Discovery and Characterization of Potent, Efficacious, Orally Available Antimalarial Plasmepsin X Inhibitors and Preclinical Safety Assessment of UCB7362.

Plasmepsin X (PMX) is an essential aspartyl protease controlling malaria parasite egress and invasion of erythrocytes, development of functional liver merozoites (prophylactic activity), and blocking transmission to mosquitoes, making it a potential multistage drug target. We report the optimization of an aspartyl protease binding scaffold and the discovery of potent, orally active PMX inhibitors with in vivo antimalarial efficacy. Incorporation of safety evaluation early in the characterization of PMX inhibitors precluded compounds with a long human half-life (t1/2) to be developed. Optimization focused on improving the off-target safety profile led to the identification of UCB7362 that had an improved in vitro and in vivo safety profile but a shorter predicted human t1/2. UCB7362 is estimated to achieve 9 log 10 unit reduction in asexual blood-stage parasites with once-daily dosing of 50 mg for 7 days. This work demonstrates the potential to deliver PMX inhibitors with in vivo efficacy to treat malaria.

A cross-industry survey on photosafety evaluation of pharmaceuticals after implementation of ICH S10.

A cross-industry survey was conducted by EFPIA/IQ DruSafe in 2018 to provide information on photosafety evaluation of pharmaceuticals after implementation of ICH S10. This survey focused on the strategy utilized for photosafety risk assessment, the design of nonclinical (in vitro and in vivo) and clinical evaluations, the use of exposure margins in risk assessment, and regulatory interactions. The survey results indicated that a staged approach for phototoxicity assessment has been widely accepted by regulatory authorities globally. The OECD-based 3T3 NRU Phototoxicity Test is the most frequently used in vitro approach. Modifications to this assay suggested by ICH S10 are commonly applied. For in-vitro-positives, substantial margins from in vitro IC50 values under irradiation to Cmax (clinical) have enabled further development without the need for additional photosafety data. In vivo phototoxicity studies typically involve dosing rodents and exposing skin and eyes to simulated sunlight, and subsequently evaluating at least the skin for erythema and edema. However, no formal guidelines exist and protocols are less standardized across companies. A margin-of-safety approach (based on Cmax at NOAEL) has been successfully applied to support clinical development. Experience with dedicated clinical phototoxicity studies was limited, perhaps due to effective de-risking approaches employed based on ICH S10.

In Silico Approaches In Carcinogenicity Hazard Assessment: Current Status and Future Needs.

Historically, identifying carcinogens has relied primarily on tumor studies in rodents, which require enormous resources in both money and time. In silico models have been developed for predicting rodent carcinogens but have not yet found general regulatory acceptance, in part due to the lack of a generally accepted protocol for performing such an assessment as well as limitations in predictive performance and scope. There remains a need for additional, improved in silico carcinogenicity models, especially ones that are more human-relevant, for use in research and regulatory decision-making. As part of an international effort to develop in silico toxicological protocols, a consortium of toxicologists, computational scientists, and regulatory scientists across several industries and governmental agencies evaluated the extent to which in silico models exist for each of the recently defined 10 key characteristics (KCs) of carcinogens. This position paper summarizes the current status of in silico tools for the assessment of each KC and identifies the data gaps that need to be addressed before a comprehensive in silico carcinogenicity protocol can be developed for regulatory use.

Managing the challenge of drug-induced liver injury: a roadmap for the development and deployment of preclinical predictive models.

Drug-induced liver injury (DILI) is a patient-specific, temporal, multifactorial pathophysiological process that cannot yet be recapitulated in a single in vitro model. Current preclinical testing regimes for the detection of human DILI thus remain inadequate. A systematic and concerted research effort is required to address the deficiencies in current models and to present a defined approach towards the development of new or adapted model systems for DILI prediction. This Perspective defines the current status of available models and the mechanistic understanding of DILI, and proposes our vision of a roadmap for the development of predictive preclinical models of human DILI.

Genetic toxicology in silico protocol.

In silico toxicology (IST) approaches to rapidly assess chemical hazard, and usage of such methods is increasing in all applications but especially for regulatory submissions, such as for assessing chemicals under REACH as well as the ICH M7 guideline for drug impurities. There are a number of obstacles to performing an IST assessment, including uncertainty in how such an assessment and associated expert review should be performed or what is fit for purpose, as well as a lack of confidence that the results will be accepted by colleagues, collaborators and regulatory authorities. To address this, a project to develop a series of IST protocols for different hazard endpoints has been initiated and this paper describes the genetic toxicity in silico (GIST) protocol. The protocol outlines a hazard assessment framework including key effects/mechanisms and their relationships to endpoints such as gene mutation and clastogenicity. IST models and data are reviewed that support the assessment of these effects/mechanisms along with defined approaches for combining the information and evaluating the confidence in the assessment. This protocol has been developed through a consortium of toxicologists, computational scientists, and regulatory scientists across several industries to support the implementation and acceptance of in silico approaches.

