Heterogenous Gene Expression of Bicellular and Tricellular Tight Junction-Sealing Components in the Human Intestinal Tract.

A major site for the absorption of orally administered drugs is the intestinal tract, where the mucosal epithelium functions as a barrier separating the inside body from the outer environment. The intercellular spaces between adjacent epithelial cells are sealed by bicellular and tricellular tight junctions (TJs). Although one strategy for enhancing intestinal drug absorption is to modulate these TJs, comprehensive gene (mRNA) expression analysis of the TJs components has never been fully carried out in humans. In this study, we used human biopsy samples of normal-appearing mucosa showing no endoscopically visible inflammation collected from the duodenum, jejunum, ileum, colon, and rectum to examine the mRNA expression profiles of TJ components, including occludin and tricellulin and members of the claudin family, zonula occludens family, junctional adhesion molecule (JAM) family, and angulin family. Levels of claudin-3, -4, -7, -8, and -23 expression became more elevated in each segment along the intestinal tract from the upper segments to the lower segments, as did levels of angulin-1 and -2 expression. In contrast, expression of claudin-2 and -15 was decreased in the large intestine compared to the small intestine. Levels of occludin, tricellulin, and JAM-B and -C expression were unchanged throughout the intestine. Considering their segment specificity, claudin-8, claudin-15, and angulin-2 appear to be targets for the development of permeation enhancers in the rectum, small intestine, and large intestine, respectively. These data on heterogenous expression profiles of intestinal TJ components will be useful for the development of safe and efficient intestinal permeation enhancers.

Quantitative Analysis of mRNA and Protein Expression Levels of Aldo-Keto Reductase and Short-Chain Dehydrogenase/Reductase Isoforms in the Human Intestine.

Enzymes catalyzing the reduction reaction of xenobiotics are mainly members of the aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies. The intestine, together with the liver, is responsible for first-pass effects and is an organ that determines the bioavailability of orally administered drugs. In this study, we evaluated the mRNA and protein expression levels of 12 AKR isoforms (AKR1A1, AKR1B1, AKR1B10, AKR1B15, AKR1C1, AKR1C2, AKR1C3, AKR1C4, AKR1D1, AKR1E2, AKR7A2, and AKR7A3) and 7 SDR isoforms (CBR1, CBR3, CBR4, DCXR, DHRS4, HSD11B1, and HSD17B12) in each region of the human intestine using next-generation sequencing and data-independent acquisition proteomics. At both the mRNA and protein levels, most AKR isoforms were highly expressed in the upper regions of the intestine, namely the duodenum and jejunum, and then declined toward the rectum. Among the members in the SDR superfamily, CBR1 and DHRS4 were highly expressed in the upper regions, whereas the expression levels of the other isoforms were almost uniform in all regions. Significant positive correlations between mRNA and protein levels were observed in AKR1A1, AKR1B1, AKR1B10, AKR1C3, AKR7A2, AKR7A3, CBR1, and CBR3. The mRNA level of AKR1B10 was highest, followed by AKR7A3 and CBR1, each accounting for more than 10% of the sum of all AKR and SDR levels in the small intestine. This expression profile in the human intestine was greatly different from that in the human liver, where AKR1C isoforms are predominantly expressed. SIGNIFICANCE STATEMENT: In this study comprehensively determined the mRNA and protein expression profiles of aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase isoforms involved in xenobiotic metabolism in the human intestine and found that most of them are highly expressed in the upper region, where AKR1B10, AKR7A3, and CBR1 are predominantly expressed. Since the intestine is significantly involved in the metabolism of orally administered drugs, the information provided here is valuable for pharmacokinetic studies in drug development.

Regional Transcriptomics and Proteomics of Pharmacokinetics-Related Genes in Human Intestine.

