Application of Intestinal Epithelial Cells Differentiated from Human Induced Pluripotent Stem Cells for Studies of Prodrug Hydrolysis and Drug Absorption in the Small Intestine.

Cell models to investigate intestinal absorption functions, such as those of transporters and metabolic enzymes, are essential for oral drug discovery and development. The purpose of this study was to generate intestinal epithelial cells from human induced pluripotent stem cells (hiPSC-IECs) and then clarify whether the functions of hydrolase and transporters in them reflect oral drug absorption in the small intestine. The hiPSC-IECs showed the transport activities of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and peptide transporter 1 (PEPT1), revealed by using their probe substrates ([3H]digoxin, sulfasalazine, and [14C]glycylsarcosine), and the metabolic activities of CYP3A4, CES2, and CES1, which were clarified using their probe substrates (midazolam, irinotecan, and temocapril). The intrinsic clearance by hydrolysis of six ester prodrugs into the active form in hiPSC-IECs was correlated with the plasma exposure (Cmax , AUC, and bioavailability) of the active form after oral administration of these prodrugs to rats. Also, the permeability coefficients of 14 drugs, containing two substrates of P-gp (doxorubicin and [3H]digoxin), one substrate of BCRP (sulfasalazine), and 11 nonsubstrates of transporters (ganciclovir, [14C]mannitol, famotidine, sulpiride, atenolol, furosemide, ranitidine, hydrochlorothiazide, acetaminophen, propranolol, and antipyrine) in hiPSC-IECs were correlated with their values of the fraction of intestinal absorption (Fa) in human clinical studies. These findings suggest that hiPSC-IECs would be a useful cell model to investigate the hydrolysis of ester prodrugs and to predict drug absorption in the small intestine.

High Expression of UGT1A1/1A6 in Monkey Small Intestine: Comparison of Protein Expression Levels of Cytochromes P450, UDP-Glucuronosyltransferases, and Transporters in Small Intestine of Cynomolgus Monkey and Human.

Cynomolgus monkeys have been widely used for the prediction of drug absorption in humans. The purpose of this study was to clarify the regional protein expression levels of cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UGTs), and transporters in small intestine of cynomolgus monkey using liquid chromatography-tandem mass spectrometry, and to compare them with the corresponding levels in human. UGT1A1 in jejunum and ileum were >4.57- and >3.11-fold and UGT1A6 in jejunum and ileum were >16.1- and >8.57-fold, respectively, more highly expressed in monkey than in human. Also, jejunal expression of monkey CYP3A8 (homologue of human CYP3A4) was >3.34-fold higher than that of human CYP3A4. Among apical drug efflux transporters, BCRP showed the most abundant expression in monkey and human, and the expression levels of BCRP in monkey and human were >1.74- and >1.25-fold greater than those of P-gp and >2.76- and >4.50-fold greater than those of MRP2, respectively. These findings should be helpful to understand species differences of the functions of CYPs, UGTs, and transporters between monkey and human. The UGT1A1/1A6 data would be especially important because it is difficult to identify isoforms responsible for species differences of intestinal glucuronidation by means of functional studies due to overlapping substrate specificity.

Quantitative Targeted Absolute Proteomics of Transporters and Pharmacoproteomics-Based Reconstruction of P-Glycoprotein Function in Mouse Small Intestine.

The purpose of this study was to investigate whether a pharmacokinetic model integrating in vitro mdr1a efflux activity (which we previously reported) with in vitro/in vivo differences in protein expression level can reconstruct intestinal mdr1a function. In situ intestinal permeability-surface area product ratio between wild-type and mdr1a/1b (-/-) mice is one of the parameters used to describe intestinal mdr1a function. The reconstructed ratios of six mdr1a substrates (dexamethasone, digoxin, loperamide, quinidine, verapamil, vinblastine) and one nonsubstrate (diazepam) were consistent with the observed values reported by Adachi et al. within 2.1-fold difference. Thus, intestinal mdr1a function can be reconstructed by our pharmacoproteomic modeling approach. Furthermore, we evaluated regional differences in protein expression levels of mouse intestinal transporters. Sixteen (mdr1a, mrp4, bcrp, abcg5, abcg8, glut1, 4f2hc, sglt1, lat2, pept1, mct1, slc22a18, ostß, villin1, Na(+)/K(+)-ATPase, γ-gtp) out of 46 target molecules were detected by employing our established quantitative targeted absolute proteomics technique. The protein expression amounts of mdr1a and bcrp increased progressively from duodenum to ileum. Sglt1, lat2, and 4f2hc were highly expressed in jejunum and ileum. Mct1 and ostß were highly expressed in ileum. The quantitative expression profiles established here should be helpful to understand and predict intestinal transporter functions.

