RESUMO
N-Acylethanolamine acid amidase (NAAA) is an N-terminal cysteine hydrolase that stops the physiologic actions of palmitoylethanolamide, an endogenous lipid messenger that activates the transcription factor, peroxisome proliferator-activated receptor-α We have previously reported that the compound ARN19702 [(2-ethylsulfonylphenyl)-[(2S)-4-(6-fluoro-1,3-benzothiazol-2-yl)-2-methylpiperazin-1-yl]methanone] is an orally active, reversible NAAA inhibitor (IC50 on human NAAA = 230 nM) that produces remarkable protective effects against multiple sclerosis in mice. In the present study, we assessed the profile of ARN19702 in mouse and rat models of acute and neuropathic pain. Oral administration in male mice attenuated in a dose-dependent manner the spontaneous nocifensive response elicited by intraplantar formalin injection and the hypersensitivity caused by intraplantar carrageenan injection, paw incision, or sciatic nerve ligation. In male rats, ARN19702 reduced nociception associated with paclitaxel-induced neuropathy without development of subacute antinociceptive tolerance. Finally, ARN19702 (30 mg/kg, oral) did not produce place preference or alter exploratory motor behavior in male mice. The findings support the conclusion that NAAA is a suitable molecular target for the discovery of efficacious analgesic drugs devoid of rewarding potential. SIGNIFICANCE STATEMENT: This study evaluated the pharmacological profile of the orally bioavailable N-acylethanolamine acid amidase (NAAA) inhibitor (2-ethylsulfonylphenyl)-[(2S)-4-(6-fluoro-1,3-benzothiazol-2-yl)-2-methylpiperazin-1-yl]methanone (ARN19702) in mouse and rat models of neurogenic and inflammatory pain. The compound's potential rewarding and sedative effects were also examined. It is concluded that ARN19702 exhibits a broad analgesic profile that can be generalized across rodent species. The findings point to NAAA as a control node in the processing of neuropathic and inflammatory pain and to ARN19702 as a lead to uncover novel pain therapeutics devoid of addictive potential .
Assuntos
Etanolaminas , Amidoidrolases , Animais , Camundongos , RatosRESUMO
N-acylethanolamine acid amidase (NAAA), a cysteine hydrolase highly expressed in macrophages and B lymphocytes, catalyzes the degradation of palmitoylethanolamide. Palmitoylethanolamide is an agonist of PPAR-α and an important regulator of pain and innate immunity. In this study, we investigated the properties of the NAAA inhibitor, ARN077, in a mouse model of allergic contact dermatitis. Acute topical applications of ARN077 attenuated key signs of DNFB-induced dermatitis in a dose-dependent manner. Moreover, ARN077 increased tissue palmitoylethanolamide content and normalized circulating levels of cytokines and immunoglobulin E. No such effect was seen in PPAR-α-deficient mice. Moreover, mice lacking NAAA failed to develop edema or scratching behavior after challenge with DNFB, confirming that this enzyme plays an important role in dermatitis. Consistent with this conclusion, subchronic applications of ARN077 suppressed DNFB-induced inflammation when administered either before or after the DNFB challenge. The effects of subchronic ARN077 were dose dependent and comparable in size to those produced by the steroids clobetasol and dexamethasone. Unlike the latter, however, ARN077 did not cause skin atrophy. The results identify NAAA as a promising target for the development of effective and safe treatments for atopic dermatitis and other inflammatory disorders of the skin.
