Considerations from an International Regulatory and Pharmaceutical Industry (IQ MPS Affiliate) Workshop on the Standardization of Complex In Vitro Models in Drug Development.

In May 2022, there is an International Regulatory and Pharmaceutical Industry (Innovation and Quality [IQ] Microphysiological Systems [MPS] Affiliate) Workshop on the standardization of complex in vitro models (CIVMs) in drug development. This manuscript summarizes the discussions and conclusions of this joint workshop organized and executed by the IQ MPS Affiliate and the United States Food and Drug Administration (FDA). A key objective of the workshop is to facilitate discussions around opportunities and/or needs for standardization of MPS and chart potential pathways to increase model utilization in the context of regulatory decision making. Participation in the workshop included 200 attendees from the FDA, IQ MPS Affiliate, and 26 global regulatory organizations and affiliated parties representing Europe, Japan, and Canada. It is agreed that understanding global perspectives regarding the readiness of CIVM/MPS models for regulatory decision making and potential pathways to gaining acceptance is useful to align on globally. The obstacles are currently too great to develop standards for every context of use (COU). Instead, it is suggested that a more tractable approach may be to think of broadly applicable standards that can be applied regardless of COU and/or organ system. Considerations and next steps for this effort are described.

Inhibition of hepatic organic anion-transporting polypeptide by RNA interference in sandwich-cultured human hepatocytes: an in vitro model to assess transporter-mediated drug-drug interactions.

Organic anion-transporting polypeptides (OATPs), members of the SLCO/SLC21 family, mediate the transport of various endo- and xenobiotics. In human liver, OATP1B1, 1B3, and 2B1 are located at the basolateral membrane of hepatocytes and are involved in hepatic drug uptake and biliary elimination. Clinically significant drug-drug interactions (DDIs) mediated by hepatic OATPs have drawn great attention from clinical practitioners and researchers. However, there are considerable challenges to prospectively understanding the extent of OATP-mediated DDIs because of the lack of specific OATP inhibitors or substrates and the limitations of in vitro tools. In the present study, a novel RNA interference knockdown sandwich-cultured human hepatocyte model was developed and validated. Quantitative polymerase chain reaction, microarray and immunoblotting analyses, along with uptake assays, illustrated that the expression and transport activity of hepatic OATPs were reduced by small interfering (siRNA) efficiently and specifically in this model. Although OATP siRNA decreased only 20 to 30% of the total uptake of cerivastatin into human hepatocytes, it caused a 50% reduction in cerivastatin metabolism, which was observed by monitoring the formation of the two major metabolites of cerivastatin. The results suggest that coadministration of a drug that is a hepatic OATP inhibitor could significantly alter the pharmacokinetic profile of cerivastatin in clinical studies. Further studies with this novel model demonstrated that OATP and cytochrome P450 have a synergistic effect on cerivastatin-gemfibrozil interactions. The siRNA knockdown sandwich-cultured human hepatocytes may provide a new powerful model for evaluating DDIs.

Effect of an experimental proteasome inhibitor on the cytoskeleton, cytosolic protein turnover, and induction in the neuronal cells in vitro.

GT-1 murine neuronal cells exposed to an experimental proteasome inhibitor (EPI) for 24h showed increased cell death via a non-apoptotic mechanism, as assessed by TUNEL and DNA fragmentation assays. Immunofluorescence staining demonstrated that EPI induced reorganization and relocation of non-ubiquinated actin microfilaments and microtubules to the perinuclear region in EPI treated cells. Immunohistochemistry analysis also demonstrated that other non-cytoskeletal proteins became ubiquitinated and/or upregulated including ubiquitin and other stress proteins. Perinuclear-centrosomal accumulation of gamma-tubulin and vimentin, key components of aggresomes, was observed in the EPI treated cells. Biochemical analysis indicated that EPI-induced accumulation of ubiquitinated protein aggregates in GT-1 cells was detergent - and mechanical - disruption resistant, a feature of aggresomes. Similar results were observed in GT-1 cells treated with lactacystin, a prototypical proteasome inhibitor, which is structurally dissimilar to EPI indicating a pharmacologic effect. In conclusion, EPI causes cytoskeletal reorganization and accumulation of diverse ubiquitinated and non-ubiquitinated proteins in the perinuclear region and potentially overloads the endoplasmic reticulum-dependent quality control mechanism. These processes acting alone, or in combination, are hypothesized to affect axonal transport or other aspects of cellular homeostasis and thus, represent events potentially relevant to the development of peripheral neuropathy associated with administration of proteasome inhibitors in nonclinical studies.