Generation of enterocyte-like cells with pharmacokinetic functions from human induced pluripotent stem cells using small-molecule compounds.

The small intestine plays an important role in all aspects of pharmacokinetics, but there is no system for the comprehensive evaluation of small-intestinal pharmacokinetics, including drug metabolism and absorption. In this study, we aimed to construct an intestinal pharmacokinetics evaluation system and to generate pharmacokinetically functional enterocytes from human induced pluripotent stem cells. Using activin A and fibroblast growth factor 2, we differentiated these stem cells into intestinal stem cell-like cells, and the resulting cells were differentiated into enterocytes in a medium containing epidermal growth factor and small-molecule compounds. The differentiated cells expressed intestinal marker genes and drug transporters. The expression of sucrase-isomaltase, an intestine-specific marker, was markedly increased by small-molecule compounds. The cells exhibited activities of drug-metabolizing enzymes expressed in enterocytes, including CYP1A1/2, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, UGT, and sulfotransferase. Fluorescence-labeled dipeptide uptake into the cells was observed and was inhibited by ibuprofen, an inhibitor of the intestinal oligopeptide transporter solute carrier 15A1/PEPT1. CYP3A4 mRNA expression level was increased by these compounds and induced by the addition of 1α,25-dihydroxyvitamin D3. CYP3A4/5 activity was also induced by 1α,25-dihydroxyvitamin D3 in cells differentiated in the presence of the compounds. All these results show that we have generated enterocyte-like cells that have pharmacokinetic functions, and we have identified small-molecule compounds that are effective for promoting intestinal differentiation and the gain of pharmacokinetic functions. Our enterocyte-like cells would be useful material for developing a novel evaluation system to predict human intestinal pharmacokinetics.

6-(4-Pyridyl)pyrimidin-4(3H)-ones as CNS penetrant glycogen synthase kinase-3ß inhibitors.

The discovery of a series of 6-(4-pyridyl)pyrimidin-4(3H)-ones derived from a hit compound with low molecular weight and sufficient chemical space is reported. Transformation of substituents led to subnanomolar potent inhibitors with in vivo tau phoshorylation lowering activity.

Amodiaquine derivatives as inhibitors of severe fever with thrombocytopenia syndrome virus (SFTSV) replication.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne viral infection caused by a bandavirus in the family of Phenuiviridae, commonly known as SFTS virus (SFTSV). We have previously isolated SFTSV from blood samples of SFTS patients and established an antiviral assay system to identify selective inhibitors of SFTSV in vitro. Using the assay system, the antimalarial agent amodiaquine was identified as a selective inhibitor of SFTSV replication. However, due to its insufficient antiviral activity, 98 amodiaquine derivatives were newly synthesized and examined for their anti-SFTSV activity. Among the derivatives, some compounds showed selective inhibitory effect on SFTSV replication in vitro. The 50% effective concentration (EC50) and cytotoxic concentration (CC50) of the most active compound (C-90) were 2.6 ± 0.6 and >50 µM, respectively. This EC50 value was comparable to or slightly better than that of favipiravir (4.1 ± 0.6 µM). On the other hand, pharmacokinetic studies in vivo revealed that C-90 was poor in its oral bioavailability in mice. Therefore, we further designed and synthesized derivatives and obtained 2 compounds with selective anti-SFTSV activity in vitro and improved pharmacokinetics in vivo.

Pharmacokinetics, Safety, and Tolerability of NPC-21, an Anti-Cytomegalovirus Monoclonal Antibody, in Healthy Japanese and White Adult Men: A Randomized, Placebo-Controlled, First-in-Human Phase 1 Study.

NPC-21 (EV2038) is a fully human monoclonal antibody that targets the antigenic domain 1 of glycoprotein B on the human cytomegalovirus (hCMV) envelope. NPC-21 has been shown to have broadly neutralizing activity and to inhibit cell-to-cell transmission of hCMV in preclinical studies. It is currently in development for the prophylactic or preemptive treatment of hCMV in patients receiving a solid-organ transplant or hematopoietic stem cell transplant. A first-in-human phase 1 study was conducted to assess the pharmacokinetics, safety, and tolerability of NPC-21 in healthy adult men. Forty participants (Japanese, n = 32; White, n = 8) were randomly assigned to receive a single intravenous dose of NPC-21 1, 3, 10, or 20 mg/kg or placebo. Six Japanese participants were included in each dose group and six White participants received a 10-mg/kg dose. The placebo group included 8 Japanese participants and 2 White participants. All 40 participants completed the study. Serum concentration, maximum serum concentration, area under the plasma concentration-time curve from time 0 to the last measurable concentration, and area under the plasma concentration-time curve from time 0 to infinity increased dose dependently; dose proportionality was linear. NPC-21 demonstrated a biphasic elimination pattern, with an estimated half-life between 612 and 790 hours. NPC-21 was safe and well tolerated up to 20 mg/kg. All adverse events were mild, and none led to treatment discontinuation or were considered related to the study drug. There were no differences in pharmacokinetics or safety between Japanese and White participants. These results support further investigation of NPC-21.

