Functional intestinal monolayers from organoids derived from human iPS cells for drug discovery research.

BACKGROUND: Human induced pluripotent stem (iPS) cell-derived enterocyte-like cells (ELCs) are expected to be useful for evaluating the intestinal absorption and metabolism of orally administered drugs. However, it is difficult to generate large amounts of ELCs with high quality because they cannot proliferate and be passaged. METHODS: To solve the issue above, we have established intestinal organoids from ELCs generated using our protocol. Furthermore, monolayers were produced from the organoids. We evaluated the usefulness of the monolayers by comparing their functions with those of the original ELCs and the organoids. RESULTS: We established organoids from ELCs (ELC-org) that could be passaged and maintained for more than a year. When ELC-org were dissociated into single cells and seeded on cell culture inserts (ELC-org-mono), they formed a tight monolayer in 3 days. Both ELC-org and ELC-org-mono were composed exclusively of epithelial cells. Gene expressions of many drug-metabolizing enzymes and drug transporters in ELC-org-mono were enhanced, as compared with those in ELC-org, to a level comparable to those in adult human small intestine. The CYP3A4 activity level in ELC-org-mono was comparable or higher than that in primary cryopreserved human small intestinal cells. ELC-org-mono had the efflux activities of P-gp and BCRP. Importantly, ELC-org-mono maintained high intestinal functions without any negative effects even after long-term culture (for more than a year) or cryopreservation. RNA-seq analysis showed that ELC-org-mono were more mature as intestinal epithelial cells than ELCs or ELC-org. CONCLUSIONS: We have successfully improved the function and convenience of ELCs by utilizing organoid technology.

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.

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.

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.

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.

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.

Using ultrasonography in evaluating the intramuscular injection techniques used for administering drug treatments to schizophrenic patients in Japan.

This study was conducted with six patients with schizophrenia, four of whom received the atypical antipsychotic risperidone long-acting injectable (RLAI), and two patients receiving the typical depot injection (TDI). The purpose of this study was to determine the location (gluteus medius or maximus; deltoid muscles) and diffusion of typical and atypical antipsychotic medications administered intramuscularly using ultrasonography. When using the standardized depth of needle insertion, in some cases, the drug was injected into the gluteus maximus instead of the gluteus medius. Similarly, in some cases the TDI was not visible in the ultrasonographic images until sixteen days after the injection. This verifies how hard the injection site becomes when microspheres of RLAI is injected as compared to other muscle areas. These results confirmed that the gluteus muscle structure was the ideal muscle for depot injection as evidenced by the injection solution being dispersed and rendered not visible immediately after intramuscular injection (IM). With the use of ultrasonography, injection sites and drug dispersions were evaluated under a direct visual guidance, suggesting that ultrasonography is a useful method for establishing evidence for determining correct insertion of IM injection, diffusion of medications, and the effective administration of IM injections.

The conformation and orientation of a 27-residue CCR5 peptide in a ternary complex with HIV-1 gp120 and a CD4-mimic peptide.

Interaction of CC chemokine receptor 5 (CCR5) with the human immunodeficiency virus type 1 (HIV-1) gp120/CD4 complex involves its amino-terminal domain (Nt-CCR5) and requires sulfation of two to four tyrosine residues in Nt-CCR5. The conformation of a 27-residue Nt-CCR5 peptide, sulfated at Y10 and Y14, was studied both in its free form and in a ternary complex with deglycosylated gp120 and a CD4-mimic peptide. NMR experiments revealed a helical conformation at the center of Nt-CCR5(1-27), which is induced upon gp120 binding, as well as a helical propensity for the free peptide. A well-defined structure for the bound peptide was determined for residues 7-23, increasing by 2-fold the length of Nt-CCR5's known structure. Two-dimensional saturation transfer experiments and measurement of relaxation times highlighted Nt-CCR5 residues Y3, V5, P8-T16, E18, I23 and possibly D2 as the main binding determinant. A calculated docking model for Nt-CCR5(1-27) suggests that residues 2-22 of Nt-CCR5 interact with the bases of V3 and C4, while the C-terminal segment of Nt-CCR5(1-27) points toward the target cell membrane, reflecting an Nt-CCR5 orientation that differs by 180° from that of a previous model. A gp120 site that could accommodate (CCR5)Y3 in a sulfated form has been identified. The present model attributes a structural basis for binding interactions to all gp120 residues previously implicated in Nt-CCR5 binding. Moreover, the strong interaction of sulfated (CCR5)Tyr14 with (gp120)Arg440 revealed by the model and the previously found correlation between E322 and R440 mutations shed light on the role of these residues in HIV-1 phenotype conversion, furthering our understanding of CCR5 recognition by HIV-1.

Effects of N- and C-terminal addition of oligolysines or native loop residues on the biophysical properties of transmembrane domain peptides from a G-protein coupled receptor.

