Experiences and Translatability of In Vitro and In Vivo Models to Evaluate Caprate as a Permeation Enhancer.

Transient permeation enhancers (PEs) have been widely used to improve the oral absorption of macromolecules. During pharmaceutical development, the correct selection of the macromolecule, PE, and the combination needs to be made to maximize oral bioavailability and ensure successful clinical development. Various in vitro and in vivo methods have been investigated to optimize this selection. In vitro methods are generally preferred by the pharmaceutical industry to reduce the use of animals according to the "replacement, reduction, and refinement" principle commonly termed "3Rs," and in vitro methods typically have a higher throughput. This paper compares two in vitro methods that are commonly used within the pharmaceutical industry, being Caco-2 and an Ussing chamber, to two in vivo models, being in situ intestinal instillation to rats and in vivo administration via an endoscope to pigs. All studies use solution formulation of sodium caprate, which has been widely used as a PE, and two macromolecules, being FITC-dextran 4000 Da and MEDI7219, a GLP-1 receptor agonist peptide. The paper shares our experiences of using these models and the challenges with the in vitro models in mimicking the processes occurring in vivo. The paper highlights the need to consider these differences when translating data generated using these in vitro models for evaluating macromolecules, PE, and combinations thereof for enabling oral delivery.

A novel multi-parametric high content screening assay in ciPTEC-OAT1 to predict drug-induced nephrotoxicity during drug discovery.

Drug-induced nephrotoxicity is a major concern in the clinic and hampers the use of available treatments as well as the development of innovative medicines. It is typically discovered late during drug development, which reflects a lack of in vitro nephrotoxicity assays available that can be employed readily in early drug discovery, to identify and hence steer away from the risk. Here, we report the development of a high content screening assay in ciPTEC-OAT1, a proximal tubular cell line that expresses several relevant renal transporters, using five fluorescent dyes to quantify cell health parameters. We used a validation set of 62 drugs, tested across a relevant concentration range compared to their exposure in humans, to develop a model that integrates multi-parametric data and drug exposure information, which identified most proximal tubular toxic drugs tested (sensitivity 75%) without any false positives (specificity 100%). Due to the relatively high throughput (straight-forward assay protocol, 96-well format, cost-effective) the assay is compatible with the needs in the early drug discovery setting to enable identification, quantification and subsequent mitigation of the risk for nephrotoxicity.

Significantly Different Covalent Binding of Oxidative Metabolites, Acyl Glucuronides, and S-Acyl CoA Conjugates Formed from Xenobiotic Carboxylic Acids in Human Liver Microsomes.

Xenobiotic carboxylic acids may be metabolized to oxidative metabolites, acyl glucuronides, and/or S-acyl-CoA thioesters (CoA conjugates) in vitro, e.g., in hepatocytes, and in vivo. These metabolites can potentially be reactive species and bind covalently to tissue proteins and are generally considered to mediate adverse drug reactions in humans. Acyl glucuronide metabolites have been the focus of reactive metabolite research for decades, whereas drug-CoA conjugates, which have been shown to be up to 40-70 times more reactive, have been given much less attention. In an attempt to dissect the contribution of different pathways to covalent binding, we utilized human liver microsomes supplemented with NADPH, uridine 5'-diphosphoglucuronic acid (UDPGA), or CoA to evaluate the reactivity of each metabolite separately. Seven carboxylic acid drugs were included in this study. While ibuprofen and tolmetin are still on the market, ibufenac, fenclozic acid, tienilic acid, suprofen, and zomepirac were stopped before their launch or withdrawn. The reactivities of the CoA conjugates of ibuprofen, ibufenac, fenclozic acid, and tolmetin were higher compared to those of their corresponding oxidative metabolites and acyl glucuronides, as measured by the level of covalent binding to human liver microsomal proteins. The highest covalent binding was observed for ibuprofenyl-CoA and ibufenacyl-CoA, to levels of 1000 and 8600 pmol drug eq/mg protein, respectively. In contrast and in agreement with the proposed P450-mediated toxicity for these drug molecules, the reactivities of oxidative metabolites of suprofen and tienilic acid were higher compared to the reactivities of their conjugated metabolites, with NADPH-dependent covalent binding of 250 pmol drug eq/mg protein for both drugs. The seven drugs all formed UDPGA-dependent acyl glucuronides, but none of these resulted in covalent binding. This study shows that, unlike studies with hepatocytes or in vivo, human liver microsomes provide an opportunity to investigate the reactivity of individual metabolites.

Excised segments of rat small intestine in Ussing chamber studies: A comparison of native and stripped tissue viability and permeability to drugs.

Excised rat intestinal tissue mounted in an Ussing chamber can be used for intestinal permeability assessments in drug development. The outer layer of the intestine, the serosa and part of the muscle layer, is traditionally removed since it is considered a barrier to the diffusion of nutrients and oxygen as well as to that of pharmaceutical substances. However, the procedure for removing the serosal-muscle layer, i.e. stripping, is a technically challenging process in the pre-experimental preparation of the tissue which may result in tissue damage and reduced viability of the segment. In this study, the viability of stripped and native (non-stripped) rat small intestine tissue segments mounted in Ussing chambers was monitored and the apparent permeability of the tissue to a set of test compounds across both tissue preparations was determined. Electrical measurements, in particular the potential difference (PD) across the intestinal membrane, were used to evaluate the viability. In this study, there were no differences in initial PD (health status of the tissue) or PD over time (viability throughout the experiment) between native and stripped rat jejunum segments. Overall, there were also no significant differences in permeability between stripped and native rat intestinal tissue for the compounds in this study. Based on these results, we propose that stripping can be excluded from the preparation procedures for rat jejunal tissue for permeability studies when using the Ussing chamber technique.