In vitro and in vivo anti-inflammatory active copper(II)-lawsone complexes

Abstract

We report in vitro and in vivo anti-inflammatory activities of a series of copper(II)-lawsone complexes of the general composition [Cu(Law)2(LN)x(H2O)(2-x)]·yH2O; where HLaw = 2-hydroxy-1,4-naphthoquinone, x = 1 when LN = pyridine (1) and 2-aminopyridine (3) and x = 2 when LN = imidazole (2), 3-aminopyridine (4), 4-aminopyridine (5), 3-hydroxypyridine (6), and 3,5-dimethylpyrazole (7). The compounds were thoroughly characterized by physical techniques, including single crystal X-ray analysis of complex 2. Some of the complexes showed the ability to suppress significantly the activation of nuclear factor κB (NF-κB) both by lipopolysaccharide (LPS) and TNF-alpha (complexes 3-7 at 100 nM level) in the similar manner as the reference drug prednisone (at 1 μM level). On the other hand, all the complexes 1-7 decreased significantly the levels of the secreted TNF-alpha after the LPS activation of THP-1 cells, thus showing the anti-inflammatory potential via both NF-κB moderation and by other mechanisms, such as influence on TNF-alpha transcription and/or translation and/or secretion. In addition, a strong intracellular pro-oxidative effect of all the complexes has been found at 100 nM dose in vitro. The ability to suppress the inflammatory response, caused by the subcutaneous application of λ-carrageenan, has been determined by in vivo testing in hind-paw edema model on rats. The most active complexes 1-3 (applied in a dose corresponding to 40 μmol Cu/kg), diminished the formation of edema simalarly as the reference drug indomethacine (applied in 10 mg/kg dose). The overall effect of the complexes, dominantly 1-3, shows similarity to anti-inflammatory drug benoxaprofen, known to induce intracellular pro-oxidative effects.

Fig 1

The general formula of 1,4-naphthoquinone showing most usual positions of substitutions, as indicated by arrows (A), and the formulas of selected 1,4-naphthoquinone derivatives isolated from natural sources: lawsone (B), juglone (C), and lapachol (D).

Fig 2. The general pathway of the preparation of complexes 1–7.

Fig 3. The result of temperature-dependent SQUID magnetometry measurements for complex 5.

The curves represent the best fit of experimental data according to the Curie-Weiss law (the best-fit parameters are g_iso = 2.18, C = 5.585×10⁻⁶ m³.mol^-1, and θ = -0.181 K with a temperature-independent paramagnetism term a_TIP = 3.40×10⁻⁸ m³.mol^-1). C₀ = 4.7141997×10⁻⁶ m³.mol^-1.

Fig 4. The molecular structure of complex 2.

Symmetry code used: ⁱ = -x+1,-y,-z+1.

Fig 5. Effect of complexes 1–7 on the NF-κB activity.

THP1-XBlue™-MD2-CD14 cell line were pre-treated with the complexes (100 nM, dissolved in PBS), [Cu(Law)₂(H₂O)₂]·0.5H₂O, HLaw and prednisone (1 μM, dissolved in DMSO) for 1 h. Subsequently, LPS (1 μg/mL) (A) or TNF-α (10 ng/mL) (B) or Pam3CSK4 (100 ng/mL) (C) was added [except for the control cells (basal)] to trigger the activation of NF-κB. After 24 h, the activity of NF-κB was evaluated based on the amount of the secreted alkaline phosphatase measured spectrophotometrically. The results are expressed as the mean ± S.E.M. for six independent experiments. ** Indicates a significant difference in comparison with the vehicle-treated cells p < 0.01, and **** indicates a significant difference in comparison with the vehicle-treated cells p < 0.0001.

Fig 6. The pro-oxidative effect of complexes 1–7, reference aqua-complex and lawsone determined by dichlorofluorescein method.

THP1-XBlue™-MD2-CD14 cell line were treated with the complexes, or standard antioxidant Trolox® at the final concentration of 100 nM for 1 h (left diagram) and 24 h (right diagram). The data represent the relative increase of fluorescence intensity after the addition of DCFH-DA in comparison with the untreated control. The results are expressed as the mean values ± S.E.M. for three independent experiments.

Fig 7. The effect of the tested complexes on the secretion of TNF-α.

THP-1 macrophages were pre-treated with the compounds 1–7, [Cu(Law)₂(H₂O)₂]·0.5H₂O (aqua-complex), HLaw (100 nM, dissolved in PBS) and prednisone (1 μM, dissolved in DMSO) for 1 h. Subsequently, LPS (1 μg/mL) was added [except for the control cells (basal)] to trigger the secretion of the pro-inflammatory cytokine TNF-α. After 24 h, the amount of the secreted TNF-α was evaluated by ELISA. The results are expressed as the mean values ± S.E.M. for three independent experiments. * Indicates a significant difference in comparison with the vehicle-treated cells p < 0.05, ** indicates a significant difference in comparison with the vehicle-treated cells p < 0.01, *** indicates a significant difference in comparison with the vehicle-treated cells p < 0.001, and **** indicates a significant difference in comparison with the vehicle-treated cells p < 0.0001.

Fig 8. The time-resolved changes in the average volume of the hind paws of rats.

The values are expressed as average values ± S.E.M. calculated for 7 animals in each experimental group. The copper(II) complexes were applied i.p. at the dose corresponding to 40 mmol Cu/kg (ca. 20 mg/kg), and indomethacin at the i.p. dose of 5 mg/kg.

Fig 9. Histological evaluation of inflammatory response in tissue sections of the hind paw, stained by hematoxylin/eosin (40x magnification).

The section of plantar tissues from the groups pretreated with complex 1 (A) or indomethacin (C) with the weak inflammatory response in the hypodermis with scarce PMN infiltrate. The tissue sections from the group exposed to complex 7 (B) and 10% DMF solution (control, D) with the strong inflammatory reaction in the hypodermis with massive focal PMN infiltrate.