Unveiling the intra-tumor fate of trastuzumab deruxtecan in a xenograft model to support its mechanism of action.

Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate used for cancer treatment comprising an anti-human epidermal growth factor receptor type 2 (HER2) antibody and the topoisomerase I inhibitor DXd. The present study investigated the intratumor fate of T-DXd. Fluorescence-labeled T-DXd was found to accumulate in tumors of HER2-positive tumor xenograft mice and was observed to be distributed within lysosomes of in vitro tumor cells in accordance with their HER2 expression. DXd was released by cysteine proteases, including cathepsins, in lysosomal fractions in vitro in response to the pH. Tumor slices obtained from HER2-positive tumor xenograft mice treated with T-DXd were examined by semi-quantitative and three-dimensional immunohistochemical assays using phosphor-integrated dots, which visualized DXd-related signals in the nucleus, the site of topoisomerase I inhibition. In addition, based on the data showing the antibody component of T-DXd barely distributed in the nucleus, it was suggested that the DXd-related signals detected in the nucleus were predominantly derived from free DXd. These observations help support the mode of action of T-DXd from the perspective of drug disposition.

Transition of average drug-to-antibody ratio of trastuzumab deruxtecan in systemic circulation in monkeys using a hybrid affinity capture liquid chromatography-tandem mass spectrometry.

Trastuzumab deruxtecan (T-DXd, DS-8201a) is an antibody-drug conjugate, comprising an anti-HER2 antibody at a drug-to-antibody ratio of 7-8 with the topoisomerase I inhibitor DXd. In this study, the concentrations of antibody-conjugated DXd and total antibody were determined and observed to decrease over time following intravenous administration of T-DXd to monkeys. The drug-to-antibody ratio of T-DXd also decreased in a time-dependent manner, which reached approximately 2.5 in 21 days after administration. It was suggested that antibody-conjugated DXd of T-DXd was relatively stable in vivo compared with that of other reported antibody-drug conjugates.

Drug Metabolism and Pharmacokinetics of Antisense Oligonucleotide Therapeutics: Typical Profiles, Evaluation Approaches, and Points to Consider Compared with Small Molecule Drugs.

Oligonucleotide therapeutics are attracting attention as a new treatment modality for a range of diseases that have been difficult to target using conventional approaches. Technical advances in chemical modification and drug delivery systems have led to the generation of compounds with excellent profiles as pharmaceuticals, and 16 oligonucleotide therapeutics have been marketed to date. There is a growing need to develop optimal and efficient approaches to evaluate drug metabolism and pharmacokinetics (DMPK) and drug-drug interactions (DDIs) of oligonucleotide therapeutics. The DMPK/DDI profiles of small molecule drugs are highly diverse depending on their structural and physicochemical characteristics, whereas oligonucleotide therapeutics share similar DMPK profiles within each chemistry type. Most importantly, the mechanisms and molecules involved in the distribution and metabolism of oligonucleotides differ from those of small molecules. In addition, there are considerations regarding experimental approaches in the evaluation of oligonucleotides, such as bioanalytical challenges, the use of radiolabeled tracers, materials for in vitro metabolism/DDI studies, and methods to study biodistribution. In this review, we attempt to summarize the DMPK characteristics of antisense oligonucleotide (ASO) therapeutics and discuss some of the issues regarding how to optimize the evaluation and prediction of the DMPK and DDI of ASOs.

Physiologically Based Pharmacokinetic Modeling for Quantitative Prediction of Exposure to a Human Disproportionate Metabolite of the Selective NaV1.7 Inhibitor DS-1971a, a Mixed Substrate of Cytochrome P450 and Aldehyde Oxidase, Using Chimeric Mice With Humanized Liver.

