Evaluation of Collection and Processing Conditions for Gene Expression Analysis Using Human Myeloid Cells.

Background: The population of blast cells among peripheral blood mononuclear cells (PBMCs) obtained from patients is a desirable specimen for analyzing gene expression in diseases including acute myeloid leukemia. Although the enrichment of blast cells often needs to be performed at a central laboratory, acceptable conditions for sample transport from clinical sites remain to be established. Methods: We evaluated storage temperature, duration, and tube type before initiating sample processing for the analysis of cluster of differentiation (CD)33+ myeloid cells among PBMCs as an alternative to CD34+/CD33+ blast cells. Results: CD33+ myeloid cells were successfully purified by MACS. The cell viability and the RNA integrity were sustained during storage up to 48 hours before sample processing. Storage at 4°C had minimal effects on gene expression, whereas storage at room temperature induced the senescence pathway, characterized by the expression of stress-inducible genes. A CPT tube was also better than an ethylenediaminetetraacetic acid tube for minimizing gene expression change. Conclusions: Our study provided important clues for establishing a sample handling approach for gene expression analysis with purified cell fractions from human PBMCs. To keep the variation of gene expression to a minimum, samples should be delivered at 4°C within 48 hours before processing.

Intestinal Membrane Function in Inflammatory Bowel Disease.

Inflammatory bowel disease is a set of chronic inflammatory diseases that mainly develop in the gastrointestinal mucosa, including ulcerative colitis and Crohn's disease. Gastrointestinal membrane permeability is an important factor influencing the pharmacological effects of pharmaceuticals administered orally for treating inflammatory bowel disease and other diseases. Understanding the presence or absence of changes in pharmacokinetic properties under a disease state facilitates effective pharmacotherapy. In this paper, we reviewed the gastrointestinal membrane function in ulcerative colitis and Crohn's disease from the perspective of in vitro membrane permeability and electrophysiological parameters. Information on in vivo permeability in humans is summarized. We also overviewed the inflammatory bowel disease research using gut-on-a-chip, in which some advances have recently been achieved. It is expected that these findings will be exploited for the development of therapeutic drugs for inflammatory bowel disease and the optimization of treatment options and regimens.

Recent Advances in the Gastrointestinal Complex in Vitro Model for ADME Studies.

Intestinal absorption is a complex process involving the permeability of the epithelial barrier, efflux transporter activity, and intestinal metabolism. Identifying the key factors that govern intestinal absorption for each investigational drug is crucial. To assess and predict intestinal absorption in humans, it is necessary to leverage appropriate in vitro systems. Traditionally, Caco-2 monolayer systems and intestinal Ussing chamber studies have been considered the 'gold standard' for studying intestinal absorption. However, these methods have limitations that hinder their universal use in drug discovery and development. Recently, there has been an increasing number of reports on complex in vitro models (CIVMs) using human intestinal organoids derived from intestinal tissue specimens or iPSC-derived enterocytes plated on 2D or 3D in microphysiological systems. These CIVMs provide a more physiologically relevant representation of key ADME-related proteins compared to conventional in vitro methods. They hold great promise for use in drug discovery and development due to their ability to replicate the expressions and functions of these proteins. This review highlights recent advances in gut CIVMs employing intestinal organoid model systems compared to conventional methods. It is important to note that each CIVM should be tailored to the investigational drug properties and research questions at hand.

Oral Absorption of Middle-to-Large Molecules and Its Improvement, with a Focus on New Modality Drugs.