CYP3A4*16 and CYP3A4*18 alleles found in East Asians exhibit differential catalytic activities for seven CYP3A4 substrate drugs.

CYP3A4, the major form of cytochrome P450 (P450) expressed in the adult human liver, is involved in the metabolism of approximately 50% of commonly prescribed drugs. Several genetic polymorphisms in CYP3A4 are known to affect its catalytic activity and to contribute in part to interindividual differences in the pharmacokinetics and pharmacodynamics of CYP3A4 substrate drugs. In this study, catalytic activities of the two alleles found in East Asians, CYP3A4*16 (T185S) and CYP3A4*18 (L293P), were assessed using the following seven substrates: midazolam, carbamazepine, atorvastatin, paclitaxel, docetaxel, irinotecan, and terfenadine. The holoprotein levels of CYP3A4.16 and CYP3A4.18 were significantly higher and lower, respectively, than that of CYP3A4.1 when expressed in Sf21 insect cell microsomes together with human NADPH-P450 reductase. CYP3A4.16 exhibited intrinsic clearances (V(max)/K(m)) that were lowered considerably (by 84-60%) for metabolism of midazolam, carbamazepine, atorvastatin, paclitaxel, and irinotecan compared with CYP3A4.1 due to increased K(m) with or without decreased V(max) values, whereas no apparent decrease in intrinsic clearance was observed for docetaxel. On the other hand, K(m) values for CYP3A4.18 were comparable to those for CYP3A4.1 for all substrates except terfenadine; but V(max) values were lower for midazolam, paclitaxel, docetaxel, and irinotecan, resulting in partially reduced intrinsic clearance values (by 34-52%). These results demonstrated that the impacts of both alleles on CYP3A4 catalytic activities depend on the substrates used. Thus, to evaluate the influences of both alleles on the pharmacokinetics of CYP3A4-metabolized drugs and their drug-drug interactions, substrate drug-dependent characteristics should be considered for each drug.

Inhibition of mitogen-activated protein kinase kinase, DNA methyltransferase, and transforming growth factor-ß promotes differentiation of human induced pluripotent stem cells into enterocytes.

We previously reported that small-molecule compounds were effective in generating pharmacokinetically functional enterocytes from human induced pluripotent stem (iPS) cells. In this study, to determine whether the compounds promote the differentiation of human iPS cells into enterocytes, we investigated the effects of a combination of mitogen-activated protein kinase kinase (MEK), DNA methyltransferase (DNMT), and transforming growth factor (TGF)-ß inhibitors on intestinal differentiation. Human iPS cells cultured on feeder cells were differentiated into endodermal cells by activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, the cells were differentiated into enterocyte cells by epidermal growth factor and small-molecule compounds. After differentiation, mRNA expression levels and drug-metabolizing enzyme activities were measured. The mRNA expression levels of the enterocyte marker sucrase-isomaltase and the major drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 were increased by a combination of MEK, DNMT, and TGF-ß inhibitors. The mRNA expression of CYP3A4 was markedly induced by 1α,25-dihydroxyvitamin D3. Metabolic activities of CYP1A1/2, CYP2B6, CYP2C9, CYP2C19, CYP3A4/5, UDP-glucuronosyltransferase, and sulfotransferase were also observed in the differentiated cells. In conclusion, MEK, DNMT, and TGF-ß inhibitors can be used to promote the differentiation of human iPS cells into pharmacokinetically functional enterocytes.

Functional characterization of single nucleotide polymorphisms with amino acid substitution in CYP1A2, CYP2A6, and CYP2B6 found in the Japanese population.

