Injection Of Platelet-activating Factor Research Articles

Replacement of L-amino acid peptides with D-amino acid peptides mitigates anti-PEG antibody generation against polymer-peptide conjugates in mice

The generation of anti-PEG antibodies in response to PEGylated proteins, peptides, and carriers significantly limits their clinical applicability. IgM antibodies mediate the clearance of these therapeutics upon repeat injection, resulting in toxicity and hindered therapeutic efficacy. We observed this phenomenon in our polymer platform, virus-inspired polymer for endosomal release (VIPER), which employs pH-sensitive triggered display of a lytic peptide, melittin, to facilitate endosomal escape. While the polymer-peptide conjugate was well tolerated after a single injection, we observed unexpected mortality upon repeat injection. Thus, the goal of this work was to enhance the safety and tolerability of VIPER for frequent dosing. Based on previous reports on anti-PEG antibodies and the adjuvant activity of melittin, we characterized the antibody response to polymer, peptide, and polymer-peptide conjugates after repeat-dosing and measured high IgM titers that bound PEG. By substituting the L-amino acid peptide for its D-amino acid enantiomer, we significantly attenuated the anti-PEG antibody generation and toxicity, permitting repeat-injections. We attempted to rescue mice from L-melittin induced toxicity by prophylactic injection of platelet activating factor (PAF) antagonist CV-6209, but observed minimal effect, suggesting that PAF is not the primary mediator of the observed hypersensitivity response. Overall, we demonstrated that the D-amino acid polymer-peptide conjugates, unlike L-amino acid polymer-peptide conjugates, exhibit good tolerability in vivo, even upon repeat administration, and do not elicit the generation of anti-PEG antibodies.

Reversible cross-tolerance to platelet-activating factor signaling by bacterial toxins

Bacterial toxins signaling through Toll-like receptors (TLRs) are implicated in the pathogenesis of many inflammatory diseases. Among the toxins, lipopolysaccharide (LPS) exerts its action via TLR-4 while lipoteichoic acid (LTA) and bacterial lipoproteins such as Braun lipoprotein (BLP) or its synthetic analogue Pam3CSK4 act through TLR-2. Part of the TLR mediated pathogenicity is believed to stem from endogenously biosynthesized platelet-activating factor (PAF)- a potent inflammatory phospholipid acting through PAF-receptor (PAF-R). However, the role of PAF in inflammatory diseases like endotoxemia is controversial. In order to test the direct contribution of PAF in TLR-mediated pathogenicity, we intraperitoneally injected PAF to Wistar albino mice in the presence or absence of bacterial toxins. Intraperitoneal injection of PAF (5 μg/mouse) causes sudden death of mice, that can be delayed by simultaneously or pre-treating the animals with high doses of bacterial toxins- a phenomenon known as endotoxin cross-tolerance. The bacterial toxins- induced tolerance to PAF can be reversed by increasing the concentration of PAF suggesting the reversibility of cross-tolerance. We did similar experiments using human platelets that express both canonical PAF-R and TLRs. Although bacterial toxins did not induce human platelet aggregation, they inhibited PAF-induced platelet aggregation in a reversible manner. Using rabbit platelets that are ultrasensitive to PAF, we found bacterial toxins (LPS and LTA) and Pam3CSK4 causing rabbit platelet aggregation via PAF-R dependent way. The physical interaction of PAF-R and bacterial toxins is also demonstrated in a human epidermal cell line having stable PAF-R expression. Thus, we suggest the possibility of direct physical interaction of bacterial toxins with PAF-R leading to cross-tolerance.