To meet unmet medical needs, middle-to-large molecules, including peptides and oligonucleotides, have emerged as new therapeutic modalities. Owing to their middle-to-large molecular sizes, middle-to-large molecules are not suitable for oral absorption, but there are high expectations around orally bioavailable macromolecular drugs, since oral administration is the most convenient dosing route. Therefore, extensive efforts have been made to create bioavailable middle-to-large molecules or develop absorption enhancement technology, from which some successes have recently been reported. For example, Rybelsus® tablets and Mycapssa® capsules, both of which contain absorption enhancers, were approved as oral medications for type 2 diabetes and acromegaly, respectively. The oral administration of Rybelsus and Mycapssa exposes their pharmacologically active peptides with molecular weights greater than 1000, namely, semaglutide and octreotide, respectively, into systemic circulation. Although these two medications represent major achievements in the development of orally absorbable peptide formulations, the oral bioavailability of peptides after taking Rybelsus and Mycapssa is still only around 1%. In this article, we review the approaches and recent advances of orally bioavailable middle-to-large molecules and discuss challenges for improving their oral absorption.

The change of the electrophysiological parameters using human intestinal tissues from ulcerative colitis and Crohn's disease.

The purpose of this study was to investigate how disease state of the UC and CD patients affect tissue function determined from electrophysiology viewpoint the electrophysiological parameters on normal, ulcerative colitis (UC) and Crohn's disease (CD) patients. Potential differences (PD), short circuit current (Isc) and resistance (R) as electrophysiological parameters were determined using human large intestinal tissues. The measure of autoptical abnormality was quantified on an arbitrary scale of 0-2. A severe effect of ulcer and thickened mucosa by fibrosis was scored as Grade 2. The larger number of autopsy grade on both UC and CD tissues, the lower values of PD and R than those of normal tissues were observed, although Isc values were not statistically changed irrespective of autopsy grade. This electrophysiological observation of reduced PD indicated functional impairment of active ion transport via ion pumps. Additionally, the R values of CD tissues on each autopsy grade tended to be lower than those of UC tissues. These results suggest that the effect of inflammatory bowel disease on barrier function is different between UC and CD tissues. Therefore, the fibrosis on CD patients might affect the electrophysiological parameters than that of UC patients.

Tenodesis with bone marrow venting under local anesthesia for recalcitrant lateral epicondylitis: results of 2 years of follow-up.

Hypothesis: We hypothesized that the treatment of recalcitrant lateral epicondylitis requires accurate identification of the painful area to promote remodeling of the degenerated extensor insertion and to stabilize the tendon origin during tendon healing. Thus, we performed tenodesis with bone marrow venting under local anesthesia for recalcitrant lateral epicondylitis. Methods: Twenty patients (21 elbows) were treated with bone marrow venting at the painful area of the lateral epicondyle of the elbow and tenodesis using 2 soft anchors lateral to the capitellum (immediately distal to the painful area) and were followed up for ≥2 years. Patients were assessed using the numerical rating scale for pain and the Quick Disabilities of the Arm, Shoulder, and Hand questionnaire, and objective evaluation included active range of motion. Results: The mean preoperative and postoperative pain scores were 7.5 and 0.5, respectively, indicating significant pain relief (P < .001). The mean preoperative and postoperative Quick Disabilities of the Arm, Shoulder, and Hand questionnaire scores were 44.2 and 1.0, respectively (P < .001). Two elbows had a slightly positive Thomsen test at the final visit. No recurrence of intra-articular symptoms induced by synovial fringe impingement was observed. Patients experienced more pain at the bone-tendon junction of extensors than at the tendon parenchyma. Conclusion: Tenodesis with bone marrow venting under local anesthesia was effective for subjective patient satisfaction and positive clinical outcomes at ≥2 years of follow-up in patients with recalcitrant lateral epicondylitis. Intra-articular symptoms can be improved by stabilization of the lateral soft tissue without treatment for intra-articular lesions.

Species differences between rats and primates (humans and monkeys) in complex cleavage pathways of DS-8500a characterized by 14C-ADME studies in humans and monkeys after administration of two radiolabeled compounds and in vitro studies.

