Application of Covalent Binding Body Burden in the HµREL Human Hepatocyte Coculture Model for Reactivity Risk Assessment of Metabolically Low Turnover Drugs.

The human hepatocyte suspension model has been a valuable tool to study covalent binding (CVB) for compounds that form reactive metabolites. However, accurately measuring CVB values with the suspension model becomes challenging for metabolically low turnover compounds. In this study, we evaluated the HµREL human hepatocyte coculture model relative to existing literature using human hepatocyte suspension for drugs of known drug-induced liver injury category. Our results indicate that this coculture model provides ample metabolic turnover to reproducibly measure CVB. It is sufficiently robust to apply a predefined 1 mg/day CVB body burden threshold for risk assessment to guide our discovery programs, allowing for expanded coverage to include metabolically low turnover compounds.

Survey Outcome on Immunogenicity Risk Assessment Tools for Biotherapeutics: an Insight into Consensus on Methods, Application, and Utility in Drug Development.

A survey conducted by the Therapeutic Product Immunogenicity (TPI) community within the American Association of Pharmaceutical Scientists (AAPS) posed questions to the participants on their immunogenicity risk assessment strategies prior to clinical development. The survey was conducted in 2 phases spanning 5 years, and queried information about in silico algorithms and in vitro assay formats for immunogenicity risk assessments and how the data were used to inform early developability effort in discovery, chemistry, manufacturing and control (CMC), and non-clinical stages of development. The key findings representing the trends from a majority of the participants included the use of high throughput in silico algorithms, human immune cell-based assays, and proteomics based outputs, as well as specialized assays when therapeutic mechanism of action could impact risk assessment. Additional insights into the CMC-related risks could also be gathered with the same tools to inform future process development and de-risk critical quality attributes with uncertain and unknown risks. The use of the outputs beyond supporting early development activities was also noted with participants utilizing the risk assessments to drive their clinical strategy and streamline bioanalysis.

T-Cell Dependent Immunogenicity of Protein Therapeutics Pre-clinical Assessment and Mitigation-Updated Consensus and Review 2020.

Immune responses to protein and peptide drugs can alter or reduce their efficacy and may be associated with adverse effects. While anti-drug antibodies (ADA) are a standard clinical measure of protein therapeutic immunogenicity, T cell epitopes in the primary sequences of these drugs are the key drivers or modulators of ADA response, depending on the type of T cell response that is stimulated (e.g., T helper or Regulatory T cells, respectively). In a previous publication on T cell-dependent immunogenicity of biotherapeutics, we addressed mitigation efforts such as identifying and reducing the presence of T cell epitopes or T cell response to protein therapeutics prior to further development of the protein therapeutic for clinical use. Over the past 5 years, greater insight into the role of regulatory T cell epitopes and the conservation of T cell epitopes with self (beyond germline) has improved the preclinical assessment of immunogenic potential. In addition, impurities contained in therapeutic drug formulations such as host cell proteins have also attracted attention and become the focus of novel risk assessment methods. Target effects have come into focus, given the emergence of protein and peptide drugs that target immune receptors in immuno-oncology applications. Lastly, new modalities are entering the clinic, leading to the need to revise certain aspects of the preclinical immunogenicity assessment pathway. In addition to drugs that have multiple antibody-derived domains or non-antibody scaffolds, therapeutic drugs may now be introduced via viral vectors, cell-based constructs, or nucleic acid based therapeutics that may, in addition to delivering drug, also prime the immune system, driving immune response to the delivery vehicle as well as the encoded therapeutic, adding to the complexity of assessing immunogenicity risk. While it is challenging to keep pace with emerging methods for the preclinical assessment of protein therapeutics and new biologic therapeutic modalities, this collective compendium provides a guide to current best practices and new concepts in the field.

Can Galactose Be Converted to Glucose in HepG2 Cells? Improving the in Vitro Mitochondrial Toxicity Assay for the Assessment of Drug Induced Liver Injury.