Principles and procedures for handling out-of-domain and indeterminate results as part of ICH M7 recommended (Q)SAR analyses.

The International Council for Harmonization (ICH) M7 guideline describes a hazard assessment process for impurities that have the potential to be present in a drug substance or drug product. In the absence of adequate experimental bacterial mutagenicity data, (Q)SAR analysis may be used as a test to predict impurities' DNA reactive (mutagenic) potential. However, in certain situations, (Q)SAR software is unable to generate a positive or negative prediction either because of conflicting information or because the impurity is outside the applicability domain of the model. Such results present challenges in generating an overall mutagenicity prediction and highlight the importance of performing a thorough expert review. The following paper reviews pharmaceutical and regulatory experiences handling such situations. The paper also presents an analysis of proprietary data to help understand the likelihood of misclassifying a mutagenic impurity as non-mutagenic based on different combinations of (Q)SAR results. This information may be taken into consideration when supporting the (Q)SAR results with an expert review, especially when out-of-domain results are generated during a (Q)SAR evaluation.

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.

Evidence-based selection of training compounds for use in the mechanism-based integrated prediction of drug-induced liver injury in man.

The current test systems employed by pharmaceutical industry are poorly predictive for drug-induced liver injury (DILI). The 'MIP-DILI' project addresses this situation by the development of innovative preclinical test systems which are both mechanism-based and of physiological, pharmacological and pathological relevance to DILI in humans. An iterative, tiered approach with respect to test compounds, test systems, bioanalysis and systems analysis is adopted to evaluate existing models and develop new models that can provide validated test systems with respect to the prediction of specific forms of DILI and further elucidation of mechanisms. An essential component of this effort is the choice of compound training set that will be used to inform refinement and/or development of new model systems that allow prediction based on knowledge of mechanisms, in a tiered fashion. In this review, we focus on the selection of MIP-DILI training compounds for mechanism-based evaluation of non-clinical prediction of DILI. The selected compounds address both hepatocellular and cholestatic DILI patterns in man, covering a broad range of pharmacologies and chemistries, and taking into account available data on potential DILI mechanisms (e.g. mitochondrial injury, reactive metabolites, biliary transport inhibition, and immune responses). Known mechanisms by which these compounds are believed to cause liver injury have been described, where many if not all drugs in this review appear to exhibit multiple toxicological mechanisms. Thus, the training compounds selection offered a valuable tool to profile DILI mechanisms and to interrogate existing and novel in vitro systems for the prediction of human DILI.

Cytotoxicity evaluation using cryopreserved primary human hepatocytes in various culture formats.

Sixteen training compounds selected in the IMI MIP-DILI consortium, 12 drug-induced liver injury (DILI) positive compounds and 4 non-DILI compounds, were assessed in cryopreserved primary human hepatocytes. When a ten-fold safety margin threshold was applied, the non-DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes (n=13 donors) in suspension and 14-days following repeat dose exposure (3 treatments) to an established 3D-microtissue co-culture (3D-MT co-culture, n=1 donor) consisting of human hepatocytes co-cultured with non-parenchymal cells (NPC). In contrast, only 5/12 DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes in suspension. Exposure of the 2D-sandwich culture human hepatocyte monocultures (2D-sw) for 3days resulted in the correct identification of 11/12 DILI-positive compounds, whereas exposure of the human 3D-MT co-cultures for 14days resulted in identification of 9/12 DILI-compounds; in addition to ximelagatran (also not identified by 2D-sw monocultures, Sison-Young et al., 2016), the 3D-MT co-cultures failed to detect amiodarone and bosentan. The sensitivity of the 2D human hepatocytes co-cultured with NPC to ximelagatran was increased in the presence of lipopolysaccharide (LPS), but only at high concentrations, therefore preventing its classification as a DILI positive compound. In conclusion (1) despite suspension human hepatocytes having the greatest metabolic capacity in the short term, they are the least predictive of clinical DILI across the MIP-DILI test compounds, (2) longer exposure periods than 72h of human hepatocytes do not allow to increase DILI-prediction rate, (3) co-cultures of human hepatocytes with NPC, in the presence of LPS during the 72h exposure period allow the assessment of innate immune system involvement of a given drug.

