Modelling and Prevention of Acute Kidney Injury through Ischemia and Reperfusion in a Combined Human Renal Proximal Tubule/Blood Vessel-on-a-Chip.

Background: Renal ischemia/reperfusion injury (rIRI) is one of the major causes of AKI. Although animal models are suitable for investigating systemic symptoms of AKI, they are limited in translatability. Human in vitro models are crucial in giving mechanistic insights into rIRI; however, they miss out on crucial aspects such as reperfusion injury and the multitissue aspect of AKI. Methods: We advanced the current renal proximal tubule-on-a-chip model to a coculture model with a perfused endothelial vessel separated by an extracellular matrix. The coculture was characterized for its three-dimensional structure, protein expression, and response to nephrotoxins. Then, rIRI was captured through control of oxygen levels, nutrient availability, and perfusion flow settings. Injury was quantified through morphologic assessment, caspase-3/7 activation, and cell viability. Results: The combination of low oxygen, reduced glucose, and interrupted flow was potent to disturb the proximal tubules. This effect was strongly amplified upon reperfusion. Endothelial vessels were less sensitive to the ischemia-reperfusion parameters. Adenosine treatment showed a protective effect on the disruption of the epithelium and on the caspase-3/7 activation. Conclusions: A human in vitro rIRI model was developed using a coculture of a proximal tubule and blood vessel on-a-chip, which was used to characterize the renoprotective effect of adenosine. The robustness of the model and assays in combination with the throughput of the platform make it ideal to advance pathophysiological research and enable the development of novel therapeutic modalities.

Perspectives on the evaluation and adoption of complex in vitro models in drug development: Workshop with the FDA and the pharmaceutical industry (IQ MPS Affiliate).

Complex in vitro models (CIVM) offer the potential to improve pharmaceutical clinical drug attrition due to safety and/ or efficacy concerns. For this technology to have an impact, the establishment of robust characterization and qualifi­cation plans constructed around specific contexts of use (COU) is required. This article covers the output from a workshop between the Food and Drug Administration (FDA) and Innovation and Quality Microphysiological Systems (IQ MPS) Affiliate. The intent of the workshop was to understand how CIVM technologies are currently being applied by pharma­ceutical companies during drug development and are being tested at the FDA through various case studies in order to identify hurdles (real or perceived) to the adoption of microphysiological systems (MPS) technologies, and to address evaluation/qualification pathways for these technologies. Output from the workshop includes the alignment on a working definition of MPS, a detailed description of the eleven CIVM case studies presented at the workshop, in-depth analysis, and key take aways from breakout sessions on ADME (absorption, distribution, metabolism, and excretion), pharmacology, and safety that covered topics such as qualification and performance criteria, species differences and concordance, and how industry can overcome barriers to regulatory submission of CIVM data. In conclusion, IQ MPS Affiliate and FDA scientists were able to build a general consensus on the need for animal CIVMs for preclinical species to better determine species concordance. Furthermore, there was acceptance that CIVM technologies for use in ADME, pharmacology and safety assessment will require qualification, which will vary depending on the specific COU.

Reconstituted Human Organ Models as a Translational Tool for Human Organ Response: Definition, Expectations, Cases, and Strategies for Implementation in Drug Discovery and Development.

Recent progress in the fields of tissue engineering, micro-electro mechanical systems, and materials science have greatly improved cell culture systems, which were traditionally performed in a static two-dimensional manner. This progress has led to a number of new cell culture concepts represented by organ-on-a-chip, three dimensional (3D)-tissues, and microphysiological systems, among others. In this review, these culture models are categorized as reconstituted human organ models, which recapitulate human organ-like structure, function, and responses with physiological relevance. In addition, we also describe the expectations of reconstituted organ models from the viewpoint of a pharmaceutical company based on recent concerns expressed in drug discovery and development. These models can be used to assess the pharmacokinetics, safety and efficacy of new molecular entities (NMEs) prior to clinical trials. They can also be used to conduct mechanistic investigations of events that arise due to administration of NMEs in humans. In addition, monitoring biomarkers of organ function in these models will aid in the translation of their changes in humans. As the majority of reconstituted human organ models show improved functional characteristics and long-term maintenance in culture, they are valuable for modeling human events. An example is described using the three-dimensional bioprinted human liver tissue model in this article. Implementation of reconstituted human organ models in drug discovery and development can be accelerated by encouraging collaboration between developers and users. Such efforts will provide significant benefits for delivering new and improved medicines to patients.

Recent Progress in Hepatocyte Culture Models and Their Application to the Assessment of Drug Metabolism, Transport, and Toxicity in Drug Discovery: The Value of Tissue Engineering for the Successful Development of a Microphysiological System.