In a previous study on the human mass balance of DS-1971a, a selective NaV1.7 inhibitor, its CYP2C8-dependent metabolite M1 was identified as a human disproportionate metabolite. The present study assessed the usefulness of pharmacokinetic evaluation in chimeric mice grafted with human hepatocytes (PXB-mice) and physiologically based pharmacokinetic (PBPK) simulation of M1. After oral administration of radiolabeled DS-1971a, the most abundant metabolite in the plasma, urine, and feces of PXB-mice was M1, while those of control SCID mice were aldehyde oxidase-related metabolites including M4, suggesting a drastic difference in the metabolism between these mouse strains. From a qualitative perspective, the metabolite profile observed in PXB-mice was remarkably similar to that in humans, but the quantitative evaluation indicated that the area under the plasma concentration-time curve (AUC) ratio of M1 to DS-1971a (M1/P ratio) was approximately only half of that in humans. A PXB-mouse-derived PBPK model was then constructed to achieve a more accurate prediction, giving an M1/P ratio (1.3) closer to that in humans (1.6) than the observed value in PXB-mice (0.69). In addition, simulated maximum plasma concentration and AUC values of M1 (3429 ng/ml and 17,116 ng·h/ml, respectively) were similar to those in humans (3180 ng/ml and 18,400 ng·h/ml, respectively). These results suggest that PBPK modeling incorporating pharmacokinetic parameters obtained with PXB-mice is useful for quantitatively predicting exposure to human disproportionate metabolites. SIGNIFICANCE STATEMENT: The quantitative prediction of human disproportionate metabolites remains challenging. This paper reports on a successful case study on the practical estimation of exposure (C max and AUC) to DS-1971a and its CYP2C8-dependent, human disproportionate metabolite M1, by PBPK simulation utilizing pharmacokinetic parameters obtained from PXB-mice and in vitro kinetics in human liver fractions. This work adds to the growing knowledge regarding metabolite exposure estimation by static and dynamic models.

Oral Absorption of Middle-to-Large Molecules and Its Improvement, with a Focus on New Modality Drugs.

To meet unmet medical needs, middle-to-large molecules, including peptides and oligonucleotides, have emerged as new therapeutic modalities. Owing to their middle-to-large molecular sizes, middle-to-large molecules are not suitable for oral absorption, but there are high expectations around orally bioavailable macromolecular drugs, since oral administration is the most convenient dosing route. Therefore, extensive efforts have been made to create bioavailable middle-to-large molecules or develop absorption enhancement technology, from which some successes have recently been reported. For example, Rybelsus® tablets and Mycapssa® capsules, both of which contain absorption enhancers, were approved as oral medications for type 2 diabetes and acromegaly, respectively. The oral administration of Rybelsus and Mycapssa exposes their pharmacologically active peptides with molecular weights greater than 1000, namely, semaglutide and octreotide, respectively, into systemic circulation. Although these two medications represent major achievements in the development of orally absorbable peptide formulations, the oral bioavailability of peptides after taking Rybelsus and Mycapssa is still only around 1%. In this article, we review the approaches and recent advances of orally bioavailable middle-to-large molecules and discuss challenges for improving their oral absorption.

Species differences between rats and primates (humans and monkeys) in complex cleavage pathways of DS-8500a characterized by 14C-ADME studies in humans and monkeys after administration of two radiolabeled compounds and in vitro studies.

Our previous study in rats demonstrated that the metabolic pathways of DS-8500a, a novel GPR119 agonist, include cleavage pathways: reductive cleavage of the oxadiazole ring in the liver and hydrolysis of the amide side chain. In the present study, in vivo metabolic profiling in humans and monkeys after the oral administration of two 14C-labeled compounds was performed to investigate species differences of the cleavage pathways. In monkeys, the oxadiazole ring-cleaved metabolites were mainly detected in feces, but not observed in bile, unlike in rats, suggesting that the reductive ring-opening metabolism occurs in the gastrointestinal tract. In vitro incubation with enterobacterial culture media demonstrated that the reductive cleavage of the oxadiazole ring in humans and monkeys was considerably faster than that in rats. The other cleavage metabolite (M20), produced via hydrolysis of the amide side chain, was detected as the major plasma metabolite in humans and monkeys, and its subsequent metabolite (M21) was excreted in feces, whereas M21 was not a major component in rats, indicating a notable species difference in the amide hydrolysis. In conclusion, this study comprehensively revealed the pronounced species difference of the cleavage pathways: reductive ring-opening by intestinal microflora and liver, and amide hydrolysis.

