged drug in both animals and humans. We then investigate the PK of viltolarsen in mice, rats, cynomolgus monkeys, and dogs and used the results, with body weight, to extrapolate to humans by several methods. The estimate of human total clearance obtained from cynomolgus monkeys was the best, and body weight may be the key factor in accurately predicting human total clearance. In contrast, all of the well-known prediction methods for the volume of distribution at steady state gave underestimates. However, the human PK profiles predicted from the PK parameters in cynomolgus monkeys fit the observed human plasma concentrations well. These results are expected to contribute to the further development of PMOs. SIGNIFICANCE STATEMENT: We investigated how to predict the human PK of phosphorodiamidate morpholino oligomers from nonclinical data. The estimates of human PK parameters and profiles determined from cynomolgus monkeys by an allometric scaling approach were the most suitable, and the cynomolgus monkey body weight may be the key factor in accurately predicting human total clearance.

In Vitro Metabolism of 2-Methoxy-N-[3-[4-[3-methyl-4-[(6-methyl- 3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) by Aldehyde Oxidase and Predicting Its Percent Contribution Relative to CYP-Mediated Metabolism.

2-methoxy-N-[3-[4-[3-methyl-4-[(6-methyl-3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) is an anticancer drug that was discontinued due to hepatotoxicity found in clinical studies. Metabolite analysis of CP-724,714 was conducted using human hepatocytes, in which twelve oxidative metabolites and one hydrolyzed metabolite were formed. Among the three mono-oxidative metabolites, the formation of two was inhibited by adding 1-aminobenzotriazole, a pan-CYP inhibitor. In contrast, the remaining one was not affected by this inhibitor but partially inhibited by hydralazine, indicating that aldehyde oxidase (AO) was involved in metabolizing CP-724,714, which contains a quinazoline substructure, a heterocyclic aromatic quinazoline ring, known to be preferably metabolized by AO. One of the oxidative metabolites of CP-724,714 observed in human hepatocytes was also generated in recombinant human AO. Although CP-724,714 is metabolized by both CYPs and AO in human hepatocytes, the contribution level of AO could not be evaluated using its specific inhibitors because of low AO activity in in vitro human materials. Here, we present a metabolic pathway for CP-724,714 in human hepatocytes and the involvement of AO in CP-724,714 metabolism. We showed here a plausible workflow for predicting AO contribution to the metabolism of CP-724,714 based on DMPK screening data. SIGNIFICANCE STATEMENT: 2-methoxy-N-[3-[4-[3-methyl-4-[(6-methyl-3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) was identified as a substrate of aldehyde oxidase (AO) rather than xanthine oxidase. Since CP-724,714 is also metabolized by cytochrome P450s (CYPs), the contribution levels of AO and CYPs in the metabolism of CP-724,714 were estimated simultaneously based on in vitro drug metabolism screening data.

Disposition and Metabolism of [14C]Lemborexant in Healthy Human Subjects and Characterization of Its Circulating Metabolites.

Lemborexant is a novel dual orexin receptor antagonist recently approved for the treatment of insomnia in the United States and Japan. Here, disposition and metabolic profiles were investigated in healthy human subjects. After single oral administration of 10 mg [14C]lemborexant (100 µCi), plasma concentrations of lemborexant and radioactivity peaked at 1 hour postdose and decreased biphasically. Cumulative recovery of the administered radioactivity within 480 hours was 86.5% of the dose, with 29.1% in urine and 57.4% in feces. Unchanged lemborexant was not detected in urine but accounted for 13.0% of the dose in feces, suggesting that the main elimination pathway of lemborexant was metabolism. Metabolite analyses revealed that the major metabolic pathways of lemborexant are oxidation of the dimethylpyrimidine moiety and subsequent further oxidation and/or glucuronidation. In plasma, lemborexant was the dominant component, accounting for 26.5% of total drug-related exposure. M4, M9, M10, and M18 were detected as the major radioactive components; M10 was the only metabolite exceeding 10% of total drug-related exposure. Although M4, M9, and M10 showed binding affinity for orexin receptors comparable to that of lemborexant, their contributions to the sleep-promoting effects of lemborexant are likely low because of the limited brain penetration by P-glycoprotein. Exposure comparison between humans and nonclinical toxicology species confirmed that plasma exposure of M10 was higher in at least one animal species compared with that in humans, indicating that there is no disproportionate metabolite in humans, as defined by International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use M3(R2) and U.S. Food and Drug Administration Metabolite in Safety Testing guidance; therefore, no additional toxicology studies are needed. SIGNIFICANCE STATEMENT: This study provides detailed data of the disposition and metabolism of lemborexant, a novel therapeutic drug for insomnia, in humans, as well as a characterization of the circulating metabolites and assessment of their contributions to efficacy and safety. The information presented herein furthers our understanding of the pharmacokinetic profiles of lemborexant and its metabolites and will promote the safe and effective use of lemborexant in the clinic.

