Hepatotoxic Liability Research Articles

Comprehensive Evaluation of Organotypic and Microphysiological Liver Models for Prediction of Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) is a major concern for the pharmaceutical industry and constitutes one of the most important reasons for the termination of promising drug development projects. Reliable prediction of DILI liability in preclinical stages is difficult, as current experimental model systems do not accurately reflect the molecular phenotype and functionality of the human liver. As a result, multiple drugs that passed preclinical safety evaluations failed due to liver toxicity in clinical trials or postmarketing stages in recent years. To improve the selection of molecules that are taken forward into the clinics, the development of more predictive in vitro systems that enable high-throughput screening of hepatotoxic liabilities and allow for investigative studies into DILI mechanisms has gained growing interest. Specifically, it became increasingly clear that the choice of cell types and culture method both constitute important parameters that affect the predictive power of test systems. In this review, we present current 3D culture paradigms for hepatotoxicity tests and critically evaluate their utility and performance for DILI prediction. In addition, we highlight possibilities of these emerging platforms for mechanistic evaluations of selected drug candidates and present current research directions towards the further improvement of preclinical liver safety tests. We conclude that organotypic and microphysiological liver systems have provided an important step towards more reliable DILI prediction. Furthermore, we expect that the increasing availability of comprehensive benchmarking studies will facilitate model dissemination that might eventually result in their regulatory acceptance.

Prediction of Drug-Induced Hepatotoxicity Using Long-Term Stable Primary Hepatic 3D Spheroid Cultures in Chemically Defined Conditions.

High failure rates of drug candidates in the clinics, restricted-use warnings as well as withdrawals of drugs in postmarketing stages are of substantial concern for the pharmaceutical industry and drug-induced liver injury (DILI) constitutes one of the most frequent reasons for such safety failures. Importantly, as DILI cannot be accurately predicted using animal models, animal safety tests are commonly complemented with assessments in human in vitro systems. 3D spheroid cultures of primary human hepatocytes in chemically defined conditions, hereafter termed CD-spheroids, have recently emerged as a microphysiological model system in which hepatocytes retain their molecular phenotypes and hepatic functions for multiple weeks in culture. However, their predictive power for the detection of hepatotoxic liabilities has not been systematically assessed. Therefore, we here evaluated the hepatotoxicity of 123 drugs with or without direct implication in clinical DILI events. Importantly, using ATP quantifications as the single endpoint, the model accurately distinguished between hepatotoxic and nontoxic structural analogues and exceeded both sensitivity and specificity of all previously published in vitro assays at substantially lower exposure levels, successfully detecting 69% of all hepatotoxic compounds without producing any false positive results (100% specificity). Furthermore, the platform supports the culture of spheroids of primary hepatocytes from preclinical animal models, thereby allowing the identification of animal-specific toxicity events. We anticipate that CD-spheroids represent a powerful and versatile tool in drug discovery and preclinical drug development that can reliably flag hepatotoxic drug candidates and provide guidance for the selection of the most suitable animal models.

Isothiocyanates: cholinesterase inhibiting, antioxidant, and anti-inflammatory activity

Finding a new type of cholinesterase inhibitor that would overcome the brain availability and pharmacokinetic parameters or hepatotoxic liability has been a focus of investigations dealing with the treatment of Alzheimer’s disease. Isothiocyanates have not been previously investigated as potential cholinesterase inhibitors. These compounds can be naturally produced from their glucosinolate precursors, secondary metabolites widely distributed in our daily Brassica vegetables. Among 11 tested compounds, phenyl isothiocyanate and its derivatives showed the most promising inhibitory activity. 2-Methoxyphenyl ITC showed best inhibition on acetylcholinesterase with IC50 of 0.57 mM, while 3-methoxyphenyl ITC showed the best inhibition on butyrylcholinesterase having 49.2% at 1.14 mM. Assessment of the antioxidant efficacy using different methods led to a similar conclusion. The anti-inflammatory activity was also tested using human COX-2 enzyme, ranking phenyl isothiocyanate, and 3-methoxyphenyl isothiocyanate as most active, with ∼99% inhibition at 50 μM.