Assuntos
Amidoidrolases/antagonistas & inibidores , Carbamatos/uso terapêutico , Dermatite Alérgica de Contato/tratamento farmacológico , Éteres Cíclicos/uso terapêutico , Inflamação/tratamento farmacológico , Prurido/tratamento farmacológico , Amidas , Amidoidrolases/fisiologia , Animais , Dermatite Alérgica de Contato/etiologia , Dinitrofluorbenzeno , Modelos Animais de Doenças , Etanolaminas/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Palmíticos/análiseRESUMO
Macrophages are multi-faceted phagocytic effector cells that derive from circulating monocytes and undergo differentiation in target tissues to regulate key aspects of the inflammatory process. Macrophages produce and degrade a variety of lipid mediators that stimulate or suppress pain and inflammation. Among the analgesic and anti-inflammatory lipids released from these cells are the fatty acid ethanolamides (FAEs), which produce their effects by engaging nuclear peroxisome proliferator activated receptor-α (PPAR-α). Two members of this lipid family, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), have recently emerged as important intrinsic regulators of nociception and inflammation. These substances are released from the membrane precursor, N-acylphosphatidylethanolamine (NAPE), by the action of a NAPE-specific phospholipase D (NAPE-PLD), and in macrophage are primarily deactivated by the lysosomal cysteine amidase, N-acylethanolamine acid amidase (NAAA). NAPE-PLD and NAAA regulate FAE levels, exerting a tight control over the ability of these lipid mediators to recruit PPAR-α and attenuate the inflammatory response. This review summarizes recent findings on the contribution of the FAE-PPAR-α signaling complex in inflammation, and on NAAA inhibition as a novel mechanistic approach to treat chronic inflammatory disorders.
Assuntos
Inflamação/prevenção & controle , Lipídeos/farmacologia , Macrófagos/metabolismo , PPAR alfa/agonistas , Animais , Humanos , Lipídeos/fisiologiaRESUMO
Among the growing family of ribosomally synthesized, post-translationally modified peptides, particularly intriguing are class III lanthipeptides containing the triamino acid labionin. In the course of a screening program aimed at finding bacterial cell wall inhibitors, we discovered a new lanthipeptide produced by an Actinoplanes sp. The molecule, designated NAI-112, consists of 22 amino acids and contains an N-terminal labionin and a C-terminal methyl-labionin. Unique among lanthipeptides, it carries a 6-deoxyhexose moiety N-linked to a tryptophan residue. Consistently, the corresponding gene cluster encodes, in addition to the LanKC enzyme characteristic of this lanthipeptide class, a glycosyl transferase. Despite possessing weak antibacterial activity, NAI-112 is effective in experimental models of nociceptive pain, reducing pain symptoms in mice in both the formalin and the chronic constriction injury tests. Thus, NAI-112 represents, after the labyrinthopeptins, the second example of a lanthipeptide effective against nociceptive pain.
Assuntos
Analgésicos/química , Antibacterianos/química , Bacteriocinas/química , Micromonosporaceae/química , Peptídeos/química , Sequência de Aminoácidos , Analgésicos/metabolismo , Analgésicos/uso terapêutico , Animais , Antibacterianos/metabolismo , Antibacterianos/uso terapêutico , Bacteriocinas/genética , Bacteriocinas/uso terapêutico , Genes Bacterianos , Glicosilação , Masculino , Camundongos , Micromonosporaceae/genética , Dados de Sequência Molecular , Família Multigênica , Dor/tratamento farmacológico , Peptídeos/genética , Peptídeos/uso terapêuticoRESUMO
Fatty acid ethanolamides (FAEs), which include palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), are endogenous agonists of peroxisome proliferator-activated receptor-α (PPAR-α) and important regulators of the inflammatory response. They are degraded in macrophages by the lysosomal cysteine amidase, N-acylethanolamine acid amidase (NAAA). Previous studies have shown that pharmacological inhibition of NAAA activity suppresses macrophage activation in vitro and causes marked anti-inflammatory effects in vivo, which is suggestive of a role for NAAA in the control of inflammation. It is still unknown, however, whether NAAA-mediated FAE deactivation might regulate pain signaling. The present study examined the effects of ARN077, a potent and selective NAAA inhibitor recently disclosed by our group, in rodent models of hyperalgesia and allodynia caused by inflammation or nerve damage. Topical administration of ARN077 attenuated, in a dose-dependent manner, heat hyperalgesia and mechanical allodynia elicited in mice by carrageenan injection or sciatic nerve ligation. The antinociceptive effects of ARN077 were prevented by the selective PPAR-α antagonist GW6471 and did not occur in PPAR-α-deficient mice. Furthermore, topical ARN077 reversed the allodynia caused by ultraviolet B radiation in rats, and this effect was blocked by pretreatment with GW6471. Sciatic nerve ligation or application of the proinflammatory phorbol ester 12-O-tetradecanoylphorbol 13-acetate decreased FAE levels in sciatic nerve and skin tissue, respectively. ARN077 reversed these biochemical effects. The results identify ARN077 as a potent inhibitor of intracellular NAAA activity, which is active in vivo by topical administration. The findings further suggest that NAAA regulates peripheral pain initiation by interrupting endogenous FAE signaling at PPAR-α.