Bioactivation of diclofenac in human hepatocytes and the proposed human hepatic proteins modified by reactive metabolites.

To reveal putative bioactivation pathways of diclofenac, in vitro human liver materials such as microsomal fractions and hepatocytes were used to confirm metabolic activation of diclofenac by 35S-cysteine trapping assay and covalent binding assay. Candidate human liver proteins possibly targeted by 14C-diclofenac via bioactivation were investigated using two-dimensional gel electrophoresis followed by detection of remaining radioactivity on the modified proteins with bio-imaging analyzer.In the 35S-cysteine trapping assay, three and two adducts with 35S-cysteine were observed in NADPH-fortified and UDPGA-fortified human liver microsomes, respectively. In the covalent binding assay using 14C-diclofenac in human hepatocytes, the extent of covalent binding of diclofenac to human hepatic proteins increased time-dependently. Addition of glutathione attenuated the extent of covalent binding of 14C-diclofenac to human liver microsomal proteins.Fifty-nine proteins from human hepatocytes were proposed as the candidate proteins targeted by reactive metabolites of diclofenac. Proteins modified by cytochrome P450-mediated reactive metabolites were identified by using a cytochrome P450 inhibitor, 1-aminobenzyltriazole and seven of the nine radioactive protein spots were removed by 1-aminobenzyltriazole treatment.In contrast, the remaining two radioactive protein spots, mainly containing human serum albumin and heat shock proteins, were not affected by the addition of 1-aminobenzyltriazole, which suggested the involvement of the acyl glucuronide of diclofenac, formed via uridine diphosphate-glucuronosyl transferases, in the covalent modifications induced by diclofenac.

Prediction of Human Hepatic Clearance for Cytochrome P450 Substrates via a New Culture Method Using the Collagen Vitrigel Membrane Chamber and Fresh Hepatocytes Isolated from Liver Humanized Mice.

In drug discovery, hepatocytes have been widely utilized as in vitro tools for predicting the in vivo hepatic clearance (CL) of drug candidates. However, conventional hepatocyte models do not always reproduce in vivo physiological function, and CYP activities in particular decrease quite rapidly during culture. Furthermore, conventional in vitro assays have limitations in their ability to predict hepatic CL of metabolically stable drug candidates. In order to accurately predict hepatic CL of candidate drugs, a new method of culturing hepatocytes that activates their functional properties, including CYP activities, is in high demand. In the previous study, we established a novel long-term culture method for PXB-cells® using a collagen vitrigel membrane (CVM) chamber, which can maintain CYP activity and liver specific functions at high levels for several weeks. In this study, the vitrigel culture method was applied to predictions of hepatic CL for 22 CYP typical substrates with low to middle CL, and the prediction accuracy by this method was assessed by comparing CL data between predicted (in vitro intrinsic CL using the dispersion model) and observed (in vivo clinical data) values. The results of this study showed that in vitro CL values for approximately 60% (13/22) and 80% (18/22) of the compounds were predicted within a 2- and 3-fold difference with in vivo CL, respectively. These results suggest that the new culture method using the CVM chamber and PXB-cells is a promising in vitro system for predicting human hepatic CL with high accuracy for CYP substrates, including metabolically stable drug candidates.

Disposition and metabolism of [14C]lemborexant, a novel dual orexin receptor antagonist, in rats and monkeys.