Our previous study in rats demonstrated that the metabolic pathways of DS-8500a, a novel GPR119 agonist, include cleavage pathways: reductive cleavage of the oxadiazole ring in the liver and hydrolysis of the amide side chain. In the present study, in vivo metabolic profiling in humans and monkeys after the oral administration of two 14C-labeled compounds was performed to investigate species differences of the cleavage pathways. In monkeys, the oxadiazole ring-cleaved metabolites were mainly detected in feces, but not observed in bile, unlike in rats, suggesting that the reductive ring-opening metabolism occurs in the gastrointestinal tract. In vitro incubation with enterobacterial culture media demonstrated that the reductive cleavage of the oxadiazole ring in humans and monkeys was considerably faster than that in rats. The other cleavage metabolite (M20), produced via hydrolysis of the amide side chain, was detected as the major plasma metabolite in humans and monkeys, and its subsequent metabolite (M21) was excreted in feces, whereas M21 was not a major component in rats, indicating a notable species difference in the amide hydrolysis. In conclusion, this study comprehensively revealed the pronounced species difference of the cleavage pathways: reductive ring-opening by intestinal microflora and liver, and amide hydrolysis.

Discovery of 3-amino-4-{(3S)-3-[(2-ethoxyethoxy)methyl]piperidin-1-yl}thieno[2,3-b]pyridine-2-carboxamide (DS96432529): A potent and orally active bone anabolic agent.

The continuing investigation of SAR of 3-aminothieno[2,3-b]pyridine-2-carboxamide derivatives has been described. In this study, C4-piperidine derivatives with polar functional groups were synthesized to develop orally available bone anabolic agents. The optimized compound 9o (DS96432529), which exhibited the best PK profile and high in vitro activity, showed the highest in vivo efficacy in this series. Moreover, significant synergistic effects were observed following co-administration of DS96432529 and alendronate or parathyroid hormone. The mechanism of action is most likely mediated through CDK8 inhibition.

Safety and efficacy of uninterrupted treatment with edoxaban or warfarin during the peri-procedural period of catheter ablation for atrial fibrillation.

BACKGROUND: The real-world safety and efficacy of uninterrupted anticoagulation treatment with edoxaban (EDX) or warfarin (WFR) during the peri-procedural period of catheter ablation (CA) for atrial fibrillation (AF) are yet to be investigated. METHODS: We conducted a two-center experience, observational study to retrospectively investigate consecutive patients who underwent CA for AF and received EDX or WFR. We examined the incidence of thromboembolic and bleeding complications during the peri-procedural period. RESULTS: The EDX and WFR groups included 153 and 103 patients, respectively (total: 256 patients). Demise or thromboembolic events did not occur in either of the groups. The incidence of major bleeding in the EDX and WFR groups was 0.7% and 2.9%, respectively. The total incidence of major/minor bleeding in the EDX and WFR groups was 7.8% and 8.7%, respectively. Of note, the incidence of bleeding complications in the uninterrupted WFR strategy group was markedly high in patients with an estimated glomerular filtration rate (eGFR) <30 (75%) or a HAS-BLED score ≥3 (60%). Patients with eGFR ≥30 and a HAS-BLED score ≤2 had a lower incidence of bleeding (<10%), regardless of the administered anticoagulation drug (EDX or WFR). CONCLUSIONS: This study confirmed the safety and efficacy of uninterrupted anticoagulation therapy using EDX or WFR in real-world patients undergoing CA for AF. Patients with severely impaired renal function and/or a higher bleeding risk during uninterrupted therapy with WFR were at a prominent risk of bleeding. Therefore, particular attention should be paid in the treatment of these patients.

Comparison of the Intestinal Drug Permeation and Accumulation Between Normal Human Intestinal Tissues and Human Intestinal Tissues With Ulcerative Colitis.

