Thomas K. Baker

The future of drug safety testing: expanding the view and narrowing the focus.

Drug safety remains a high profile issue at a time when the cost and time required to develop a new drug are at an all time high. Balancing risk against the expected clinical benefit is the primary purpose of preclinical and clinical testing. We offer an expanded view on the application of predictive strategies and technologies to early safety decisions and suggestions to narrow the focus for improving preclinical safety testing to the problems that contribute most to adverse drug reactions.

Relating molecular properties and in vitro assay results to in vivo drug disposition and toxicity outcomes.

A primary goal of lead optimization is to identify compounds with improved absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. A number of reports have linked computed molecular properties to desirable in vivo ADMET outcomes, but a significant limitation of these analyses is the failure to control statistically for possible covariates. We examine the relationship between molecular properties and in vitro surrogate assays vs in vivo properties within 173 chemical series from a database of 3773 compounds with rodent pharmacokinetic and toxicology data. This approach identifies the following pairs of surrogates as most predictive among those examined: rat primary hepatocyte (RPH) cytolethality/volume of distribution (V(d)) for in vivo toxicology outcomes, scaled microsome metabolism/calculated logP for in vivo unbound clearance, and calculated logD/kinetic aqueous solubility for thermodynamic solubility. The impact of common functional group substitutions is examined and provides insights for compound design.

Involvement of calcium channels in the sexual dimorphism of cadmium-induced hepatotoxicity

Cadmium toxicity has been evaluated in a number of in vivo and in vitro toxicological studies. In vivo Cd toxicity exhibits sexual dimorphism with females being more susceptible to Cd uptake, accumulation, and toxicity in the liver. Research to date does not explain why females are more sensitive to Cd-induced hepatotoxicity. Recent studies demonstrate that progesterone sensitizes female F(344) rats and TRL-1215 cells to Cd toxicity, however the mode of action is still unclear. Approximately one half of the Cd entering the cytoplasm does so through receptor operated Ca(2+) channels. Progesterone treatment of human spermatozoa and Xenopus laevis oocytes causes a rapid influx of Ca(2+) suggesting a possible mechanism. Since hepatocytes have progesterone receptors on their cellular membrane and Ca(2+) influx into the cytoplasm occurs following progesterone treatment we evaluated the hypothesis that progesterone facilitates the uptake and accumulation of Cd via Ca(2+) channels, leading to enhanced toxicity. Primary isolated rat hepatocytes were treated with Cd, progesterone, and/or verapamil for 4 h and cytolethality was measured. Pretreatment with the Ca(2+) channel blocker verapamil increased the Cd concentration producing 50% lethality (LC(50)) by 2-fold, thus decreasing Cd cytolethality. In contrast, pretreatment with progesterone decreased the Cd LC(50) by 2-fold resulting in enhanced Cd cytolethality. Verapamil treatment reversed the progesterone enhanced Cd cytolethality. Verapamil and/or progesterone in the absence of Cd did not affect hepatocyte viability. Overall, the results of this study demonstrate that inhibition of progesterone-induced Ca(2+) influx with the Ca(2+) channel blocker verapamil, decreases Cd cytolethality in primary isolated rat hepatocytes. These findings indicate that progesterone activation of receptor-mediated Ca(2+) channels is involved in the sexually dimorphic hepatotoxicity seen following acute Cd exposure.

Toxicogenomics and cancer risk assessment: a framework for key event analysis and dose-response assessment for nongenotoxic carcinogens.

In order to determine a threshold for nongenotoxic carcinogens, the traditional risk assessment approach has been to identify a mode of action (MOA) with a nonlinear dose-response. The dose-response for one or more key event(s) linked to the MOA for carcinogenicity allows a point of departure (POD) to be selected from the most sensitive effect dose or no-effect dose. However, this can be challenging because multiple MOAs and key events may exist for carcinogenicity and oftentimes extensive research is required to elucidate the MOA. In the present study, a microarray analysis was conducted to determine if a POD could be identified following short-term oral rat exposure with two nongenotoxic rodent carcinogens, fenofibrate and methapyrilene, using a benchmark dose analysis of genes aggregated in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) biological processes, which likely encompass key event(s) for carcinogenicity. The gene expression response for fenofibrate given to rats for 2days was consistent with its MOA and known key events linked to PPARα activation. The temporal response from daily dosing with methapyrilene demonstrated biological complexity with waves of pathways/biological processes occurring over 1, 3, and 7days; nonetheless, the benchmark dose values were consistent over time. When comparing the dose-response of toxicogenomic data to tumorigenesis or precursor events, the toxicogenomics POD was slightly below any effect level. Our results suggest that toxicogenomic analysis using short-term studies can be used to identify a threshold for nongenotoxic carcinogens based on evaluation of potential key event(s) which then can be used within a risk assessment framework.

