Anti-Hyperuricemic, Anti-Arthritic, Hemolytic Activity and Therapeutic Safety of Glycoconjugated Triazole-Phthalimides

Abstract

Hyperuricemia, the metabolic alteration that leads to gout or gouty arthritis, is increasing worldwide. Glycoconjugated triazole-phthalimides show potent anti-inflammatory activity. The aim of this study was to evaluate the anti-hyperuricemia effect of glycoconjugated triazole-phthalimides. To develop hyperuricemia, groups of mice received orally potassium oxonate (250 mg/kg) for 7 days, and F2, F3 and F4 glycoconjugated triazole-phthalimides (20 mg/kg), allopurinol (300 mg/kg), and 1% carboxymethylcellulose; indomethacin (2 and 4 mg/kg) was the positive control for anti-arthritic effect. Genotoxic and mutagenic effects were evaluated by the comet and micronucleus assays, respectively. The hemolytic action of the compounds was evaluated. Phthalimides F2, F3 and F4 significantly reduced the levels of serum uric acid, creatinine and urea in hyperuricemic animals. In addition, the compounds were efficient in reducing protein denaturation in a dose-dependent manner. In an interesting way, the histopathological analysis of kidneys from groups treated with F2, F3 and F4 showed a glomerular architecture, with the Bowman's capsule and renal tubules having a normal appearance and without inflammatory changes. Also, F2 and F4 showed a small increase in micronuclei, indicating a low mutagenic effect, whilst by comet assay only, we could infer that F4 affected the frequency and damage index, thus indicating a very small genotoxic action. Similarly, the phthalimides showed a low degree of erythrocyte hemolysis (<3%). Our data demonstrate that the new glycoconjugate triazole-phthalimides have potential to treat hyperuricemia and its secondary complications, such as gouty arthritis, with a low to non-significant rate of erythrocytes hemolysis, genotoxicity and mutagenicity making these molecules strong candidates as pharmaceutical agents for treatment requiring uric-acid-lowering therapy.

Keywords: arthritis; gouty arthritis; hyperuricemia; medicinal chemistry; uric acid.

Figure 1

Schematic delineation of the hyperuricemia model. All animals were treated with 250 mg/kg of potassium oxonate for 7 days. G1, negative control and 1% carboxymethylcellulose. G2, F2 test: treated with 250 mg/kg/day of potassium oxonate, and 20 mg/kg/day F2. G3, F3 test: treated with 250 mg/kg of potassium oxonate and F3 20 mg/kg. G4, F4 test: treated with 250 mg/kg of potassium oxonate and F4 20 mg/kg. G5, positive control: treated with 250 mg/kg of potassium oxonate and allopurinol 300 mg/kg. The potassium oxonate, glycoconjugated triazole-phthalimides and 1% CMC vehicle were administered orally for seven days. On the eighth day, potassium oxonate was removed and the animals continued to receive the glycoconjugated phthalimides with triazoles, 1% CMC and allopurinol until the tenth day of research.

Scheme 1

Synthesis of glycoconjugate triazole-phthalimides F2, F3 and F4.

Figure 2

Serum levels of some biochemical parameters from hyperuricemic mice treated with Phthalimides F2, F3, F4 and allopurinol. (A) Serum levels of uric acid. (B) Serum levels of creatinine. (C) Serum levels of urea. The statistical differences of the control were determined by multivariate ANOVA followed by the Bonferroni test, * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. negative control.

Figure 3

Representative photomicrographs. Kidneys: renal glomeruli and convoluted tubules without morphological changes are visible in the following groups of mice—negative control (A), treated with phthalimides F2 (B), F3 (C), F4 (D) and treated with allopurinol (E). All images are with 400× magnification.

Figure 4

Effect of glycoconjugated triazole-phthalimides on protein denaturation. (A) Indomethacin (2 mg/kg). (B), 4 mg/kg.

Figure 5

Comet essay. (A) micronucleus test. (B) Comet test to evaluate damage index and (C) comet test to evaluate damage frequency. The statistical differences of the control were determined by Wilcoxon’s non-parametric test. * p < 0.05 vs. negative control (red asterisk means the non-parametric mean of each group).

Figure 6

Percentage of hemolysis caused by glycoconjugated triazole-phthalimides.