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.

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.

CYP2C8-Mediated Formation of a Human Disproportionate Metabolite of the Selective NaV1.7 Inhibitor DS-1971a, a Mixed Cytochrome P450 and Aldehyde Oxidase Substrate.

Predicting human disproportionate metabolites is difficult, especially when drugs undergo species-specific metabolism mediated by cytochrome P450s (P450s) and/or non-P450 enzymes. This study assessed human metabolites of DS-1971a, a potent Nav1.7-selective blocker, by performing human mass balance studies and characterizing DS-1971a metabolites, in accordance with the Metabolites in Safety Testing guidance. In addition, we investigated the mechanism by which the major human disproportionate metabolite (M1) was formed. After oral administration of radiolabeled DS-1971a, the major metabolites in human plasma were P450-mediated monoxidized metabolites M1 and M2 with area under the curve ratios of 27% and 10% of total drug-related exposure, respectively; the minor metabolites were dioxidized metabolites produced by aldehyde oxidase and P450s. By comparing exposure levels of M1 and M2 between humans and safety assessment animals, M1 but not M2 was found to be a human disproportionate metabolite, requiring further characterization under the Metabolites in Safety Testing guidance. Incubation studies with human liver microsomes indicated that CYP2C8 was responsible for the formation of M1. Docking simulation indicated that, in the formation of M1 and M2, there would be hydrogen bonding and/or electrostatic interactions between the pyrimidine and sulfonamide moieties of DS-1971a and amino acid residues Ser100, Ile102, Ile106, Thr107, and Asn217 in CYP2C8, and that the cyclohexane ring of DS-1971a would be located near the heme iron of CYP2C8. These results clearly indicate that M1 is the predominant metabolite in humans and a human disproportionate metabolite due to species-specific differences in metabolism. SIGNIFICANCE STATEMENT: This report is the first to show a human disproportionate metabolite generated by CYP2C8-mediated primary metabolism. We clearly demonstrate that DS-1971a, a mixed aldehyde oxidase and cytochrome P450 substrate, was predominantly metabolized by CYP2C8 to form M1, a human disproportionate metabolite. Species differences in the formation of M1 highlight the regio- and stereoselective metabolism by CYP2C8, and the proposed interaction between DS-1971a and CYP2C8 provides new knowledge of CYP2C8-mediated metabolism of cyclohexane-containing substrates.

Evaluation of species differences in the metabolism of the selective NaV1.7 inhibitor DS-1971a, a mixed substrate of cytochrome P450 and aldehyde oxidase.

Nonclinical metabolite profiling of DS-1971a, a potent selective NaV1.7 inhibitor, was performed to predict human metabolites.After the oral administration of radiolabelled DS-1971a, the predominant metabolite in mouse plasma was M4, a monoxide at the pyrimidine ring, while the major metabolites with the first and second highest exposure in monkey plasma were M2, a monoxide at the cyclohexane ring, and M11, a demethylated pyrazole metabolite.Incubation studies with liver cytosolic and microsomal fractions in the absence or presence of NADPH indicated that the metabolising enzyme responsible for M4 formation was aldehyde oxidase (AO), while cytochrome P450s (P450s) were responsible for M2 and M11 formation. These results suggest that DS-1971a is a substrate for both AO and P450.When DS-1971a was incubated with liver S9 fractions and NADPH, the most abundant metabolites were M4 in mice, and M2 and M11 in monkeys, indicating that the results of in vitro incubation studies could provide information reflecting the in vivo plasma metabolite profiles in mice and monkeys. The results obtained from the incubation with the human liver S9 fraction and NADPH suggested that a major circulating metabolite in humans is M1, a regioisomer of M2.

Pharmacokinetics of trastuzumab deruxtecan (T-DXd), a novel anti-HER2 antibody-drug conjugate, in HER2-positive tumour-bearing mice.

Trastuzumab deruxtecan (T-DXd, DS-8201a) is an antibody-drug conjugate (ADC), comprising an anti-HER2 antibody (Ab) at a drug-to-Ab ratio of 7-8 with the topoisomerase I inhibitor DXd. In this study, we investigated the pharmacokinetics (PK), biodistribution, catabolism, and excretion profiles of T-DXd in HER2-positive tumour-bearing mice.Following intravenous (iv) administration of T-DXd, the PK profiles of T-DXd and total Ab (the sum of conjugated and unconjugated Ab) were almost similar, indicating that the linker is stable during circulation. Biodistribution studies using radiolabelled T-DXd demonstrated tumour-specific distribution and long-term retention. DXd was the main catabolite released from T-DXd in tumours, with exposure levels at least five times higher than those in normal tissues and seven times higher than those achieved by non-targeted control ADC. Following iv administration of DXd, it was rapidly cleared from the circulation (T1/2; 1.35 h) and excreted mainly through faeces as its intact form.The PK profiles reveal that T-DXd effectively delivers the expected payload, DXd, to tumours, while minimising payload exposure to the systemic circulation and normal tissues. The released DXd is rapidly cleared from systemic circulation, presumably via the bile with negligible metabolism, and excreted through the faeces.

In vivo multiple metabolic pathways for a novel G protein-coupled receptor 119 agonist DS-8500a in rats: involvement of the 1,2,4-oxadiazole ring-opening reductive reaction in livers under anaerobic conditions.

