Identification of 2,2-Dimethylbutanoic Acid (HST5040), a Clinical Development Candidate for the Treatment of Propionic Acidemia and Methylmalonic Acidemia.

Propionic acidemia (PA) and methylmalonic acidemia (MMA) are rare autosomal recessive disorders of propionyl-CoA (P-CoA) catabolism, caused by a deficiency in the enzymes P-CoA carboxylase and methylmalonyl-CoA (M-CoA) mutase, respectively. PA and MMA are classified as intoxication-type inborn errors of metabolism because the intramitochondrial accumulation of P-CoA, M-CoA, and other metabolites results in secondary inhibition of multiple pathways of intermediary metabolism, leading to organ dysfunction and failure. Herein, we describe the structure-activity relationships of a series of short-chain carboxylic acids which reduce disease-related metabolites in PA and MMA primary hepatocyte disease models. These studies culminated in the identification of 2,2-dimethylbutanoic acid (10, HST5040) as a clinical candidate for the treatment of PA and MMA. Additionally, we describe the in vitro and in vivo absorption, distribution, metabolism, and excretion profile of HST5040, data from preclinical studies, and the synthesis of the sodium salt of HST5040 for clinical trials.

Non-alcoholic fatty liver disease (NAFLD) models in drug discovery.

INTRODUCTION: The progressive disease spectrum of non-alcoholic fatty liver disease (NAFLD), which includes non-alcoholic steatohepatitis (NASH), is a rapidly emerging public health crisis with no approved therapy. The diversity of various therapies under development highlights the lack of consensus around the most effective target, underscoring the need for better translatable preclinical models to study the complex progressive disease and effective therapies. Areas covered: This article reviews published literature of various mouse models of NASH used in preclinical studies, as well as complex organotypic in vitro and ex vivo liver models being developed. It discusses translational challenges associated with both kinds of models, and describes some of the studies that validate their application in NAFLD. Expert opinion: Animal models offer advantages of understanding drug distribution and effects in a whole body context, but are limited by important species differences. Human organotypic in vitro and ex vivo models with physiological relevance and translatability need to be used in a tiered manner with simpler screens. Leveraging newer technologies, like metabolomics, proteomics, and transcriptomics, and the future development of validated disease biomarkers will allow us to fully utilize the value of these models to understand disease and evaluate novel drugs in isolation or combination.

Drug-induced steatohepatitis.

INTRODUCTION: Drug induced steatohepatitis (DISH), a form of drug induced liver injury (DILI) is characterized by intracellular accumulation of lipids in hepatocytes and subsequent inflammatory events, in some ways similar to the pathology seen with other metabolic, viral and genetic causes of non alcoholic fatty liver disease and steatohepatitis (NAFLD and NASH). Areas covered: This paper provides a comprehensive review of the main underlying mechanisms by which various drugs cause DISH, and outlines existing preclinical tools to predict it and study underlying pathways involved. The translational hurdles of these models are discussed, with the example of an organotypic liver system designed to address them. Finally, we describe the clinical assessment and management of DISH. Expert Opinion: The complexity of the interconnected mechanistic pathways underlying DISH makes it important that preclinical evaluation of drugs is done in a physiologically and metabolically relevant context. Advanced organotypic tissue models, coupled with translational functional biomarkers and next-generational pan-omic measurements, may offer the best shot at gathering mechanistic knowledge and potential of a drug causing steatohepatitis. Ultimately this information could also help predict, detect or guide the development of specific treatments for DISH, which is an unmet need as of today.

Transcriptional profiling suggests that Nevirapine and Ritonavir cause drug induced liver injury through distinct mechanisms in primary human hepatocytes.

