Eric A.G. Blomme

Preclinical Characterization of A‐582941: A Novel α7 Neuronal Nicotinic Receptor Agonist with Broad Spectrum Cognition‐Enhancing Properties

Among the diverse sets of nicotinic acetylcholine receptors (nAChRs), the α7 subtype is highly expressed in the hippocampus and cortex and is thought to play important roles in a variety of cognitive processes. In this review, we describe the properties of a novel biaryl diamine α7 nAChR agonist, A‐582941. A‐582941 was found to exhibit high‐affinity binding and partial agonism at α7 nAChRs, with acceptable pharmacokinetic properties and excellent distribution to the central nervous system (CNS). In vitro and in vivo studies indicated that A‐582941 activates signaling pathways known to be involved in cognitive function such as ERK1/2 and CREB phosphorylation. A‐582941 enhanced cognitive performance in behavioral models that capture domains of working memory, short‐term recognition memory, memory consolidation, and sensory gating deficit. A‐582941 exhibited a benign secondary pharmacodynamic and tolerability profile as assessed in a battery of assays of cardiovascular, gastrointestinal, and CNS function. The studies summarized in this review collectively provide preclinical validation that α7 nAChR agonism offers a mechanism with potential to improve cognitive deficits associated with various neurodegenerative and psychiatric disorders.

Trovafloxacin-Induced Gene Expression Changes in Liver-Derived in Vitro Systems: Comparison of Primary Human Hepatocytes to HepG2 Cells

Primary human hepatocytes (PHH) are a main instrument in drug metabolism research and in the prediction of drug-induced phase I/II enzyme induction in humans. The HepG2 liver-derived cell line is commonly used as a surrogate for human hepatocytes, but its use in absorption, distribution, metabolism, and excretion and toxicity studies can be limited because of lowered basal levels of metabolizing enzymes. Despite the widespread use of HepG2 cells, a comparison of their transcriptomes with those of PHH has not been well characterized. In this study, microarray analysis was conducted to ascertain the differences and similarities in mRNA expression between HepG2 cells and human hepatocytes before and after exposure to a panel of fluoroquinolone compounds. Comparison of the naive HepG2 cell and PHH transcriptomes revealed a substantial number of basal gene expression differences. When HepG2 cells were dosed with a series of fluoroquinolones, trovafloxacin (TVX), which has been associated with human idiosyncratic hepatotoxicity, induced substantially more gene expression changes than the other quinolones, similar to previous observations with PHH. Although TVX-treatment resulted in many gene expression differences between HepG2 cells and PHH, there were also a number of TVX-induced commonalities, including genes involved in RNA processing and mitochondrial function. Taken together, these results provide insight for interpretation of results from drug metabolism and toxicity studies conducted with HepG2 cells in lieu of PHH and could provide further insight into the mechanistic evaluation of TVX-induced hepatotoxicity.

Gene Expression Analysis in Rats Treated with Experimental Acetyl-Coenzyme A Carboxylase Inhibitors Suggests Interactions with the Peroxisome Proliferator-Activated Receptor α Pathway

Acetyl CoA carboxylase (ACC) 2, which catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, has been identified as a potential target for type 2 diabetes and obesity. Small-molecule inhibitors of ACC2 would be expected to reduce de novo lipid synthesis and increase lipid oxidation. Treatment of ob/ob mice with compound A-908292 (S) ({(S)-3-[2-(4-isopropoxy-phenoxy)-thiazol-5-yl]-1-methyl-prop-2-ynyl}-carbamic acid methyl ester), a small-molecule inhibitor with an IC50 of 23 nM against ACC2, resulted in a reduction of serum glucose and triglyceride levels. However, compound A-875400 (R) ({(R)-3-[2-(4-isopropoxy-phenoxy)-thiazol-5-yl]-1-methyl-prop-2-ynyl}-carbamic acid methyl ester), an inactive enantiomer of A-908292 (S) with approximately 50-fold less activity against ACC2, also caused a similar reduction in glucose and triglycerides, suggesting that the glucose-lowering effects in ob/ob mice may be mediated by other metabolic pathways independent of ACC2 inhibition. To characterize the pharmacological activity of these experimental compounds at a transcriptional level, rats were orally dosed for 3 days with either A-908292 (S) or A-875400 (R), and gene expression analysis was performed. Gene expression analysis of livers showed that treatment with A-908292 (S) or A-875400 (R) resulted in gene expression profiles highly similar to known peroxisome proliferator-activated receptor (PPAR)-α activators. The results suggest that, in vivo, both A-908292 (S) and A-875400 (R) stimulated the PPAR-α-dependent signaling pathway. These results were further supported by both an in vitro genomic evaluation using rat hepatocytes and immunohistochemical evaluation using 70-kDa peroxisomal membrane protein. Overall, the gene expression analysis suggests a plausible mechanism for the similar pharmacological findings with active and inactive enantiomers of an ACC2 inhibitor.