The purpose of this study was to examine drug absorption profile utilizing human intestinal tissues from ulcerative colitis (UC) patients and to compare with normal tissues from intestinal cancer patients. Human intestinal tissues from UC and cancer patients mounted in a mini-Ussing chamber were used to evaluate the permeation of drugs, including FD-4, a very low permeable marker, rebamipide, a low permeable marker, and metoprolol, a high permeable marker. The transport index, an index of sum of permeated and tissue-accumulated molecules, of the model drugs was in accordance with their absorption rank order, and was almost kept constant irrespective of autopsy grade based on tissue fibrosis. On the other hand, UC tissues of grade 2 showed the decreased Xcorr, an index of permeated amount of molecules and increased Tcorr, an index of tissue-accumulated molecules for every tested compound. Our finding of the transport characteristics in intestinal tissues of severe UC patients in mini-Ussing chamber system demonstrated that autopsy grade of UC patients did not drastically change membrane permeability of the tested compounds. Furthermore, it was suggested that morphological changes of intestinal tissues caused by fibrosis led to limited permeation and subsequently increased accumulation with little change of total absorption.

ATF7 mediates TNF-α-induced telomere shortening.

Telomeres maintain the integrity of chromosome ends and telomere length is an important marker of aging. The epidemiological studies suggested that many types of stress including psychosocial stress decrease telomere length. However, it remains unknown how various stresses induce telomere shortening. Here, we report that the stress-responsive transcription factor ATF7 mediates TNF-α-induced telomere shortening. ATF7 and telomerase, an enzyme that elongates telomeres, are localized on telomeres via interactions with the Ku complex. In response to TNF-α, which is induced by various stresses including psychological stress, ATF7 was phosphorylated by p38, leading to the release of ATF7 and telomerase from telomeres. Thus, a decrease of ATF7 and telomerase on telomeres in response to stress causes telomere shortening, as observed in ATF7-deficient mice. These findings give credence to the idea that various types of stress might shorten telomere.

Inhibitory Potency of Marketed Drugs for Ulcerative Colitis and Crohn's Disease on PEPT1.

We investigate the inhibitory effect of marketed drugs for treatment of inflammatory bowel disease (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD) on the uptake transporters of peptide transporter 1 (PEPT1), which are up-regulated under the inflamed condition. The uptake transport of glycylsarcosine, a typical substrate for PEPT1, was reduced to 60% only by 5-aminosalicylate at the clinically relevant concentration among tested marketed drugs in PEPT1 transfected HEK293 cell lines. These findings suggest that the inhibition of PEPT1, which were up-regulated in inflamed or non-inflamed site on UC and CD patients, contribute to the clinical effect of commercially available drugs for IBD patients through the inhibition of uptake of antigenic proinflammatory oligopeptides such as formyl-methionine (Met)-leucine (Leu)-phenylalanine (Phe) via PEPT1.

Observation of Clinically Relevant Drug Interaction in Chimeric Mice with Humanized Livers: The Case of Valproic Acid and Carbapenem Antibiotics.

BACKGROUND AND OBJECTIVE: Human in vitro and dog in vitro/in vivo researches indicate that the drug-drug interaction (DDI) of decreased plasma valproic acid (VPA) concentration by co-administration of carbapenem antibiotics is caused by inhibition of acylpeptide hydrolase (APEH)-mediated VPA acylglucuronide (VPA-G) hydrolysis by carbapenems. In this study, we investigated VPA disposition and APEH activities in TK-NOG chimeric mice, whose livers were highly replaced with human hepatocytes, to evaluate the utility of this animal model and the clinical relevance of the DDI mechanism. METHODS: VPA and VPA-G concentrations in plasma, urinary excretion of VPA-G and APEH activity in humanized livers were measured after co-administration of VPA with meropenem (MEPM) to chimeric mice. RESULTS: After co-administration with MEPM to the chimeric mice, plasma VPA concentration more rapidly decreased than without the co-administration. An increase in plasma AUC and urinary excretion of VPA-G was also observed. APEH activity in humanized livers was strongly inhibited even at 24 h after co-administration of MEPM to the chimeric mice. CONCLUSION: The DDI of VPA with carbapenems was successfully observed in chimeric mice with humanized livers. The DDI was caused by long-lasting inhibition of hepatic APEH-mediated VPA-G hydrolysis by carbapenems, which strongly supports the APEH-mediated mechanism of the clinical DDI. This is the first example showing the usefulness of chimeric mice with humanized livers for evaluation of a DDI via non-cytochrome P450 enzyme.