Renadirsen, a Novel 2'OMeRNA/ENA® Chimera Antisense Oligonucleotide, Induces Robust Exon 45 Skipping for Dystrophin In Vivo.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by out-of-frame or nonsense mutation in the dystrophin gene. It begins with a loss of ambulation between 9 and 14 years of age, followed by various other symptoms including cardiac dysfunction. Exon skipping of patients' DMD pre-mRNA induced by antisense oligonucleotides (AOs) is expected to produce shorter but partly functional dystrophin proteins, such as those possessed by patients with the less severe Becker muscular dystrophy. We are working on developing modified nucleotides, such as 2'-O,4'-C-ethylene-bridged nucleic acids (ENAs), possessing high nuclease resistance and high affinity for complementary RNA strands. Here, we demonstrate the preclinical characteristics (exon-skipping activity in vivo, stability in blood, pharmacokinetics, and tissue distribution) of renadirsen, a novel AO modified with 2'-O-methyl RNA/ENA chimera phosphorothioate designed for dystrophin exon 45 skipping and currently under clinical trials. Notably, systemic delivery of renadirsen sodium promoted dystrophin exon skipping in cardiac muscle, skeletal muscle, and diaphragm, compared with AOs with the same sequence as renadirsen but conventionally modified by PMO and 2'OMePS. These findings suggest the promise of renadirsen sodium as a therapeutic agent that improves not only skeletal muscle symptoms but also other symptoms in DMD patients, such as cardiac dysfunction.

Pharmacokinetics, Metabolism, and Excretion of [14C]Esaxerenone, a Novel Mineralocorticoid Receptor Blocker in Humans.

Esaxerenone (CS-3150) is a novel, nonsteroidal, selective mineralocorticoid receptor blocker. The absorption, metabolism, distribution, and excretion of esaxerenone were assessed in in vitro studies and in a clinical study, where [14C]esaxerenone (150 µCi/20 mg) was administered orally to six healthy male subjects. The plasma concentrations of esaxerenone and its metabolites (M4, M11, and M1) were measured using liquid chromatography-tandem mass spectrometry. The recovery of radioactivity was 92.5%, with 38.5% and 54.0% excreted in the urine and feces, respectively. The half-life of radioactivity in blood and plasma was approximately 30 hours, similar to that of the unchanged form in plasma. The blood-to-plasma ratio was 0.628, demonstrating low partitioning to blood components. In plasma, esaxerenone was the most abundant moiety (40.8%), followed by O-glucuronide (21.4%; M4), acyl-glucuronide of amide-bond hydrolysate (8.0%; M11), and the deshydroxyethyl form (1.7%; M1). In vitro studies showed that esaxerenone was a substrate of CYP3A and multiple UDP-glucuronosyltransferase isoforms. Oxidation contributed approximately 30% to its clearance, as indicated by the excretion ratio of oxidized metabolites into urine and feces. Caco-2 studies showed that esaxerenone was a substrate of P-glycoprotein and breast cancer resistance protein; however, the excretion ratios of the unchanged form in the feces and urine were 18.7% and 1.6%, respectively, indicating that these transporters were not important for the absorption and elimination of esaxerenone. Low urinary excretion of esaxerenone suggested that the plasma exposure of esaxerenone was not affected by renal dysfunction. Multiple elimination pathways including oxidation, glucuronidation, and hydrolysis, and the low contribution of transporters, indicated limited drug-drug interaction potential.

Identification of a novel hormone sensitive lipase inhibitor with a reduced potential of reactive metabolites formation.

Hormone sensitive lipase (HSL) has emerged as an attractive target for the treatment of dyslipidemia. We previously reported compound 1 as a potent and orally active HSL inhibitor. Although an attractive profile was demonstrated, subsequent studies revealed that compound 1 has a bioactivation liability. The oxygen-carbon linker in compound 1 was identified as being potentially responsible for reactive metabolite formation. By exchanging of this susceptible fragment was feasible, and a benzanilide derivative 6b with a decreased bioactivation liability was obtained. Further modification of the novel benzanilide scaffold resulted in the identification of compound 24b. Compound 24b exhibited potent HSL inhibitory activity (IC50=2nM) with a significantly reduced bioactivation potential. Oral administration of compound 24b exhibited an antilipolytic effect on rats at 3mg/kg.