Optimization of Cyclic Peptide Property Using Chromatographic Capacity Factor on Permeability of Passive Cell Membrane and Human Induced Pluripotent Stem Cell-Derived Intestinal Membrane.

Cyclic peptides have attracted increasing attention as a privileged class of molecules addressing undruggable targets. Cell permeability of cyclic peptides has remained a challenging issue owing to their molecular properties. Various efficiency metrics have emerged to assess this issue. Among them, the lipophilic permeability efficiency (LPE) metric is the difference between an experimental 1,9-decadiene-water partition coefficient at pH 7.4 (log Ddec/w) and calculated octanol/water partition coefficients (ALogP). This metric provides insight into how structural changes affect permeability. Here, we demonstrate the chromatographic capacity factor (log k') of cyclic peptides using reversed-phase liquid chromatography as an alternative to log Ddec/w, which enables efficient and reliable experimental lipophilicity for the adoption of LPE in early drug discovery. The log k' indicates the passive membrane permeability of cyclic peptides and can be used to optimize passive membrane permeability in combination with other parameters. In addition, intestinal membrane permeability of cyclic peptides on human induced pluripotent stem cell-derived intestinal epithelial cells was achieved with log k' and high passive membrane permeability, although cyclic peptides are P-glycoprotein substrates. These approaches could facilitate optimization of properties of cyclic peptides for oral administration and contribute to the successful discovery and development of cyclic peptides.

Species difference in brain penetration of P-gp and BCRP substrates among monkey, dog and mouse.

The brain penetration of 19 drugs, including P-glycoprotein (P-gp) and/or breast cancer resistance protein (BCRP) substrates, was compared among mice, cynomolgus monkeys and beagle dogs. The brain-to-plasma concentration ratios (Kp,brain) of the tested compounds in monkey and dog showed good correlation, whereas species differences were observed between non-rodents (monkey/dog) and rodents (mouse). In particular, the Kp,brain values of 7 compounds out of 12 P-gp substrates (Kp,brain ratio in P-gp knockout mice versus wild-type mice ≥3) in monkey and dog were more than three-fold higher than those in mice and a similar trend was observed in the brain-to-plasma unbound concentration ratios (Kp,uu,brain). The cerebral spinal fluid (CSF) drug concentrations (CCSF), a surrogate for unbound brain concentration (Cu,brain), were also compared between dog and monkey, and the CSF-to-plasma unbound concentration ratios (Kp,uu,CSF) of BCRP substrates in dog were notably higher than those in monkey, although non-bcrp substrates showed good correlation. Also, the Kp,uu,CSF values of BCRP substrates in dog were clearly higher than the Kp,uu,brain values, indicating that the dog CCSF of BCRP substrates was not suitable as a surrogate of Cu,brain. These observations should be useful when selecting the appropriate animal models for CNS drug discovery.

Successful Prediction of Human Pharmacokinetics After Oral Administration by Optimized Physiologically Based Pharmacokinetics Approach and Permeation Assay Using Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Cells.

None of the physiologically based pharmacokinetic (PBPK) approaches using preclinical data show high predictability of human pharmacokinetic (PK) profiles for drugs affected by the intestinal first-pass effect. Here we report a novel PBPK approach that incorporated the findings of a permeation study using human induced pluripotent stem cell-derived intestinal epithelial cells (hiPSC-IECs) to predict human PK profiles after oral administration of drugs. In hiPSC-IECs, gene expression levels of cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) are enhanced by rifampicin and 1,25-dihydroxyvitamin D3. The permeability of 24 drugs (10 test drugs and 14 reference drugs), including CYP3A4 and P-gp substrates, correlated highly with gastrointestinal availability (Fa × Fg), and could be converted to the apparent absorption rate constant (ka, app) based on the correlation between Fa × Fg and in vivo ka of 27 drugs. The ka, app was input into the PBPK model which contained the optimized calculation processes of metabolism and tissue distribution. The absolute average fold error of PK parameters such as maximum plasma concentration and bioavailability for test drugs was less than 2, suggesting that human PK profiles could be predicted with high accuracy. Our robust PBPK approach enables quick decision-making in drug discovery based on human PK profiles.