Science-based assessment of source materials for cell-based medicines: report of a stakeholders workshop.

Human pluripotent stem cells (hPSCs) have the potential to transform medicine. However, hurdles remain to ensure safety for such cellular products. Science-based understanding of the requirements for source materials is required as are appropriate materials. Leaders in hPSC biology, clinical translation, biomanufacturing and regulatory issues were brought together to define requirements for source materials for the production of hPSC-derived therapies and to identify other key issues for the safety of cell therapy products. While the focus of this meeting was on hPSC-derived cell therapies, many of the issues are generic to all cell-based medicines. The intent of this report is to summarize the key issues discussed and record the consensus reached on each of these by the expert delegates.

Application of in Vitro T Cell Assay Using Human Leukocyte Antigen-Typed Healthy Donors for the Assessment of Drug Immunogenicity.

It is unclear whether priming of naïve T cells to drugs is detectable in healthy human donors expressing different human leukocyte antigen (HLA) alleles. Thus, we examined T cell priming with drugs associated with HLA risk alleles and control compounds in 14 HLA-typed donors. Nitroso sulfamethoxazole and piperacillin activated T cells from all donors, whereas responses to carbamazepine and oxypurinol were only seen in donors expressing HLA-B*15:02 and HLA-B*58:01, respectively. Weak flucloxacillin-specific T cell responses were detected in donors expressing HLA-B*57:01 and HLA-B*58:01. These data show that the priming of T cells with certain drugs is skewed toward donors expressing specific HLA alleles.

Innovative organotypic in vitro models for safety assessment: aligning with regulatory requirements and understanding models of the heart, skin, and liver as paradigms.

The development of improved, innovative models for the detection of toxicity of drugs, chemicals, or chemicals in cosmetics is crucial to efficiently bring new products safely to market in a cost-effective and timely manner. In addition, improvement in models to detect toxicity may reduce the incidence of unexpected post-marketing toxicity and reduce or eliminate the need for animal testing. The safety of novel products of the pharmaceutical, chemical, or cosmetics industry must be assured; therefore, toxicological properties need to be assessed. Accepted methods for gathering the information required by law for approval of substances are often animal methods. To reduce, refine, and replace animal testing, innovative organotypic in vitro models have emerged. Such models appear at different levels of complexity ranging from simpler, self-organized three-dimensional (3D) cell cultures up to more advanced scaffold-based co-cultures consisting of multiple cell types. This review provides an overview of recent developments in the field of toxicity testing with in vitro models for three major organ types: heart, skin, and liver. This review also examines regulatory aspects of such models in Europe and the UK, and summarizes best practices to facilitate the acceptance and appropriate use of advanced in vitro models.

Assessment of Antipiperacillin IgG Binding to Structurally Related Drug Protein Adducts.

The risk of developing hypersensitivity to alternative antibiotics is a concern for penicillin hypersensitive patients and healthcare providers. Herein we use piperacillin hypersensitivity as a model to explore the reactivity of drug-specific IgG against alternative ß-lactam protein adducts. Mass spectrometry was used to show the drugs (amoxicillin, flucloxacillin, benzyl penicillin, aztreonam, and piperacillin) bind to similar lysine residues on the protein carrier bovine serum albumin. However, the hapten-specific IgG antibodies found in piperacillin hypersensitive patient plasma did not bind to other ß-lactam protein conjugates. These data outline the fine specificity of piperacillin-specific IgG antibodies that circulate in patients with hypersensitivity.

Dynamic and accurate assessment of acetaminophen-induced hepatotoxicity by integrated photoacoustic imaging and mechanistic biomarkers in vivo.

