Receptor binding mode and pharmacological characterization of a potent and selective dual CXCR1/CXCR2 non-competitive allosteric inhibitor.

BACKGROUND AND PURPOSE: DF 2156A is a new dual inhibitor of IL-8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential. EXPERIMENTAL APPROACH: The binding mode, molecular mechanism of action and selectivity were investigated using chemotaxis of L1.2 transfectants and human leucocytes, in addition to radioligand and [(35) S]-GTPγS binding approaches. The therapeutic potential of DF 2156A was evaluated in acute (liver ischaemia and reperfusion) and chronic (sponge-induced angiogenesis) experimental models of inflammation. KEY RESULTS: A network of polar interactions stabilized by a direct ionic bond between DF 2156A and Lys(99) on CXCR1 and the non-conserved residue Asp(293) on CXCR2 are the key determinants of DF 2156A binding. DF 2156A acted as a non-competitive allosteric inhibitor blocking the signal transduction leading to chemotaxis without altering the binding affinity of natural ligands. DF 2156A effectively and selectively inhibited CXCR1/CXCR2-mediated chemotaxis of L1.2 transfectants and leucocytes. In a murine model of sponge-induced angiogenesis, DF 2156A reduced leucocyte influx, TNF-α production and neovessel formation. In vitro, DF 2156A prevented proliferation, migration and capillary-like organization of HUVECs in response to human IL-8. In a rat model of liver ischaemia and reperfusion (I/R) injury, DF 2156A decreased PMN and monocyte-macrophage infiltration and associated hepatocellular injury. CONCLUSION AND IMPLICATIONS: DF 2156A is a non-competitive allosteric inhibitor of both IL-8 receptors CXCR1 and CXCR2. It prevented experimental angiogenesis and hepatic I/R injury in vivo and, therefore, has therapeutic potential for acute and chronic inflammatory diseases.

Protective effect of an inhibitor of interleukin-8 (meraxin) from ischemia and reperfusion injury in a rat model of kidney transplantation.

INTRODUCTION: Since the ischemia and reperfusion injury is one of the main causes of delayed graft function after transplantation, research efforts have focused on studying the molecules involved in this inflammatory process. The chemokine interleukin-8 (IL-8) seems to be the main one responsible through a chemoattractive action toward neutropils. Therefore, one of the strategies adopted to prevent this process is blocking the binding between IL-8 and its receptors. The aim of our study was to test the effect of meraxin, a new derivative from repertaxin, to protect the renal graft from ischemia and reperfusion injury. MATERIALS AND METHODS: Eighty male syngenic rats were divided into four groups. The control group underwent only kidney transplantation, while the other groups were treated with meraxin at various dosages 2 hours before graft reperfusion. Blood and histological samples were taken at sacrifice 24 hours after transplantation. RESULTS: Creatinine was significantly lower in the group treated with the high dosage of meraxin. Histological observation of the grafted tissue showed instead only a mild and not significant neutrophilic infiltration, equal in each group. CONCLUSIONS: Graft function was improved by the administration of meraxin at high dosage, but this effect did not seem to be connected to a reduction in inflammatory infiltration in the parechymal tissue. Maybe the cause is in the mechanisms of clotting activation, due to alteration of adhesion molecules and endothelial cells.

Neutrophil recruitment in the reperfused-injured rat liver was effectively attenuated by repertaxin, a novel allosteric noncompetitive inhibitor of CXCL8 receptors: a therapeutic approach for the treatment of post-ischemic hepatic syndromes.

Hepatic reperfusion injury represents a crucial problem in several clinical situations including liver transplantation, extensive hepatectomy and hypovolemic shock with resuscitation. Repertaxin is a new non-competitive allosteric blocker of interleukin-8 (CXCL8) receptors, which by locking CXCR1/R2 in an inactive conformation, prevents receptor signaling and polymorphonuclear leukocyte (PMN) chemotaxis. The present study shows that repertaxin dramatically prevents rat post-ischemic hepatocellular necrosis (80% of inhibition) and PMN infiltration (96% of inhibition) at a clinically-relevant time (24 h) of reperfusion. Treatment with repertaxin by continuous infusion is demonstrated to be the optimal route of administration of the compound especially in view of its clinical therapeutic use. Because repertaxin has proven to be safe and well tolerated in different animal studies and in phase I studies in human volunteers, it is in fact a candidate novel therapeutic agent for the prevention and treatment of hepatic post-ischemic injury.

Evidence of an increased nitric oxide production in primary biliary cirrhosis.

OBJECTIVE: Although possible implications of nitric oxide in the pathophysiology of liver cirrhosis have been extensively studied, until now few articles have addressed the assessment of nitric oxide production in primary biliary cirrhosis. This study was directed to evaluate circulating nitrosyl-hemoglobin levels as well as neutrophil elastase and soluble adhesion molecule concentrations in this condition, by assuming these parameters as possible markers of either inflammatory response or neutrophil activation. METHODS: Laboratory investigations were performed in 30 patients with primary biliary cirrhosis, in 13 patients with postviral and/or alcoholic cirrhosis, and in a group of eight subjects with chronic hepatitis. RESULTS: Although no difference was detected with respect to chronic hepatitis subjects, higher levels of nitrosyl-hemoglobin adducts were found in primary biliary cirrhosis patients than in postviral or alcoholic cirrhotics and in normal subjects (3.55+/-1.75 arbitrary units vs 1.95+/-0.57 and 0.84+/-0.34, p = 0.0004 and p < 0.0001, respectively). Similarly, more elevated concentrations of neutrophil elastase (213.7+/-192.0 microg/L vs 51.1+/-34.3 and 38.0+/-11.5, p < 0.0001 and p < 0.0001, respectively) as well as of soluble forms of intercellular adhesion molecule 1 and endothelial-leukocyte adhesion molecule 1 were shown in primary biliary cirrhosis patients than in subjects with cirrhosis of other etiologies and in controls. CONCLUSIONS: Highly enhanced nitric oxide production in primary biliary cirrhosis could be related to the development of strong inflammation and at least partially to neutrophil activation, thus suggesting a putative role of these cellular mediators in the development of liver damage owing to their ability to synthesize and release a wide variety of important factors, including elastase and nitric oxide.

Lidocaine inhibition of the hyperpolarization-activated current (I(f)) in sinoatrial myocytes.

The aim of this study was to provide information on the dose dependence and biophysical details of lidocaine blockade of the hyperpolarization-activated current (I(f)) in the sinoatrial node. Isolated rabbit sinoatrial myocytes were patch-clamped in the whole-cell configuration at 36+/-0.5 degrees C, in the presence of 1 mM Ba2+ and 2 mM Mn2+ to minimize contamination by K+ and Ca2+ currents, respectively. Lidocaine inhibited I(f) dose-dependently with a maximal inhibition of 69.5% at 75 microM and a half-maximal effect at 38.2 microM. Lidocaine reduced the conductance of fully activated I(f), without affecting the current reversal potential; the blocking effect was independent of membrane potential. Voltage dependence of I(f) activation gating was not affected by lidocaine, whose effect was independent of use and rate. Lidocaine did not modify the time course of I(f) activation. At therapeutic concentrations, lidocaine significantly inhibited I(f) by reducing fully activated channel conductance. Lack of voltage and rate dependence of effect differentiates lidocaine from most of other blockers of this current.