Difference in Mechanism-Based Inhibition of Cytochrome P450 3A4 and 3A5 by a Series of Fluoroquinolone Antibacterial Agents.

A series of fluoroquinolone antibacterial compounds were found to be irreversible (compounds 1-5) and quasi-irreversible (compounds 6-9) inhibitors of CYP3A4. The purpose of this study was to evaluate their mechanism-based inhibition (MBI) potency against CYP3A5. Compounds 1-5 were also irreversible inhibitors of CYP3A5, whereas compounds 6-9 showed neither irreversible nor quasi-irreversible inhibition of CYP3A5. Compounds 6 and 8 did not form a metabolite-intermediate complex with the heme of CYP3A5 during incubation. The structural analysis of the metabolites after incubation of compounds 1 and 6 with CYP3A5 revealed that their metabolites were identical to those produced by CYP3A4, including the precursors of which are speculated to account for the MBI of CYP3A4. The homology modeling of CYP3A5 suggests that four residues around the nitroso intermediate of compound 6 in the substrate-binding pocket of CYP3A4 correspond with the bulkier residues in CYP3A5-especially Phe210 in CYP3A5-which might contribute to the steric hindrance with the nitroso intermediate of compound 6. The substrate-binding pocket structure of CYP3A5 might prevent the nitroso intermediate from coordinate binding with the heme, thereby preventing quasi-irreversible inhibition. Our study may provide new insights into the observable differences between the inhibition of CYP3A4 and CYP3A5.

Pharmacokinetics, distribution, and disposition of esaxerenone, a novel, highly potent and selective non-steroidal mineralocorticoid receptor antagonist, in rats and monkeys.

1. Esaxerenone (CS-3150) is a novel non-steroidal mineralocorticoid receptor antagonist. The pharmacokinetics, tissue distribution, excretion, and metabolism of esaxerenone were evaluated in rats and monkeys. 2. Following intravenous dosing of esaxerenone at 0.1-3 mg/kg, the total body clearance and the volume of distribution were 3.53-6.69 mL/min/kg and 1.47-2.49 L/kg, respectively, in rats, and 2.79-3.69 mL/min/kg and 1.34-1.54 L/kg, respectively, in monkeys. The absolute oral bioavailability was 61.0-127% in rats and 63.7-73.8% in monkeys. 3. After oral administration of [14C]esaxerenone, the radioactivity was distributed widely to tissues, with the exception of a low distribution to the central nervous system. Both in rats and in monkeys, following oral administration of [14C]esaxerenone the main excretion route of the radioactivity was feces. 4. Five initial metabolic pathways in rats and monkeys were proposed to be N-dealkylation, carboxylation, hydroxymethylation, O-glucuronidation, and O-sulfation. The oxidized metabolism was predominant in rats, while both oxidation and glucuronidation were predominant in monkeys.

Analysis of Mechanism-Based Inhibition of CYP 3A4 by a Series of Fluoroquinolone Antibacterial Agents.

A series of fluoroquinolone compounds (compounds 1-9), which contain a common quinolone scaffold, inactivated the metabolic activity of CYP3A. The purpose of this study was to identify mechanism-based inhibition (MBI) among these fluoroquinolone compounds by metabolite profiling to elucidate the association of the substructure and MBI potential. Reversibility of MBI after incubation with potassium ferricyanide differed among the test compounds. Representative quasi-irreversible inhibitors form a metabolite-intermediate (MI) complex with the heme of CYP3A4 according to absorption analysis. Metabolite profiling identified the cyclopropane ring-opened metabolites from representative irreversible inhibitors, suggesting irreversible binding of the carbon-centered radical species with CYP3A4. On the other hand, the oxime form of representative quasi-irreversible inhibitors was identified, suggesting generation of a nitroso intermediate that could form the MI complex. Metabolites of compound 10 with a methyl group at the carbon atom at the root of the amine moiety of compound 8 include the oxime form, but compound 10 did not show quasi-irreversible inhibition. The docking study with CYP3A4 suggested that a methyl moiety introduced at the carbon atom at the root of the primary amine disrupts formation of the MI complex between the heme and the nitroso intermediate because of steric hindrance. This study identified substructures of fluoroquinolone compounds associated with the MBI mechanism; introduction of substituted groups inducing steric hindrance with the heme of P450 can prevent formation of an MI complex. Our series of experiments may be broadly applicable to prevention of MBI at the drug discovery stage.