The prediction and understanding of acetaminophen (APAP)-induced liver injury (APAP-ILI) and the response to therapeutic interventions is complex. This is due in part to sensitivity and specificity limitations of currently used assessment techniques. Here we sought to determine the utility of integrating translational non-invasive photoacoustic imaging of liver function with mechanistic circulating biomarkers of hepatotoxicity with histological assessment to facilitate the more accurate and precise characterization of APAP-ILI and the efficacy of therapeutic intervention. Perturbation of liver function and cellular viability was assessed in C57BL/6J male mice by Indocyanine green (ICG) clearance (Multispectral Optoacoustic Tomography (MSOT)) and by measurement of mechanistic (miR-122, HMGB1) and established (ALT, bilirubin) circulating biomarkers in response to the acetaminophen and its treatment with acetylcysteine (NAC) in vivo. We utilised a 60% partial hepatectomy model as a situation of defined hepatic functional mass loss to compared acetaminophen-induced changes to. Integration of these mechanistic markers correlated with histological features of APAP hepatotoxicity in a time-dependent manner. They accurately reflected the onset and recovery from hepatotoxicity compared to traditional biomarkers and also reported the efficacy of NAC with high sensitivity. ICG clearance kinetics correlated with histological scores for acute liver damage for APAP (i.e. 3h timepoint; r=0.90, P<0.0001) and elevations in both of the mechanistic biomarkers, miR-122 (e.g. 6h timepoint; r=0.70, P=0.005) and HMGB1 (e.g. 6h timepoint; r=0.56, P=0.04). For the first time we report the utility of this non-invasive longitudinal imaging approach to provide direct visualisation of the liver function coupled with mechanistic biomarkers, in the same animal, allowing the investigation of the toxicological and pharmacological aspects of APAP-ILI and hepatic regeneration.

Test systems in drug discovery for hazard identification and risk assessment of human drug-induced liver injury.

INTRODUCTION: The liver is an important target for drug-induced toxicities. Early detection of hepatotoxic drugs requires use of well-characterized test systems, yet current knowledge, gaps and limitations of tests employed remains an important issue for drug development. Areas Covered: The current state of the science, understanding and application of test systems in use for the detection of drug-induced cytotoxicity, mitochondrial toxicity, cholestasis and inflammation is summarized. The test systems highlighted herein cover mostly in vitro and some in vivo models and endpoint measurements used in the assessment of small molecule toxic liabilities. Opportunities for research efforts in areas necessitating the development of specific tests and improved mechanistic understanding are highlighted. Expert Opinion: Use of in vitro test systems for safety optimization will remain a core activity in drug discovery. Substantial inroads have been made with a number of assays established for human Drug-induced Liver Injury. There nevertheless remain significant gaps with a need for improved in vitro tools and novel tests to address specific mechanisms of human Drug-Induced Liver Injury. Progress in these areas will necessitate not only models fit for application, but also mechanistic understanding of how chemical insult on the liver occurs in order to identify translational and quantifiable readouts for decision-making.

A multicenter assessment of single-cell models aligned to standard measures of cell health for prediction of acute hepatotoxicity.

Assessing the potential of a new drug to cause drug-induced liver injury (DILI) is a challenge for the pharmaceutical industry. We therefore determined whether cell models currently used in safety assessment (HepG2, HepaRG, Upcyte and primary human hepatocytes in conjunction with basic but commonly used endpoints) are actually able to distinguish between novel chemical entities (NCEs) with respect to their potential to cause DILI. A panel of thirteen compounds (nine DILI implicated and four non-DILI implicated in man) were selected for our study, which was conducted, for the first time, across multiple laboratories. None of the cell models could distinguish faithfully between DILI and non-DILI compounds. Only when nominal in vitro concentrations were adjusted for in vivo exposure levels were primary human hepatocytes (PHH) found to be the most accurate cell model, closely followed by HepG2. From a practical perspective, this study revealed significant inter-laboratory variation in the response of PHH, HepG2 and Upcyte cells, but not HepaRG cells. This variation was also observed to be compound dependent. Interestingly, differences between donors (hepatocytes), clones (HepG2) and the effect of cryopreservation (HepaRG and hepatocytes) were less important than differences between the cell models per se. In summary, these results demonstrate that basic cell health endpoints will not predict hepatotoxic risk in simple hepatic cells in the absence of pharmacokinetic data and that a multicenter assessment of more sophisticated signals of molecular initiating events is required to determine whether these cells can be incorporated in early safety assessment.