Minor compensatory changes in SAGE Mdr1a (P-gp), Bcrp, and Mrp2 knockout rats do not detract from their utility in the study of transporter-mediated pharmacokinetics.

Mdr1a-, Bcrp-, and Mrp2-knockout rats are a more practical species for absorption, distribution, metabolism, and excretion (ADME) studies than murine models and previously demonstrated expected alterations in the pharmacokinetics of various probe substrates. At present, gene expression and pathology changes were systematically studied in the small intestine, liver, kidney, and brain tissue from male SAGE Mdr1a, Bcrp, and Mrp2 knockout rats versus wild-type Sprague-Dawley controls. Gene expression data supported the relevant knockout genotype. As expected, Mrp2 knockout rats were hyperbilirubinemic and exhibited upregulation of hepatic Mrp3. Overall, few alterations were observed within 112 ADME-relevant genes. The two potentially most consequential changes were upregulation of intestinal carboxylesterase in Mdr1a knockouts and catechol-O-methyltransferase in all tissues of Bcrp knockout rats. Previously reported upregulation of hepatic Mdr1b P-glycoprotein in proprietary Wistar Mdr1a knockout rats was not observed in the SAGE counterpart investigated herein. Relative liver and kidney weights were 22–53% higher in all three knockouts, with microscopic increases in hepatocyte size in Mdr1a and Mrp2 knockout rats and glomerular size in Bcrp and Mrp2 knockouts. Increased relative weight of clearing organs is quantitatively consistent with reported increases in the clearance of drugs that are not substrates of the knocked-out transporter. Overall, SAGE knockout rats demonstrated modest compensatory changes, which do not preclude their general application to study transporter-mediated pharmacokinetics. However, until future studies elucidate the magnitude of functional change, caution is warranted in rare instances of extensive metabolism by catechol-O-methyltransferase in Bcrp knockouts and intestinal carboxylesterase in Mdr1a knockout rats, specifically for molecules with free catechol groups and esters subject to gut-wall hydrolysis.

In Vitro L6 Irritation Assay Predicts Clinical Injection Site Reactions for Small Molecules

Injection site reactions (ISRs) are commonly encountered in the development of parenteral drugs, and severe ISRs can lead to preclinical and clinical dose limiting toxicities. Tools to assess the risk of clinical ISRs during drug development are not well established. We developed an in vitro ISR screen using L6 rat myotubes to assess compounds for irritation risk. Reference compounds that were either known to induce ISRs or were non-irritating in the clinical setting were used to validate this method. We evaluated three compounds, two with known clinical ISRs (mitoxantrone and doxorubicin) and one without clinical ISR (metoprolol), using a preclinical in vivo rat model and the L6 in vitro model at clinically relevant concentrations, and showed that the L6 assay is a better prognostic indicator for clinical ISR risk. We then utilized this assay during early preclinical development to guide optimization of structure activity relationship (SAR), selection of dose concentrations for pre-clinical in vivo experiments, and prioritization of alternative formulations to minimize ISR risk. Our studies indicate that the L6 assay is a better measure of clinical ISR risk than current in vivo preclinical models, and that it can help guide not only compound selection, but also selection of dose concentration and formulation.

Use of a rat ex-vivo testis culture method to assess toxicity of select known male reproductive toxicants

Due to the complex physiology of the testes, in vitro models have been largely unsuccessful at modeling testicular toxicity in vivo. We conducted a pilot study to evaluate the utility of the Durand ex vivo rat seminiferous tubule culture model [1-3] that supports spermatogenesis through meiosis II, including the formation of round spermatids. We used this system to evaluate the toxicity of four known testicular toxicants: 1,3-dinitrobenzene (DNB), 2-methoxyacetic acid (MAA), bisphenol A (BPA), and lindane over 21 days of culture. This organotypic culture system demonstrated the ability to successfully model in vivo testicular toxicity (Sertoli cell toxicity and disruption of meiosis) for all four compounds. These findings support the application of this system to study molecules and evaluate mechanisms of testicular toxicity.

Discover Toxicology: An Early Safety Assessment Approach

Early safety assessment efforts from target identification to lead development have undergone rapid growth and evolution over the last 10 years. In this chapter, we will discuss the current development trends driving the need for early safety assessment practices. We will discuss the key areas of focus which include target-related, off-target-related, and chemical property-related toxicities. We will offer an overview of the various scientific approaches being utilized in each of these focus areas along with an organizational framework that has proven effective in de-risking the early portfolio. We will conclude with some perspectives on application within the project team setting and traps associated with data over interpretation.