Long-enduring primary hepatocyte-based co-cultures improve prediction of hepatotoxicity

The failure of drug candidates during clinical trials and post-marketing withdrawal due to Drug Induced Liver Injury (DILI), results in significant late-stage attrition in the pharmaceutical industry. Animal studies have proven insufficient to definitively predict DILI in the clinic, therefore a variety of in vitro models are being tested in an effort to improve prediction of human hepatotoxicity. The model system described here consists of cryopreserved primary rat, dog or human hepatocytes co-cultured together with a fibroblast cell line, which aids in the hepatocytes' maintenance of more in vivo-like characteristics compared to traditional hepatic mono-cultures, including long term viability and retention of activity of cytochrome P450 isozymes. Cell viability was assessed by measurement of ATP following treatment with 29 compounds having known hepatotoxic liabilities. Hμrelrat™, Hμreldog™, and Hμrelhuman™ hepatic co-cultures were treated for 24h, or under repeat-dosing for 7 or 13days, and compared to rat and human hepatic mono-cultures following single-dose exposure for 24h. The results allowed for a comparison of cytotoxicity, species-specific responses and the effect of repeat compound exposure on the prediction of hepatotoxic potential in each model. Results show that the co-culture model had greater sensitivity compared to that of the hepatic mono-cultures. In addition, “time-based ratios” were determined by dividing the compounds' 24-hour TC50/Cmax values by TC50/Cmax values measured after dosing for either 7 or 13days. The results suggest that this approach may serve as a useful adjunct to traditional measurements of hepatotoxicity, improving the predictive value of early screening studies.

Setting Clinical Exposure Levels of Concern for Drug-Induced Liver Injury (DILI) Using Mechanistic in vitro Assays.

Severe drug-induced liver injury (DILI) remains a major safety issue due to its frequency of occurrence, idiosyncratic nature, poor prognosis, and diverse underlying mechanisms. Numerous experimental approaches have been published to improve human DILI prediction with modest success. A retrospective analysis of 125 drugs (70 = most-DILI, 55 = no-DILI) from the Food and Drug Administration Liver Toxicity Knowledge Base was used to investigate DILI prediction based on consideration of human exposure alone or in combination with mechanistic assays of hepatotoxic liabilities (cytotoxicity, bile salt export pump inhibition, or mitochondrial inhibition/uncoupling). Using this dataset, human plasma Cmax,total ≥ 1.1 µM alone distinguished most-DILI from no-DILI compounds with high sensitivity/specificity (80/73%). Accounting for human exposure improved the sensitivity/specificity for each assay and helped to derive predictive safety margins. Compounds with plasma Cmax,total ≥ 1.1 µM and triple liabilities had significantly higher odds ratio for DILI than those with single/dual liabilities. Using this approach, a subset of recent pharmaceuticals with evidence of liver injury during clinical development was recognized as potential hepatotoxicants. In summary, plasma Cmax,total ≥ 1.1 µM along with multiple mechanistic liabilities is a major driver for predictions of human DILI potential. In applying this approach during drug development the challenge will be generating accurate estimates of plasma Cmax,total at efficacious doses in advance of generating true exposure data from clinical studies. In the meantime, drug candidates with multiple hepatotoxic liabilities should be deprioritized, since they have the highest likelihood of causing DILI in case their efficacious plasma Cmax,total in humans is higher than anticipated.

