Tsuyoshi Mikkaichi

Edoxaban Transport via P-Glycoprotein Is a Key Factor for the Drug’s Disposition

Edoxaban (the free base of DU-176b), an oral direct factor Xa inhibitor, is mainly excreted unchanged into urine and feces. Because active membrane transport processes such as active renal secretion, biliary excretion, and/or intestinal secretion, and the incomplete absorption of edoxaban after oral administration have been observed, the involvement of drug transporters in the disposition of edoxaban was investigated. Using a bidirectional transport assay in human colon adenocarcinoma Caco-2 cell monolayers, we observed the vectorial transport of [14C]edoxaban, which was completely inhibited by verapamil, a strong P-glycoprotein (P-gp) inhibitor. In an in vivo study, an increased distribution of edoxaban to the brain was observed in Mdr1a/1b knockout mice when compared with wild-type mice, indicating that edoxaban is a substrate for P-gp. However, there have been no observations of significant transport of edoxaban by renal or hepatic uptake transporters, organic anion transporter (OAT)1, OAT3, organic cation transporter (OCT)2, or organic anion transporting polypeptide (OATP)1B1. Edoxaban exhibited no remarkable inhibition of OAT1, OAT3, OCT1, OCT2, OATP1B1, OATP1B3, or P-gp up to 30 μM; therefore, the risk of clinical drug–drug interactions due to any edoxaban-related transporter inhibition seems to be negligible. Our results demonstrate that edoxaban is a substrate of P-gp but not of other major uptake transporters tested. Because metabolism is a minor contributor to the total clearance of edoxaban and strong P-gp inhibitors clearly impact edoxaban transport, the P-gp transport system is a key factor for edoxaban’s disposition.

Identification of the fused bicyclic 4-amino-2-phenylpyrimidine derivatives as novel and potent PDE4 inhibitors

2-Phenyl-4-piperidinyl-6,7-dihydrothieno[3,4-d]pyrimidine derivative (2) was found to be a new PDE4 inhibitor with moderate PDE4B activity (IC50=150 nM). A number of derivatives with a variety of 4-amino substituents and fused bicyclic pyrimidines were synthesized. Among these, 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivative (18) showed potent PDE4B inhibitory activity (IC50=25 nM). Finally, N-propylacetamide derivative (31b) was determined as a potent inhibitor for both PDE4B (IC50=7.5 nM) and TNF-α production in mouse splenocytes (IC50=9.8 nM) and showed good in vivo anti-inflammatory activity in the LPS-induced lung inflammation model in mice (ID50=18 mg/kg). The binding mode of the new inhibitor (31e) in the catalytic site of PDE4B is presented based on an X-ray crystal structure of the ligand-enzyme complex.

Interaction of angiotensin II type 1 receptor blockers with P-gp substrates in Caco-2 cells and hMDR1-expressing membranes

AIMS The inhibitory effect of angiotensin II type 1 receptor blockers (ARBs) on P-glycoprotein (P-gp) was examined to evaluate their clinical drug-drug interaction (DDI) potential. MAIN METHODS We performed an inhibition study on the vectorial transport of digoxin, a typical substrate for P-gp, using a human colonic adenocarcinoma cell line, Caco-2 cells, and verapamil-stimulated ATPase activity using human multidrug resistance 1 (hMDR1)-expressing membrane. KEY FINDINGS The vectorial transport of digoxin was inhibited by candesartan cilexetil, irbesartan and telmisartan with the IC(50) values of 14.7, 34.0 and 2.19microM, respectively. Those values were 7.4-426-fold higher than their theoretical clinical gastrointestinal concentration [I] at doses in clinical DDI studies. Other ARBs failed to show interaction with P-gp. SIGNIFICANCE It was demonstrated that candesartan cilexetil, irbesartan and telmisartan had the potential to inhibit the transport of various drugs via P-gp. Telmisartan, which caused an increase in the serum digoxin concentration in humans, had a sufficiently high [I]/IC(50) value, suggesting that DDI between digoxin and telmisartan was caused by the inhibition of digoxin efflux via intestinal P-gp.