Tissue engineering technology has provided many useful culture models. This article reviews the merits of this technology in a hepatocyte culture system and describes the applications of the sandwich-cultured hepatocyte model in drug discovery. In addition, we also review recent investigations of the utility of the 3-dimensional bioprinted human liver tissue model and spheroid model. Finally, we present the future direction and developmental challenges of a hepatocyte culture model for the successful establishment of a microphysiological system, represented as an organ-on-a-chip and even as a human-on-a-chip. A merit of advanced culture models is their potential use for detecting hepatotoxicity through repeated exposure to chemicals as they allow long-term culture while maintaining hepatocyte functionality. As a future direction, such advanced hepatocyte culture systems can be connected to other tissue models for evaluating tissue-to-tissue interaction beyond cell-to-cell interaction. This combination of culture models could represent parts of the human body in a microphysiological system.

Species differences in sinusoidal and canalicular efflux transport of mycophenolic acid 7-O-glucuronide in sandwich-cultured hepatocytes.

Metabolism and sinusoidal/canalicular efflux of mycophenolic acid (MPA) was investigated using sandwich-cultured hepatocytes (SCHs). After applying MPA to SCHs from humans, wild-type rats, and multidrug resistance-associated protein (Mrp) 2-deficient rats, the MPA metabolites 7-O-glucuronide (MPAG) and acyl glucuronide (AcMPAG) were detected in the intracellular compartment of the SCHs. Sinusoidal efflux of MPAG was detected in all SCH preparations including Mrp2-deficient rat SCHs, whereas canalicular efflux of MPAG was observed in wild-type rat and human SCHs but not in Mrp2-deficient rat SCHs. The ratio of canalicular efflux to net (canalicular plus sinusoidal) efflux was 37 ± 8% in wild-type rat SCHs, while the ratio in human SCHs was significantly lower (20 ± 2%, P < 0.05), indicating species differences in the direction of hepatic MPAG transport. This 20% ratio in human SCHs corresponds to a high sinusoidal MPAG efflux (80%) that can in part account for the urine-dominated recovery of MPAG in humans. Both sinusoidal and canalicular MPAG efflux in rat SCHs shows a good correspondence to urinary and biliary recovery of MPAG after MPA dosing. The sinusoidal efflux of AcMPAG in human SCHs was detected from one out of three donors, suggesting donor-to-donor variation. In conclusion, this study demonstrates the predictive value of SCHs for elucidating the interplay of metabolism and efflux transport, in addition to demonstrating a species difference between rat and human in sinusoidal and canalicular efflux of MPAG.

Optimized methods for targeted peptide-based quantification of human uridine 5'-diphosphate-glucuronosyltransferases in biological specimens using liquid chromatography-tandem mass spectrometry.

The aim of this study was to optimize methods for quantifying 13 uridine 5'-diphosphate-glucuronosyltransferase (UGT) isoforms (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, and 2B17) in human liver, intestinal, and kidney microsomes, and in recombinant human UGT-expressing insect cell membranes (rhUGTs) by targeted peptide-based quantification using liquid chromatography-tandem mass spectrometry. Production of targeted peptides was compared by combining three denaturing agents (urea, sodium deoxycholate, and octyl glucoside) and three denaturing temperatures (37°C, 60°C, and 95°C) followed by tryptic digestion for 2-20 hours. Denaturing conditions and digestion times yielding high production efficiency varied markedly among isoforms and specimens, indicating the importance of specific optimization. Each UGT isoform was quantified using the methods found to be optimal. The expression of 10 (1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10, 2B15, and 2B17), 6 (1A1, 1A3, 1A4, 1A10, 2B7, and 2B17), and 3 (1A6, 1A9, and 2B7) isoforms was detected in human liver, intestinal, and kidney microsomes, respectively, and levels were reproducible using multiple protocols. All isoforms were quantified in rhUGTs. Determining the levels of UGTs in human tissue specimens and those in rhUGTs is important for estimating the contribution of glucuronidation to body clearance based on in vitro-in vivo extrapolation.

Species differences in intestinal glucuronidation activities between humans, rats, dogs and monkeys.

1. Glucuronidation via UDP-glucuronosyltransferase (UGT) in the intestine has been reported to influence the pharmacokinetics (PK) of drugs; however, information concerning the differences in activity between species is limited. Here, we investigated the in vitro and in vivo activities of intestinal glucuronidation for 17 UGT substrates in humans, rats, dogs and monkeys. 2. Although in vitro intrinsic clearance (CLint,u,UGT) in intestinal microsomes showed a good correlation between humans and laboratory animals, values tended to be lower in humans than in laboratory animals. The ratio of CLint,u,UGT in the absence and presence of bovine serum albumin differed between species. In vivo, the fraction of drug absorbed (FaFg) in humans correlated with that in dogs and monkeys, but not in rats. 3. While an inverse correlation between CLint,u,UGT and FaFg was observed in each species, the CLint,u,UGT values in the intestinal microsomes corresponding to FaFg values in dogs were three to four times higher than in other animals. 4. These results indicate the need for a degree of caution when extrapolating PK data from laboratory animals to humans.