The intestine is an organ responsible for the absorption and metabolism of orally administered drugs. To predict pharmacokinetics behavior in the small intestine, it is necessary to examine the human intestinal expression profiles of the genes related to drug absorption, distribution, metabolism, and excretion (ADME). In this study, to obtain more accurate expression profiles in various regions of the human intestine, biopsy samples were collected from endoscopically noninflamed mucosa of the duodenum, jejunum, ileum, colon, and rectum from Japanese including Crohn's disease or ulcerative colitis patients, and both RNA-seq and quantitative proteomics analyses were performed. We also analyzed the expression of drug-metabolizing enzymes (cytochromes P450 (CYPs) and non-CYP enzymes), drug transporters, and nuclear receptors. Overall, the mRNA expression levels of these ADME-related genes correlated highly with the protein expression levels. The characteristics of the expression of ADME-related genes differed significantly between the small and large intestines, including the expression levels of CYP enzymes, which were higher and lower in the small and large intestines, respectively. Most CYPs were expressed dominantly in the small intestine, especially the jejunum, but were rarely expressed in the large intestine. On the other hand, non-CYP enzymes were expressed in the large intestine but at lower expression levels than in the small intestine. Moreover, the expression levels of drug metabolizing enzyme genes differed even between the proximal and distal small intestine. Transporters were expressed most highly in the ileum. The data in the present study will enhance understanding of the intestinal ADME of drug candidates and would be useful for drug discovery research.

Functional coupling of organic anion transporter OAT10 (SLC22A13) and monocarboxylate transporter MCT1 (SLC16A1) influencing the transport function of OAT10.

OAT10 (SLC22A13) is a transporter highly expressed in renal tubules and transporting organic anions including nicotinate, ß-hydroxybutyrate, p-aminohippurate, and orotate. In transport assays using Xenopus oocytes and HEK293 cells, we found that apparent substrate selectivity of OAT10 was different between the expression systems, particularly less pronounced uptake of ß-hydroxybutyrate in HEK293 cells. Because functional coupling between transporters may interfere with functional properties of the transporter, we searched for endogenous transporters in HEK293 cells that could affect OAT10. By means of comprehensive approach with co-immunoprecipitation followed by LC-MS/MS analysis, we identified monocarboxylate transporter MCT1 (SLC16A1) as physically coupled with OAT10. The knockdown of MCT1 in OAT10-expressing HEK293 cells increased the uptake of ß-hydroxybutyrate and nicotinate, common substrates of OAT10 and MCT1, whereas the uptake of orotate, a substrate of only OAT10, was not affected. MCT1 is supposed to act as an escape route and mediate the efflux of nicotinate and ß-hydroxybutyrate taken up by OAT10 localized nearby MCT1, as suggested by co-immunoprecipitation. This functional coupling would explain altered apparent substrate selectivity in HEK293 cells compared with Xenopus oocytes. The findings in this study warn in transporter studies that the expression system can interfere with assessing correct transport properties due to unexpected interactions with endogenous transporters.

[Perforin gene mutation in middle-age onset hemophagocytic lymphohistiocytosis].

A 54-year-old woman was referred to our hospital for pancytopenia and liver dysfunction, and with no personal or family history of hemophagocytic lymphohistiocytosis (HLH). Although the etiology was unknown, she was diagnosed with HLH. She experienced exacerbation of HLH even after initiating systemic chemotherapy with etoposide, dexamethasone, and cyclosporine. Furthermore, flow cytometric analysis of the natural killer cells revealed a reduction in perforin expression, and DNA sequencing of the perforin gene (PRF1) revealed two known mutations, confirming the diagnosis of late-onset familial HLH type 2. She received an allogeneic stem cell transplant from an unrelated human leukocyte antigens identical donor, but developed thrombotic microangiopathy, and succumbed to septic shock shortly after the transplant. Previously, HLH in adults was believed to develop from underlying diseases. However, as in our case, several reports demonstrated that HLH gene mutations could be found even after adolescence. Adult with HLH with no underlying disorders should undergo early HLH-associated gene testing for confirmatory diagnosis.