Alternative to LRRK2-IN-1 for Pharmacological Studies of Parkinson's Disease.

BACKGROUND/AIMS: LRRK2 (leucine-rich repeat protein kinase 2) is one of the most commonly accepted genes associated with Parkinson's disease (PD). The overexpression of disease-associated mutations in LRRK2 is toxic to the cells, while reduction or elimination of LRRK2 expression promotes cell health and growth. Thus, the identification of an LRRK2 inhibitor with good physiochemical and pharmacokinetic properties is of great interest for the treatment of PD. METHODS: In this study, we have investigated LRRK2 compounds, LRRK2-IN-1 and Compound 1, in vitro and in vivo to determine how suitable they are as a selective LRRK2 tool compound. RESULTS: We report that Compound 1, patented by GSK, is a potent and selective LRRK2 inhibitor with good blood-brain barrier permeability as reflected by its high brain to plasma ratio in rats. In addition, Compound 1 can significantly promote neurite outgrowth in a primary cortical culture, indicating an optimistic cellular function of this compound in a biological system. In contrast, LRRK2-IN-1 is a less selective LRRK2 inhibitor and has low brain penetration. Furthermore, LRRK2-IN-1 is cyto- and genotoxic, while Compound 1 does not exhibit any toxicity. CONCLUSIONS: These results suggest that Compound 1 may be a superior tool compound than LRRK2-IN-1 to advance future pharmacological research on LRRK2.

Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans.

Drug induced liver injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine model along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n=40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n=11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n=14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies.

Evaluation of impedance-based label-free technology as a tool for pharmacology and toxicology investigations.

The use of label-free technologies based on electrical impedance is becoming more and more popular in drug discovery. Indeed, such a methodology allows the continuous monitoring of diverse cellular processes, including proliferation, migration, cytotoxicity and receptor-mediated signaling. The objective of the present study was to further assess the usefulness of the real-time cell analyzer (RTCA) and, in particular, the xCELLigence platform, in the context of early drug development for pharmacology and toxicology investigations. In the present manuscript, four cellular models were exposed to 50 compounds to compare the cell index generated by RTCA and cell viability measured with a traditional viability assay. The data revealed an acceptable correlation (ca. 80%) for both cell lines (i.e., HepG2 and HepaRG), but a lack of correlation (ca. 55%) for the primary human and rat hepatocytes. In addition, specific RTCA profiles (signatures) were generated when HepG2 and HepaRG cells were exposed to calcium modulators, antimitotics, DNA damaging and nuclear receptor agents, with a percentage of prediction close to 80% for both cellular models. In a subsequent experiment, HepG2 cells were exposed to 81 proprietary UCB compounds known to be genotoxic or not. Based on the DNA damaging signatures, the RTCA technology allowed the detection of ca. 50% of the genotoxic compounds (n = 29) and nearly 100% of the non-genotoxic compounds (n = 52). Overall, despite some limitations, the xCELLigence platform is a powerful and reliable tool that can be used in drug discovery for toxicity and pharmacology studies.

The use of real-time cell analyzer technology in drug discovery: defining optimal cell culture conditions and assay reproducibility with different adherent cellular models.

The use of impedance-based label-free technology applied to drug discovery is nowadays receiving more and more attention. Indeed, such a simple and noninvasive assay that interferes minimally with cell morphology and function allows one to perform kinetic measurements and to obtain information on proliferation, migration, cytotoxicity, and receptor-mediated signaling. The objective of the study was to further assess the usefulness of a real-time cell analyzer (RTCA) platform based on impedance in the context of quality control and data reproducibility. The data indicate that this technology is useful to determine the best coating and cellular density conditions for different adherent cellular models including hepatocytes, cardiomyocytes, fibroblasts, and hybrid neuroblastoma/neuronal cells. Based on 31 independent experiments, the reproducibility of cell index data generated from HepG2 cells exposed to DMSO and to Triton X-100 was satisfactory, with a coefficient of variation close to 10%. Cell index data were also well reproduced when cardiomyocytes and fibroblasts were exposed to 21 compounds three times (correlation >0.91, p < 0.0001). The data also show that a cell index decrease is not always associated with cytotoxicity effects and that there are some confounding factors that can affect the analysis. Finally, another drawback is that the correlation analysis between cellular impedance measurements and classical toxicity endpoints has been performed on a limited number of compounds. Overall, despite some limitations, the RTCA technology appears to be a powerful and reliable tool in drug discovery because of the reasonable throughput, rapid and efficient performance, technical optimization, and cell quality control.