Evaluation of the Usefulness of Breast Cancer Resistance Protein (BCRP) Knockout Mice and BCRP Inhibitor-Treated Monkeys to Estimate the Clinical Impact of BCRP Modulation on the Pharmacokinetics of BCRP Substrates

ABSTRACTPurposeTo evaluate whether the impact of functional modulation of the breast cancer resistance protein (BCRP, ABCG2 421C>A) on human pharmacokinetics after oral administration is predictable using Bcrp knockout mice and cynomolgus monkeys pretreated with a BCRP inhibitor, elacridar.MethodsThe correlation of the changes of the area under the plasma concentration-time curve (AUC) caused by ABCG2 421C>A with those caused by the Bcrp knockout in mice, or BCRP inhibition in monkeys, was investigated using well-known BCRP substrates (rosuvastatin, pitavastatin, fluvastatin, and sulfasalazine).ResultsIn mice, the bioavailability changes, which corrected the effect of systemic clearance by Bcrp knockout, correlated well with the AUC changes in humans, whereas the correlation was weak when AUC changes were directly compared. In monkeys, the AUC changes pretreated with elacridar resulted in a good estimation of those in humans within approximately 2-fold ranges.ConclusionsThis study suggests that pharmacokinetics studies that use the correction of the bioavailability changes in Bcrp knockout mice are effective for estimating clinical AUC changes in ABCG2 421C>A variants for BCRP substrate drugs and those studies in monkeys that use a BCRP inhibitor serve for the assessment of BCRP impact on the gastrointestinal absorption in a non-rodent model.

Integrated Approach of In Vivo and In Vitro Evaluation of the Involvement of Hepatic Uptake Organic Anion Transporters in the Drug Disposition in Rats Using Rifampicin as an Inhibitor

Cumulative studies describe the importance of drug transporters as one of the key determinants of pharmacokinetics that necessitate investigation and assessment of the involvement of drug transporters in drug discovery and development. The present study investigated an integrated in vivo and in vitro approach to determine the involvement of organic anion transporting polypeptides (Oatps) in the disposition of drugs in rats using rifampicin as an inhibitor. When bromosulfophthalein (BSP) and HMG-CoA reductase inhibitors (statins), which were used as model substrates for Oatps, were administered intravenously (3 and 1 mg/kg, respectively) to rats pretreated with rifampicin orally (30 mg/kg), the total plasma clearance of BSP and statins was attenuated compared with that in control rats, suggesting the involvement of Oatps in the disposition of these drugs in vivo. On the other hand, the pharmacokinetics of midazolam, used as a model substrate of cytochrome P450 3a (Cyp3a), was unchanged between control rats and rifampicin-pretreated rats. The involvement of Oatps in the disposition of statins observed in vivo was further clarified by employing an in vitro hepatic uptake study and media-loss assay in the presence or absence of 100 μM rifampicin. Hepatic intrinsic clearance was reduced in the presence of rifampicin in both the media-loss assay and hepatocyte uptake study. The present study suggests in vivo investigations in rats using rifampicin together with in vitro investigations with a media-loss assay and/or uptake assay using rat hepatocytes can help determine whether a clinical drug-drug interaction study is necessary in drug development.

Identification of the 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives as highly selective PDE4B inhibitors

A PDE4B subtype selective inhibitor is expected to have a wider therapeutic window than non-selective PDE4 inhibitors. In this Letter, two series of 7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives and 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives were evaluated for their PDE4B subtype selectivity using human PDE4B2 and PDE4D2 full length enzymes. To improve their PDE4B selectivity over PDE4D, we optimized the substituents on the pyrimidine ring and the side chain phenyl ring, resulting in several derivatives with more than 100-fold selectivity for PDE4B. Consequently, we identified 2-(3-chloro-4-methoxy-phenyl)-5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivative 54 as a highly selective PDE4B inhibitor, which had potent hPDE4B inhibitory activity with an IC50 value of 3.0 nM and 433-fold PDE4B selectivity over PDE4D.

Synthesis and biological evaluation of 5-carbamoyl-2-phenylpyrimidine derivatives as novel and potent PDE4 inhibitors.