Effect of proinflammatory cytokine IL-6 on efflux transport of rebamipide in Caco-2 cells.

1. Effect of IL-6, a pro-inflammatory cytokine, on efflux transport of rebamipide, an antiulcer drug, was investigated in Caco-2 cells. 2. Rebamipide had a greater basal-to-apical than apical-to-basal transport rate. Efflux transport of rebamipide was inhibited by cyclosporine A, a P-gp inhibitor, and probenecid, which is a general MRP inhibitor, but not by Ko143, a BCRP inhibitor. 3. By the addition of IL-6, mannitol transport was slightly increased in a concentration-dependent manner in both directions of absorption and efflux. The addition of IL-6 did not change efflux transport of rebamipide even though efflux transport of digoxin, a typical substrate of P-gp, was significantly decreased by the addition of IL-6, indicating decrease of the function of P-gp. 4. Therefore, it was suggested that increase of MRP(s)-mediated transport compensates for the decrease of P-gp mediated transport of rebamipide. These findings suggested that rebamipide absorption is unlikely to be changed in IBD patients.

The Pro-inflammatory Cytokine Interleukin-6 Regulates Nanoparticle Transport Across Model Follicle-Associated Epithelium Cells.

The aim of this study was to investigate whether the pro-inflammatory cytokines improved the function of the cell monolayer model of the human follicle-associated epithelium (FAE) of co-culture of Caco-2 cells on permeable filters with Raji B-cells underneath from the viewpoint of particle transport. Exposure to tumor necrosis factor-α resulted in an almost maintained epithelial integrity/paracellular permeability combined with an increased nanoparticle transport in a dose-dependent manner while the effects of interleukin (IL)-1ß were limited. Exposure to IL-6 significantly enhanced the nanoparticle transport with the limited disruption of the cell monolayer integrity. The addition of IL-6 or tumor necrosis factor-α to Caco-2 monolayers without Raji B-cells did not enhance nanoparticle transport. In our IL-6 treated FAE model, the nanoparticle transport almost disappeared at 4°C or after the addition of 5-(N-ethyl-N-isopropyl) amiloride, an inhibitor of macropinocytosis. Furthermore, IgA binding, presumably by a secretory IgA receptor, a marker of M-cells was observed on the apical side of our model FAE. These results indicate that the combined effect of IL-6 with unknown factors from Raji-B cells made the FAE model more functional with regard to nanoparticle transport. The IL-6 enhanced FAE model will be a useful platform for nanoparticle drug delivery research across the intestinal epithelium.

Stereoselective hydroxylation by CYP2C19 and oxidation by ADH4 in the in vitro metabolism of tivantinib.

1. In prior studies, it has been shown that tivantinib is extensively metabolized in humans to many oxidative metabolites and glucuronides. In order to identify the responsible enzymes, we investigated the in vitro metabolism of tivantinib and its four major circulating metabolites. 2. The primary isoforms involved in the elimination of tivantinib were CYP2C19 and CYP3A4/5. CYP2C19 showed catalytic activity for the formation of M5 (hydroxylated metabolite), but not for M4 (a stereoisomer of M5), whereas CYP3A4/5 catalyzed the formation of both metabolites. For the elimination of M4, M5 and M8 (keto-metabolite), CYP3A4/5 was the major cytochrome P450 isoform and UGT1A9 was mainly involved in the glucuronidation of M4 and M5. 3. ADH4 was identified as one of the major alcohol dehydrogenase isoforms contributing to the formation of M6 (sequential keto-metabolite of M4 and M5) and M8. The substrate preference of ADH for M4, and not M5, was observed in the formation of M6. 4. In conclusion, CYP2C19, CYP3A4/5, UGT1A9 and ADH4 were the primary drug metabolizing enzymes involved in the in vitro metabolism of tivantinib and its metabolites. The stereoselective hydroxylation by CYP2C19 and substrate stereoselectivity of ADH4-catalyzed oxidation in the in vitro metabolism of tivantinib was discovered.