The new approach in development of anti-Alzheimer's disease drugs via the cholinergic hypothesis

A wide range of evidences show that cholinesterase (ChE) inhibitors can interfere with the progression of Alzheimer's disease (AD). The earliest known ChE inhibitors, namely, physostigmine and tacrine, showed modest improvement in the cognitive function of AD patients. However, clinical studies show that physostigmine has poor oral activity, brain penetration and pharmacokinetic parameters while tacrine has hepatotoxic liability. Studies were then focused on finding a new type of acetylcholinesterase (AChE) inhibitor that would overcome the disadvantages of these two compounds. During the study, by chance we found a seed compound. We then conducted a structure–activity relationship (SAR) study of this compound. After four years of exploratory research, we found donepezil hydrochloride (donepezil). Recently, acetylcholinesterase inhibitors (AChEIs) have been studied for other mechanisms of action, such as neuroprotective action and lowering of beta-amyloid (β-amyloid). Donepezil also reduced beta-amyloid plaque in in vitro. The amyloid hypothesis is believed to be the most promising approach in the development of anti-AD drugs. We speculate the mechanism of lowering beta-amyloid by donepezil implicate alpha-secretase (α-secretase) enhancer.

Research and Development of Donepezil Hydrochloride, a New Type of Acetylcholinesterase Inhibitor

A wide range of evidence shows that Cholinesterase (ChE) inhibitors can interfere with the progression of Alzheimer’s disease (AD). The earliest known ChE inhibitors, namely, physostigmine and tacrine, showed modest improvement in the cognitive function of AD patients. However, clinical studies show that physostigmine has poor oral activity, brain penetration and pharmacokinetic parameters, while tacrine has hepatotoxic liability. Studies were then focused on finding a new type of acetylcholinesterase (AChE) inhibitor that would overcome the disadvantages of these two compounds. During the study, by chance we found a seed compound. We then conducted a structure-activity relationship study of this compound. After four years of exploratory research, we found donepezil hydrochloride (donepezil). Donepezil showed several positive characteristics including the following: 1) It has a novel structure compared to other conventional ChE inhibitors; 2) It shows strong anti-AChE activity and has long lasting efficacy; 3) The inhibitory characteristic of donepezil shows that it is highly selective for AChE as compared to butyryl-cholinesterase (BuChE) and showed reversibility; 4) The results of clinical studies on donepezil show a very high significant difference on ADAS cog and CIBIC plus scores of AD patients. Donepezil is currently marketed in 56 countries all over the world.

Donepezil hydrochloride: a treatment drug for Alzheimer's disease.

The role of the cholinergic system with respect to cognitive deficits characteristic of Alzheimer's disease (AD) has led to a number of studies focusing on the development of acetylcholinesterase (AChE) inhibitors as a drug for treating this disease. The earliest known AChE inhibitors, namely, physostigmine and tacrine, performed poorly in clinical trials (e.g., poor oral activity, brain penetration, and hepatotoxic liability). Studies were then focused on finding a new type of acetylcholinesterase inhibitor that would overcome the disadvantages of these two compounds. Donepezil hydrochloride inaugurates a new class of AChE inhibitors with longer and more selective action and with manageable adverse effects.

Donepezil hydrochloride (E2020) and other acetylcholinesterase inhibitors.

A wide range of evidence shows that acetylcholinesterase (AChE) inhibitors can interfere with the progression of Alzheimer's disease (AD). The successful development of these compounds was based on a well-accepted theory that the decline in cognitive and mental functions associated with AD is related to the loss of cortical cholinergic neurotransmission. The earliest known AChE inhibitors, namely, physostigmine and tacrine, showed modest improvement in the cognitive function of Alzheimer's patients. However, clinical studies show that physostigmine has poor oral activity, brain penetration and pharmacokinetic parameters while tacrine has hepatotoxic liability. Studies were then focused on finding a new type of acetylcholinesterase inhibitor that would overcome the disadvantages of these two compounds. Donepezil hydrochloride inaugurates a new class of AChE inhibitors with longer and more selective action with manageable adverse effects. Currently, there are about 19 new Alzheimer's drugs in various phases of clinical development.

Structure-activity relationships of acetylcholinesterase inhibitors: Donepezil hydrochloride for the treatment of Alzheimer’s Disease

Abstract