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.

Tapinarof Is a Natural AhR Agonist that Resolves Skin Inflammation in Mice and Humans.

Tapinarof (GSK2894512) is a naturally derived topical treatment with demonstrated efficacy for patients with psoriasis and atopic dermatitis, although the biologic target and mechanism of action had been unknown. We demonstrate that the anti-inflammatory properties of tapinarof are mediated through activation of the aryl hydrocarbon receptor (AhR). We show that tapinarof binds and activates AhR in multiple cell types, including cells of the target tissue-human skin. In addition, tapinarof moderates proinflammatory cytokine expression in stimulated peripheral blood CD4+ T cells and ex vivo human skin, and impacts barrier gene expression in primary human keratinocytes; both of these processes are likely to be downstream of AhR activation based on current evidence. That the anti-inflammatory properties of tapinarof derive from AhR agonism is conclusively demonstrated using the mouse model of imiquimod-induced psoriasiform skin lesions. Topical treatment of AhR-sufficient mice with tapinarof leads to compound-driven reductions in erythema, epidermal thickening, and tissue cytokine levels. In contrast, tapinarof has no impact on imiquimod-induced skin inflammation in AhR-deficient mice. In summary, these studies identify tapinarof as an AhR agonist and confirm that its efficacy is dependent on AhR.

Hepatitis C replication inhibitors that target the viral NS4B protein.

We describe the preclinical development and in vivo efficacy of a novel chemical series that inhibits hepatitis C virus replication via direct interaction with the viral nonstructural protein 4B (NS4B). Significant potency improvements were realized through isosteric modifications to our initial lead 1a. The temptation to improve antiviral activity while compromising physicochemical properties was tempered by the judicial use of ligand efficiency indices during lead optimization. In this manner, compound 1a was transformed into (+)-28a which possessed an improved antiviral profile with no increase in molecular weight and only a modest elevation in lipophilicity. Additionally, we employed a chimeric "humanized" mouse model of HCV infection to demonstrate for the first time that a small molecule with high in vitro affinity for NS4B can inhibit viral replication in vivo. This successful proof-of-concept study suggests that drugs targeting NS4B may represent a viable treatment option for curing HCV infection.

Screening New Drugs for Immunotoxic Potential: II. Assessment of the Effects of Selective and Nonselective COX-2 Inhibitors on Complement Activation, Superoxide Anion Production and Leukocyte Chemotaxis and Migration Through Endothelial Cells.

Results from earlier experiments in our laboratories revealed that both selective and nonselective inhibitors of cyclooxygenase-2 possess little potential for decreasing in vitro phagocytosis by rat macrophages or canine neutrophils and no potential for decreasing in vivo phagocytosis by the intact murine immune system. We now report the results of studies to assess in vitro and ex vivo effects of the drugs on 1) canine complement activation, 2) generation of superoxide anion and hydrogen peroxide (oxidative burst) by canine neutrophils, and 3) leukocytic chemotaxis and transmigration through endothelial cell monolayers. In vitro concentrations of naproxen sodium, SC-236, SC-245, and SC-791 ranging from 0.1 to 10 muM were tested for their abilities to inhibit canine complement-mediated hemolysis of opsonized sheep erythrocytes and to block phorbol myristate acetate-induced oxidative burst in canine neutrophils. Both models responded to known inhibitory agents, leupeptin in the complement activation test and staurosporine in the superoxide anion assay. In contrast, tested nonsteroidal anti-inflammatory drugs produced only trivial changes in complement activation and superoxide anion production. Experiments on plasma and neutrophils isolated from dogs administered an experimental selective COX-2 inhibitor during a 28-day toxicology study revealed no evidence of drug-associated changes in complement activation or formation of superoxide anion. SC-791 reduced chemotaxis of canine leukocytes toward zymosan-activated dog plasma, but not toward leukotriene B(4). None of the other drugs tested significantly affected leukocytic chemotaxis. Ibuprofen, SC-245 and SC-791 but not SC-236, reduced transmigration of canine leukocytes through endothelial cell monolayers. Based on the results of these experiments and our earlier studies we have concluded that, although high (suprapharmacologic) concentrations of the drugs may induce in vitro evidence of apparent immunomodulation of the innate immune system, the findings are unlikely to represent a significant human health risk.

Screening new drugs for immunotoxic potential: III. assessment of the effects of selective and nonselective COX-2 inhibitors on T-cell-dependent antibody and natural killer cell responses in the rat.