The disposition and metabolism of lemborexant, a novel dual orexin receptor antagonist currently under development as a therapeutic agent for insomnia disorder, were evaluated after a single oral administration of [14C]lemborexant in Sprague-Dawley rats (10 mg/kg) and cynomolgus monkeys (3 mg/kg). In both species, [14C]lemborexant was rapidly absorbed: radioactivity concentration in blood peaked at 0.83-1.8 h, and decreased with elimination half-life of 110 h. The radioactivity administered was excreted primarily into faeces, with relatively little excreted into urine. Lemborexant was not detected in bile, urine or faeces, indicating that lemborexant administered orally was completely absorbed from the gastrointestinal tract and that the main elimination pathway was metabolism in both species. In rats, lemborexant was found to be minor in plasma (≤5.2% of total radioactivity), and M9 (hydroxylated form) was the major circulating metabolite. In monkeys, the major circulating components were lemborexant, M4 (N-oxide metabolite), M13 (di-oxidised form), M14 (di-oxidised form) and M16 (glucuronide of mono-oxidised form). In both species, lemborexant was metabolised to various metabolites by multiple pathways, the primary of which was oxidation of the dimethylpyrimidine or fluorophenyl moiety.

Comparison of the Reactivity of Trapping Reagents toward Electrophiles: Cysteine Derivatives Can Be Bifunctional Trapping Reagents.

Trapping reagents are powerful tools to detect unstable reactive metabolites. There are a variety of trapping reagents based on chemical reactivity to electrophiles, and we investigated the reactivity of thiol and amine trapping reagents to metabolically generated electrophiles and commercially available electrophilic compounds. Glutathione (GSH) and N-acetylcysteine (Nac) trapped soft electrophiles, and amine derivatives such as semicarbazide (SC) and methoxyamine (MeA) reacted as hard nucleophiles to trap aldehydes as imine derivatives. Cysteine (Cys) and homocysteine (HCys) captured both soft electrophiles and hard electrophilic aldehydes. There were no qualitative differences in trapping soft electrophiles among Cys, HCys, GSH, and Nac, although quantitative reactivity to trap soft electrophiles varied likely depending on the pKa values of their thiol group. In the reactivity with aldehydes, Cys and HCys showed relatively lower reactivity as compared with SC and MeA. Nonetheless, they can trap aldehydes, and the resulting conjugates were stable and detected easily because their amino group formed imines after reaction with aldehydes, which are successively attacked by the intramolecular thiol group to form stable ring structures. This report demonstrated that Cys and HCys are advantageous to evaluate the formations of both soft electrophiles and aldehyde-type derivatives from a lot of drug candidates at early drug discovery by their unique structural characteristics.

HPLC with fluorescence detection assay of perampanel, a novel AMPA receptor antagonist, in human plasma for clinical pharmacokinetic studies.

Perampanel (Fycompa®), a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, is registered for the adjunctive treatment of patients (aged ≥12 years) with refractory partial-onset seizures. To support therapeutic drug monitoring, a simple high-performance liquid chromatography (HPLC) assay with fluorescence detection was developed to determine perampanel concentrations in human plasma and validated to support clinical trials. Human plasma samples (1.0 mL) were processed by liquid extraction using diethyl ether, followed by chromatographic separation on a YMC Pack Pro C18 column (150 × 4.6 mm i.d., 5 µm) with isocratic elution of acetonitrile-water-acetic acid-sodium acetate (840:560:3:1.8, v/v/v/w) at a flow rate of 1.0 mL/min. Column eluent was monitored at excitation and emission wavelengths of 290 and 430 nm, respectively. The assay was linear (range 1.0-500 ng/mL) and this could be extended to 25 µg/mL by 50-fold dilution integrity. No endogenous peaks were detected in the elution of analytes in drug-free blank human plasma from six individuals and no interference was observed with co-medications tested. Intra- and inter-batch reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. Validation data demonstrated that our assay is simple, selective, reproducible and suitable for therapeutic drug monitoring of perampanel.

Validation of an ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of flecainide in human plasma and its clinical application.