As a part of the studies conducted by the Pharma SNPs Consortium (PSC), the enzyme activities of CYP1A2, CYP2A6 and CYP2B6 variants with altered amino acids as a result of single nucleotide polymorphisms (SNPs) found among the Japanese population were analyzed under a unified protocol using the same lots of reagents by the laboratories participating in the PSC. Mutations in CYP1A2, CYP2A6 and CYP2B6 were introduced by site-directed mutagenesis and the wild type and mutated CYP molecules were expressed in Escherichia coli. The expressed cytochrome P450s were purified and the enzyme activities were measured in reconstitution systems. CYP1A2 and CYP1A2Gln478His did not show any differences in 7-ethoxyresorufin O-deethylase activity. CYP2A6 and CYP2A6Glu419Asp metabolized coumarin to form 7-hydroxycoumarin in a similar manner, whereas CYP2A6Ile471Thr showed low activity compared to the wild-type CYP2A6. CYP2B6, CYP2B6Pro167Ala and CYP2B6Arg487Cys showed the same activity for 7-ethoxy-4-triflouromethyl-coumarin O-deethylation. However, CYP2B6Gln172His was roughly twice as active as CYP2B6 and the other CYP2B6 variants for 7-ethoxy-4-triflouromethylcoumarin O-deethylation activity. Although higher inter- and intra-laboratory variations were observed for the calculated Km and V(max) values because the studies were conducted in several different laboratories, the degree of variations was reduced by the increased number of analyses and the adoption of a simple analysis system.

Histone deacetylase inhibitor valproic acid promotes the differentiation of human induced pluripotent stem cells into hepatocyte-like cells.

In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells.

Evaluation of the role of breast cancer resistance protein (BCRP/ABCG2) and multidrug resistance-associated protein 4 (MRP4/ABCC4) in the urinary excretion of sulfate and glucuronide metabolites of edaravone (MCI-186; 3-methyl-1-phenyl-2-pyrazolin-5-one).

Edaravone (MCI-186; 3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, is used for the treatment of acute cerebral infarction. Edaravone is mainly excreted into the urine after conjugation to glucuronide or sulfate. Previous studies have demonstrated that edaravone sulfate is a good substrate of human organic anion transporter (OAT) 1 (SLC22A6) and human OAT3 (SLC22A8). In this study, we examined the involvement of breast cancer resistance protein [BCRP (ABCG2)] and [multidrug resistance-associated protein 4 MRP4 (ABCC4)] in the luminal efflux in the kidney. Increased ATP-dependent uptake of edaravone sulfate but not edaravone glucuronide was observed in BCRP-expressing membrane vesicles compared with control vesicles (Km = 16.5 microM). In contrast, edaravone glucuronide, but not edaravone sulfate, exhibited greater ATP-dependent uptake in MRP4-expressing membrane vesicles than that in control vesicles (Km = 9.85 microM). Unlike taurocholate uptake, S-methylglutathione had no effect on the ATP-dependent uptake of edaravone glucuronide by MRP4. The functional importance of BCRP and MRP4 in the urinary excretion of edaravone sulfate and edaravone glucuronide, respectively, was investigated using Bcrp and Mrp4 knockout mice. The renal clearance with respect to the kidney concentration of edaravone sulfate was reduced significantly but not abolished in Bcrp knockout mice compared with wild-type mice (3.62 versus 4.85 ml/min/kg b.wt.). The renal clearance of edaravone glucuronide was lower in Mrp4 knockout mice than wild-type mice (2.01 versus 5.06 ml/min/kg BW). Our results suggest that Bcrp and Mrp4 are partly involved in the luminal efflux of edaravone sulfate and edaravone glucuronide, respectively.

Human organic anion transporters 1 (hOAT1/SLC22A6) and 3 (hOAT3/SLC22A8) transport edaravone (MCI-186; 3-methyl-1-phenyl-2-pyrazolin-5-one) and its sulfate conjugate.

3-Methyl-1-phenyl-2-pyrazolin-5-one (MCI-186; edaravone), a novel free radical scavenger, is used for the treatment of acute cerebral infarction. After marketing, a few cases of acute renal failure were reported in patients following treatment with this drug. Because edaravone is mainly excreted into the urine following conjugation to glucuronide or sulfate, the renal excretion mechanisms of edaravone should help provide important information when considering the clinical cases. We examined the transport of edaravone and its sulfate and glucuronide conjugates via human organic anion transporter 1 (hOAT1) and 3 (hOAT3), expressed on the basolateral membranes of proximal tubules. The hOAT1- and hOAT3-transfected human embryonic kidney (HEK)-293 cells exhibited a markedly higher uptake of edaravone sulfate and a slightly higher uptake of edaravone than vector-transfected cells. The K(m) values of edaravone sulfate uptake by hOAT1 and hOAT3 were 11 and 15 microM, respectively. Estimation of the relative contribution of hOAT1 and hOAT3 using reference compounds suggested that hOAT1 and hOAT3 might contribute to the renal uptake of edaravone sulfate to the same extent. However, edaravone and its sulfate showed no cytotoxicity toward both hOAT1-HEK and control cells, suggesting that higher uptake in hOAT1-HEK did not associate with cytotoxicity of these compounds. In conclusion, our results suggest that both hOAT1 and hOAT3 are responsible for the basolateral uptake of edaravone sulfate in the kidney.