Results from earlier experiments in our laboratories revealed that both selective and non-selective inhibitors of cyclooxygenase-2 possess little potential for decreasing in vitro phagocytosis by rat macrophages or canine neutrophils, and no potential for decreasing in vivo phagocytosis by the intact murine immune system. We have also demonstrated that pharmacologically relevant doses and concentrations of these drugs do not reduce canine complement activation, superoxide anion generation, leukocytic chemotaxis or transmigration of leukocytes through endothelial monolayers. We now report the results of immunotoxicology studies to assess the effects of the drugs on cell-mediated immunity. Male and female Sprague-Dawley rats were administered daily oral gavage doses of naproxen (10 mg/kg), SC-791 (2.5 mg/kg), or SC-245 (17 mg/kg) for 28 consecutive days or treated with cyclophosphamide or anti-asialo GM1 antibody as positive immunomodulatory controls (for T-dependent antibody response and natural killer cell assay, respectively). All rats, except those treated with GM1 antibody or used in toxicokinetic analyses, were immunized on study day 25 with sheep red blood cells to induce a primary T-dependent antibody response. The doses of test agents were chosen to be either supra-pharmacologic or limited by anticipated systemic toxicity. Hematologic changes consistent with gastrointestinal (GI) blood loss due to mild GI mucosal toxicity were seen with naproxen and SC-791. Both positive control agents produced anticipated immunomodulatory effects confirming the validity of the assay system. In the antibody-forming cell assay, naproxen, SC-791 and SC-245 were without effects on splenic cellularity, splenocyte viability or the number of sheep red blood cell antibody-forming cells. Cyclophosphamide reduced both splenic cellularity and antibody-forming cell counts. In the natural killer cell assay, naproxen, SC-791 and SC-245 were all without effects on natural killer cell activity, while anti-asialo antibody reduced natural killer cell activity up to 85%. In considering the sum total of scientific information relative to the immunotoxicological potential of non-selective and selective cyclooxygenase-2 inhibitors, we conclude that, although high (supra-pharmacologic) concentrations of these drugs may induce some in vitro immunomodulatory effects on the innate immune system, the findings are of doubtful predictive significance with respect to human health implications.

Screening New Drugs for Immunotoxic Potential: I. Assessment of the Effects of Conventional Nonsteroidal Anti-Inflammatory Drugs and Selective COX-2 Inhibitors on In Vitro and In Vivo Phagocytic Activity.

Although both experimental and clinical literature contain reports suggestive of associations between enhanced susceptibility to soft tissue infections and nonsteroidal anti-inflammatory drug (NSAID) use, the immunotoxicological potential of this class of therapeutic agents has not been thoroughly investigated. In consideration of the widespread clinical use of these agents, we have initiated studies of the interaction between NSAIDs (both nonselective and selective COX-2 inhibitors) and the immune system. This communication describes the conduct and results of assessments of the effects of NSAIDs on the in vitro phagocytic activity of rat macrophages and canine neutrophils and on the functional activity of the intact murine mononuclear phagocytic system. During in vitro experiments 0.1 to 10 muM concentrations of naproxen, indomethacin, and experimental selective COX-2 inhibitors, SC-236, SC-245 and SC-791, caused marginal, but statistically significant, reductions in phagocytic activity of resident rat peritoneal macrophages. The effects were consistently small and there was no evidence of concentration-response relationships. An in vitro concentration of 10 muM of either SC-236 or SC-791 was required to decrease phagocytosis by dog neutrophils. Repeated oral doses of either naproxen or SC-236 (3, 10, or 30 mg/kg twice daily for 3 days) were without effect on the intact phagocytic system of the mouse. A potential immunotoxicologic effect based on direct impairment of phagocytic processes seems an unlikely explanation for drug-induced susceptibility to infection reported earlier. However, the results of these experiments do not support an unequivocal conclusion relative to immunotoxicological potential of either conventional NSAIDs or selective COX-2 inhibitors. Further studies on other components of the immune system are needed to fully explore possible immunomodulatory effects of NSAIDs.

Effect of oral gavage dosing regimens in female Tg rasH2 transgenic mice.

Female Tg rasH2 (CB6F1/Jic-TgrasH2@Tac) mice were administered water once daily, water twice daily with 8 or 12 hours between doses, 1% sodium dodecyl sulfate in water (1% SDS) once daily, or 1% SDS twice daily with 12 hours between doses by oral gavage at volumes of 10 ml/kg/day for 28 or 29 consecutive days. A control group of mice received no treatment and no sham manipulation. There were no significant differences in body weight or food consumption between treated groups and untreated control mice. Mean weights of spleens, livers, and thymuses were lower than control values in most groups of mice subjected to gavage. Focal or multifocal loss of thymic cortical architecture was observed in 13 of 50 mice distributed among all groups (including naïve controls), however only in one instance was this finding suggestive of a precursor to neoplasia. This study demonstrated that Tg rasH2 mice can tolerate once or twice daily gavage dosing with water or vehicle containing 1% SDS. Loss of thymic cortical architecture was a common incidental finding in female Tg rasH2 mice.