In vivo inhibition of acylpeptide hydrolase by carbapenem antibiotics causes the decrease of plasma concentration of valproic acid in dogs.

1. Our previous in vitro studies suggest that inhibition of the acylpeptide hydrolase (APEH) activity as valproic acid glucuronide (VPA-G) hydrolase by carbapenems in human liver cytosol is a key process for clinical drug-drug interaction (DDI) of valproic acid (VPA) with carbapenems. Here, we investigated whether in vivo DDI of VPA with meropenem (MEPM) was caused via inhibition of APEH in dogs. 2. More rapid decrease of plasma VPA levels and increased urinary excretion of VPA-G were observed after co-administration with MEPM compared with those after without co-administration, whereas the plasma level and bile excretion of VPA-G showed no change. 3. Dog VPA-G hydrolase activity, inhibited by carbapenems, was mainly located in cytosol from both the liver and kidney. APEH-immunodepleted cytosols lacked VPA-G hydrolase activity. Hepatic and renal APEH activity was negligible even at 24 h after dosing of MEPM to a dog. 4. In conclusion, DDI of VPA with carbapenems in dogs is caused by long-lasting inhibition of APEH-mediated VPA-G hydrolysis by carbapenems, which could explain the delayed recovery of plasma VPA levels to the therapeutic window even after discontinuation of carbapenems in humans.

Liver-selective distribution in rats supports the importance of active uptake into the liver via organic anion transporting polypeptides (OATPs) in humans.

Organic anion transporting polypeptide (OATP) 1B1 and 1B3 are key molecules that are involved in hepatic uptake related to drug elimination, and OATP-mediated drug interactions are of clinical concern. In this study, with an aim to determine a cutoff value for the potential involvement of OATP, we collected data on the distribution of 12 human OATP and 24 non-OATP radiolabeled substrates in rats. The OATP substrates exhibited a higher tissue-to-plasma ratio (Kp) in the liver than that in the other tissues. As an index of liver-specific distribution, a hepatic Kp ratio (the ratio of Kp in the liver to that in other tissues) was introduced, and a hepatic Kp ratio <10 was proposed as a criterion for excluding the involvement of OATP in vivo. Approximately 20% of the non-OATP substrates as well as 100% of the OATP substrates exceeded the cutoff value of 10; therefore, further in vitro transport studies will be required to decide whether to conduct clinical drug interaction studies. Since distribution studies are usually conducted in rats during drug development, the use of a hepatic Kp ratio is practical and could refine the current decision tree for selecting OATP substrates in the drug interaction guidance/guidelines.

Discovery and structure-guided optimization of tert-butyl 6-(phenoxymethyl)-3-(trifluoromethyl)benzoates as liver X receptor agonists.

To obtain potent liver X receptor (LXR) agonists, a structure-activity relationship study was performed on a series of tert-butyl benzoate analogs. As the crystal structure analysis suggested applicable interactions between the LXR ligand-binding domain and the ligands, two key functional groups were introduced. The introduction of the hydroxyl group on the C6-position of the benzoate part enhanced the agonistic activity in a cell-based assay, and the carboxyl group in terminal improved the pharmacokinetic profile in mice, respectively. The obtained compound 32b increased blood ABCA1 mRNA expression without plasma TG elevation in both mice and cynomolgus monkeys.