A 1,2,4-oxadiazole ring-containing compound DS-8500a was developed as a novel G protein-coupled receptor 119 agonist. In vivo metabolic fates of [14C]DS-8500a differently radiolabeled in the benzene ring or benzamide side carbon in rats were investigated. Differences in mass balances were observed, primarily because after the oxadiazole ring-opening and subsequent ring-cleavage small-molecule metabolites containing the benzene side were excreted in the urine, while those containing the benzamide side were excreted in the bile. DS-8500a was detected at trace levels in urine and bile, demonstrating extensive metabolism prior to urinary/biliary excretion. At least 16 metabolite structures were proposed in plasma, urine, and bile samples from rats treated with [14C]DS-8500a. Formation of a ring-opened metabolite (reduced DS-8500a) in hepatocytes of humans, monkeys, and rats was confirmed; however, it was not affected by typical inhibitors of cytochrome P450s, aldehyde oxidases, or carboxylesterases in human hepatocytes. Extensive formation of the ring-opened metabolite was observed in human liver microsomes fortified with an NADPH-generating system under anaerobic conditions. These results suggest an in vivo unique reductive metabolism of DS-8500a is mediated by human non-cytochrome P450 enzymes.

Comprehensive preclinical pharmacokinetic evaluations of trastuzumab deruxtecan (DS-8201a), a HER2-targeting antibody-drug conjugate, in cynomolgus monkeys.

Trastuzumab deruxtecan (DS-8201a) is an antibody-drug conjugate (ADC) composed of a monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2) conjugated to a topoisomerase I inhibitor (DXd) at a drug-to-antibody ratio (DAR) of 7-8. Here, we examined the pharmacokinetic (PK) profiles of DS-8201a and DXd in cynomolgus monkeys, a cross-reactive species. Following intravenous (iv) administration of DS-8201a, the linker was stable in plasma, and systemic DXd exposure was low. DXd was rapidly cleared following iv dosing. Biodistribution studies revealed that intact DS-8201a was present mostly in the blood without tissue-specific retention. The major pathway of excretion for DXd was the faecal route following iv administration of radiolabelled DS-8201a. The only detectable metabolite in the urine and faeces was unmetabolized DXd. DXd is a substrate of organic anion transporting polypeptides, P-gp, and breast cancer resistance protein. In conclusion, the stable linker in circulation and the high clearance of DXd upon release resulted in the low systemic exposure to DXd. Furthermore, the minimal tissue-specific retention and rapid excretion of DXd into faeces as its unmetabolized form with potentially limited impact on drug - drug interaction as a victim were also critical elements of the PK profile of DS-8201a.

Identification of metals from osteoblastic ST-2 cell supernatants as novel OGR1 agonists.

Ovarian cancer G-protein-coupled receptor 1 (OGR1) is a G-protein-coupled receptor (GPCR), which has previously been identified as a receptor for protons. It has been reported in this and previous studies that OGR1 expression was markedly up-regulated during osteoclast differentiation. We predicted the possibility of other molecules activating OGR1 in neutral pH, and that osteoblasts might release OGR1 agonistic molecules and activate OGR1 expressed in osteoclasts such as RANKL. We screened for cell supernatants and organ extracts and discovered OGR1 agonistic activity in ST-2 osteoblastic cell supernatants and pancreatic tissues. Finally, we partially purified and identified essential metals, Fe, Zn, Co, Ni and Mn, as novel OGR1 agonists. These OGR1 agonistic metals induce intracellular Gq-coupled inositol phosphate signals in OGR1-expressing cells and primary osteoclasts through OGR1. We also confirmed that these OGR1 agonistic metals activated OGR1 through the same residues which act with protons. Here, we demonstrate that metals, Fe, Zn, Co, Ni and Mn are the novel OGR1 agonists, which can singly activate OGR1 in neutral pH.

Kinetic barriers and ordering of non-covalently bound states.

Binding of a dimer of a glycopeptide antibiotic to two molecules of a ligand that are bound to a membrane surface (by a hydrocarbon anchor) has been investigated. This binding on a surface is cooperatively enhanced (surface enhancement) relative to the binding in solution, because the former occurs intramolecularly on a template. Previously a correlation between surface enhancement and thermodynamic stability of the dimer in free solution (Kdimsol) was hypothesised. However, we found that two weakly dimerising antibiotics (vancomycin and ristocetin A) with similar Kdimsol give very different surface enhancements. We propose a model to explain the data correlating surface enhancement to the kinetic barrier to dissociation of the dimer. The surface enhancement of binding can be expected to increase with increasing tightness of the non-covalent interactions formed at the dimer interface. The effect should be found in general where cooperativity is exercised within an organised template (e.g., DNA duplexes and proteins).

Cooperative binding interactions of glycopeptide antibiotics.

Glycopeptide antibiotics of the vancomycin group bind to bacterial cell wall analogue precursors, and typically also form dimers. We have studied the interplay between these two sets of noncovalent bonds formed at separate interfaces. Indole-2-carboxylic acid (L) forms a set of hydrogen bonds to the glycopeptide antibiotic chloroeremomycin (CE) that are analogous to those formed by N-Ac-D-Ala. The ligand/CE dimer interactions (in L/CE/CE/L) are shown to occur with positive cooperativity and structural tightening at the dimer interface. From theoretical considerations and from other data, it is inferred, but not proven, that in the exercise of positive cooperativity, the interface that will be tightened to the greatest degree is the one that lies in the shallowest free energy well.