Dermal toxicity studies: factors impacting study interpretation and outcome.

The field of dermal toxicity continues to evolve in order to accurately predict dermal (and systemic) responses in humans to topically applied chemicals. Although the testing methods have undergone extensive refinements, idiosyncrasies and unexpected issues during the conduct of these studies are not unusual due to the plethora of new vehicles available for formulating test substances, changing regulatory requirements, and introducting new strain and/or species of laboratory animals as no single species or method seems to suffice for evaluating skin toxicity. The objective of this article is to illustrate some pragmatic issues that should be considered during the conduct as well as interpretation of dermal toxicity studies. Routine procedure-related issues such as hair clipping, tape stripping, and wrapping the animal's torso to prevent oral ingestion can influence the interpretation. Excipients used in dermal toxicity studies may be nontoxic when used alone but complex dermal formulations can result in unexpected irritation and toxicity. In conclusion, interpretation and risk assessment of dermal toxicity studies should be done in a comprehensive manner, taking into account procedure-related impact on study results, unique species susceptibility, limitation of gross visual (naked eye) observation for evidence of toxicity, and normal anatomical variation.

Discovery of a highly potent, nonabsorbable apical sodium-dependent bile acid transporter inhibitor (GSK2330672) for treatment of type 2 diabetes.

The apical sodium-dependent bile acid transporter (ASBT) transports bile salts from the lumen of the gastrointestinal (GI) tract to the liver via the portal vein. Multiple pharmaceutical companies have exploited the physiological link between ASBT and hepatic cholesterol metabolism, which led to the clinical investigation of ASBT inhibitors as lipid-lowering agents. While modest lipid effects were demonstrated, the potential utility of ASBT inhibitors for treatment of type 2 diabetes has been relatively unexplored. We initiated a lead optimization effort that focused on the identification of a potent, nonabsorbable ASBT inhibitor starting from the first-generation inhibitor 264W94 (1). Extensive SAR studies culminated in the discovery of GSK2330672 (56) as a highly potent, nonabsorbable ASBT inhibitor which lowers glucose in an animal model of type 2 diabetes and shows excellent developability properties for evaluating the potential therapeutic utility of a nonabsorbable ASBT inhibitor for treatment of patients with type 2 diabetes.

Effects of Solutol (Kolliphor) and cremophor in polyethylene glycol 400 vehicle formulations in Sprague-Dawley rats and beagle dogs.

When conventional vehicles (eg, methylcellulose and water) impart inadequate physical, chemical, and/or biological properties for proper toxicological assessment of test article formulations, nonconventional vehicles may be considered. Often toxicity data for nonconventional vehicle formulations are limited. Studies were conducted to collect toxicity data from a rodent and a non-rodent species given 2 nonconventional vehicles, Solutol HS15/polyethylene glycol (PEG) 400 and Cremophor RH40/PEG 400, with differing formulations and dose volumes (10 mL/kg for rats; 2 or 5 mL/kg for dogs). In rats, both vehicles caused increase in kidney weights (males only) and decrease in thymic weights (males only) without concurrent microscopic findings; altered urine electrolytes, minimally decreased serum electrolytes (males only), and increased serum total cholesterol (females only) were also present. The Cremophor formulation was also associated with increased serum urea (males only) and urine phosphorus: creatinine. For rats given the Solutol formulation, both genders had decreased urine glucose parameters and males had increased urine volume. In dogs, loose/watery feces and emesis were present given either vehicle, and mucus-cell hyperplasia of the ileum was present given the Solutol formulation. Increased red blood cell mass and decreased urine volume in dogs given 30% Solutol/70% PEG 400 (5 mL/kg/d) were likely due to subclinical dehydration and hemoconcentration. For the Cremophor formulations, dose volume-dependent increased incidence of minimal subepithelial gastric hemorrhage was noted in dogs, and dogs given 5 mL/kg/d showed increased serum urea nitrogen. Overall, regardless of the formulation or dose volume, neither vehicle produced overt toxicity in either species, but the Solutol formulation produced fewer effects in rats. Generally, lower dose volumes minimized the severity and/or incidence of findings.

Etomoxir-induced oxidative stress in HepG2 cells detected by differential gene expression is confirmed biochemically.

Although they are known to be effective antidiabetic agents, little is published about the toxic effects of carnitine palmitoyltransferase-1 (CPT-1) inhibitors, such as etomoxir (ET). These compounds inhibit mitochondrial fatty acid beta-oxidation by irreversibly binding to CPT-1 and preventing entry of long chain fatty acids into the mitochondrial matrix. Treatment of HepG2 cells with 1 mM etomoxir for 6 h caused significant modulations in the expression of several redox-related and cell cycle mRNAs as measured by microarray analysis. Upregulated mRNAs included heme oxygenase 1 (HO1), 8-oxoguanine DNA glycosylase 1 (OGG1), glutathione reductase (GSR), cyclin-dependent kinase inhibitor 1A (CDKN1 [p21(waf1)]) and Mn+ superoxide dismutase precursor (SOD2); while cytochrome P450 1A1 (CYP1A1) and heat shock 70kD protein 1 (HSPA1A) were downregulated. Real time quantitative PCR (RT-PCR) confirmed the significant changes in 4 of 4 mRNAs assayed (CYP1A1, HO1, GSR, CDKN1), and identified 3 additional mRNA changes; 2 redox-related genes, gamma-glutamate-cysteine ligase modifier subunit (GCLM) and thioredoxin reductase (TXNRD1) and 1 DNA replication gene, topoisomerase IIalpha (TOP2A). Temporal changes in selected mRNA levels were examined by RT-PCR over 11 time points from 15 min to 24 h postdosing. CYP1A1 exhibited a 38-fold decrease by 4 h, which rebounded to a 39-fold increase by 20 h. GCLM and TXNRD1 exhibited 13- and 9-fold increases, respectively at 24 h. Etomoxir-induced oxidative stress and impaired mitochondrial energy metabolism were confirmed by a significant decrease in reduced glutathione (GSH), reduced/oxidized glutathione ratio (GSH/GSSG), mitochondrial membrane potential (MMP), and ATP levels, and by concurrent increase in oxidized glutathione (GSSG) and superoxide generation. This is the first report of oxidative stress caused by etomoxir.