A longitudinal assessment of miR-122 and GLDH as biomarkers of drug-induced liver injury in the rat.

CONTEXT: There is an ongoing search for specific and translational biomarkers of drug-induced liver injury (DILI). MicroRNA-122 (miR-122) has previously shown potential as a sensitive, specific, and translational biomarker of DILI in both rodent, and human studies. OBJECTIVE: To build on previous work within the field, we examined biomarker kinetics in a rat model of acetaminophen (APAP)-induced liver injury to confirm the sensitivity, and specificity of miR-122 and glutamate dehydrogenase (GLDH). MATERIALS AND METHODS: qRT-PCR and a standard enzymatic assay were used for biomarker analysis. RESULTS: Both miR-122 and GLDH were demonstrated to be more readily-detectable biomarkers of APAP-DILI than alanine aminotransferase (ALT). Peak levels for all biomarkers were detected at 2 days after APAP. At day 3, miR-122 had returned to baseline; however, other biomarkers remained elevated between 3 and 4 days. We were also able to demonstrate that, although miR-122 is present in greater quantities in exosome-free form, both exosome-bound and non-vesicle bound miR-122 are released in a similar profile throughout the course of DILI. DISCUSSION AND CONCLUSIONS: Together, this study demonstrates that both GLDH and miR-122 could be used during preclinical drug-development as complementary biomarkers to ALT to increase the chance of early detection of hepatotoxicity.

New Approaches to Investigate Drug-Induced Hypersensitivity.

The workshop on "New Approaches to Investigate Drug-Induced Hypersensitivity" was held on June 5, 2014 at the Foresight Center, University of Liverpool. The aims of the workshop were to (1) discuss our current understanding of the genetic, clinical, and chemical basis of small molecule drug hypersensitivity, (2) highlight the current status of assays that might be developed to predict potential drug immunogenicity, and (3) identify the limitations, knowledge gaps, and challenges that limit the use of these assays and utilize the knowledge gained from the workshop to develop a pathway to establish new and improved assays that better predict drug-induced hypersensitivity reactions during the early stages of drug development. This perspective reviews the clinical and immunological bases of drug hypersensitivity and summarizes various experts' views on the different topics covered during the meeting.

Challenges and approaches for the development of safer immunomodulatory biologics.

Immunomodulatory biologics, which render their therapeutic effects by modulating or harnessing immune responses, have proven their therapeutic utility in several complex conditions including cancer and autoimmune diseases. However, unwanted adverse reactions--including serious infections, malignancy, cytokine release syndrome, anaphylaxis and hypersensitivity as well as immunogenicity--pose a challenge to the development of new (and safer) immunomodulatory biologics. In this article, we assess the safety issues associated with immunomodulatory biologics and discuss the current approaches for predicting and mitigating adverse reactions associated with their use. We also outline how these approaches can inform the development of safer immunomodulatory biologics.

Managing the challenge of chemically reactive metabolites in drug development.

The normal metabolism of drugs can generate metabolites that have intrinsic chemical reactivity towards cellular molecules, and therefore have the potential to alter biological function and initiate serious adverse drug reactions. Here, we present an assessment of the current approaches used for the evaluation of chemically reactive metabolites. We also describe how these approaches are being used within the pharmaceutical industry to assess and minimize the potential of drug candidates to cause toxicity. At early stages of drug discovery, iteration between medicinal chemistry and drug metabolism can eliminate perceived reactive metabolite-mediated chemical liabilities without compromising pharmacological activity or the need for extensive safety evaluation beyond standard practices. In the future, reactive metabolite evaluation may also be useful during clinical development for improving clinical risk assessment and risk management. Currently, there remains a huge gap in our understanding of the basic mechanisms that underlie chemical stress-mediated adverse reactions in humans. This review summarizes our views on this complex topic, and includes insights into practices considered by the pharmaceutical industry.

Generating hepatic cell lineages from pluripotent stem cells for drug toxicity screening.