Stereoselective hydroxylation by CYP2C19 and oxidation by ADH4 in the in vitro metabolism of tivantinib.

1. In prior studies, it has been shown that tivantinib is extensively metabolized in humans to many oxidative metabolites and glucuronides. In order to identify the responsible enzymes, we investigated the in vitro metabolism of tivantinib and its four major circulating metabolites. 2. The primary isoforms involved in the elimination of tivantinib were CYP2C19 and CYP3A4/5. CYP2C19 showed catalytic activity for the formation of M5 (hydroxylated metabolite), but not for M4 (a stereoisomer of M5), whereas CYP3A4/5 catalyzed the formation of both metabolites. For the elimination of M4, M5 and M8 (keto-metabolite), CYP3A4/5 was the major cytochrome P450 isoform and UGT1A9 was mainly involved in the glucuronidation of M4 and M5. 3. ADH4 was identified as one of the major alcohol dehydrogenase isoforms contributing to the formation of M6 (sequential keto-metabolite of M4 and M5) and M8. The substrate preference of ADH for M4, and not M5, was observed in the formation of M6. 4. In conclusion, CYP2C19, CYP3A4/5, UGT1A9 and ADH4 were the primary drug metabolizing enzymes involved in the in vitro metabolism of tivantinib and its metabolites. The stereoselective hydroxylation by CYP2C19 and substrate stereoselectivity of ADH4-catalyzed oxidation in the in vitro metabolism of tivantinib was discovered.

Metabolism and disposition of [(14)C]tivantinib after oral administration to humans, dogs and rats.

1. The biotransformation and disposition of tivantinib in humans, dogs and rats was examined after a single oral administration of [(14)C]tivantinib. Tivantinib constituted no more than one-third of the plasma radioactivity in all species, demonstrating significant contribution of the metabolites to plasma radioactivity. The major circulating metabolites in all species were M4 and M5, hydroxylated metabolites at the benzyl position of the tricyclic ring, accounting for 19.3 and 12.2% of the AUC of the total radioactivity, respectively, in humans. 2. The majority of radioactivity was excreted to the feces via bile. Tivantinib was detected at trace levels in urine, feces and bile, demonstrating extensive metabolism prior to biliary excretion and nearly complete tivantinib absorption under fed conditions. 3. Seven metabolic pathways were identified for tivantinib and included six oxidations (M4, M5, M7, M8, M9 and M11) and one glucuronidation (M23). The major metabolic and excretory pathways were found to be common among all species. Species differences in the metabolic pathways included lactam metabolite (M8) formation in humans and dehydrogenated metabolite (M11) formation in animals. 4. None of the metabolites identified in this work are believed to significantly impact the efficacy or toxicity of tivantinib in humans.

Tissue distribution and identification of radioactivity components at elimination phase after oral administration of [¹4C]CS-1036, an α-amylase inhibitor, to rats.