Comparison of culture media for human intestinal organoids from the viewpoint of pharmacokinetic studies.

Human intestinal organoids are expected to be applied in pharmaceutical research. Various culture media for human intestinal organoids have been developed, but it remains unclear which media are preferable for pharmacokinetic studies. Here, we cultured human intestinal organoids with three major culture media that are already used widely around the world: the medium of Sato et al. (S-medium; reported in 2011), Fujii et al. (F-medium; 2018), and Miyoshi et al. (M-medium; 2013). The growth of human intestinal organoids cultured in S-medium was faster than that in F- or M-medium. The gene expression levels of most pharmacokinetic-related enzymes or transporters in human intestinal organoids cultured in M-medium were higher than those in S- or F-medium, and comparable to those in the adult human small intestine. The level of cytochrome P450 (CYP) 3A4 activity was also highest in human intestinal organoids cultured in M-medium. Collectively, the results underscored the importance of selection and optimization of culture medium for various applications using human intestinal organoids, including pharmacokinetic studies.

In Vivo Gene Expression Profile of Human Intestinal Epithelial Cells: From the Viewpoint of Drug Metabolism and Pharmacokinetics.

Orally administered drugs are absorbed and metabolized in the intestine. To accurately predict pharmacokinetics in the intestine, it is essential to understand the intestinal expression profiles of the genes related to drug absorption, distribution, metabolism, and excretion (ADME). However, in many previous studies, gene expression analysis in the intestine has been carried out using specimens from patients with cancer. In this study, to obtain more accurate gene expression profiles, biopsy samples were collected under endoscopic observation from the noninflammatory regions of 14 patients with inflammatory bowel disease, and RNA-seq analysis was performed. Gene expression analysis of drug-metabolizing enzymes (cytochromes P450), non-cytochrome P450 enzymes, nuclear receptors, drug-conjugating enzymes (UDP-glucuronosyltransferases and sulfotransferases), and apical and basolateral drug transporters was performed in biopsy samples from the duodenum, ileum, colon, and rectum. The proportions of the cytochromes P450 expressed in the ileum were 25% (CYP3A4), 19% (CYP2C18), and 14% (CYP3A5). CYP3A4 and CYP2C19 were highly expressed in the duodenum and ileum, but not in the colon and rectum. In the ileum, apical transporters such as P-gp, peptide transporter 1, breast cancer resistance protein, MRP2, and ASBT were strongly expressed, and the expression levels of P-gp and ASBT in the ileum were higher than those in other regions. In the ileum, basolateral transporters such as OSTα, OSTß, and MRP3 were strongly expressed. We succeeded in obtaining gene expression profiles of ADME-related genes in human intestinal epithelial cells in vivo. We expect that this information would be useful for accurate prediction of the pharmacokinetics of oral drugs. SIGNIFICANCE STATEMENT: To obtain gene expression profiles of ADME-related genes in human intestinal epithelial cells in vivo, biopsy samples were collected under endoscopic observation from the noninflammatory regions of 14 patients with inflammatory bowel disease, and RNA-seq analysis was performed. Gene expression profiles of drug-metabolizing enzymes (cytochromes P450), non-cytochrome P450 enzymes, nuclear receptors, drug-conjugating enzymes (UDP-glucuronosyltransferases and sulfotransferases), and apical and basolateral drug transporters in biopsy samples from the duodenum, ileum, colon, and rectum were obtained in this study.

[BCR-ABL1-positive myelodysplastic syndrome with neutropenia and anemia treated successfully with imatinib mesylate].

