Inhibition of C5aR1 as a promising approach to treat taxane-induced neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of several antitumor agents resulting in progressive and often irreversible damage of peripheral nerves. In addition to their known anticancer effects, taxanes, including paclitaxel, can also induce peripheral neuropathy by activating microglia and astrocytes, which release pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1ß), and chemokine (C-C motif) ligand 2 (CCL-2). All these events contribute to the maintenance of neuropathic or inflammatory response. Complement component 5a (C5a)/C5a receptor 1 (C5aR1) signaling was very recently shown to play a crucial role in paclitaxel-induced peripheral neuropathy. Our recent findings highlighted that taxanes have the previously unreported property of binding and activating C5aR1, and that C5aR1 inhibition by DF3966A is effective in preventing paclitaxel-induced peripheral neuropathy (PIPN) in animal models. Here, we investigated if C5aR1 inhibition maintains efficacy in reducing PIPN in a therapeutic setting. Furthermore, we characterized the role of C5aR1 activation by paclitaxel and the CIPN-associated activation of nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome. Our results clearly show that administration of the C5aR1 inhibitor strongly reduced cold and mechanical allodynia in mice when given both during the onset of PIPN and when neuropathy is well established. C5aR1 activation by paclitaxel was found to be a key event in the induction of inflammatory factors in spinal cord, such as TNF-α, ionized calcium-binding adapter molecule 1 (Iba-1), and glial fibrillary acidic protein (GFAP). In addition, C5aR1 inhibition significantly mitigated paclitaxel-induced inflammation and inflammasome activation by reducing IL-1ß and NLRP3 expression at both sciatic and dorsal root ganglia level, confirming the involvement of inflammasome in PIPN. Moreover, paclitaxel-induced upregulation of C5aR1 was significantly reduced by DF3966A treatment in central nervous system. Lastly, the antinociceptive effect of C5aR1 inhibition was confirmed in an in vitro model of sensory neurons in which we focused on receptor channels usually activated upon neuropathy. In conclusion, C5aR1 inhibition is proposed as a therapeutic option with the potential to exert long-term protective effect on PIPN-associated neuropathic pain and inflammation.

IL-1 beta primes IL-8-activated human neutrophils for elastase release, phospholipase D activity, and calcium flux.

Interleukin-8 (IL-8), the prototype of the alpha (e.i., C-X-C branch) chemokine family, induced elastase release in a concentration-dependent manner (50-1000 ng/mL) in cytochalasin B-treated human polymorphonuclear leukocytes (PMNs). This response was potentiated about twofold if PMNs were preexposed to interleukin-1 beta (IL-1 beta) at concentrations that were by themselves inactive. The effect of IL-1 beta was clearly observed after 5 min and was maximal after a 30-min preincubation of the cells. The effect was present over the whole active concentration range of IL-8 and was completely blocked by the presence of IL-1 receptor antagonist. Priming of elastase release by IL-1 beta was not associated with a change in receptor number or affinity for IL-8. On the contrary, it was correlated with priming of phospholipase D activity and calcium flux activated by IL-8. Preincubation of the cells with ethanol and/or La3+ inhibited IL-8-induced degranulations, suggesting that activation of phospholipase D and increase of [Ca2+]i were important for this response. In contrast, ethanol and La3+ did not decrease the priming effect of IL-1 beta. IL-8 and IL-1 beta have been shown to be released by the same cell types and may be concomitantly present at sites of inflammation, giving rise to an amplification of the inflammatory response.

Role of tumor necrosis factor-alpha in endotoxin-induced lung parenchymal hyporesponsiveness in mice.

