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.

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.

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.

Discovery of 1-[2-(1-methyl-1H-pyrazol-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-6-yl]-3-(pyridin-4-ylmethyl)urea as a potent NAMPT (nicotinamide phosphoribosyltransferase) activator with attenuated CYP inhibition.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of the NAD+ salvage pathway. Since NAD+ plays a pivotal role in many biological processes including metabolism and aging, activation of NAMPT is an attractive therapeutic target for treatment of diverse array of diseases. Herein, we report the continued optimization of novel urea-containing derivatives which were identified as potent NAMPT activators. Early optimization of HTS hits afforded compound 12, with a triazolopyridine core, as a lead compound. CYP direct inhibition (DI) was identified as an issue of concern, and was resolved through modulation of lipophilicity to culminate in 1-[2-(1-methyl-1H-pyrazol-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-6-yl]-3-(pyridin-4-ylmethyl)urea (21), which showed potent NAMPT activity accompanied with attenuated CYP DI towards multiple CYP isoforms.

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.

Discovery of DS68702229 as a Potent, Orally Available NAMPT (Nicotinamide Phosphoribosyltransferase) Activator.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of the nicotinamide adenine dinucleotide (NAD+) salvage pathway. Because NAD+ plays a pivotal role in energy metabolism and boosting NAD+ has positive effects on metabolic regulation, activation of NAMPT is an attractive therapeutic approach for the treatment of various diseases, including type 2 diabetes and obesity. Herein we report the discovery of 1-(2-phenyl-1,3-benzoxazol-6-yl)-3-(pyridin-4-ylmethyl)urea 12c (DS68702229), which was identified as a potent NAMPT activator. Compound 12c activated NAMPT, increased cellular NAD+ levels, and exhibited an excellent pharmacokinetic profile in mice after oral administration. Oral administration of compound 12c to high-fat diet-induced obese mice decreased body weight. These observations indicate that compound 12c is a promising anti-obesity drug candidate.

Discovery of a Novel Class of State-Dependent NaV1.7 Inhibitors for the Treatment of Neuropathic Pain.

The discovery of a novel class of state-dependent voltage-gated sodium channel (NaV)1.7 inhibitors is described. By the modification of amide or urethane bond in NaV1.7 blocker III, structure-activity relationship studies that led to the identification of novel NaV1.7 inhibitor 2i (DS01171986) were performed. Compound 2i exhibited state-dependent inhibition of NaV1.7 without NaV1.1, NaV1.5 or human ether-a-go-go related gene (hERG) liabilities at concentrations up to 100 µM. Further biological profiling successfully revealed that 2i possessed potent analgesic properties in a murine model of neuropathic pain (ED50: 3.4 mg/kg) with an excellent central nervous system (CNS) safety margin (> 600 fold).

Drug-Like Properties in Macrocycles above MW 1000: Backbone Rigidity versus Side-Chain Lipophilicity.

Large macrocyclic peptides can achieve surprisingly high membrane permeability, although the properties that govern permeability in this chemical space are only beginning to come into focus. We generated two libraries of cyclic decapeptides with stable cross-ß conformations, and found that peptoid substitutions within the ß-turns of the macrocycle preserved the rigidity of the parent scaffold, whereas peptoid substitutions in the opposing ß-strands led to "chameleonic" species that were rigid in nonpolar media but highly flexible in water. Both rigid and chameleonic compounds showed high permeability over a wide lipophilicity range, with peak permeabilities differing significantly depending on scaffold rigidity. Our findings indicate that modulating lipophilicity can be used to engineer favorable ADME properties into both rigid and flexible macrocyclic peptides, and that scaffold rigidity can be used to tune optimal lipophilicity.

Discovery of (phenoxy-2-hydroxypropyl)piperidines as a novel class of voltage-gated sodium channel 1.7 inhibitors.

A novel class of NaV1.7 inhibitors has been identified by high-throughput screening followed by structure activity relationship studies. Among this series of compounds, piperidine 9o showed potent human and mouse NaV1.7 inhibitory activities with fair subtype selectivity over NaV1.5. Compound 9o successfully demonstrated analgesic efficacy in mice comparable to that of the currently used drug, mexiletine, but with an expanded central nervous system safety margin.

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.

N-Aryl Indoles as a Novel Class of Potent NaV1.7 Inhibitors.

A novel class of potent NaV1.7 inhibitors has been discovered. The replacement of diaryl ether in compound I was investigated to enhance mouse NaV1.7 inhibitory activity, which resulted in the discovery of N-aryl indoles. The introduction of the 3-methyl group is crucial for high NaV1.7 in vitro potency. The adjustment of lipophilicity led to the discovery of 2e. Compound 2e (DS43260857) demonstrated high in vitro potencies against both human and mouse NaV1.7 with high selectivity over NaV1.1, NaV1.5, and hERG. In vivo evaluations revealed 2e demonstrating potent efficacy in PSL mice with excellent pharmacokinetics.