(2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-ß-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036) is a potent inhibitor of pancreatic and salivary α-amylase. After oral administration of [¹4C]CS-1036 to rats, the radioactivity was still detectable up to 7-14 days after administration in various tissues, and its terminal phase in plasma could be explained neither by the exposure of CS-1036 nor its major metabolite M1. The slow elimination of radioactivity in various tissues was hypothesized to be caused by covalent binding to macromolecules or use for biogenic components. To assess the use for biogenic components, amino acid analysis of plasma proteins and lipid analysis of adipose tissue were conducted after repeated oral administration of [¹4C]CS-1036 by high-performance liquid chromatography and accelerated mass spectrometry and by thin layer chromatography and liquid chromatography/mass spectrometry, respectively. In amino acid analysis, glutamic acid, aspartic acid, alanine, and proline were identified as major radioactive amino acids, and radioactive nonessential amino acids occupied 76.0% of the radioactivity. In lipid analysis, a part of the radioactive lipids were identified as the fatty acids constituting the neutral lipids by lipase-hydrolysis. The radioactive fatty acids from neutral lipids were identified as palmitic acid, oleic acid, and 8,11,14-eicosatrienoic acid. Intestinal flora were involved in CS-1036 metabolism and are indicated to be involved in the production of small molecule metabolites, which are the sources for amino acids and fatty acids, from [¹4C]CS-1036. In conclusion, radioactivity derived from [¹4C]CS-1036 was incorporated as the constituents of amino acids of plasma proteins and fatty acids of neutral lipids.

Discovery and optimization of novel fatty acid transport protein 1 (FATP1) inhibitors.

The discovery, optimization and structure-activity relationship of novel FATP1 inhibitors have been described. The detailed SAR studies of each moiety of the inhibitors combined with metabolite analysis led to the identification of the potent inhibitors 11p and 11q with improved blood stability.

Protein tyrosine nitration of mitochondrial carbamoyl phosphate synthetase 1 and its functional consequences.

Mitochondria are the primary locus for the generation of reactive nitrogen species including peroxynitrite and subsequent protein tyrosine nitration. Protein tyrosine nitration may have important functional and biological consequences such as alteration of enzyme catalytic activity. In the present study, mouse liver mitochondria were incubated with peroxynitrite, and the mitochondrial proteins were separated by 1D and 2D gel electrophoresis. Nitrotyrosinylated proteins were detected with an anti-nitrotyrosine antibody. One of the major proteins nitrated by peroxynitrite was carbamoyl phosphate synthetase 1 (CPS1) as identified by LC-MS protein analysis and Western blotting. The band intensity of nitration normalized to CPS1 was increased in a peroxynitrite concentration-dependent manner. In addition, CPS1 activity was decreased by treatment with peroxynitrite in a peroxynitrite concentration- and time-dependent manner. The decreased CPS1 activity was not recovered by treatment with reduced glutathione, suggesting that the decrease of the CPS1 activity is due to tyrosine nitration rather than cysteine oxidation. LC-MS analysis of in-gel digested samples, and a Popitam-based modification search located 5 out of 36 tyrosine residues in CPS1 that were nitrated. Taken together with previous findings regarding CPS1 structure and function, homology modeling of mouse CPS1 suggested that nitration at Y1450 in an α-helix of allosteric domain prevents activation of CPS1 by its activator, N-acetyl-l-glutamate. In conclusion, this study demonstrated the tyrosine nitration of CPS1 by peroxynitrite and its functional consequence. Since CPS1 is responsible for ammonia removal in the urea cycle, nitration of CPS1 with attenuated function might be involved in some diseases and drug-induced toxicities associated with mitochondrial dysfunction.

Combination of GSH trapping and time-dependent inhibition assays as a predictive method of drugs generating highly reactive metabolites.

Covalent binding (CB) of reactive metabolites (RMs) is potentially involved in severe adverse drug reactions. Because the CB assay is of low throughput and costly, a qualitative trapping assay using agents such as [(35)S]GSH is often performed in the early stages of drug discovery. However, trapping methods alone cannot replace the CB assay. We hypothesized that the time-dependent inhibition (TDI) assay might be complementary to the [(35)S]GSH trapping assay in detecting RMs. We performed CB assays, [(35)S]GSH trapping assays, and TDI assays for 42 structurally diverse compounds. First, we showed that the [(35)S]GSH trapping assay alone does not correlate with the extent of CB. Four compounds that the [(35)S]GSH trapping assay failed to detect but that showed high extent of CB were inactivators of the enzyme in the TDI assay. There was a tendency for compounds judged as positive in the TDI assay to show a high degree of CB irrespective of the result of the [(35)S]GSH trapping assay. Finally, to combine parameters from the two assays, we introduced intrinsic clearance to describe the formation of RMs (CL(int, RMs)). The Spearman rank correlation coefficient between the extent of CB and CL(int, RMs) was 0.77 (p < 0.0001), which was better than that for the formation rates of [(35)S]GSH adducts. Therefore, we demonstrated that a combination of the [(35)S]GSH trapping and TDI assays is an effective method for detecting compounds potentially capable of generating highly reactive metabolites in the early stages of drug discovery.

