Effect of 2-Chloro-Substitution of Adenine Moiety in Mixed-Ligand Gold(I) Triphenylphosphine Complexes on Anti-Inflammatory Activity: The Discrepancy between the <i>In Vivo</i> and <i>In Vitro</i> Models

<div><p>A series of gold(I) triphenylphosphine (PPh₃) complexes (1–9) involving 2-chloro-<i>N</i>6-(substituted-benzyl)adenine derivatives as N-donor ligands was synthesized and thoroughly characterized by relevant methods, including electrospray-ionization (ESI) mass spectrometry and multinuclear NMR spectroscopy. The anti-inflammatory and antiedematous effects of three representatives <b>1</b>, 5 and 9 were evaluated by means of <i>in vitro</i> model based on the expression of pro- and anti-inflammatory cytokines and influence of the complexes on selected forms of matrix metalloproteinases secreted by LPS-activated THP-1 monocytes and <i>in vivo</i> model evaluating the antiedematous effect of the complexes in the carrageenan-induced rat hind-paw edema model. In addition to the pharmacological observations, the affected hind paws were <i>post mortem</i> subjected to histological and immunohistochemical evaluations. The results of both <i>in vivo</i> and <i>ex vivo</i> methods revealed low antiedematous and anti-inflammatory effects of the complexes, even though the <i>in vitro</i> model identified them as promising anti-inflammatory acting compounds. The reason for this discrepancy lies probably in low stability of the studied complexes in biological environment, as demonstrated by the solution interaction studies with sulfur-containing biomolecules (cysteine and reduced glutathione) using the ESI mass spectrometry.</p> </div

Effects of the gold(I) complexes and the reference drug Auranofin on the LPS-induced IL-1β secretion.

<p>The cells were pre-treated with complexes 1, 5 and 9 (100 nM - depicted in dark blue; 600 nM - depicted in light blue) and Auranofin (100 nM), or the vehicle (DMSO) only. After 1 h of the incubation, the inflammatory response was induced by the LPS [except for the control cells]. The secretion was measured 24 h after the LPS addition. The results are expressed as means for three independent experiments. *** Significant difference in comparison to vehicle-treated cells (<i>p</i> < 0.001), # significant difference in comparison to Auranofin-treated cells (<i>p</i> < 0.05), ## significant difference in comparison to Auranofin-treated cells (<i>p</i> < 0.01), ### significant difference in comparison to Auranofin-treated cells (<i>p</i> < 0.001).</p

Histologic sections of the hind paw, stained with Gömöri trichrome staining (a) and Hematoxylin-eosin (b-d) (photographed at 40x magnification).

<p>Tissue exposed to 25% DMSO (control; a), and complex 5 (b) with the massive acute inflammatory reaction in the transition from dermis to hypodermis with the massive infiltrate of neurophils (PMN) in connective tissues; tissue exposed to complex 9 (c) with the massive acute inflammatory reaction reaching from the hypodermis up to the papillary layer of dermis with a massive infiltrate of neurophils (PMN); tissue exposed to Auranofin (d) with the inflammatory reaction in the hypodermis with a slight PMN infiltrate. 1 - PMN infiltrate, 2 - arteriola, 3 - dilated veins with haemostasis, 4 - vein with PMN presence in the blood stream and haemostasis, 5 - collagen connective tissue, 6 - haemorrhage characterised by the presence of erythrocytes in interstitium, 7 - muscle fibres. </p

The time-dependent profile of antiedematous effect of tested compounds on carrageenan induced hind paw edema in rats.

<p>The time-dependent profile of antiedematous effect of tested compounds on carrageenan induced hind paw edema in rats.</p

Cytotoxicity of complexes 1, 5, and 9 on THP-1 cells.

<p>THP-1 cells were treated with the decreasing concentration (10–0.039 μM) of 1, 5, and 9, respectively. The number of metabolically active cells was determined by the WST-1 test after 24 h of incubation. The viability was calculated in comparison to the vehicle-treated cells. The results are expressed as means ± S.E. for three independent experiments.</p

Schematic representation of complexes 1–9.

<p>R symbolizes hydrogen for HL₁ and 1, 3-fluoro for HL₂ and 2, 2-chloro for HL₃ and 3, 3-chloro for HL₄ and 4, 2-methoxy for HL₅ and 5, 3-methoxy for HL₆ and 6, 4-methoxy for HL₇ and 7, 4-hydroxy for HL₈ and 8 and 4-methyl for HL₉ and 9.</p

Effects of the gold(I) complexes and the reference drug Auranofin on the LPS-induced TNF-α secretion.

<p>The cells were pretreated with complexes 1, 5 and 9 (100 nM - depicted in dark blue; 600 nM - depicted in light blue), and Auranofin (100 nM), or the vehicle (DMSO) only. After 1 h of the incubation, the inflammatory response was induced by LPS [except for the control cells]. The secretion was measured 24 h after the LPS addition. The results are expressed as means for three independent experiments. * Significant difference in comparison to vehicle-treated cells (p < 0.05), *** significant difference in comparison to vehicle-treated cells (p < 0.001), ### significant difference in comparison to Auranofin-treated cells (p < 0.001).</p

The results of <i>in vitro</i> cytotoxicity of complexes1–5 and <i>cisplatin</i> against variety of human cancer and healthy cell lines.

<p>Cells were treated with the tested compounds for 24 h; measurements were performed in triplicate, and cytotoxicity experiments were repeated on three different cell passages; data are expressed as IC₅₀±S.E. (µM).</p><p>n.d. – not determined; asterisk (*) symbolizes significant difference (p<0.05) in <i>in vitro</i> cytotoxicity of <b>1</b>–<b>5</b> as compared with <i>cisplatin</i>.</p><p>The results of <i>in vitro</i> cytotoxicity of complexes1–5 and <i>cisplatin</i> against variety of human cancer and healthy cell lines.</p