Glucuronidation and subsequent biliary excretion of mycophenolic acid in rat sandwich-cultured hepatocytes.

Rat sandwich-cultured hepatocytes (SCH) were used to correlate the in vitro hepatic disposition of mycophenolic acid (MPA) with published in vivo data, as well as mechanistic studies on drug-drug interaction. The major metabolite of MPA in SCH was 7-O-glucuronide (MPAG) followed by acyl-glucuronide (AcMPAG). MPAG and AcMPAG, but not MPA, showed significant in vitro biliary excretion with biliary excretion indexes (BEI) of 40% for MPAG and 45% for AcMPAG. While these BEIs were similar, the biliary excretion amount (BEA) of MPAG (120 pmol/mg protein) was orders of magnitude higher than that of AcMPAG (0.34 pmol/mg protein). Since MPAG is the major metabolite in in vivo bile, we propose that BEA is a better qualifier of biliary excretion. Quercetin inhibited MPAG and AcMPAG production, while chrysin inhibited only MPAG production, showing that chrysin is not a pan-glucuronidation inhibitor. Cyclosporin A (CysA) reduced the BEI of MPAG and increased intracellular MPA accumulation without changing MPAG amounts. These results suggest that CysA causes inhibition of biliary excretion of MPAG, as well as a mixed inhibition of glucuronidation of MPA and sinusoidal efflux of MPA/MPAG. In conclusion, the present study demonstrates a good agreement of hepatic MPA disposition between SCH and in vivo rats.

HDAC inhibitors restore the capacity of aged mice to respond to haloperidol through modulation of histone acetylation.

Antipsychotic drugs are widely prescribed to elderly patients for the treatment of a variety of psychopathological conditions, including psychosis and the behavioral disturbances associated with dementia. However, clinical experience suggests that these drugs may be less efficacious in the elderly individuals than in the young. Recent studies suggest that aging may be associated with epigenetic changes and that valproic acid (VPA), a histone deacetylase inhibitor, may reverse such changes. However, it is not yet known whether HDAC inhibitors can modulate age-related epigenetic changes that may impact antipsychotic drug action. In this study, we analyzed conditioned avoidance response (CAR) and c-Fos expression patterns to elucidate the effect of HDAC inhibitors VPA and entinostat (MS-275) on behavioral and molecular markers of the effects of haloperidol (HAL) in aged mice. Our results showed that HAL administration failed to suppress the avoidance response during the CAR test, suggesting an age-related decrease in drug efficacy. In addition, HAL-induced c-Fos expression in the nucleus accumbens shell and prefrontal cortex was significantly lower in aged mice as compared with young mice. Pretreatment with VPA and MS-275 significantly improved HAL effects on the CAR test in aged mice. Also, VPA and MS-275 pretreatment restored HAL-induced increases in c-Fos expression in the nucleus accumbens shell and prefrontal cortex of aged mice to levels comparable with those observed in young mice. Lastly, but most importantly, increases in c-Fos expression and HAL efficacy in the CAR test of the HAL+VPA and HAL+MS-275 groups were correlated with elevated histone acetylation at the c-fos promoter region in aged mice. These findings suggest that pretreatment with VPA or MS-275 increases the behavioral and molecular effects of HAL in aged mice and that these effects occur via modulation of age-related histone hypoacetylation in the nucleus accumbens shell and prefrontal cortex.

Quantitative prediction of human intestinal glucuronidation effects on intestinal availability of UDP-glucuronosyltransferase substrates using in vitro data.

We investigated whether the effects of intestinal glucuronidation on the first-pass effect can be predicted from in vitro data for UDP-glucuronosyltransferase (UGT) substrates. Human in vitro intrinsic glucuronidation clearance (CL(int, UGT)) for 11 UGT substrates was evaluated using pooled intestinal microsomes (4.00-4620 µl · min⁻¹ · mg⁻¹) and corrected by the free fraction in the microsomal mixture (CLu(int), (UGT) = 5.2-5133 µl · min⁻¹ · mg⁻¹). Eleven UGT substrates were stable against intestinal cytochrome P450, indicating intestinal glucuronidation has a main effect on human intestinal availability. Oral absorbability intestinal availability (F(a)F(g)) values were calculated from in vivo pharmacokinetic parameters in the literature (F(a)F(g) = 0.01-1.0). It was found that CLu(int, UGT) and human F(a)F(g) have an inverse relationship that can be fitted to a simplified intestinal availability model. Experiments using Supersomes from insect cells expressing UGT isoforms showed that the substrates used were conjugated by various UGT isoforms. These results suggest that combining the simplified intestinal availability model and in vitro conjugation assay make it possible to predict human F(a)F(g) regardless of UGT isoform.