A simple and reproducible bioanalytical method for the determination of flecainide in human plasma was developed and validated using an ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) to obtain higher sensitivity than the current available methods. After simple protein precipitation, flecainide and a stable isotope-labeled internal standard (IS) were chromatographed on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm) with isocratic elution of mobile phase consisting of 45% methanol containing 0.1% formic acid at a flow rate 0.25 mL/min. Detection was performed in positive electrospray ionization by monitoring the selected ion transitions at m/z 415.4/301.1 for flecainide and m/z 419.4/305.1 for the IS. The method was validated according to current bioanalytical method validation guidelines. The calibration standard curve was linear from 2.5 to 1000 ng/mL using 0.1 mL of plasma. No significant interferences were detected in blank human plasma. Accuracy and precision in the intra- and inter-batch reproducibility study were within acceptance criteria. Neither hemolysis effects nor matrix effects were observed. The UPLC-MS/MS method developed was successfully applied to determine plasma flecainide concentrations to support clinical studies and incurred sample reanalysis also ensured the reproducibility of the method.

Validation of a hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of gemcitabine in human plasma with tetrahydrouridine.

A simple and reproducible bioanalytical method for the determination of gemcitabine in human plasma treated with tetrahydrouridine (THU) was developed and validated using a hydrophilic interaction ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). To prevent deamination of gemcitabine, blood was treated with THU, and the plasma samples obtained after centrifugation were used in this study. Gemcitabine and gemcitabine-(13)C, (15)N2 used as an internal standard, were extracted from human plasma treated with THU using a 96-well Hybrid SPE-Precipitation plate. Extracts were chromatographed on a hydrophilic interaction chromatography column with isocratic elution. Detection was performed using Quattro Premier with positive electrospray ionization multiple reaction monitoring mode. The standard curve ranged from 10 to 10,000 ng/mL without carryover. No significant interferences were detected in blank plasma and no interferences by 2'-2'-difluoro-2'-deoxyuridine, a metabolite of gemcitabine. Accuracy and precision in the intra-batch reproducibility study using quality control samples with three THU levels did not exceed ±5.4 and 7.3%, respectively, and the inter-batch reproducibility results also met the criteria. Stability of gemcitabine was ensured in whole blood and plasma as well as stability of THU in solutions. The UPLC-MS/MS method developed was successfully validated and can be applied for gemcitabine bioanalysis in clinical studies.

Oxidative metabolic pathway of lenvatinib mediated by aldehyde oxidase.

Lenvatinib is a multityrosine kinase inhibitor that inhibits vascular endothelial growth factor receptors, and is being developed as an anticancer drug. P450s are involved in one of the elimination pathways of lenvatinib, and mono-oxidized metabolites, such as N-oxide (M3) and desmethylated metabolite (M2), form in rats, dogs, monkeys, and humans. Meanwhile, two other oxidative metabolites are produced only in monkey and human liver S9 fractions, and their structures have been identified using high-resolution mass spectrometry as a quinolinone form of lenvatinib (M3') and a quinolinone form of desmethylated lenvatinib (M2'). The formation of M3' from lenvatinib occurred independently of NADPH and was effectively inhibited by typical inhibitors of aldehyde oxidase, indicating the involvement of aldehyde oxidase, but not P450s, in this pathway. M2' was a dioxidized metabolite arising from a combination of mono-oxidation and desmethylation and could only be produced from M2 in a NADPH-independent manner; M2' could not be generated from M3 or M3'. These results suggested that M2' is formed from lenvatinib by a unique two-step pathway through M2. Although both lenvatinib and M2 were substrates for aldehyde oxidase, an enzyme kinetic study indicated that M2 was a much more favorable substrate than lenvatinib. No inhibitory activities of lenvatinib, M2', or M3' and no significant inhibitory activities of M2 or M3 on aldehyde oxidase were observed, suggesting a low possibility of drug-drug interactions in combination therapy with substrates of aldehyde oxidase.

Antipruritic effect of the topical phosphodiesterase 4 inhibitor E6005 ameliorates skin lesions in a mouse atopic dermatitis model.