Discovery of DS-1971a, a Potent, Selective NaV1.7 Inhibitor.

A highly potent, selective NaV1.7 inhibitor, DS-1971a, has been discovered. Exploration of the left-hand phenyl ring of sulfonamide derivatives (I and II) led to the discovery of novel series of cycloalkane derivatives with high NaV1.7 inhibitory potency in vitro. As the right-hand heteroaromatic ring affected the mechanism-based inhibition liability of CYP3A4, replacement of this moiety resulted in the generation of 4-pyrimidyl derivatives. Additionally, GSH adducts formation, which can cause idiosyncratic drug toxicity, was successfully avoided by this modification. An additional optimization led to the discovery of DS-1971a. In preclinical studies, DS-1971a demonstrated highly potent selective in vitro profile with robust efficacy in vivo. DS-1971a exhibited a favorable toxicological profile, which enabled multiple-dose studies of up to 600 mg bid or 400 mg tid (1200 mg/day) administered for 14 days to healthy human males. DS-1971a is expected to exert potent efficacy in patients with peripheral neuropathic pain, with a favorable safety profile.

Therapeutic effects of a 186Re-complex-conjugated bisphosphonate for the palliation of metastatic bone pain in an animal model.

UNLABELLED: Previously, based on the concept of bifunctional radiopharmaceuticals, we developed a highly stable (186)Re-mercaptoacetylglycylglycylglycine (MAG3) complex-conjugated bisphosphonate, [[[[(4-hydroxy-4,4-diphosphonobutyl)carbamoylmethyl]carbamoylmethyl]carbamoylmethyl]carbamoylmethanethiolate] oxorhenium(V) ((186)Re-MAG3-HBP), for the treatment of painful bone metastases. This agent showed a superior biodistribution as a bone-seeking agent in normal mice when compared with (186)Re-1-hydroxyethylidene-1,1-diphosphonate ((186)Re-HEDP). In this study, we evaluated the therapeutic effects of (186)Re-MAG3-HBP using an animal model of bone metastasis. METHODS: The model was prepared by injecting syngeneic MRMT-1 mammary tumor cells into the left tibia of female Sprague-Dawley rats. (186)Re-MAG3-HBP (55.5, 111, or 222 MBq/kg) or (186)Re-HEDP (55.5 MBq/kg) was then administered intravenously 21 d later. To evaluate the therapeutic effects and side effects, tumor size and peripheral blood cell counts were determined. Palliation of bone pain was evaluated by a von Frey filament test. RESULTS: In the rats treated with (186)Re-HEDP, tumor growth was comparable with that in untreated rats. In contrast, when (186)Re-MAG3-HBP was administered, tumor growth was significantly inhibited. Allodynia induced by bone metastasis was attenuated by treatment with (186)Re-MAG3-HBP or (186)Re-HEDP, but (186)Re-MAG3-HBP tended to be more effective. CONCLUSION: These results indicate that (186)Re-MAG3-HBP could be useful as a therapeutic agent for the palliation of metastatic bone pain.

Chemical design of a radiolabeled gelatinase inhibitor peptide for the imaging of gelatinase activity in tumors.

Since elevated levels of gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] are associated with a poor prognosis in cancer patients, these enzymes are potential targets for tumor imaging. In the present study, a cyclic decapeptide, cCTTHWGFTLC (CTT), was selected as a mother compound because of its selective inhibitory activity toward gelatinases. For imaging gelatinase activity in tumors, we designed a CTT-based radiopharmaceutical taking into consideration that (1) the HWGF motif of the peptide is important for the activity, (2) hydrophilic radiolabeled peptides show low-level accumulation in the liver and (3) an increase in the negative charge of radiolabeled peptides is effective in reducing renal accumulation. Thus, a highly hydrophilic and negatively charged radiolabel, indiun-111-diethylenetriaminepentaacetic acid ((111)In-DTPA), was attached to an N-terminal residue distant from the HWGF motif ((111)In-DTPA-CTT). In MMP-2 inhibition assays, In-DTPA-CTT significantly inhibited the proteolytic activity in a concentration-dependent fashion. When injected into normal mice, (111)In-DTPA-CTT showed low levels of radioactivity in the liver and kidney. A comparison of the pharmacokinetic characteristics of (111)In-DTPA-CTT with those of other CTT derivatives having different physicochemical properties revealed that the increase in hydrophilicity and negative charge caused by the conjugation of (111)In-DTPA reduced levels of radioactivity in the liver and kidney. In tumor-bearing mice, a significant correlation was observed between the accumulation in the tumor as well as tumor-to-blood ratio of (111)In-DTPA-CTT and gelatinase activity. These findings support the validity of the chemical design of (111)In-DTPA-CTT for reducing accumulation in nontarget tissues and maintaining the inhibitory activity of the mother compound. Furthermore, (111)In-DTPA-CTT derivatives would be potential radiopharmaceuticals for the imaging of gelatinase activity in metastatic tumors in vivo.