Use of Traditional End Points and Gene Dysregulation to Understand Mechanisms of Toxicity: Toxicogenomics in Mechanistic Toxicology

Microarray technologies can be used to generate massive amounts of gene expression information as an initial step to decipher the molecular mechanisms of toxicologic changes. Identifying genes whose expression is associated with specific toxic end points is an initial step in predicting, characterizing, and understanding toxicity. Analysis of gene function and the chronology of gene expression changes represent additional methods to generate hypotheses of the mechanisms of toxicity. Follow-up experiments are typically required to confirm or refute hypotheses derived from toxicogenomic data. Understanding the mechanism of toxicity for a compound is a critical step in forming a rational plan for developing counterscreens for toxicity and for increasing productivity of research and development while decreasing the risk of late-stage failure in pharmaceutical development.

Analysis of gene expression profiles in rat hippocampus following treatment with nicotine and an α7 nAChR selective agonist

The nicotinic acetylcholine receptors (nAChRs) play critical roles in neuronal transmission and modulation. Among the diverse nAChRs, the alpha7 subtype has been considered as a potential therapeutic target for treating cognitive deficits associated with neuropsychiatric and neurodegenerative diseases. Although a number of mechanisms including neurotransmitter and biochemical effects linking alpha7 nAChR activation and cognitive function are beginning to be described, the underlying molecular processes especially following repeated administration remain unclear. To address this, we have performed gene expression analysis in rats treated with nicotine and a selective alpha7 nAChR agonist, PNU-282987. Our results showed significant overlap in gene expression changes induced by PNU-282987 and nicotine, suggesting convergent pathways triggered by these compounds. Treatment with nicotine also resulted in regulation of a number of genes that were not regulated by PNU-282987, consistent with the interaction of nicotine with other nAChRs beyond the alpha7 subtype. Interestingly, these gene expression changes were observed 24 h post-dose, suggesting that both nicotine and PNU-282987 cause protracted changes in gene expression. Overall, our results identify gene expression changes that may contribute to further defining the roles of nAChR activation in cognitive function.

A multitargeted receptor tyrosine kinase inhibitor, SU6668, does not affect the healing of cutaneous full-thickness incisional wounds in SKH-1 mice.

Disturbances of angiogenesis have been suggested to result in the impaired healing of skin wounds. Using a murine incisional wound model, we evaluated the effects of SU6668, an inhibitor of the receptors for vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF), on the healing of skin wounds. Mice were administered vehicle, SU6668 (100 or 400 mg/kg/day, b.i.d.), or dexamethasone (1 mg/kg/day, b.i.d.), and wound healing was monitored histologically and using a tensiometer. SU6668 at a fully efficacious dose of 100 mg/kg/day had no significant effect on the healing process, while at a supratherapeutic dose of 400 mg/kg/day, there were subtle transient histologic changes and slight decreases in tensile strength, suggesting a slight delay in the wound healing process. In conclusion, these data indicate that inhibition of the receptors for VEGF, PDGF, and FGF at levels necessary to inhibit tumor growth in mouse xenograft models does not affect the healing of incisional wounds in mice. Redundant pathways likely compensate for inhibition of VEGF, PDGF, and FGF signaling pathways in the skin healing process.

Potential functional and pathological side effects related to off-target pharmacological activity

Most pharmaceutical companies test their discovery-stage proprietary molecules in a battery of in vitro pharmacology assays to try to determine off-target interactions. During all phases of drug discovery and development, various questions arise regarding potential side effects associated with such off-target pharmacological activity. Here we present a scientific literature curation effort undertaken to determine and summarize the most likely functional and pathological outcomes associated with interactions at 70 receptors, enzymes, ion channels and transporters with established links to adverse effects. To that end, the scientific literature was reviewed using an on-line database, and the most commonly reported effects were summarized in tabular format. The resultant table should serve as a practical guide for research scientists and clinical investigators for the prediction and interpretation of adverse side effects associated with molecules interacting with components of this screening battery.