Although changes in airway responsiveness in pulmonary inflammation are commonly related to the action of infiltrated leukocytes, our previous report suggested a direct role of inflammatory cytokines in LPS-induced lung hyporesponsiveness. The aim of this study was to define if cytokines detected in the BALF (bronchoalveolar lavage fluid) of intratracheal LPS-treated mice could be, at least in part, responsible for 5-HT (5-hydroxytryptamine) lung hyporeactivity. Our results show that intratracheal instillation of LPS induced a time-dependent increase in IL-(interleukin-)1beta, IL-6, and TNF (tumor necrosis factor)alpha in the BALF. Cytokine production was paralleled by 5-HT lung hyporesponsiveness, and intratracheal administration of TNFalpha proved to be very efficient in inhibiting 5-HT responsiveness. In addition, systemic treatment with rolipram, an inhibitor of TNFalpha production, was paralleled by a significant recovery of lung responsiveness. On the contrary, IL-1beta and IL-6 were not demonstrated to play a relevant role in 5-HT hyporesponsiveness. It is concluded that TNFalpha could be a crucial mediator of LPS-induced lung hyporesponsiveness.

Selective inhibition of interleukin-8-induced neutrophil chemotaxis by ketoprofen isomers.

Although it is commonly accepted that the anti-inflammatory effect of nonsteroidal anti-inflammatory drugs (NSAIDs) is mainly associated to their ability to inhibit the cyclooxygenase (COX) enzyme system, several results indicate that non-COX mechanisms could be important in the therapeutical effect of these drugs. The aim of this study was to define if NSAIDs could exert, at least in part, their anti-inflammatory effect by inhibiting the activities of human polymorphonuclear leukocytes (PMNs) triggered by chemotactic stimuli and, if so, to understand the relationship of this effect with COX inhibition. A unique opportunity to dissociate the inhibition of prostaglandin (PG) synthesis from other therapeutical properties of NSAIDs is constituted by ketoprofen isomers being the S-isomer 100 time more potent than R-isomer on COX inhibition. Our results show that R- and S-ketoprofen, independently of their potency as PG inhibitors, proved very efficacious in selective inhibition of interleukin-8 (IL-8) chemotaxis. Inhibition of IL-8 chemotaxis was not restricted to ketoprofen isomer as it could be observed also with drugs belonging to different classes of NSAIDs and it was obtained at drug concentration superimposable to plasma levels after therapeutic administration in patients. Reduction of IL-8 migration by ketoprofen isomers was paralleled by selective inhibition of PMN response in terms of intracellular calcium concentration ([Ca(2+)]i) increase and extracellular signal regulated kinase(ERK)-2 activation, two intracellular mediators reported to be critical for PMN activities. It is concluded that inhibition of IL-8 chemotaxis could represent a new clinical target for ketoprofen isomers and, in fact, contribute to the anti-inflammatory activity of NSAIDs.

Interleukin-1 beta primes interleukin-8-stimulated chemotaxis and elastase release in human neutrophils via its type I receptor.

Interleukin-1 (IL-1) is a pleiotropic proinflammatory cytokine which binds to human neutrophils (PMN) and can directly or indirectly activate their functions. In this study we show that a brief exposure to IL-1 beta induces a potentiation of both PMN elastase release and chemotactic response to interleukin-8 (IL-8), the prototype of C-X-C chemokines. Priming by IL-1 beta was maximal at 100 ng/ml, was completely blocked in the presence of IL-1 receptor antagonist (IL-1ra) and, in the chemotaxis assay, was best observed at suboptimal (3-6 ng/ml) or inactive (0.75 ng/ml) concentrations of IL-8. Priming of PMN by IL-1 beta was completely blocked by M1, a specific antibody against the type I IL-1 receptor (IL-1RI). On the other hand M22, an antibody directed against the IL-1 decoy type II IL-1 receptor did not affect IL-1 beta action and slightly increased the priming effect. Thus, exclusively via its type I receptor, IL-1 beta can act on PMN at multiple levels, by promoting their accumulation in tissues through the induction of chemotactic factors (e.g. IL-8) and the upregulation of adhesion molecules, and by priming their response to chemotactic agonists.

Effect of a mucoactive compound (CO 1408) on airway hyperreactivity and inflammation induced by passive cigarette smoke exposure in guinea-pigs.