Multiplexing cell viability assays.

Today, obtaining mechanistic insights into biological, toxicological, and pathological processes is of upmost importance. Researchers aim to obtain as many as possible data from one cell sample to understand the biological processes under study. Multiplexing, which is the ability to gather more than one set of data from the same sample, fulfills completely this objective. Obviously, multiplexing has several advantages compared to single plex experiments and probably the most important one is that data on various parameters at exactly the same time point on the same cells or group of cells can be obtained and consequently this may contribute to saving time and effort and a reduction of the costs.In this chapter, different endpoints were measured starting from two-seeded multiwell plates, namely, cell viability, caspase-3/7 activity, lactate dehydrogenase (LDH), adenosine triphosphate (ATP), aspartate aminotransferase (AST), and glutamate dehydrogenase (GLDH) measurements. These -different endpoints were analyzed together to determine the cytotoxic properties of pharmaceutical compounds and/or reference compounds. A 96-well plate was designed to allow appropriate measurement of five doses of a compound in triplicate to determine the effect of the compound on the six different endpoints. The first four endpoints (cell viability, caspase-3/7 activity, LDH, and ATP) are discussed in detail in this chapter. AST and GLDH measurements are not discussed in detail as these are fully automatic measurements and thus behind the scope of this chapter.As an illustrating example, the reference compound tamoxifen was used to evaluate its cytotoxic properties using the hepatocellular carcinoma cell line HepG2 cells.

Determination of phospholipidosis potential based on gene expression analysis in HepG2 cells.

Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and the concurrent development of concentric lamellar bodies. Recently, H. Sawada et al. (2005, Toxicol. Sci. 83, 282-292) identified 17 genes as potential biomarkers of PLD in HepG2 cells. The present study was undertaken to determine if this set of genes measured by quantitative PCR could be validated in the same cell line. The objective was also to investigate the dose-response relationship to further validate the assay and to select the concentrations to use for screening activities. In a first experiment (one concentration tested), out of the 17 genes, the best gene biomarkers of PLD (i.e., 11 genes) were selected for practical screening reasons. Based on these genes, 91.6% (i.e., 11 of 12) of the compounds known to induce PLD were identified as positive and all the negative compounds (i.e., five of five) were also confirmed. When the data obtained in the first experiment were compared to the data by Sawada et al., (2005) the coefficient of correlation calculated was slightly higher than 75%. In the second experiment (26 compounds [all 17 compounds from the first experiment plus 9 other compounds] tested at a minimum of three concentrations), 93.3% (14/15) of the compounds known to induce PLD were identified as such and all the negative controls (six compounds) were also confirmed. Three compounds likely to induce PLD were identified as positive in our assay. Finally, two compounds for which no data are available were also tested. When both experiments 1 and 2 were compared, the coefficient of correlation for 16 compounds tested at the same concentrations reached 87.7%. In conclusion, the present study further confirms the utility of gene expression in HepG2 cells to identify a potential to induce PLD. Finally, based on the data presented, researchers are encouraged to use a range of minimum three concentrations (e.g., 12.5, 25, and 50 microM) to screen for PLD in the human HepG2 cell line.

Molecular cloning and differential expression of the cat immediate early gene c-fos.

Recently, the effect of binocular central retinal lesions on the expression of immediate early genes in the visual system of adult cats was demonstrated using in situ hybridization and immunocytochemistry. The present study was undertaken to quantify cat c-fos mRNA expression differences in the cat primary visual cortex after sensory deafferentation. Prior to quantification, DNA fragments obtained using reverse transcription-polymerase chain reaction (RT-PCR) in combination with rapid amplification of complementary DNA ends (RACE) were cloned and sequenced. This provided us with the necessary sequence(1) information to prepare cat-specific c-fos primers for the development of a new quantitative RT-PCR assay. We optimized a reverse transcription-competitive polymerase chain reaction (RT-cPCR) method with a heterologous DNA fragment (competitor) as external standard to quantify relative amounts of cat c-fos mRNA expression levels. Internal standardization was accomplished by quantifying, in a parallel RT-cPCR, a well-characterized housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This cat-specific RT-cPCR assay allowed us to measure c-fos mRNA expression levels in central and peripheral regions of primary visual cortex in normal and retinal lesion cats.