Phosphodiesterase (PDE) 4 inhibition is a well-known anti-inflammatory mechanism, but the development of PDE4 inhibitors has been hampered by side effects such as nausea and emesis. Local delivery of a PDE4 inhibitor to the site of inflammation may overcome these issues. The purpose of this study was to assess the therapeutic potential of E6005 (methyl 4-[({3-[6,7-dimethoxy-2-(methylamino)quinazolin-4-yl]phenyl}amino)carbonyl]benzoate), a novel PDE4 inhibitor developed as a topical agent for atopic dermatitis (AD). E6005 potently and selectively inhibited human PDE4 activity with an IC50 of 2.8 nM and suppressed the production of various cytokines from human lymphocytes and monocytes with IC50 values ranging from 0.49 to 3.1 nM. In mice models, the topical application of E6005 produced an immediate antipruritic effect as well as an anti-inflammatory effect with reduced expression of cytokines/adhesion molecules. On the basis of these observed effects, topical E6005 ameliorated the appearance of atopic dermatitis-like skin lesions in two types of AD models, hapten- and mite-elicited models, exhibiting inhibitory effects comparable to that of tacrolimus. The use of ¹4C-labeled E6005 showed rapid clearance from the blood and low distribution to the brain, contributing to the low emetic potential of this compound. These results suggest that E6005 may be a promising novel therapeutic agent with antipruritic activity for the treatment of AD.

Species difference in the mechanism of nonlinear pharmacokinetics of E2074, a novel sodium channel inhibitor, in rats, dogs, and monkeys.

New chemical entities often exhibit nonlinear pharmacokinetics (PK) profiles in experimental animals. However, the number of studies that have focused on species differences in nonlinear PK is very limited; thus, the aim of this study was to clarify the mechanism of the nonlinear PK of E2074 (2-[(2R)-2-fluoro-3-{(3r)-[(3-fluorobenzyl)oxy]-8-azabicyclo[3.2.1]oct-8-yl}propyl]-4,5-dimethyl-2,4-dihydro-3H-1,2,4-triazol-3-one), a novel sodium channel inhibitor, in rats, dogs, and monkeys. Nonlinear PK profiles with more than dose-proportional increases of Cmax and area under the plasma concentration curve were observed in all species after oral administration. The Michaelis-Menten constant (Km) values of hepatic microsomal metabolism were 7.23 and 0.41 µM in rats and dogs in vitro, respectively, which were lower than the unbound maximum plasma concentrations after oral administration in vivo, indicating that the nonlinear PK in rats and dogs was attributable to the saturation of hepatic metabolism. However, we do not believe that the saturation of hepatic metabolism was the mechanism of nonlinearity in monkeys because of the high Km value (42.44 µM) observed in liver microsomes. Intestinal metabolism was observed in monkey intestinal microsomes but not in rats and dogs, and the nonlinear PK in monkeys was diminished by inhibition of intestinal metabolism with a concomitant oral dose of ketoconazole. These results suggest that saturation of the intestinal metabolism is the potential mechanism of nonlinearity in monkeys. P-glycoprotein was not involved in the nonlinear PK profiles in any species. In conclusion, the mechanism of the nonlinear PK of E2074 is species dependent, with the saturation of hepatic metabolism in rats and dogs and that of intestinal metabolism in monkeys being the primary cause.

Substrate-dependent inhibition of organic anion transporting polypeptide 1B1: comparative analysis with prototypical probe substrates estradiol-17ß-glucuronide, estrone-3-sulfate, and sulfobromophthalein.

Organic anion transporting polypeptide (OATP) 1B1 plays an important role in the hepatic uptake of many drugs, and the evaluation of OATP1B1-mediated drug-drug interactions (DDIs) is emphasized in the latest DDI (draft) guidance documents from U.S. and E.U. regulatory agencies. It has been suggested that some OATP1B1 inhibitors show a discrepancy in their inhibitory potential, depending on the substrates used in the cell-based assay. In this study, inhibitory effects of 14 compounds on the OATP1B1-mediated uptake of the prototypical substrates [³H]estradiol-17ß-glucuronide (E2G), [³H]estrone-3-sulfate (E1S), and [³H]sulfobromophthalein (BSP) were studied in OATP1B1-transfected cells. Inhibitory potencies of tested compounds varied depending on the substrates. Ritonavir, gemfibrozil, and erythromycin caused remarkable substrate-dependent inhibition with up to 117-, 14-, and 13-fold difference in their IC50 values, respectively. Also, the clinically relevant OATP inhibitors rifampin and cyclosporin A exhibited up to 12- and 6-fold variation in their IC50 values, respectively. Regardless of the inhibitors tested, the most potent OATP1B1 inhibition was observed when [³H]E2G was used as a substrate. Mutual inhibition studies of OATP1B1 indicated that E2G and E1S competitively inhibited each other, whereas BSP noncompetitively inhibited E2G uptake. In addition, BSP inhibited E1S in a competitive manner, but E1S caused an atypical kinetics on BSP uptake. This study showed substrate-dependent inhibition of OATP1B1 and demonstrated that E2G was the most sensitive in vitro OATP1B1 probe substrate among three substrates tested. This will give us an insight into the assessment of clinically relevant OATP1B1-mediated DDI in vitro with minimum potential of false-negative prediction.