Quantitative prediction of intestinal glucuronidation of drugs in rats using in vitro metabolic clearance data.

UDP-glucuronosyltransferase (UGT) is highly expressed in the small intestine and catalyzes the glucuronidation of small molecules, which may affect the oral bioavailability of drugs. However, no method of predicting the in vivo observed fraction of absorbed drug (F(a)F(g)) affected by UGT has yet been established. Here, we investigated the relationship between F(a)F(g) and in vitro clearance of nine UGT substrates (ketoprofen, tolcapone, telmisartan, raloxifene, entacapone, resveratrol, buprenorphine, quercetin, and ezetimibe) via UGT in intestinal microsomes (CL(int, UGT)) in rats. F(a)F(g) was calculated from pharmacokinetic parameters after intravenous and oral administration or using the portal-systemic concentration difference method, with values ranging from 0.027 (ezetimibe) to 1 (tolcapone). Glucuronides of model compounds were observed in the portal plasma after oral administration, with CL(int, UGT) values ranging from 57.8 (tolcapone) to 19,200 µL/min/mg (resveratrol). An inverse correlation between F(a)F(g) and CL(int, UGT) was observed for most compounds and was described using a simplified intestinal availability model reported previously. This model gave accurate predictions of F(a)F(g) values for three in-house compounds. Our results show that F(a)F(g) in rats is affected by UGT and can be predicted using CL(int, UGT). This work should hasten the development of a method to predict F(a)F(g) in humans.

The l-isomer-selective transport of aspartic acid is mediated by ASCT2 at the blood-brain barrier.

Aspartic acid (Asp) undergoes l-isomer-selective efflux transport across the blood-brain barrier (BBB). This transport system appears to play an important role in regulating l- and d-Asp levels in the brain. The purpose of this study was to identify the responsible transporters and elucidate the mechanism for l-isomer-selective Asp transport at the BBB. The l-isomer-selective uptake of Asp by conditionally immortalized mouse brain capillary endothelial cells used as an in vitro model of the BBB took place in an Na+- and pH-dependent manner. This process was inhibited by system ASC substrates such as l-alanine and l-serine, suggesting that system ASC transporters, ASCT1 and ASCT2, are involved in the l-isomer selective transport. Indeed, l-Asp uptake by oocytes injected with either ASCT1 or ASCT2 cRNA took place in a similar manner to that in cultured BBB cells, whereas no significant d-Asp uptake occurred. Although both ASCT1 and ASCT2 mRNA were expressed in the cultured BBB cells, the expression of ASCT2 mRNA was 6.7-fold greater than that of ASCT1. Moreover, immunohistochemical analysis suggests that ASCT2 is localized at the abluminal side of the mouse BBB. These results suggest that ASCT2 plays a key role in l-isomer-selective Asp efflux transport at the BBB.

Newly developed rat brain pericyte cell line, TR-PCT1, responds to transforming growth factor-beta1 and beta-glycerophosphate.

Brain pericytes form an incomplete envelope around endothelial cells and within the microvascular basement membrane of capillaries and postcapillary venules. Recently, it has been reported that brain pericytes exhibit pluripotency, regulation of endothelial cell activity, and macrophage activity. However, many molecular and cellular aspects of brain pericytes remain unclear. In this study, we have tried to establish a conditionally immortalized brain pericyte cell line (TR-PCT) derived from the brain capillary of a transgenic rat harboring a temperature-sensitive simian virus 40 T antigen gene. One of the clones was named TR-PCT1, and we established 6 clones of pericyte-like cells from a 16 week-old tsA58 transgenic rat. For comparison, primary pericytes from a Wistar rat were also studied. The expression of platelet-derived growth factor receptor beta, angiopoietin-1, osteopontin, and intercellular adhesion molecule-1 in TR-PCT1 was determined by reverse transcription-polymerase chain reaction. Transforming growth factor-beta1 enhanced a-smooth muscle actin expression in TR-PCT1, but this expression was reduced by subsequent treatment with basic fibroblast growth factor. When TR-PCT1 was seeded on type I collagen plates and treated with beta-glycerophosphate, nodules developed in the cells and these nodules reacted positively to von Kossa stain used as a marker of calcification. We believe that TR-PCT1 will help us gain a better understanding of the physiological and/or pathophysiological role of pericytes.