Global transcriptomic profiling using small volumes of whole blood: a cost-effective method for translational genomic biomarker identification in small animals.

Blood is an ideal tissue for the identification of novel genomic biomarkers for toxicity or efficacy. However, using blood for transcriptomic profiling presents significant technical challenges due to the transcriptomic changes induced by ex vivo handling and the interference of highly abundant globin mRNA. Most whole blood RNA stabilization and isolation methods also require significant volumes of blood, limiting their effective use in small animal species, such as rodents. To overcome these challenges, a QIAzol-based RNA stabilization and isolation method (QSI) was developed to isolate sufficient amounts of high quality total RNA from 25 to 500 μL of rat whole blood. The method was compared to the standard PAXgene Blood RNA System using blood collected from rats exposed to saline or lipopolysaccharide (LPS). The QSI method yielded an average of 54 ng total RNA per μL of rat whole blood with an average RNA Integrity Number (RIN) of 9, a performance comparable with the standard PAXgene method. Total RNA samples were further processed using the NuGEN Ovation Whole Blood Solution system and cDNA was hybridized to Affymetrix Rat Genome 230 2.0 Arrays. The microarray QC parameters using RNA isolated with the QSI method were within the acceptable range for microarray analysis. The transcriptomic profiles were highly correlated with those using RNA isolated with the PAXgene method and were consistent with expected LPS-induced inflammatory responses. The present study demonstrated that the QSI method coupled with NuGEN Ovation Whole Blood Solution system is cost-effective and particularly suitable for transcriptomic profiling of minimal volumes of whole blood, typical of those obtained with small animal species.

Acyl glucuronide metabolites: Implications for drug safety assessment

Acyl glucuronides are important metabolites of compounds with carboxylic acid moieties and have unique properties that distinguish them from other phase 2 metabolites. In particular, in addition to being often unstable, acyl glucuronide metabolites can be chemically reactive leading to covalent binding with macromolecules and toxicity. While there is circumstantial evidence that drugs forming acyl glucuronide metabolites can be associated with rare, but severe idiosyncratic toxic reactions, many widely prescribed drugs with good safety records are also metabolized through acyl glucuronidation. Therefore, there is a need to understand the various factors that can affect the safety of acyl glucuronide-producing drugs including the rate of acyl glucuronide formation, the relative reactivity of the acyl glucuronide metabolite formed, the rate of elimination, potential proteins being targeted, and the rate of aglucuronidation. In this review, these factors are discussed and various approaches to de-risk the safety liabilities of acyl glucuronide metabolites are evaluated.

N-vinylpyrrolidone dimer, a novel formulation excipient, causes hepatic and thyroid hypertrophy through the induction of hepatic microsomal enzymes in rats.

N-vinylpyrrolidone dimer (VPD) is a novel experimental formulation excipient intended for preclinical toxicology studies. In a previous 4-week toxicity study, VPD induced dose-dependent hepatocellular and thyroid gland hypertrophy in Sprague-Dawley (SD) rats. The objectives of the current investigation were to define the underlying molecular mechanisms of these changes. Two separate studies were conducted using male SD rats, daily doses of 300, 1000 or 3,000 mg/kg of VPD, and a positive control (phenobarbital at 75 mg/kg/day): (1) a 28-day study to monitor thyroid hormone levels after 7 and 28 days of dosing; (2) a 5-day study to evaluate hepatic and thyroid gland transcriptomic changes, as well as hepatic UGT activity levels. At VPD dosages of 300 mg/kg/day and higher, 2-fold increases of serum thyroid stimulating hormone (TSH) levels were observed in male SD rats after 28 days of dosing, while serum thyroxine (T4) and triiodothyronine (T3) levels were unchanged. Liver UGT enzyme activity levels were increased in VPD-treated rats after 5 days. In addition, in the 5-day study, VPD caused increased hepatic mRNA levels of a panel of drug metabolizing enzymes (DMEs) and transporters, including Cyp3a1, Cyp2b1, Ugt 2b1, and Abcc3. Similar patterns of induction were observed in primary rat hepatocytes exposed to VPD. Transcriptomic changes in the thyroid gland were identified for genes involved in thyroid hormone biosynthesis and in the FAK, PTEN, and Wnt/β-catenin signaling pathways. Collectively, these data indicate that VPD acts as an inducer of hepatic DMEs in SD rats and that this likely leads to enhanced peripheral metabolism of T3/T4, resulting in a feedback response characterized by increased serum TSH levels, and thyroid gland hypertrophy and hyperplasia.