Assuntos
Amidoidrolases/antagonistas & inibidores , Analgésicos/uso terapêutico , Carbamatos/uso terapêutico , Endocanabinoides/fisiologia , Inibidores Enzimáticos/uso terapêutico , Éteres Cíclicos/uso terapêutico , Hiperalgesia/tratamento farmacológico , Ácidos Oleicos/fisiologia , PPAR alfa/fisiologia , Percepção da Dor/efeitos dos fármacos , Amidas , Amidoidrolases/genética , Amidoidrolases/fisiologia , Analgésicos/administração & dosagem , Analgésicos/farmacologia , Animais , Queimaduras/tratamento farmacológico , Queimaduras/etiologia , Carbamatos/administração & dosagem , Carbamatos/farmacologia , Carragenina/toxicidade , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Etanolaminas , Éteres Cíclicos/administração & dosagem , Éteres Cíclicos/farmacologia , Células HEK293 , Humanos , Hiperalgesia/induzido quimicamente , Hiperalgesia/fisiopatologia , Lisossomos/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , PPAR alfa/agonistas , PPAR alfa/deficiência , Percepção da Dor/fisiologia , Ácidos Palmíticos , Lesões por Radiação/tratamento farmacológico , Lesões por Radiação/etiologia , Ratos , Proteínas Recombinantes de Fusão/fisiologia , Nervo Isquiático/lesões , Acetato de Tetradecanoilforbol/toxicidade , Raios Ultravioleta/efeitos adversosRESUMO
Microglia respond rapidly to injury, increasing their synthesis and release of inflammatory mediators, many of which contribute to the maintenance of persistent pain following CNS or PNS injury. We have recently shown that the lysosomal cysteine protease Cathepsin S (CatS) expressed by spinal microglia is vital for the full expression of neuropathic pain. Here we evaluated the mechanisms by which CatS release occurs from primary microglia in culture. Stimulation of microglia with lipopolysaccharide (LPS) or adenosine tri-phosphate (ATP) alone was insufficient to induce release of enzymatically active CatS in extracellular media. However, following priming with LPS, ATP at 1 mM but not 50 µM resulted in significant release of CatS in the media and maturation of CatS protein in cell extracts. The enzymatic activity measured in media at neutral pH was specific for CatS as it was completely prevented by the CatS inhibitor LHVS. ATP-induced release of CatS required potassium efflux and both extracellular calcium influx and mobilization of intracellular calcium. Pharmacological modulation of ATP-induced release of CatS enzymatic activity revealed that this was dependent on activation of the P2X7 receptor and intracellular phospholipase C and phospholipase A(2). In addition, ATP-induced CatS release involved p38 mitogen activated protein kinase (MAPK) phosphorylation, but not ERK and PI3K signalling pathways. Thus, as high concentration of extracellular ATP promotes release of active CatS from microglia via P2X7 receptor activation, we suggest that the inhibition of CatS release is one of the mechanisms responsible for P2X7 antagonist efficacy in neuropathic pain.