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.

Improvement of Protein Expression Profile in Three-Dimensional Renal Proximal Tubular Epithelial Cell Spheroids Selected Based on OAT1 Gene Expression: A Potential In Vitro Tool for Evaluating Human Renal Proximal Tubular Toxicity and Drug Disposition.

The proximal tubule plays an important role in the kidney and is a major site of drug interaction and toxicity. Analysis of kidney toxicity via in vitro assays is challenging, because only a few assays that reflect functions of drug transporters in renal proximal tubular epithelial cells (RPTECs) are available. In this study, we aimed to develop a simple and reproducible method for culturing RPTECs by monitoring organic anion transporter 1 (OAT1) as a selection marker. Culturing RPTECs in spherical cellular aggregates increased OAT1 protein expression, which was low in the conventional two-dimensional (2D) culture, to a level similar to that in human renal cortices. By proteome analysis, it was revealed that the expression of representative two proximal tubule markers was maintained and 3D spheroid culture improved the protein expression of approximately 7% of the 139 transporter proteins detected, and the expression of 2.3% of the 4,800 proteins detected increased by approximately fivefold that in human renal cortices. Furthermore, the expression levels of approximately 4,800 proteins in three-dimensional (3D) RPTEC spheroids (for 12 days) were maintained for over 20 days. Cisplatin and adefovir exhibited transporter-dependent ATP decreases in 3D RPTEC spheroids. These results indicate that the 3D RPTEC spheroids developed by monitoring OAT1 gene expression are a simple and reproducible in vitro experimental system with improved gene and protein expressions compared with 2D RPTECs and were more similar to that in human kidney cortices. Therefore, it can potentially be used for evaluating human renal proximal tubular toxicity and drug disposition. SIGNIFICANCE STATEMENT: This study developed a simple and reproducible spheroidal culture method with acceptable throughput using commercially available RPTECs by monitoring OAT1 gene expression. RPTECs cultured using this new method showed improved mRNA/protein expression profiles to those in 2D RPTECs and were more similar to those of human kidney cortices. This study provides a potential in vitro proximal tubule system for pharmacokinetic and toxicological evaluations during 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.

Development and Validation of a Proteomic Correlation Profiling Technique to Detect and Identify Enzymes Involved in Metabolism of Drugs of Concern.

To predict the variation of pharmacological or toxicological effect caused by pharmacokinetic variance, it is important to be able to detect previously unknown and unsuspected enzymes involved in drug metabolism. We investigated the use of proteomic correlation profiling (PCP) as a technique to identify the enzymes involved in metabolism of drugs of concern. By evaluating the metabolic activities of each enzyme (including isoforms of cytochrome P450, uridine 5' diphospho-glucuronosyltransferase, and hydrolases, plus aldehyde oxidase and carbonyl reductase) on their typical substrates using a panel of human liver samples, we were able to show the validity of PCP for this purpose. R or Rs and P values were calculated for the association between the protein abundance profile of each protein and the metabolic rate profile of each typical substrate. For the 18 enzymatic activities examined, 13 of the enzymes reported to be responsible for the reactions had correlation coefficients higher than 0.7 and were ranked first to third. For the remaining five activities, the responsible enzymes had correlation coefficients lower than 0.7 and lower rankings. The reasons for this were diverse, including confounding resulting from low protein abundance ratios, artificially high correlations of other enzymes due to limited sample numbers, the presence of inactive enzyme forms, and genetic polymorphisms. Overall, PCP was able to identify the majority of responsible drug-metabolizing enzymes across several enzyme classes (oxidoreductase, transferase, hydrolase); use of this methodology could allow more timely and accurate identification of unknown drug-metabolizing enzymes. SIGNIFICANCE STATEMENT: Proteomic correlation profiling using samples from individual human donors was proven to be a useful methodology for the identification of enzymes responsible for drug-metabolism. This methodology could accelerate the identification of unknown drug-metabolizing enzymes in the future.

Characterization of Enzymes Involved in Nintedanib Metabolism in Humans.