Metabolic intermediate complex formation of human cytochrome P450 3A4 by lapatinib.

Lapatinib, an oral breast cancer drug, has recently been reported to be a mechanism-based inactivator of cytochrome P450 (P450) 3A4 and also an idiosyncratic hepatotoxicant. It was suggested that formation of a reactive quinoneimine metabolite was involved in mechanism-based inactivation (MBI) and/or hepatotoxicity. We investigated the mechanism of MBI of P450 3A4 by lapatinib. Liquid chromatography-mass spectrometry analysis of P450 3A4 after incubation with lapatinib did not show any peak corresponding to irreversible modifications. The enzymatic activity inactivated by lapatinib was completely restored by the addition of potassium ferricyanide. These results indicate that the mechanism of MBI by lapatinib is quasi-irreversible and mediated via metabolic intermediate complex (MI complex) formation. This finding was verified by the increase in a signature Soret absorbance at approximately 455 nm. Two amine oxidation products of the metabolism of lapatinib by P450 3A4 were characterized: N-hydroxy lapatinib (M3) and the oxime form of N-dealkylated lapatinib (M2), suggesting that a nitroso or another related intermediate generated from M3 is involved in MI complex formation. In contrast, P450 3A5 was much less susceptible to MBI by lapatinib via MI complex formation than P450 3A4. In addition, P450 3A5 had a significantly lower ability than 3A4 to generate M3, consistent with N-hydroxylation as the initial step in the pathway to MI complex formation. In conclusion, our results demonstrate that the primary mechanism for MBI of P450 3A4 by lapatinib is not irreversible modification by the quinoneimine metabolite, but quasi-irreversible MI complex formation mediated via oxidation of the secondary amine group of lapatinib.

CS-8958, a prodrug of the novel neuraminidase inhibitor R-125489, demonstrates a favorable long-retention profile in the mouse respiratory tract.

CS-8958 is a prodrug of the pharmacologically active form R-125489, a selective neuraminidase inhibitor, and has long-acting anti-influenza virus activity in vivo. In this study, the tissue distribution profiles after a single intranasal administration of CS-8958 (0.5 micromol/kg of body weight) to mice were investigated, focusing especially on the retention of CS-8958 in the respiratory tract by comparing it with R-125489 and a marketed drug, zanamivir. After administration of [(14)C]CS-8958, radioactivity was retained in the respiratory tract over long periods. At 24 h postdose, the radioactivity concentrations after administration of [(14)C]CS-8958 were approximately 10-fold higher in both the trachea and the lung than those of [(14)C]R-125489 and [(14)C]zanamivir. The [(14)C]CS-8958-derived radioactivity present in these two tissues consisted both of unchanged CS-8958 and of R-125489 at 1 h postdose, while only R-125489, and no other metabolites, was detected at 24 h postdose. After administration of unlabeled CS-8958, CS-8958 was rapidly eliminated from the lungs, whereas the lung R-125489 concentration reached a maximum at 3 h postdose and gradually declined, with an elimination half-life of 41.4 h. The conversion of CS-8958 to R-125489 was observed in mouse trachea and lung S9 fractions and was inhibited by esterase inhibitors, such as diisopropylfluorophosphate and bis-p-nitrophenylphosphate. These results demonstrated that CS-8958 administered intranasally to mice was efficiently converted to R-125489 by a hydrolase(s) such as carboxylesterase, and then R-125489 was slowly eliminated from the respiratory tract. These data support the finding that CS-8958 has potential as a long-acting neuraminidase inhibitor, leading to significant efficacy as an anti-influenza drug by a single treatment.