Environmental exposure to tobacco smoke contributes to the onset of several lung diseases, e.g. chronic bronchitis and asthma, including an increase in airway reactivity. We have investigated the effect of a new mucoactive compound, CO 1408, on airway hyperreactivity and lung inflammation induced in guinea-pigs by passive cigarette smoke exposure. Animals were exposed to cigarette smoke in a Plexi-glass box, three times a day for four days. Airway reactivity to histamine was assessed ex-vivo in lung parenchymal strips. As a measure of lung inflammation, the number of leucocytes was evaluated in bronchoalveolar lavage (BAL) fluids and histological sections. Passive smoke exposure potentiated histamine-induced contraction in lung parenchymal strips, a phenomenon associated with an increase in proinflammatory cells in the BAL fluids and enhanced eosinophil infiltration into parenchymal tissues. Pretreatment with oral CO 1408 at 400 mg.kg-1 but not 100 mg.kg-1, completely prevented the cigarette smoke-induced airway hyperreactivity. 400 mg.kg-1 CO 1408 also inhibited the increase in cell numbers in the BAL fluids, but not eosinophil recruitment in parenchymal tissues. The present data indicate the ability of CO 1408 to modulate smoke-induced airway hyperreactivity and, to some extent, lung inflammation, an effect which might be of value in the therapy of obstructive pulmonary diseases.

Pharmacological studies on the new mucoactive agent (-)-6(S)-hydroxy-4(R)-(1-hydroxy-1-methylethyl)-1-cyclohexene-1-ethanol .

The pharmacological activity and acute toxicity of (-)-6(S)-hydroxy-4(R)-(1-hydroxy-1-methylethyl)-1-cyclohexene-1-ethanol (CO/1408, CAS 103079-06-7), a new mucoactive drug, were evaluated. After oral and intravenous administration CO/1408 increased the pulmonary secretion of fluorescein in rats, as an index of bronchosecretogogue activity. In addition, CO/1408 markedly increased the mucociliary transport rate. In in vitro study CO/1408 did not modify the viscosity of pig gastric mucin. Acute toxicity studies showed a very low toxicity after single dose indicating a high safety level for the doses used in this report. The results obtained point out the potential usefulness of CO/1408 to ameliorate the symptoms observed in some obstructive pulmonary diseases.

Synthesis and in vitro and in vivo evaluation of the 2-(6'methoxy-3',4'-dihydro-1'-naphtyl)-4H-3,1-benzoxazin-4- one as a new potent substrate inhibitor of human leukocyte elastase.

The title 2-vinyl-4H-3,1-benzoxazin-4-one has been synthesised and tested for inhibitory activity against human leukocyte elastase. The compound has shown activity both in vitro towards human sputum elastase and in vivo in an hemorrhagic assay.

Lipopolysaccharide-induced lung injury in mice. II. Evaluation of functional damage in isolated parenchyma strips.

Pulmonary inflammatory diseases are characterized by changes in airway responsiveness. This phenomenon is commonly related to the action of inflammatory mediators produced by infiltrated leukocytes. The aim of this study was to investigate in an ex vivo experimental model the effect of acute instillation of lipopolysaccharide (bacterial endotoxin; LPS) on lung parenchyma contractility. We firstly characterized the responsiveness of isolated murine lung to airway stimuli. Murine parenchymal strips were found to be mainly sensitive to 5-hydroxytryptamine (5-HT) while the cholinergic agonist, methacholine (MCh), evoked a smaller contractile response. 5-HT responsiveness was inhibited by methysergide. No significant parenchymal contraction was evoked by histamine, substance P and bradykinin. Lung responsiveness to 5-HT was significantly reduced by in vivo LPS treatment and this effect was only partially paralleled by leukocyte infiltration. In addition, LPS-induced hyporesponsiveness was significantly inhibited by betamethasone (BMS) or pentoxifylline (PTX) pretreatment suggesting that 5-HT lung hyporesponsiveness could be mediated by LPS-induced inflammatory mediators such as inflammatory cytokines.