5-Carbamoyl-2-phenylpyrimidine derivative 2 has been identified as a phosphodiesterase 4 (PDE4) inhibitor with moderate PDE4B inhibitory activity (IC50=200 nM). Modification of the carboxylic acid moiety of 2 gave N-neopentylacetamide derivative 10f, which had high in vitro PDE4B inhibitory activity (IC50=8.3 nM) and in vivo efficacy against lipopolysaccharide (LPS)-induced pulmonary neutrophilia in mice (ID50=16 mg/kg, ip). Furthermore, based on the X-ray crystallography of 10f bound to the human PDE4B catalytic domain, we designed 7,8-dihydro-6H-pyrido[4,3-d]pyrimidin-5-one derivative 39 which has a fused bicyclic lactam scaffold. Compound 39 exhibited excellent inhibitory activity against LPS-induced tumor necrosis factor alpha (TNF-α) production in mouse splenocytes (IC50=0.21 nM) and in vivo anti-inflammatory activity against LPS-induced pulmonary neutrophilia in mice (41% inhibition at a dose of 1.0 mg/kg, i.t.).

Pharmacokinetics of DS-5565, a novel α2δ ligand, in rats and monkeys and assessment of DDI risk

OBJECTIVE: We elucidated the pharmacokinetic profiles of DS-5565 in animals and drug interaction risk as perpetrator. BACKGROUND: DS-5565 is an oral analgesic drug that binds to the α 2 δ subunit of voltage-dependent Ca 2+ channels. DESIGN/METHODS: DS-5565 is the salt and the free form of DS-5565 is active moiety. Plasma concentration of the active moiety was determined by validated LC-MS/MS method. The profiles were investigated in F344 rats, streptozotocin-induced diabetic BN rats as neuropathic pain model, and cynomolgus monkeys. Distribution was assessed by whole body autoradioluminography following an oral administration of 14 C-labeled active moiety in F344 rats. Induction potential for CYP1A2 and CYP3A4 was assessed using fresh human hepatocytes. Inhibition potential for various CYP isoforms and drug transporters was tested using human liver microsomes and transporter overexpressing cells, respectively. RESULTS: The plasma exposure increased proportionally with the investigated dose in both strain and species. The bioavailability was higher than 85% in both species. The radioactivity was detected in almost tissues at 30 min but was primarily detectable in limited organs at 24 h post-dose. The plasma protein binding in rats, monkeys and humans in vitro was low. A few metabolites, which have not pharmacological activity, were detected qualitatively at low levels in plasma in both after oral administration. The primary excretion route of the radioactivity was urine; 蠅 87% of the dose was recovered within 7 days after an oral administration of the 14 C-labeled compound in rats and monkeys. Renal clearance of DS-5565 was higher than GFR in both animals, suggesting active secretion in the kidney. DS-5565 did not induce CYP1A2/3A4 in human hepatocytes, and not inhibit various CYP isoforms in human liver microsomes and drug transporters in overexpressing cells. CONCLUSIONS: Pharmacokinetics of DS-5565 in animals was favorable. DS-5565 has low potential to be perpetrator in drug-drug interaction. Disclosure: Dr. Yamamura has received personal compensation for activities with Daiichi Sankyo. Dr. Yamada has received personal compensation for activities with Daiichi Sankyo. Dr. Takahashi has received personal compensation for activities with Daiichi Pharmaceutical Corp. as an employee. Dr. Uchiyama has received personal compensation for activities with Daiichi Phamaceutical Corp. Dr. Koda has received personal compensation for activities with Daiichi Pharmaceutical Corp. Dr. Mikkaichi has received personal compensation for activities with Daiichi Pharmaceutical Corp. Dr. Nishiya has received personal compensation for activities with Daiichi Pharmaceutical Corp. as an employee. Dr. Honda has received personal compensation for activities with Daiichi Pharmaceutical Corp. Dr. Arakawa has received personal compensation for activities with Daiichi Pharmaceutical Corporation as an employee. Dr. Domon has received personal compensation for activities with Daiichi Pharmaceutical Corp. as an employee. Dr. Fischer has received personal compensation for activities with Daiichi Pharmaceutical Corp. as an employee. Dr. Mueller has received personal compensation for activities with Daiichi Pharmaceutical Corp.

Design and evaluation of an extended-release matrix tablet formulation; the combination of hypromellose acetate succinate and hydroxypropylcellulose

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