An 81-year-old female was referred to our hospital with progressive neutropenia and anemia of unknown etiology. We performed a bone marrow biopsy which was notable for hypercellularity, multinucleated megakaryocytes and hypo-granular neutrophils with 2.6% blasts. A diagnosis of myelodysplastic syndrome with multilineage dysplasia (MDS-MLD) was made. Karyotype analysis revealed a t (9;22)(q34;q11.2) BCR-ABL1 fusion with no additional chromosomal abnormalities. BCR-ABL1 was also detected in transcripts from peripheral blood cells as well as in polynuclear leukocytes via FISH. Within one year, her peripheral blood neutrophil count had declined to 403/µl; further analysis was notable for increasing dysplasia including enlarged platelets and hypo-granular neutrophils. Platelet counts gradually increased over time and reached 100×104/µl. A second bone marrow examination revealed similar cell morphology and the BCR-ABL1 translocation. Her condition deteriorated and blood transfusions were required. Treatment with low doses of the tyrosine kinase inhibitor (TKI), imatinib mesylate (100 mg), was initiated. Thereafter, both the neutropenia and anemia resolved gradually, platelet counts returned to normal levels, and dysplasia eventually disappeared. Detection of the BCR-ABL fusion in mRNA decreased to < 0.0007% (IS%) after 16 months of treatment. Several cases of BCR-ABL1-positive myelodysplastic syndrome treated with TKIs have been reported. Our results suggest that complete hematologic recovery in response to imatinib mesylate suggests a critical role for the BCR-ABL1 fusion in the pathogenesis of this disease.

Sex-, Species-, and Tissue-Specific Metabolism of Empagliflozin in Male Mouse Kidney Forms an Unstable Hemiacetal Metabolite (M466/2) That Degrades to 4-Hydroxycrotonaldehyde, a Reactive and Cytotoxic Species.

Following oral administration of empagliflozin (1000 mg/kg/day) to male and female CD-1 mice for 2 years, renal tubular injury was identified in male mice. Renal injury was not detected in male mice (≤300 mg/kg/day), in female mice (1000 mg/kg/day), or in male or female Han Wistar rats (700 mg/kg/day). Using transfected HEK293 cells and Xenopus oocytes, empagliflozin was found to be a substrate of various mouse and rat organic anion transporters (oat/Oat) and organic anion transporting polypeptide (oatp/Oatp) transporters: mouse oat3, rat Oat3, mouse oatp1a1, and rat Oatp1a1. However, using isolated kidney slices from male and female mice and rats, no sex-based difference in the extent of uptake of empagliflozin occurred. Metabolism studies using hepatic and renal microsomes from male and female mice, rats, and humans revealed a hemiacetal metabolite of empagliflozin (M466/2), predominantly formed in male mouse kidney microsomes. Formation of M466/2 in male mouse kidney microsomes was 31-fold higher compared to that in female mouse kidney microsomes and was ∼29- and ∼20-fold higher compared to that in male and female mouse liver microsomes, respectively. M466/2 is unstable and degrades to form a phenol metabolite (M380/1) and 4-hydroxycrotonaldehyde (4-OH CTA). Formed 4-OH CTA was trapped by reduced GSH, and the structure of the GSH adduct was confirmed by mass spectrometry. Stoichiometric formation of M380/1 from M466/2 was observed (93-96% at 24 h); however, formation of 4-OH CTA was considerably lower (∼17.5% at 40 h), which is consistent with 4-OH CTA being a highly reactive species. These data represent a highly selective tissue-, species-, and sex-specific lesion in male CD-1 mice associated with a cytotoxic metabolite product, 4-OH CTA. In humans, glucuronidation of empagliflozin is the most prevalent metabolic pathway, and oxidation is a minor pathway. Thus, renal toxicity due to the formation of 4-OH CTA from empagliflozin is not expected in humans.

Brain penetration of WEB 2086 (Apafant) and dantrolene in Mdr1a (P-glycoprotein) and Bcrp knockout rats.