Hepatotoxicity is an enormous and increasing problem for the pharmaceutical industry. Early detection of problems during the drug discovery pathway is advantageous to minimize costs and improve patient safety. However, current cellular models are sub-optimal. This review addresses the potential use of pluripotent stem cells in the generation of hepatic cell lineages. It begins by highlighting the scale of the problem faced by the pharmaceutical industry, the precise nature of drug-induced liver injury and where in the drug discovery pathway the need for additional cell models arises. Current research is discussed, mainly for generating hepatocyte-like cells rather than other liver cell-types. In addition, an effort is made to identify where some of the major barriers remain in translating what is currently hypothesis-driven laboratory research into meaningful platform technologies for the pharmaceutical industry.

Cost-effectiveness analysis of HLA B*5701 genotyping in preventing abacavir hypersensitivity.

OBJECTIVE: Abacavir, a human immunodeficiency virus-1 (HIV-1) nucleoside-analogue reverse transcriptase inhibitor, causes severe hypersensitivity in 4-8% of patients. HLA B*5701 is a known genetic risk factor for abacavir hypersensitivity in Caucasians. Our aim was to confirm the presence of this genetic factor in our patients, and to determine whether genotyping for HLA B*5701 would be a cost-effective use of healthcare resources. METHODS: Patients with and without abacavir hypersensitivity were identified from a UK HIV clinic. Patients were genotyped for HLA B*5701, and pooled data used for calculation of test characteristics. The cost-effectiveness analysis incorporated the cost of testing, cost of treating abacavir hypersensitivity, and the cost and selection of alternative antiretroviral regimens. A probabilistic decision analytic model (comparing testing versus no testing) was formulated and Monte Carlo simulations performed. RESULTS: Of the abacavir hypersensitive patients, six (46%) were HLA B*5701 positive, compared to five (10%) of the non-hypersensitive patients (odds ratio 7.9 [95% confidence intervals 1.5-41.4], P = 0.006). Pooling of our data on HLA B*5701 with published data resulted in a pooled odds ratio of 29 (95% CI 6.4-132.3; P < 0.0001). The cost-effectiveness model demonstrated that depending on the choice of comparator, routine testing for HLA B*5701 ranged from being a dominant strategy (less expensive and more beneficial than not testing) to an incremental cost-effectiveness ratio (versus no testing) of Euro 22,811 per hypersensitivity reaction avoided. CONCLUSIONS: Abacavir hypersensitivity is associated with HLA B*5701, and pre-prescription pharmacogenetic testing for this appears to be a cost-effective use of healthcare resources.

Adverse drug reactions as cause of admission to hospital: prospective analysis of 18 820 patients.

OBJECTIVE: To ascertain the current burden of adverse drug reactions (ADRs) through a prospective analysis of all admissions to hospital. DESIGN: Prospective observational study. SETTING: Two large general hospitals in Merseyside, England. PARTICIPANTS: 18 820 patients aged > 16 years admitted over six months and assessed for cause of admission. MAIN OUTCOME MEASURES: Prevalence of admissions due to an ADR, length of stay, avoidability, and outcome. RESULTS: There were 1225 admissions related to an ADR, giving a prevalence of 6.5%, with the ADR directly leading to the admission in 80% of cases. The median bed stay was eight days, accounting for 4% of the hospital bed capacity. The projected annual cost of such admissions to the NHS is 466m pounds sterling (706m Euros, 847m dollars). The overall fatality was 0.15%. Most reactions were either definitely or possibly avoidable. Drugs most commonly implicated in causing these admissions included low dose aspirin, diuretics, warfarin, and non-steroidal anti-inflammatory drugs other than aspirin, the most common reaction being gastrointestinal bleeding. CONCLUSION: The burden of ADRs on the NHS is high, accounting for considerable morbidity, mortality, and extra costs. Although many of the implicated drugs have proved benefit, measures need to be put into place to reduce the burden of ADRs and thereby further improve the benefit:harm ratio of the drugs.