Nintedanib, which is used to treat idiopathic pulmonary fibrosis and non-small cell lung cancer, is metabolized to a pharmacologically inactive carboxylate derivative, BIBF1202, via hydrolysis and subsequently by glucuronidation to BIBF1202 acyl-glucuronide (BIBF1202-G). Since BIBF1202-G contains an ester bond, it can be hydrolytically cleaved to BIBF1202. In this study, we sought to characterize these metabolic reactions in the human liver and intestine. Nintedanib hydrolysis was detected in human liver microsomes (HLMs) (Clearance [CL int]: 102.8 ± 18.9 µL/min per mg protein) but not in small intestinal preparations. CES1 was suggested to be responsible for nintedanib hydrolysis according to experiments using recombinant hydrolases and hydrolase inhibitors as well as proteomic correlation analysis using 25 individual HLM. BIBF1202 glucuronidation in HLM (3.6 ± 0.3 µL/min per mg protein) was higher than that in human intestinal microsomes (1.5 ± 0.06 µL/min per mg protein). UGT1A1 and gastrointestinal UGT1A7, UGT1A8, and UGT1A10 were able to mediate BIBF1202 glucuronidation. The impact of UGT1A1 on glucuronidation was supported by the finding that liver microsomes from subjects homozygous for the UGT1A1*28 allele showed significantly lower activity than those from subjects carrying the wild-type UGT1A1 allele. Interestingly, BIBF1202-G was converted to BIBF1202 in HLS9 at 70-fold higher rates than the rates of BIBF1202 glucuronidation. An inhibition study and proteomic correlation analysis suggested that ß-glucuronidase is responsible for hepatic BIBF1202-G deglucuronidation. In conclusion, the major metabolic reactions of nintedanib in the human liver and intestine were quantitatively and thoroughly elucidated. This information could be helpful to understand the inter- and intraindividual variability in the efficacy of nintedanib. SIGNIFICANCE STATEMENT: To our knowledge, this is the first study to characterize the enzymes responsible for each step of nintedanib metabolism in the human body. This study found that ß-glucuronidase may contribute to BIBF1202-G deglucuronidation.

Identification of HSD17B12 as an enzyme catalyzing drug reduction reactions through investigation of nabumetone metabolism.

Nabumetone, a nonsteroidal anti-inflammatory prodrug, is converted to a pharmacologically active metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA); however, it is 11-fold more efficiently converted to 4-(6-methoxy-2-naphthyl)butan-2-ol (MNBO) via a reduction reaction in human hepatocytes. The goal of this study was to identify the enzyme(s) responsible for MNBO formation from nabumetone in the human liver. MNBO formation by human liver microsomes (HLM) was 5.7-fold higher than in the liver cytosol. In a panel of 24 individual HLM samples with quantitative proteomics data, the 17ß-hydroxysteroid dehydrogenase 12 (HSD17B12) protein level had the high correlation coefficient (r = 0.80, P < 0.001) among 4457 proteins quantified in microsomal fractions during MNBO formation. Recombinant HSD17B12 expressed in HEK293T cells exhibited prominent nabumetone reductase activity, and the contribution of HSD17B12 to the activity in the HLM was calculated as almost 100%. MNBO formation in HepG2 and Huh7 cells was significantly decreased by the knockdown of HSD17B12. We also examined the role of HSD17B12 in drug metabolism and found that recombinant HSD17B12 catalyzed the reduction reactions of pentoxifylline and S-warfarin, suggesting that HSD17B12 prefers compounds containing a methyl ketone group on the alkyl chain. In conclusion, our study demonstrated that HSD17B12 is responsible for the formation of MNBO from nabumetone. Together with the evidence for pentoxifylline and S-warfarin reduction, this is the first study to report that HSD17B12, which is known to metabolize endogenous compounds, such as estrone and 3-ketoacyl-CoA, plays a role as a drug-metabolizing enzyme.

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.

Quantitative Expression of Hepatobiliary Transporters and Functional Uptake of Substrates in Hepatic Two-Dimensional Sandwich Cultures: A Comparative Evaluation of Upcyte and Primary Human Hepatocytes.