Transporter gene knockout rat models are attracting increasing interest for mechanistic studies of new drugs as transporter substrates or inhibitors in vivo. However, limited data are available on the functional validity of such models at the blood-brain barrier. Therefore, the present study evaluated Mdr1a [P-glycoprotein (P-gp)], Bcrp, and combined Mdr1a/Bcrp knockout rat strains for the influence of P-gp and breast cancer resistance protein (BCRP) transport proteins on brain penetration of the selective test substrates [(14)C]WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f][1,2,4]triazolo-[4,3-a][1,4]-diazepin-2-yl]-1-(4-morpholinyl)-1-propanon) for P-gp and dantrolene for BCRP. Brain-to-plasma concentration ratios (BPR) were measured after intravenous coinfusions of 5.5 µmol/kg per hour [(14)C]WEB 2086 and 2 µmol/kg per hour dantrolene for 2 hours in groups of knockout or wild-type rats. Compared with wild-type controls, mean BPR of [(14)C]WEB 2086 increased 8-fold in Mdr1a knockouts, 9.5-fold in double Mdr1a/Bcrp knockouts, and 7.3-fold in zosuquidar-treated wild-type rats, but was unchanged in Bcrp knockout rats. Mean BPR of dantrolene increased 3.3-fold in Bcrp knockouts and 3.9-fold in double Mdr1a/Bcrp knockouts compared with wild type, but was unchanged in the Mdr1a knockouts. The human intestinal CaCo-2 cell bidirectional transport system in vitro confirmed the in vivo finding that [(14)C]WEB 2086 is a substrate of P-gp but not of BCRP. Therefore, Mdr1a, Bcrp, and combined Mdr1a/Bcrp knockout rats provide functional absence of these efflux transporters at the blood-brain barrier and are a suitable model for mechanistic studies on the brain penetration of drug candidates.

In vitro predictability of drug-drug interaction likelihood of P-glycoprotein-mediated efflux of dabigatran etexilate based on [I]2/IC50 threshold.

Dabigatran etexilate, an oral, reversible, competitive, and direct thrombin inhibitor, is an in vitro and in vivo substrate of P-glycoprotein (P-gp). Dabigatran etexilate was proposed as an in vivo probe substrate for intestinal P-gp inhibition in a recent guidance on drug-drug interactions (DDI) from the European Medicines Agency (EMA) and the Food and Drug Administration (FDA). We conducted transcellular transport studies across Caco-2 cell monolayers with dabigatran etexilate in the presence of various P-gp inhibitors to examine how well in vitro IC50 data, in combination with mathematical equations provided by regulatory guidances, predict DDI likelihood. From a set of potential P-gp inhibitors, clarithromycin, cyclosporin A, itraconazole, ketoconazole, quinidine, and ritonavir inhibited P-gp-mediated transport of dabigatran etexilate over a concentration range that may hypothetically occur in the intestine. IC50 values of P-gp inhibitors for dabigatran etexilate transport were comparable to those of digoxin, a well established in vitro and in vivo P-gp substrate. However, IC50 values varied depending whether they were calculated from efflux ratios or permeability coefficients. Prediction of DDI likelihood of P-gp inhibitors using IC50 values, the hypothetical concentration of P-gp inhibitors, and the cut-off value recommended by both the FDA and EMA were in line with the DDI occurrence in clinical studies with dabigatran etexilate. However, it has to be kept in mind that validity of the cut-off criteria proposed by the FDA and EMA depends on in vitro experimental systems and the IC50-calculation methods that are employed, as IC50 values are substantially influenced by these factors.

Impact of endogenous esterase activity on in vitro p-glycoprotein profiling of dabigatran etexilate in Caco-2 monolayers.