Unique metabolic pathway of [(14)C]lenvatinib after oral administration to male cynomolgus monkey.

Lenvatinib, a potent inhibitor of multiple tyrosine kinases, including vascular endothelial growth factor receptors 2 and 3, generated unique metabolites after oral administration of [(14)C]lenvatinib (30 mg/kg) to a male cynomolgus monkey. Lenvatinib was found to be transformed to a GSH conjugate, through displacement of an O-aryl moiety, at the quinoline part of the molecule in the liver and kidneys. The GSH conjugate underwent further hydrolysis by γ-glutamyltranspeptidase and dipeptidases, followed by intramolecular rearrangement, to form N-cysteinyl quinoline derivatives, which were dimerized to form disulfide dimers and also formed an N,S-cysteinyl diquinoline derivative. In urine, a thioacetic acid conjugate of the quinoline was also observed as one of the major metabolites of lenvatinib. Lenvatinib is a 4-O-aryl quinoline derivative, and such compounds have been known to undergo conjugation with GSH, accompanied by release of the O-aryl moiety. Because of intramolecular rearrangement in the case of lenvatinib, hydrolysis of the GSH conjugate yielded N-cysteinylglycine and N-cysteine conjugates instead of the corresponding S-conjugates. Because the N-substituted derivatives possess free sulfhydryl groups, dimerization through disulfide bonds and another nucleophilic substitution reaction with lenvatinib resulted in the formation of disulfanyl dimers and an N,S-cysteinyl diquinoline derivative, respectively. Characteristic product ions at m/z 235 and m/z 244, which were associated with thioquinoline and N-ethylquinoline derivatives, respectively, were used to differentiate S- and N-derivatives in this study. On the basis of accurate mass and NMR measurements, a unique metabolic pathway for lenvatinib in monkey and the proposed formation mechanism have been elucidated.

Expressions of cytochrome P450, UDP-glucuronosyltranferase, and transporter genes in monolayer carcinoma cells change in subcutaneous tumors grown as xenografts in immunodeficient nude mice.

Human tumors grown as xenografts in immunodeficient nude mice are widely used to investigate the pharmacological activities of anticancer drugs. Drug-metabolizing enzymes and transporters are expressed in tumor cell lines and changes in drug metabolism and pharmacokinetics (DMPK)-related gene expression after inoculation of the tumor cell may affect the pharmacological activity of the drug under consideration. The aims of the current study were to characterize DMPK-related gene expression profiles and responses to typical cytochrome P450 inducers in monolayer carcinoma cells grown in tissue culture versus those inoculated into a xenograft model. We used the human hepatocellular carcinoma cell line PLC/PRF/5 for this study and comprehensively assessed changes in DMPK-related gene expression by reverse transcription-polymerase chain reaction quantitation. CYP3A4 and UDP-glucuronosyltransferase 1A protein amounts were also analyzed by immunoprecipitation followed by immunoblotting. We found that the expression of many DMPK-related genes was elevated in the inoculated tumor compared with the monolayer carcinoma cells, indicating changes in their gene regulation pathways, presumably due to modulation of the nuclear receptor family of transcription factors. In addition, monolayer carcinoma versus inoculated tumor cells showed different responses to rifampicin, but similar responses to dexamethasone or 3-methylcholanthrene. These results suggest that inoculation of tumor cells results in the activation of drug metabolism and transport function, leading to changes in the responses to pregnane X receptor ligands and consequent discrepancies in the pharmacological activities between in vitro monolayer carcinoma cells and in vivo xenograft models.

Inoculation of human tumor cells alters the basal expression but not the inducibility of cytochrome P450 enzymes in tumor-bearing mouse liver.