Human hepatocellular carcinoma cells, HepG2, are often used for drug mediated mitochondrial toxicity assessments. Glucose in HepG2 culture media is replaced by galactose to reveal drug-induced mitochondrial toxicity as a marked shift of drug IC50 values for the reduction of cellular ATP. It has been postulated that galactose sensitizes HepG2 mitochondria by the additional ATP consumption demand in the Leloir pathway. However, our NMR metabolomics analysis of HepG2 cells and culture media showed very limited galactose metabolism. To clarify the role of galactose in HepG2 cellular metabolism, U-13C6-galactose or U-13C6-glucose was added to HepG2 culture media to help specifically track the metabolism of those two sugars. Conversion to U-13C3-lactate was hardly detected when HepG2 cells were incubated with U-13C6-galactose, while an abundance of U-13C3-lactate was produced when HepG2 cells were incubated with U-13C6-glucose. In the absence of glucose, HepG2 cells increased glutamine consumption as a bioenergetics source. The requirement of additional glutamine almost matched the amount of glucose needed to maintain a similar level of cellular ATP in HepG2 cells. This improved understanding of galactose and glutamine metabolism in HepG2 cells helped optimize the ATP-based mitochondrial toxicity assay. The modified assay showed 96% sensitivity and 97% specificity in correctly discriminating compounds known to cause mitochondrial toxicity from those with prior evidence of not being mitochondrial toxicants. The greatest significance of the modified assay was its improved sensitivity in detecting the inhibition of mitochondrial fatty acid ß-oxidation (FAO) when glutamine was withheld. Use of this improved assay for an empirical prediction of the likely contribution of mitochondrial toxicity to human DILI (drug induced liver injury) was attempted. According to testing of 65 DILI positive compounds representing numerous mechanisms of DILI together with 55 DILI negative compounds, the overall prediction of mitochondrial mechanism-related DILI showed 25% sensitivity and 95% specificity.

How Close Are We to Profiling Immunogenicity Risk Using In Silico Algorithms and In Vitro Methods?: an Industry Perspective.

In silico HLA-binding algorithms and in vitro T cell-based assays as predictive tools for human immunogenicity risk have made inroads in the biotherapeutic drug discovery and development process. Currently, these tools are being used only for candidate selection or characterization and not for making a go/no-go decision for further development. A clear limitation for a broader implementation is the lack of correlation between the predicted T cell epitope content/immune reactivity potential of a biotherapeutic and the subsequent ADA-related clinical immunogenicity outcome. The current state of technologies and their pros and cons were discussed as a part of the 2016 AAPS National Biotechnology Conference in a themed session. A review of the advances in the area and the session talks along with the ensuing discussions are summarized in this commentary.

Relationship between Nutritional Support and Tuberculosis Treatment Outcomes in West Bengal, India.

INTRODUCTION: Poverty and poor nutrition are associated with the risk of developing tuberculosis (TB). Socioeconomic factors may interfere with anti-tuberculosis treatment compliance and its outcome. We examined whether providing nutritional support (monthly supply of rice and lentil beans) to TB patients who live below the poverty line was associated with TB treatment outcome. METHODS: This was a retrospective cohort study of sputum smear-positive pulmonary TB patients living below the poverty line (income of <$1.25 per day) registered for anti-tuberculosis treatment in two rural districts of West Bengal, India during 2012 to 2013. We compared treatment outcomes among patients who received nutritional support with those who did not. A log-binomial regression model was used to assess the relation between nutritional support and unsuccessful treatment outcome (loss-to-follow-up, treatment failure and death). RESULTS: Of 173 TB patients provided nutritional support, 15 (9%) had unsuccessful treatment outcomes, while 84 (21%) of the 400 not provided nutrition support had unsuccessful treatment outcomes (p < 0.001). After adjusting for age, sex and previous treatment, those who received nutritional support had a 50% reduced risk of unsuccessful treatment outcome than those who did not receive nutritional support (Relative Risk: 0.51; 95% Confidence Intervals: 0.30 - 0.86). CONCLUSION: Under programmatic conditions, monthly rations of rice and lentils were associated with lower risk of unsuccessful treatment outcome among impoverished TB patients. Given the relatively small financial commitment needed per patient ($10 per patient per month), the national TB programme should consider scaling up nutritional support among TB patients living below the poverty line.