Dabigatran etexilate, a double prodrug of dabigatran, is a reversible, competitive, direct thrombin inhibitor that has been approved for use in many countries. A recent guideline from the European Medicines Agency on drug-drug interactions proposed dabigatran etexilate as a sensitive in vivo and in vitro probe substrate for intestinal P-glycoprotein (P-gp) inhibition. We therefore performed a series of in vitro studies to determine the best experimental conditions for evaluation of P-gp involvement on the transport process of dabigatran etexilate across colorectal adenocarcinoma Caco-2 cell monolayers. Experiments using expressed carboxylesterase 1 (CES1) and CES2 bactosomes revealed that dabigatran etexilate was hydrolyzed into BIBR 1087 by CES1 expressed in our Caco-2 cells. The impact of CES1-mediated BIBR 1087 formation during transcellular transport experiments was assessed by comparing several combinations of three experimental approaches: radioactivity detection using [(14)C]dabigatran etexilate as substrate, liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification of dabigatran etexilate, and in the presence and absence of a CES inhibitor bis(p-nitrophenyl) phosphate (BNPP). The experimental approach that was based on the use of nonlabeled dabigatran etexilate together with LC-MS/MS quantification and the addition of BNPP was selected as the most favorable condition in which to correctly evaluate the permeability coefficient (Papp) of dabigatran etexilate and its transcellular transport by P-gp. The in vitro Caco-2 study at the selected condition revealed that dabigatran etexilate is a P-gp substrate with an efflux ratio of 13.8 and an intrinsic Papp, which is the Papp under the condition of complete blockage of P-gp by P-gp inhibitor, of 29 × 10(-6) cm/s.

Establishment of human intestinal organoids derived from commercially available cryopreserved intestinal epithelium and evaluation for pharmacokinetic study.

Human intestinal organoids (HIOs) have been reported to exert their functions in a way that mimics living organs, and HIOs-derived monolayers are expected to be applied to in vitro intestinal pharmacokinetic studies. However, HIOs are established from human tissue, which raises issues of availability and ethics. In the present study, to solve these problems, we have established intestinal organoids using commercially available cryopreserved human intestinal epithelial cells (C-IOs), and compared their functions with biopsy-derived human intestinal organoids (B-IOs) from a pharmacokinetic point of view. Both C-IOs and B-IOs reproduced the morphological features of the intestinal tract and were shown to be composed of epithelial cells. Monolayers generated from C-IOs and B-IOs (C-IO-2D, B-IO-2D, respectively) structurally mimic the small intestine. The C-IOs showed gene expression levels comparable to those of the B-IOs, which were close to those of adult human small intestine. Importantly, the C-IOs-2D showed levels of pharmacokinetics-related protein expression and activity-including cytochrome P450 3A4 (CYP3A4) and carboxylesterase 2 (CES2) enzymatic activities and P-glycoprotein (P-gp) transporter activities -similar to those of B-IOs-2D. This study addresses the difficulties associated with B-IOs and provides fundamental characteristics for the application of C-IOs in pharmacokinetic studies.

Evaluation and prediction of potential drug-drug interactions of linagliptin using in vitro cell culture methods.

Linagliptin is a highly potent dipeptidyl peptidase-4 (DPP-4) inhibitor approved for the treatment of type 2 diabetes. Unlike other DPP-4 inhibitors, linagliptin is cleared primarily via the bile and gut. We used a panel of stably and transiently transfected cell lines to elucidate the carrier-mediated transport processes that are involved in linagliptin disposition in vivo and to assess the potential for drug-drug interactions (DDIs). Our results demonstrate that linagliptin is a substrate of organic cation transporter 2 (OCT2) and P-glycoprotein (P-gp) but not of organic anion-transporting polypeptide 1B1 and 1B3; organic anion transporter 1, 3, and 4; OCT1; or organic cation/carnitine transporter 1 and 2, suggesting that OCT2 and P-gp play a role in the disposition of linagliptin in vivo. Linagliptin inhibits transcellular transport of digoxin by P-gp with an apparent IC(50) of 66.1 µM, but it did not inhibit activity of multidrug resistance-associated protein 2 and breast cancer resistance protein as represented by transport of probe substrate into membrane vesicles from respective transporter-expressing cells. In addition, the inhibitory effect of linagliptin on major solute carrier transporter isoforms was investigated. Linagliptin showed inhibitory potency against only OCT1 and OCT2 out of all major solute carrier transporter isoforms examined, and those inhibition potencies, evaluated using three different in vitro probe substrates, were substrate-specific. Considering the low therapeutic plasma concentration of linagliptin, our data clearly suggest a very low risk for transporter-mediated DDIs with comedications in clinical practice.

