Isostearic acid is an active component of imiquimod formulations 2 used to induce psoriaform disease models

Abstract

Topical imiquimod based creams are indicated as immune stimulants for papillomas and various 13 skin neoplasms. Imiquimod is considered a TLR7 ligand. These creams are also used in research to 14 induce skin inflammation in mice as a model for psoriasis. We observed that this inflammatory 15 response was not strictly imiquimod dependent and we set out to establish which components 16 drive the proinflammatory effects. To this end, we examined the induction response in a 17 BALB/cJRj mouse model, in which 50 mg of cream is applied to 2 cm2 of skin (125 mg/kg imiquimod 18 – 5% W/V, and/or 625 mg/kg isostearic acid – 25% W/V). Comparing cream formulations contain- 19 ing isostearic acid, imiquimod and the combination, we observed that isostearic acid causes skin 20 inflammation within 2 days, whereas imiquimod requires up to 5 days for initial signs. Isostearic 21 acid activated an inflammasome response, stimulated release of proinflammatory cytokines and 22 upregulated the IL-23/17 axis. Animals treated with isostearic acid had enlarged livers (+40% 23 weight), which was not observed with imiquimod alone. Imiquimod was readily metabolized and 24 cleared from plasma and liver, but was maintained at high levels in the skin throughout the body 25 (200 mM at area of application; 200 µM in untreated skin). Imiquimod application was associated 26 with splenomegaly, cytokine induction/release and initial body weight loss over 3 days. Despite 27 high imiquimod skin levels throughout the animal, inflammation was only apparent in the treated 28 areas and was less severe than in isostearic acid groups. As the concentrations in these areas are 29 well above the 10 µM required for TLR7 responses in vitro, there is an implication that skin in- 30 flammation following imiquimod is due to effects other than TLR7 agonism (e.g., adenosine re- 31 ceptor agonism). In brain, isostearic caused no major changes in cytokine expression while 32 imiquimod alone sightly stimulated expression of IL-1β and CCL9. However, the combination of 33 both caused brain induction of CCL3, -9, CXCL10, -13, IL-1β and TNFα. The implication of these 34 data is that isostearic acid facilitates the entry of imiquimod or peripherally secreted cytokines 35 into the brain. Our data suggest that psoriaform skin responses in mice are more driven by isos- 36 tearic acid, than generally reported and that the dose and route used in the model, leads to pro- 37 found systemic effects, which may complicate the interpretation of drug effects in this model.