(18)F-FDG accumulation in atherosclerotic plaques: immunohistochemical and PET imaging study.

UNLABELLED: The rupture of atherosclerotic plaques and the subsequent formation of thrombi are the main factors responsible for myocardial and cerebral infarctions. Thus, the detection of vulnerable plaques in atherosclerotic lesions is a desirable goal, and attempts to image these plaques with (18)F-FDG have been made. In the present study, the relationship between the accumulation of (18)F-FDG and the biologic characteristics of atherosclerotic lesions was investigated. Furthermore, PET imaging of vulnerable plaques was performed with an animal model of atherosclerosis, Watanabe heritable hyperlipidemic (WHHL) rabbits. METHODS: WHHL (n = 11) and control (n = 3) rabbits were injected intravenously with (18)F-FDG, and the thoracic and abdominal aortas were removed 4 h after injection. The accumulated radioactivity was measured, and the number of macrophages and the intimal area were investigated by examination of stained sections. PET and CT images were also acquired at 210 min after injection of the radiotracer. RESULTS: (18)F-FDG accumulated to a significantly higher level in the aortas of the WHHL rabbits (mean +/- SD differential uptake ratio [DUR], 1.47 +/- 0.90) than in those of the control rabbits (DUR, 0.44 +/- 0.15); DUR was calculated as (tissue activity/tissue weight)/(injected radiotracer activity/animal body weight), with activities given in becquerels and weights given in kilograms. (18)F-FDG uptake and the number of macrophages were strongly correlated in the atherosclerotic lesions of the WHHL rabbits (R = 0.81). In the PET analysis, intense (18)F-FDG radioactivity was detected in the aortas of the WHHL rabbits, whereas little radioactivity was seen in the control rabbits. CONCLUSION: The results suggest that macrophages are responsible for the accumulation of (18)F-FDG in atherosclerotic lesions. Because vulnerable plaques are rich in macrophages, (18)F-FDG imaging should be useful for the selective detection of such plaques.

Establishment of evaluation method for siRNA delivery using stable cell line carrying the luciferase reporter gene.

We determined the influence of siRNA (short interfering RNA) for expression of plasmid DNA (pDNA), when mismatched siRNA and pDNA encoding beta-galactosidase (beta-gal) were transfected into HeLa cells by the cotransfection method in which they were simultaneously added to the cells. Cationic liposomes (Lipofectamine2000) were used as a gene transfection reagent. The knockdown effect on beta-gal was observed even when mismatched siRNA was used, and the effect depended on the amount of added mismatched siRNA. But, there was not a distinct difference of introduction of pDNA into cells between using mismatched siRNA and without using it. We considered that the cotransfection method should be avoided when we confirm RNAi efficiency. The reliable evaluation method for siRNA delivery in vitro was thus established by using NFAT reporter HeLa stable cell line or CHO (pMAM-luc) cell line that had DNA encoding luciferase. The following experimental conditions for each cell line were optimized: cell numbers seeded, total incubation times, concentrations of added inducers, and incubation times after addition of inducers. Transfection performance was compared for six commercially available reagents by this method. No commercially available transfection reagent, however, could reduce luciferase activity by less than one tenth without causing cellular cytotoxicity. Development of novel reagents providing higher transfection effects without cytotoxicity is needed.

Lectin-like oxidized LDL receptor-1 (LOX-1) expression is associated with atherosclerotic plaque instability--analysis in hypercholesterolemic rabbits.

Lectin-like oxidized LDL receptor-1 (LOX-1), a cell-surface receptor for oxidized LDL (Ox-LDL), has been implicated in vascular cell dysfunction related to atherosclerotic plaque instability, according to cell culture experiments. In the present study, we investigated the relationship between LOX-1 expression and plaque instability in hypercholesterolemic rabbits by immunohistological analyses in vivo. We prepared thirty series of cross sections of the thoracic aorta from six myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits (12-24 months), in which seventy atherosclerotic plaques were observed. LOX-1, matrix metalloproteinase-9 (MMP-9), monocyte chemoattractant protein-1 (MCP-1) expression, apoptotic events, plaque instability index (an index of the morphological destabilization of atherosclerotic plaques) and fibromuscular cap thickness in each atherosclerotic plaque were determined by immunohistochemical staining, TUNEL staining and Azan-Mallory staining. LOX-1 expression was positively correlated with the plaque instability index and MMP-9 expression. LOX-1 expression was more prominent in atherosclerotic plaques with thinner fibromuscular cap (<100 microm). Furthermore, LOX-1 expression was shown in the macrophage-rich lipid core area where MCP-1 expression and apoptotic events were prominent. These results indicate that enhanced LOX-1 expression was associated with histologically unstable atherosclerotic plaques in hypercholesterolemic rabbits, suggesting the involvement of LOX-1 in the destabilization of atherosclerotic plaques in vivo.