Establishment of MDR1-knockout human enteroids for pharmaceutical application.

In the drug development process, it is important to assess the contributions of drug-metabolizing enzymes and/or drug transporters to the intestinal pharmacokinetics of candidate compounds. For such assessments, chemical inhibitors are often used in in vitro systems. However, this practice poses two problems: one is the low expression levels of pharmacokinetic-related genes in conventional in vitro systems, such as Caco-2 cells, and the other is the off-target and less-efficient effects of their inhibitors. Here, as a model, we have established human biopsy-derived enteroids deficient in MDR1, a key efflux transporter. The expression levels and activities of other pharmacokinetic-related genes, such as CYP3A4, in the MDR1-knockout (KO) enteroid-derived monolayers were maintained at levels as high as those in the WT enteroid-derived monolayers. The contribution of MDR1 to the cytotoxicity of vinblastine, which CYP3A4 metabolized, was accurately evaluated by using the MDR1-KO enteroid-derived monolayers. In contrast, it could not be evaluated in the WT enteroid-derived monolayers treated by verapamil, a widely used MDR1 inhibitor, due to the off-target effect of verapamil, which also inhibits CYP3A4. The combination of human enteroid-derived monolayers and genome editing technology would be a powerful tool to evaluate the contributions of specific pharmacokinetic-related molecules.

Comparison of human biopsy-derived and human iPS cell-derived intestinal organoids established from a single individual.

Rodent-derived intestinal tissues or human colon cancer-derived Caco-2 cells are widely used for in vitro pharmacokinetic tests. However, both entail problems such as species differences from humans and low expression levels of specific pharmacokinetic-related factors, respectively. To solve these problems, many groups, including ours, have been focusing on human biopsy-derived intestinal organoids (b-IOs) and human iPS cell-derived intestinal organoids (i-IOs). However, no reports directly compare the two. Therefore, we established both from a single individual and conducted a comparative study. b-IOs had a shorter doubling time than i-IOs: about 59 h vs 148 h. b-IOs also had higher gene expression levels of major drug transporters and drug-metabolizing enzymes than i-IOs. To evaluate their applicability to pharmacokinetics, both organoids were two-dimensionally cultured. Although the b-IO monolayer had a lower transepithelial electrical resistance than the i-IO monolayer, it had higher gene expression levels of many drug transporters and major drug-metabolizing enzymes than the i-IO monolayer. RNA-seq analysis showed that the i-IOs monolayer had a more complex structure than the b-IOs monolayer because the former contained neuronal and vascular endothelial cells. This study provides basic information for pharmacokinetic applications of human biopsy-derived and human iPS cell-derived intestinal organoids.

Simultaneous absolute protein quantification of transporters, cytochromes P450, and UDP-glucuronosyltransferases as a novel approach for the characterization of individual human liver: comparison with mRNA levels and activities.

The purpose of the present study was to determine the absolute protein expression levels of multiple drug-metabolizing enzymes and transporters in 17 human liver biopsies, and to compare them with the mRNA expression levels and functional activities to evaluate the suitability of the three measures as parameters of hepatic metabolism. Absolute protein expression levels of 13 cytochrome P450 (P450) enzymes, NADPH-P450 reductase (P450R) and 6 UDP-glucuronosyltransferase (UGT) enzymes in microsomal fraction, and 22 transporters in plasma membrane fraction were determined using liquid chromatography/tandem mass spectrometry. CYP2C9, CYP2E1, CYP3A4, CYP2A6, UGT1A6, UGT2B7, UGT2B15, and P450R were abundantly expressed (more than 50 pmol/mg protein) in human liver microsomes. The protein expression levels of CYP3A4, CYP2B6, and CYP2C8 were each highly correlated with the corresponding enzyme activity and mRNA expression levels, whereas for other P450s, the protein expression levels were better correlated with the enzyme activities than the mRNA expression levels were. Among transporters, the protein expression level of organic anion-transporting polypeptide 1B1 was relatively highly correlated with the mRNA expression level. However, other transporters showed almost no correlation. These findings indicate that protein expression levels determined by the present simultaneous quantification method are a useful parameter to assess differences of hepatic function between individuals.