Transmembrane domains (TMDs) of G-protein coupled receptors (GPCRs) have very low water solubility and often aggregate during purification and biophysical investigations. To circumvent this problem many laboratories add oligolysines to the N- and C-termini of peptides that correspond to a TMD. To systematically evaluate the effect of the oligolysines on the biophysical properties of a TMD we synthesized 21 peptides corresponding to either the second (TPIFIINQVSLFLIILHSALYFKY) or sixth (SFHILLIMSSQSLLVPSIIFILAYSLK) TMD of Ste2p, a GPCR from Saccharomyces cerevisiae. Added to the termini of these peptides were either Lys(n) (n = 1,2,3) or the corresponding native loop residues. The biophysical properties of the peptides were investigated by circular dichroism (CD) spectroscopy in trifluoroethanol-water mixtures, sodium dodecyl sulfate (SDS) micelles and dimyristoylphosphocholine (DMPC)-dimyristoylphosphoglycerol (DMPG) vesicles, and by attenuated total reflection Fourier transform infrared (ATR-FTIR) in DMPC/DMPG multilayers. The results show that the conformation assumed depends on the number of lysine residues and the sequence of the TMD. Identical peptides with native or an equal number of lysine residues exhibited different biophysical properties and structural tendencies.

Doxorubicin-conjugated anti-midkine monoclonal antibody as a potential anti-tumor drug.

BACKGROUND: Midkine is a heparin-binding growth factor preferentially expressed in tumor cells. The present study was performed to utilize anti-midkine antibody for tumor therapy. METHODS: A monoclonal antibody to midkine was raised by immunizing mice deficient in the midkine gene. The binding site of the antibody was studied by using N-terminal half and C-terminal half of midkine, both of which were chemically synthesized. Doxorubicin (DOX)-conjugate of the antibody was produced by chemical conjugation. The effects of the antibody and the conjugate on cell growth were examined using a midkine-secreting tumor cell, i.e. human hepatocellular carcinoma cell (HepG2). RESULTS: The monoclonal antibody bound to the N-terminal half of midkine. The antibody did not inhibit the growth of HepG2 cells probably because the active domain of midkine is in the C-terminal half. We produced the antibody conjugated with DOX with the hope that the conjugate would be internalized accompanied with midkine. Indeed, the antibody-DOX conjugate significantly inhibited the growth of HepG2 cells compared with DOX-conjugated control IgG. CONCLUSION: The result raises the possibility of using anti-midkine antibody conjugated with DOX for cancer therapy.

Synthesis and NMR structure of p41icf, a potent inhibitor of human cathepsin L.

The total synthesis and structural characterization of the MHCII-associated p41 invariant chain fragment (P41icf) is described. P41icf plays a crucial role in the maturation of MHC class II molecules and antigen processing, acting as a highly selective cathepsin L inhibitor. P41icf synthesis was achieved using a combined solid-phase/solution approach. The entire molecule (65 residues, 7246 Da unprotected) was assembled in solution from fully protected peptides in the size range of 10 residues. After deprotection, oxidative folding in carefully adjusted experimental conditions led to the completely folded and functional P41icf with a disulfide pairing identical to that of native P41icf. CD, NMR, and surface plasmon resonance (SPR) were used for the structural and functional characterization of synthetic P41icf. CD thermal denaturation showed clear cooperative behavior. Tight cathepsin L binding was demonstrated by SPR. (1)H NMR spectroscopy at 800 MHz of unlabeled P41icf was used to solve the three-dimensional structure of the molecule. P41icf behaves as a well-folded protein domain with a topology very close to the crystallographic cathepsin L-bound form.

Nuclear targeting by the growth factor midkine.

Ligand-receptor internalization has been traditionally regarded as part of the cellular desensitization system. Low-density lipoprotein receptor-related protein (LRP) is a large endocytosis receptor with a diverse array of ligands. We recently showed that LRP binds heparin-binding growth factor midkine. Here we demonstrate that LRP mediates nuclear targeting by midkine and that the nuclear targeting is biologically important. Exogenous midkine reached the nucleus, where intact midkine was detected, within 20 min. Midkine was not internalized in LRP-deficient cells, whereas transfection of an LRP expression vector restored midkine internalization and subsequent nuclear translocation. Internalized midkine in the cytoplasm bound to nucleolin, a nucleocytoplasmic shuttle protein. The midkine-binding sites were mapped to acidic stretches in the N-terminal domain of nucleolin. When the nuclear localization signal located next to the acidic stretches was deleted, we found that the mutant nucleolin not only accumulated in the cytoplasm but also suppressed the nuclear translocation of midkine. By using cells that overexpressed the mutant nucleolin, we further demonstrated that the nuclear targeting was necessary for the full activity of midkine in the promotion of cell survival. This study therefore reveals a novel role of LRP in intracellular signaling by its ligand and the importance of nucleolin in this process.