Autoinduction of MKC-963 [(R)-1-(1-cyclohexylethylamino)-4-phenylphthalazine] metabolism in healthy volunteers and its retrospective evaluation using primary human hepatocytes and cDNA-expressed enzymes.

MKC-963, (R)-1-(1-cyclohexylethylamino)-4-phenylphthalazine, a potent inhibitor of platelet aggregation, was synthesized and used in clinical trials in the 1990s. In the process of clinical study, it was found that urinary excretion ratios for 6beta-hydroxycortisol and free cortisol increased significantly in parallel with decreases in the plasma concentrations of MKC-963 after repeated oral administration of the compound to healthy volunteers. These findings suggested that MKC-963 caused autoinduction (defined as the ability of a drug to induce enzymes that enhance its own metabolism, resulting in dispositional tolerance) in humans, and clinical studies using the compound were stopped. This experience prompted us to reevaluate the effects of this compound on CYP3A4 using primary human hepatocytes and cDNA-expressed human cytochrome P450 (P450) enzymes to determine whether the autoinduction of MKC-963 metabolism in humans could have been predicted if these in vitro systems had been used for the evaluation of MKC-963 in the preclinical study. The results of in vitro study showed that MKC-963 increased CYP3A4 mRNA expression level and activity of testosterone 6beta-hydroxylation to extents similar to those observed with rifampicin in primary human hepatocytes. In addition, approximately 90% of the MKC-963 metabolism in human liver microsomes was estimated to be attributable to CYP3A4. These in vitro findings are in good agreement with the results of clinical study, suggesting that studies using human hepatocytes and cDNA-expressed human P450s are useful for assessing the autoinductive nature of compounds under development before starting clinical studies.

Impact of drug transporter studies on drug discovery and development.

Drug transporters are expressed in many tissues such as the intestine, liver, kidney, and brain, and play key roles in drug absorption, distribution, and excretion. The information on the functional characteristics of drug transporters provides important information to allow improvements in drug delivery or drug design by targeting specific transporter proteins. In this article we summarize the significant role played by drug transporters in drug disposition, focusing particularly on their potential use during the drug discovery and development process. The use of transporter function offers the possibility of delivering a drug to the target organ, avoiding distribution to other organs (thereby reducing the chance of toxic side effects), controlling the elimination process, and/or improving oral bioavailability. It is useful to select a lead compound that may or may not interact with transporters, depending on whether such an interaction is desirable. The expression system of transporters is an efficient tool for screening the activity of individual transport processes. The changes in pharmacokinetics due to genetic polymorphisms and drug-drug interactions involving transporters can often have a direct and adverse effect on the therapeutic safety and efficacy of many important drugs. To obtain detailed information about these interindividual differences, the contribution made by transporters to drug absorption, distribution, and excretion needs to be taken into account throughout the drug discovery and development process.

Impaired renal excretion of 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040) sulfate in breast cancer resistance protein (BCRP1/ABCG2) knockout mice.

Murine breast cancer resistance protein 1 (Bcrp1) is expressed in the brush-border membrane of proximal tubule cells of the kidney. The purpose of the present study is to investigate whether Bcrp1 could be involved in the urinary excretion of the human BCRP substrates, 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole sulfate (E3040S) and 4-methylumbelliferone sulfate (4MUS), using Bcrp1(-/-) mice. E3040S and 4MUS were given to the mice by intravenous infusion, and plasma and kidney concentrations and the urinary excretion rate were determined. Knockout of Bcrp1 did not affect the creatinine clearance [7.17 +/- 1.00 and 8.66 +/- 2.02 ml/min/kg for Bcrp1(-/-) and wild-type mice, respectively]. The renal clearance of E3040S was 2.4-fold lower in Bcrp1 (-/-) mice compared with wild-type mice (2.74 +/- 0.41 versus 6.55 +/- 0.52 ml/min/kg). The concentration of E3040S in the kidney was increased in Bcrp1(-/-) mice compared with that in wild-type mice (55.5 +/- 10.5 versus 19.4 +/- 2.7 nmol/g kidney, respectively). In contrast, knockout of Bcrp1 did not affect the pharmacokinetic parameters of 4MUS, although 4MUS was predominantly excreted in the urine. This is to our knowledge the first demonstration of involvement of Bcrp1 in the renal secretion of organic sulfates. However, taking the results of 4MUS into consideration, the renal secretion of organic sulfates cannot be accounted for solely by Bcrp1, and transporters other than Bcrp1 are also involved.