CKIepsilon/delta-dependent phosphorylation is a temperature-insensitive, period-determining process in the mammalian circadian clock.

A striking feature of the circadian clock is its flexible yet robust response to various environmental conditions. To analyze the biochemical processes underlying this flexible-yet-robust characteristic, we examined the effects of 1,260 pharmacologically active compounds in mouse and human clock cell lines. Compounds that markedly (>10 s.d.) lengthened the period in both cell lines, also lengthened it in central clock tissues and peripheral clock cells. Most compounds inhibited casein kinase Iepsilon (CKIepsilon) or CKIdelta phosphorylation of the PER2 protein. Manipulation of CKIepsilon/delta-dependent phosphorylation by these compounds lengthened the period of the mammalian clock from circadian (24 h) to circabidian (48 h), revealing its high sensitivity to chemical perturbation. The degradation rate of PER2, which is regulated by CKIepsilon/delta-dependent phosphorylation, was temperature-insensitive in living clock cells, yet sensitive to chemical perturbations. This temperature-insensitivity was preserved in the CKIepsilon/delta-dependent phosphorylation of a synthetic peptide in vitro. Thus, CKIepsilon/delta-dependent phosphorylation is likely a temperature-insensitive period-determining process in the mammalian circadian clock.

Advanced glycation end products inhibitors from Alpinia zerumbet rhizomes.

Advanced glycation end products (AGEs) are major factors responsible for the complication of diabetes. The present study was carried out to investigate the inhibitory activities on fructosamine adduct and α-dicarbonyl formations by hexane extracts of various parts of Alpinia zerumbet. Furthermore, we isolated two previously known compounds, namely 5,6-dehydrokawain (DK) and dihydro-5,6-dehydrokawain (DDK). 8(17),12-Labdadiene-15,16-dial (labdadiene) was isolated for the first time from the rhizome of A. zerumbet. The results showed that labdadiene (IC50=51.06µg/mL) had similar activity to rutin and quercetin against fructosamine adduct. The inhibition of α-dicarbonyl compounds formation by labdadiene was significantly higher than that of DK and DDK. Our results indicate that labdadiene is a potent antiglycation agent which was found to inhibit AGEs formation in three different steps in the pathway. These data indicate that labdadiene could be used to prevent glycation-associated complications in diabetes.

Monolayer platform using human biopsy-derived duodenal organoids for pharmaceutical research.

The human small intestine is the key organ for absorption, metabolism, and excretion of orally administered drugs. To preclinically predict these reactions in drug discovery research, a cell model that can precisely recapitulate the in vivo human intestinal monolayer is desired. In this study, we developed a monolayer platform using human biopsy-derived duodenal organoids for application to pharmacokinetic studies. The human duodenal organoid-derived monolayer was prepared by a simple method in 3-8 days. It consisted of polarized absorptive cells and had tight junctions. It showed much higher cytochrome P450 (CYP)3A4 and carboxylesterase (CES)2 activities than did the existing models (Caco-2 cells). It also showed efflux activity of P-glycoprotein (P-gp) and inducibility of CYP3A4. Finally, its gene expression profile was closer to the adult human duodenum, compared to the profile of Caco-2 cells. Based on these findings, this monolayer assay system using biopsy-derived human intestinal organoids is likely to be widely adopted.