The athymic nude mouse is often used to grow tumors for in vivo oncology research, including the identification of anticancer drugs, whereas wild-type mice are usually used to assess the pharmacokinetics (PK) of new chemical entities. The relationship between PK and pharmacodynamics (PD) provides useful mechanistic information and helps guide of the clinical regimen. The aim of this study was to assess whether the inoculation of human hepatocellular carcinoma cells (PLC/PRF/5) into athymic nude mice alters the expression of genes encoding the drug-metabolizing enzymes and transporters in host liver. The livers from nontumor- and tumor-bearing mice were initially subjected to drug metabolism gene microarray analysis. Microarray analysis indicated that tumor inoculation had little effect on drug metabolism-related genes, including several cytochrome P450s: Cyp1a, Cyp2b, and Cyp3a. This result was further confirmed by reverse transcription-polymerase chain reaction (RT-PCR). However, immunoreactive proteins of Cyp1a, Cyp2b, and Cyp3a were suppressed by tumor inoculation. RT-PCR and Western immunoblotting analysis showed that the inducibility of Cyp1a, Cyp2b, and Cyp3a by 3-methylcholanthrene, phenobarbital, and dexamethasone, respectively, was similar between nontumor- and tumor-bearing mice. These results suggest that inoculation of human tumor cells into athymic nude mice suppresses the expression of certain drug-metabolizing enzymes, which may alter the PK and PD of antitumor drugs.

A long-term culture system based on a collagen vitrigel membrane chamber that supports liver-specific functions of hepatocytes isolated from mice with humanized livers.

During drug discovery, in vitro models are used to predict the in vivo pharmacokinetic and toxicological properties of drug candidates in humans. However, the conventional method of culturing human hepatocytes as monolayers does not necessarily replicate biologic reactions and does not support liver-specific functions, such as cytochrome P450 (CYP) activities, for prolonged periods. To remedy these problems and thus increase and prolong hepatic functions, we developed a culture system comprising a collagen vitrigel membrane (CVM) chamber and PXB-cells®, fresh hepatocytes isolated from liver-humanized chimeric mice (PXB-mice®). To quantitatively assess our new system, we evaluated the activities of 5 major CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A), albumin secretion, and urea synthesis. First, between Days 14 and 21, the activities of all CYP isoforms tested in vitrigel culture were equal to or higher than in conventional monolayer culture system. Second, the activities of CYP3A, CYP2C9, and CYP2C19 during Days 10 through 17 were higher in vitrigel culture than in suspended PXB-cells prepared on Day 0 (suspension assay). Third, albumin secretion and urea synthesis were higher in vitrigel culture than in conventional monolayer culture. Fourth, the vitrigel-cultured PXB-cells showed the characteristic morphology of parenchymal hepatocytes and were almost all alive in monolayer. These results indicate that our vitrigel culture method is superior to the conventional monolayer method in terms of diverse liver-specific functions, including CYP activity. Our findings suggest that the vitrigel culture method could be a powerful in vitro tool for predicting the pharmacokinetic and toxicological properties of drug candidates in humans.

Eribulin shows high concentration and long retention in xenograft tumor tissues.

PURPOSE: Eribulin, a synthetic analog of the natural product halichondrin B, is a microtubule dynamics inhibitor. In this study, we report the pharmacokinetic profiles of eribulin in mice, rats, and dogs following intravenous administrations with optimized and validated bio-analytical methods. METHODS: Eribulin was administered at 0.5 and 2 mg/kg in mice, 0.5 and 1 mg/kg in rats, and 0.08 mg/kg in dogs. Tumor and brain penetration of eribulin was also evaluated in LOX human melanoma xenograft models. Concentrations in plasma, tumor, and brain were measured by the LC-MS/MS method. RESULTS: The profiles of eribulin were characterized by extensive distribution, moderate clearance, and slow elimination in the three species. The pharmacokinetics are linear in mice and rats. In xenograft mice, the penetration into the brain was low, as expected, since eribulin is a P-glycoprotein substrate. In contrast to disposition in brain, the exposure of eribulin was approximately 20-30 times higher in tumor than that in plasma and half-lives were 17.8-35.9 h after both single and multiple dose regimens. CONCLUSIONS: Eribulin was distributed rapidly and eliminated slowly in mice, rats, and dogs. The exposure of eribulin was approximately 20-30 times higher in tumor than in plasma in xenograft mice. These results might be caused by eribulin's mechanism of action including increased perfusion in tumor by vascular remodeling effect.