Deficient functional expression of drug transporters incapacitates most hepatic cell lines as a reliable tool for evaluating transporter-mediated drug-drug interactions. Recently, genetically modified cells (referred to as upcyte hepatocytes) have emerged as an expandable, noncancerous source of human hepatic cells. Herein, we quantified mRNA and protein levels of key hepatobiliary transporters and we assessed associated uptake activity in short- and long-term cultures of upcyte human hepatocytes (UHH) in comparison to cryopreserved primary human hepatocytes (cPHH). Expression of canalicular efflux pumps, such as MRD1/ABCB1, MATE1/SLC47A1, and MRP2/ABCC2, was relatively well preserved in UHH. By contrast, long-term cultivation of UHH in a two-dimensional sandwich configuration [sandwich-cultured upcyte human hepatocytes (SCUHH)] was required to upregulate organic anion-transporting polypeptide OATP1B1/SLCO1B1, OATP2B1/SLCO2B1, NTCP/SLC10A1, and OCT1/SLC22A1 mRNA expression, which correlated well with respective protein abundances. However, mRNA and protein levels of sinusoidal solute carrier transporters, except for NTCP and OATP2B1, remained low in SCUHH compared to sandwich-cultured cPHH. OCT1- and NTCP-mediated uptake of N-methyl-4-phenylpyridinium acetate and taurocholate was demonstrated in both hepatic models, whereas active uptake of OATP1B1/1B3-selective marker substrates, paralleled by markedly reduced SLCO1B1/1B3 expression, were not detectable in SCUHH. Uptake studies under Na+-depletion and excess of taurocholate confirmed the presence of functional NTCP protein and indicated that NTCP, apart from OATP2B1, contributed substantially to the overall hepatic uptake of rosuvastatin in SCUHH. In conclusion, our data suggest that SCUHH, despite their limitation for evaluating OATP1B1/1B3-mediated transport processes, retain NTCP, OATP2B1, and OCT1 transport activities and thus may be considered as a tool for elucidating compensatory uptake pathways for OATP1B1/1B3 substrates.

Modelling Torsade de Pointes arrhythmias in vitro in 3D human iPS cell-engineered heart tissue.

Torsade de Pointes (TdP) is a lethal arrhythmia that is often drug-induced, thus there is an urgent need for development of models to test or predict the drug sensitivity of human cardiac tissue. Here, we present an in vitro TdP model using 3D cardiac tissue sheets (CTSs) that contain a mixture of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and non-myocytes. We simultaneously monitor the extracellular field potential (EFP) and the contractile movement of the CTSs. Upon treatment with IKr channel blockers, CTSs exhibit tachyarrhythmias with characteristics of TdP, including both a typical polymorphic EFP and meandering spiral wave re-entry. The TdP-like waveform is predominantly observed in CTSs with the cell mixture, indicating that cellular heterogeneity and the multi-layered 3D structure are both essential factors for reproducing TdP-like arrhythmias in vitro. This 3D model could provide the mechanistic detail underlying TdP generation and means for drug discovery and safety tests.

Effects of mutations of ABCA1 in the first extracellular domain on subcellular trafficking and ATP binding/hydrolysis.

ABCA1 mediates release of cellular cholesterol and phospholipid to form high density lipoprotein (HDL). The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. ABCA1-GFP expressed in HEK293 was fully functional for apoA-I-mediated HDL assembly. Immunostaining and confocal microscopic analyses demonstrated that ABCA1-GFP was mainly localized to the plasma membrane (PM) but also substantially in intracellular compartments. All three mutant ABCA1-GFPs showed no or little apoA-I-mediated HDL assembly. R587W and Q597R were associated with impaired processing of oligosaccharide from high mannose type to complex type and failed to be localized to the PM, whereas W590S did not show such dysfunctions. Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Photoaffinity labeling of W590S with 8-azido-[alpha-(32)P]ATP was stimulated by adding ortho-vanadate in the presence of Mn(2+) as much as in the presence of wild-type ABCA1. These results suggest that the defect of HDL assembly in R587W and Q597R is due to the impaired localization to the PM, whereas W590S has a functional defect other than the initial ATP binding and hydrolysis.