High-Throughput Assessment of Metabolism-Induced Toxicity of Compounds on a 384-Pillar Plate.

A variety of oxidative and conjugative enzymes are involved in the metabolism of compounds including drugs, which can be converted into toxic metabolites by Phase I drug-metabolizing enzymes (DMEs), such as the cytochromes P450 (CYP450s), and/or detoxified by Phase II DMEs, such as UDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), and glutathione S-transferases (GSTs). Traditionally, primary hepatocytes containing a complete set of DMEs have been widely used as a gold standard to assess metabolism-induced compound toxicity. However, primary hepatocytes are expensive, have high donor variability in expression levels of DMEs, and rapidly lose liver-specific functions when the cells are maintained under standard in vitro cell culture conditions over time. To address this issue and rapidly profile metabolism-induced drug toxicity, we have developed a 384-pillar plate, which is complementary to conventional 384-well plates. In this chapter, we provide step-by-step procedures for three-dimensional (3D) cell printing on the 384-pillar plate coupled with DMEs and compounds in the 384-well plate for high-throughput assessment of metabolism-induced toxicity.

A Decade in the MIST: Learnings from Investigations of Drug Metabolites in Drug Development under the "Metabolites in Safety Testing" Regulatory Guidance.

Since the introduction of metabolites in safety testing (MIST) guidance by the Food and Drug Administration in 2008, major changes have occurred in the experimental methods for the identification and quantification of metabolites, ways to evaluate coverage of metabolites, and the timing of critical clinical and nonclinical studies to generate this information. In this cross-industry review, we discuss how the increased focus on human drug metabolites and their potential contribution to safety and drug-drug interactions has influenced the approaches taken by industry for the identification and quantitation of human drug metabolites. Before the MIST guidance was issued, the method of choice for generating comprehensive metabolite profile was radio chromatography. The MIST guidance increased the focus on human drug metabolites and their potential contribution to safety and drug-drug interactions and led to changes in the practices of drug metabolism scientists. In addition, the guidance suggested that human metabolism studies should also be accelerated, which has led to more frequent determination of human metabolite profiles from multiple ascending-dose clinical studies. Generating a comprehensive and quantitative profile of human metabolites has become a more urgent task. Together with technological advances, these events have led to a general shift of focus toward earlier human metabolism studies using high-resolution mass spectrometry and to a reduction in animal radiolabel absorption/distribution/metabolism/excretion studies. The changes induced by the MIST guidance are highlighted by six case studies included herein, reflecting different stages of implementation of the MIST guidance within the pharmaceutical industry.

Simultaneous Assessment In Vitro of Transporter and Metabolic Processes in Hepatic Drug Clearance: Use of a Media Loss Approach.

Hepatocyte drug depletion-time assays are well established for determination of metabolic clearance in vitro. The present study focuses on the refinement and evaluation of a "media loss" assay, an adaptation of the conventional depletion assay involving centrifugation of hepatocytes prior to sampling, allowing estimation of uptake in addition to metabolism. Using experimental procedures consistent with a high throughput, a selection of 12 compounds with a range of uptake and metabolism characteristics (atorvastatin, cerivastatin, clarithromycin, erythromycin, indinavir, pitavastatin, repaglinide, rosuvastatin, saquinavir, and valsartan, with two control compounds-midazolam and tolbutamide) were investigated in the presence and absence of the cytochrome P450 inhibitor 1-aminobenzotriazole and organic anion transporter protein inhibitor rifamycin SV in rat hepatocytes. Data were generated simultaneously for a given drug, and provided, through the use of a mechanistic cell model, clearance terms characterizing metabolism, active and passive uptake, together with intracellular binding and partitioning parameters. Results were largely consistent with the particular drug characteristics, with active uptake, passive diffusion, and metabolic clearances ranging between 0.4 and 777, 3 and 383, and 2 and 236 µl/min per milligram protein, respectively. The same experiments provided total and unbound drug cellular partition coefficients ranging between 3.8 and 254 and 2.3 and 8.3, respectively, and intracellular unbound fractions between 0.014 and 0.263. Following in vitro-in vivo extrapolation, the lowest prediction bias was noted using uptake clearance, compared with metabolic clearance or apparent clearance from the media loss assay alone. This approach allows rapid and comprehensive characterization of hepatocyte drug disposition valuable for prediction of hepatic processes in vivo.

New Arsenate Reductase Gene (arrA) PCR Primers for Diversity Assessment and Quantification in Environmental Samples.

The extent of arsenic contamination in drinking water and its potential threat to human health have resulted in considerable research interest in the microbial species responsible for arsenic reduction. The arsenate reductase gene (arrA), an important component of the microbial arsenate reduction system, has been widely used as a biomarker to study arsenate-reducing microorganisms. A new primer pair was designed and evaluated for quantitative PCR (qPCR) and high-throughput sequencing of the arrA gene, because currently available PCR primers are not suitable for these applications. The primers were evaluated in silico and empirically tested for amplification of arrA genes in clones and for amplification and high-throughput sequencing of arrA genes from soil and groundwater samples. In silico, this primer pair matched (≥90% DNA identity) 86% of arrA gene sequences from GenBank. Empirical evaluation showed successful amplification of arrA gene clones of diverse phylogenetic groups, as well as amplification and high-throughput sequencing of independent soil and groundwater samples without preenrichment, suggesting that these primers are highly specific and can amplify a broad diversity of arrA genes. The arrA gene diversity from soil and groundwater samples from the Cache Valley Basin (CVB) in Utah was greater than anticipated. We observed a significant correlation between arrA gene abundance, quantified through qPCR, and reduced arsenic (AsIII) concentrations in the groundwater samples. Furthermore, we demonstrated that these primers can be useful for studying the diversity of arsenate-reducing microbial communities and the ways in which their relative abundance in groundwater may be associated with different groundwater quality parameters. IMPORTANCE: Arsenic is a major drinking water contaminant that threatens the health of millions of people worldwide. The extent of arsenic contamination and its potential threat to human health have resulted in considerable interest in the study of microbial species responsible for the reduction of arsenic, i.e., the conversion of AsV to AsIII In this study, we developed a new primer pair to evaluate the diversity and abundance of arsenate-reducing microorganisms in soil and groundwater samples from the CVB in Utah. We observed significant arrA gene diversity in the CVB soil and groundwater samples, and arrA gene abundance was significantly correlated with the reduced arsenic (AsIII) concentrations in the groundwater samples. We think that these primers are useful for studying the ecology of arsenate-reducing microorganisms in different environments.