Drug induced liver injury (DILI), a major cause of pre- and post-approval failure, is challenging to predict pre-clinically due to varied underlying direct and indirect mechanisms. Nevirapine, a non-nucleoside reverse transcriptase inhibitor (NNRTI) and Ritonavir, a protease inhibitor, are antiviral drugs that cause clinical DILI with different phenotypes via different mechanisms. Assessing DILI in vitro in hepatocyte cultures typically requires drug exposures significantly higher than clinical plasma Cmax concentrations, making clinical interpretations of mechanistic pathway changes challenging. We previously described a system that uses liver-derived hemodynamic blood flow and transport parameters to restore primary human hepatocyte biology, and drug responses at concentrations relevant to in vivo or clinical exposure levels. Using this system, primary hepatocytes from 5 human donors were exposed to concentrations approximating clinical therapeutic and supra-therapeutic levels of Nevirapine (11.3 and 175.0 µM) and Ritonavir (3.5 and 62.4 µM) for 48 h. Whole genome transcriptomics was performed by RNAseq along with functional assays for metabolic activity and function. We observed effects at both doses, but a greater number of genes were differentially expressed with higher probability at the toxic concentrations. At the toxic doses, both drugs showed direct cholestatic potential with Nevirapine increasing bile synthesis and Ritonavir inhibiting bile acid transport. Clear differences in antigen presentation were noted, with marked activation of MHC Class I by Nevirapine and suppression by Ritonavir. This suggests CD8+ T cell involvement for Nevirapine and possibly NK Killer cells for Ritonavir. Both compounds induced several drug metabolizing genes (including CYP2B6, CYP3A4 and UGT1A1), mediated by CAR activation in Nevirapine and PXR in Ritonavir. Unlike Ritonavir, Nevirapine did not increase fatty acid synthesis or activate the respiratory electron chain with simultaneous mitochondrial uncoupling supporting clinical reports of a lower propensity for steatosis. This in vitro study offers insights into the disparate direct and immune-mediated toxicity mechanisms underlying Nevirapine and Ritonavir toxicity in the clinic.

An In Vitro Cynomolgus Vascular Surrogate System for Preclinical Drug Assessment and Human Translation.

OBJECTIVES: The predictive value of animal and in vitro systems for drug development is limited, particularly for nonhuman primate studies as it is difficult to deduce the drug mechanism of action. We describe the development of an in vitro cynomolgus macaque vascular system that reflects the in vivo biology of healthy, atheroprone, or advanced inflammatory cardiovascular disease conditions. APPROACH AND RESULTS: We compare the responses of the in vitro human and cynomolgus vascular systems to 4 statins. Although statins exert beneficial pleiotropic effects on the human vasculature, the mechanism of action is difficult to investigate at the tissue level. Using RNA sequencing, we quantified the response to statins and report that most statins significantly increased the expression of genes that promote vascular health while suppressing inflammatory cytokine gene expression. Applying computational pathway analytics, we identified statin-regulated biological themes, independent of cholesterol lowering, that provide mechanisms for off-target effects, including thrombosis, cell cycle regulation, glycogen metabolism, and ethanol degradation. CONCLUSIONS: The cynomolgus vascular system described herein mimics the baseline and inflammatory regional biology of the human vasculature, including statin responsiveness, and provides mechanistic insight not achievable in vivo.

Organotypic systems in drug metabolism and toxicity: challenges and opportunities.

INTRODUCTION: The frequent failure of high-throughput screening cell-based tools to accurately predict in vivo responses, coupled with limitations of animal models in predicting human safety or drug efficacy, impairs the de-risking process for biotechnology/pharmaceutical companies as they make important decisions to enter human clinical trials. Organotypic systems strive to fill the gap between these screening and in vivo studies and provide a solution. AREAS COVERED: The authors examine the various approaches to recreate physiological response on the bench and trace the evolution of organotypic systems, while discussing intrinsic challenges and opportunities that lie ahead. Furthermore, they cite literature that is the foundation of several biotechnology research companies addressing this issue and discuss major government-funded initiatives to aid the development of these systems in an effort to fill this existing gap. EXPERT OPINION: Decisions from translational systems that bridge basic drug efficacy and toxicity with clinical outcome must be benchmarked against human-relevant endpoints and clinical data for early meaningful pre-clinical decisions. The use of human primary cells coupled with emerging technologies that allow precise control of the culture environment and analysis of meaningful endpoints paves the way for human organotypic systems as a major initiative in de-risking the drug discovery and development process.