An evaluation of the anti-inflammatory activity and mechanism of action of three novel auranofin derivatives

Abstract

Gold compounds have been used for the treatment of rheumatoid arthritis since the mid 20th century as a disease modifying anti-rheumatic drug. Auranofin, an oral anti-rheumatic drug, has been used for many years in the treatment of rheumatoid arthritis (RA). Although the drug has been successful in treating the symptoms of RA, many patients discontinue its use due to severe toxicity over long periods of continued treatment. Since the introduction of auranofin in 1985 there has been no new clinically approved gold drug. Drug discovery research is directing focus on overcoming these toxicity problems. Much of the problems related to the toxicity related to auranofin are due to its lipophilicity. As a result, three compounds (Asa-fin, Mpta-fin and Pta-fin) with varying substituents were synthesised and hence the lipophilic- hydrophilic balance was modulated. All compounds including auranofin were tested against normal cells to determine its toxicity as well as its anti-inflammatory activity. Three novel auranofin derivatives were compared to auranofin with regards to lipophilicity, toxicity and anti-inflammatory properties The lipophilicity of the three compounds were compared to auranofin using the octanol-water partition coefficient method. All the novel compounds showed variable lipophilicity compared to auranofin, with Pta-fin and Mpta-fin being more hydrophilic than auranofin. The cytotoxicity of these novel gold compounds Asa-fin, Mpta-fin and Pta-fin were compared to auranofin using primary porcine hepatocytes and chicken embryo fibroblasts cultures. A metabolic assay based on the reactivity of 3-[4,5-dimethylyhiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) with viable cells was done to measure the effect of the drugs on the growth of cultures. All three novel compounds proved less toxicity at comparable concentrations in primary porcine hepatocytes and in fibroblast proliferation, Asa-fin and Mpta-fin proved less toxic than Auranofin. The Anti-inflammatory activity of the experimental compounds was determined by testing the effects of the experimental compounds on human lymphocyte proliferation. The MTT assay was used to measure the effect of the drugs on the growth of the cell cultures. All three compounds inhibited the proliferation of human lymphocytes with Pta-fin having the least effect. The effect of these drugs was also evaluated on the reactive oxidant production by chemiluminescence and flow cytometry on resting, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) and Phorbol Myristate Acetate (PMA) stimulated human neutrophils. Oxidant production by neutrophils was measured after a 45-minute incubation period with luminol enhanced chemiluminescence. Treatment of neutrophils with auranofin and the three compounds showed that auranofin, Asa-fin and Mpta-fin had a biphasic activity on hydrogen peroxide production with higher concentrations decreasing hydrogen peroxide production, possibly leading to the anti-inflammatory action of these drugs. With Pta-fin no decrease in hydrogen peroxide was observed. Using flow cytometry three dyes specific to different reactive oxygen species were used. 2’, 7’-Dichloroflourescein diacetate (DCFH) is specific for detecting nitric oxide, Dihydrorhodamine 123 (DHR) is specific for detecting hydrogen peroxide and Hydroethidine (HE) is specific for detecting superoxide. Oxidant production was measured after a 30 minute incubation period with the relative dyes on a flow cytometer. Auranofin and Asa-fin decreased hydrogen peroxide and superoxide production. None of the drugs had an effect on nitric oxide production. The expression of the â2-integrin adhesion molecule, CR3, on resting and PMA stimulated neutrophils treated with the experimental compounds was measured by flow cytometry. CR3 expression by neutrophils was measured after 10 minute incubation in the dark with CD11b FITC monoclonal antibody. Treatment of neutrophils with auranofin and the three experimental compounds showed a decrease in CR3 expression on resting and stimulated neutrophils, however the effect was more marked in stimulated neutrophils. The Anti-inflammatory activity of the experimental compounds was determined by testing the effects of the experimental compounds on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX 2) in resting and lipopolysaccharide (LPS) stimulated human monocytes. COX 1 and 2 production was measured by flow cytometry. Treatment of monocytes with the experimental compounds showed a decrease in COX 2 production in stimulated monocytes but an increase in COX 2 production in resting monocytes. No effect on COX 1 production was observed with the experimental compounds. Prostaglandin E2 (PGE2) was measured with a Prostaglandin E2 Enzymeimmunoassay (ELISA) kit on human macrophages. Auranofin, Asa-fin, Mpta-fin and Pta-fin inhibited the production of PGE2. Auranofin and Asa-fin inhibited the PGE2 directly proportional to the drug concentration. The effect of these drugs was also evaluated on various inflammatory cytokines using an inflammatory cytokine kit and measured on a flow cytometer. The cytometric bead array (CBA) human inflammation kit was used to quantitatively measure interlukin-8(IL-8), interlukin-1â (IL-1â), interlukin-6 (IL-6), interlukin-10 (IL-10), tumour necrosis factor alpha (TNFá) and interlukin-12p70 (IL-12p70). Auranofin and Asa-fin decreased IL 10, TNFá, and IL1â in stimulated cells. No effect was observed on IL 8, IL-12p70 and IL 6. With Mpta-fin and Pta-fin, no significant effect was observed in the cytokines tested. Drug toxicity was evaluated in mice using all four compounds in BALB/c inbred mice. Aspartate transaminase (AST), gamma glutamine transferase (GGT), urea and creatine levels were measured in the test mice. The group receiving the highest dose of Asa-fin showed the greatest elevation of AST . The lowest dose of the auranofin treatment group showed the greatest elevation in GGT, however this increase was not seen in the subsequent higher dosing groups. None of the treatment groups indicated an increase in urea levels. Mpta-fin and Pta-fin showed no increase in the liver enzymes or in urea and creatine. The results of this work are indicative that novel gold compounds could play a promising role in anti arthritic applications. Asa-fin exhibited similar anti-inflammatory activity to auranofin but in vivo toxicity was high. Mpta-fin showed slightly inferior anti-inflammatory activity to auranofin but in vivo toxicity profiles were much more promising. Pta-fin showed the least anti-inflammatory activity of the three novel compounds tested with a similar in vivo toxicity profile as Mpta-fin.