Metabolites in safety testing: metabolite identification strategies in discovery and development.

The publication of the FDA MIST guidelines in 2008, together with the acknowledged importance of metabolism data for the progression of novel compounds through drug discovery and drug development, has resulted in a renewed focus on the metabolite identification strategies utilised throughout the pharmaceutical industry. With the plethora of existing and emerging technologies available to the metabolite identification scientist, it is argued that increased diligence should be applied to metabolism studies in the early stages of both drug discovery and drug development, in order to more routinely impact chemical design and to comply with the concepts of the MIST guidance without re-positioning the definitive radiolabelled studies from there typical place in late development.Furthermore, these strategic elements should be augmented by a broad and thorough understanding of the impact of the derived metabolism data, most notably considerations of absolute abundance, structure and pharmacological activity, such that they can be put into proper context as part of a holistic safety strategy.The combination of these approaches should ensure a metabolite identification strategy that successfully applies the principles of the MIST guidance throughout the discovery/development continuum and thereby provides appropriate confidence in support of human safety.

Noninvasive assessment of liver detoxification capacity of children, observed in children from heavily polluted industrial and clean control areas, together with assessments of air pollution and chloro-organic body burden.

The liver is an important target organ in exposure to foreign substances or their metabolites. Early changes in the metabolic capacity of the liver may be a first sign of the effect of an exposure and an indication of an early (pre)pathological process. For the noninvasive testing of this metabolic capacity, a special diagnostic tool has been developed and demonstrated under real exposure conditions. The main questions investigated were (1) whether the liver detoxification capacity of children is affected by long-term low exposure in a highly polluted area and (2) to what extent redevelopment in once heavily polluted industrial areas contributes to improvement in the health measured in the children from the changed detoxification capacity. Kindergarten children from a heavily polluted industrial area and a control area were observed over a prolonged period during redevelopment of the industrial area. The liver's detoxification capacity was assessed with a stable-isotope-based diagnostic test, the [(15)N]methacetin test. In addition, the region-specific external exposure (indicator components were chlorinated compounds) and internal load were measured. The difference in the children's internal load between the exposed and control groups reflected the differences in exposure (exposed children had an internal load 2.3 times greater than that of the control children). The exposed children showed a 6% lower liver detoxification capacity. A reduction in pollution by about 70% as a result of remediation was reflected in improved liver detoxification capacity, and the difference between the polluted and control areas was no longer significant. Prolonged exposure to low concentrations of xenobiotics can disturb hepatic functioning. The proposed test can be used as a tool to determine the effects of multicomponent exposure and is well suited for bioeffect monitoring.

Can methane suppression during digestion of woody and leafy browse compensate for energy costs of detoxification of plant secondary compounds? A test with muskoxen fed willows and birch.

Digestion and metabolism of woody and leafy browse requires detoxification of plant secondary compounds that can incur an energy cost. Browse, however, inhibits methane (CH(4)) production and therefore could offset some costs of detoxification. We measured an index of heat increment of feeding (HIFi) and CH(4) production in muskoxen (Ovibos moschatus) given a single test meal (at 10 g/kg BM(0.75)) composed of hay mixed with one of three browse species (Willow: Salix alaxensis, S. pulchra; Birch: Betula nana). Detoxification cost was estimated as HIFi of browse diet-HIFi of hay diet and CH(4) compensation as CH(4) production of hay diet-CH(4) production of browse diet. CH(4) compensation was noted in 47% of 15 trials in which a detoxification cost was evident; six trials were with woody browse and one with leafy browse. Separate controls were responsible for the difference in CH(4) compensation for leafy browse vs. woody browse. Detoxification costs for twigs and leaves of B. nana were underestimated because of their low digestibility. In only one of six treatments was CH(4) compensation documented for B. nana. We conclude that energy saved by CH(4) suppression was small (<6%) compared with detoxification costs.

Risk assessment for aflatoxin: I. Metabolism of aflatoxin B1 by different species.

A comparison of the generation and detoxification of reactive aflatoxin B1 metabolites in different species may elucidate why animal species vary widely in sensitivity to aflatoxin B1 carcinogenicity, in addition to offering some perspective on how sensitive man may be to the carcinogenic effects of this mycotoxin. Scientific literature comparing the ability of cellular fractions from different species to metabolize aflatoxin B1 is reviewed. However, in vitro studies exclude components for multiple metabolic pathways, eliminating the possibility of competition between metabolic activation and detoxification. Quantification of metabolic activation by different species from these studies is therefore limited. The species-specific carcinogenic potency of aflatoxin B1 may be reflected in levels of aflatoxin B1-DNA adducts generated in vivo. The current paucity of quantitative information on human DNA adduct levels does not permit comparisons between different species on this basis.