P38 MAP-kinase inhibitor protects against platelet-activating factor-induced death in mice

Platelet-activating factor (PAF) is a potent inflammatory agonist. In Swiss albino mice, intraperitoneal injection of PAF causes sudden death with oxidative stress and disseminated intravascular coagulation (DIC), characterized by prolonged prothrombin time, thrombocytopenia, reduced fibrinogen content, and increased levels of fibrinogen degradation products. However, the underlying mechanism(s) is unknown. The PAF-R antagonist WEB-2086 protected mice against PAF-induced death by reducing DIC and oxidative stress. Accordingly, general antioxidants such as ascorbic acid, α-tocopherol, gallic acid, and N-acetylcysteine partially protected mice from PAF-induced death. N-acetylcysteine, a clinically used antioxidant, prevented death in 67% of mice, ameliorated DIC characteristics and histological alterations in the liver, and reduced oxidative stress. WEB-2086 suppressed H2O2-mediated oxidative stress in isolated mouse peritoneal macrophages, suggesting that PAF signaling may be a downstream effector of reactive oxygen species generation. PAF stimulated all three (ERK, JNK, and p38) of the MAP-kinases, which were also inhibited by N-acetylcysteine. Furthermore, a JNK inhibitor (SP600125) and ERK inhibitor (SCH772984) partially protected mice against PAF-induced death, whereas a p38 MAP-kinase inhibitor (SB203580) provided complete protection against DIC and death. In human platelets, which have the canonical PAF-R and functional MAP-kinases, JNK and p38 inhibitors abolished PAF-induced platelet aggregation, but the ERK inhibitor was ineffective. Our studies identify p38 MAP-kinase as a critical, but unrecognized component in PAF-induced mortality in mice. These findings suggest an alternative therapeutic strategy to address PAF-mediated pathogenicity, which plays a role in a broad range of inflammatory diseases.

Characterization of platelet-activating factor-induced cutaneous edema and erythema in dogs.

OBJECTIVE To characterize platelet-activating factor (PAF)-induced edema and erythema in the skin of dogs and compare those reactions with histamine-induced cutaneous reactions. ANIMALS 6 healthy Beagles. PROCEDURES Experiments were performed at ≥ 2-week intervals. Each dog received ID injections (5 μg/site) of PAF C16, PAF C18, lyso-PAF, and histamine. Edema (mean diameter) and erythema scores (none, mild, moderate, or severe) were assessed 30 minutes after the injections. Dogs received ID injections of PAF and histamine each with various concentrations of WEB 2086 (PAF receptor antagonist) or underwent ID testing with PAF and histamine before and 3 hours after oral administration of cetirizine hydrochloride or prednisolone (at 2 doses each). RESULTS ID injections of PAF C16 and PAF C18, but not lyso-PAF, induced comparable levels of edema and erythema. The PAF-induced edema and erythema peaked at 30 minutes and lasted for 6 hours after the injection; histamine-induced edema and erythema peaked at 30 minutes and lasted for 3 hours after the injection. Edema sizes and erythema scores were significantly smaller and lower, respectively, for PAF than for histamine. The WEB 2086 inhibited PAF-induced but not histamine-induced edema and erythema. Cetirizine slightly, but significantly, repressed PAF-induced edema and erythema as well as histamine-induced cutaneous reactions. Prednisolone suppressed both PAF-induced and histamine-induced edema and erythema. CONCLUSIONS AND CLINICAL RELEVANCE In canine skin, the duration of PAF-induced inflammation was longer than that of histamine-induced inflammation. The PAF- and histamine-induced cutaneous reactions were effectively suppressed by oral administration of prednisolone. The importance of PAF in dogs with anaphylaxis and allergic disorders warrants further investigation.

Erythrocyte deformability — A partner of the inflammatory response

We aim to establish an in vivo animal model of acute inflammation using PAF (platelet activating factor) as inflammatory agent and to study the erythrocyte deformability changes induced by the inflammatory response. Counting the number of rolling and adherent neutrophils to endothelium after 2, 4 and 6h of intrascrotal injection of PAF we showed the induction of an inflammatory state. Blood samples are collected in order to measure the erythrocyte deformability and to quantify NO efflux from the red blood cells (RBCs). The results show an increased number of rolling and adherent neutrophils after 2h and 4h of inflammation as well as decreased values of erythrocyte deformability in the same time-points. This result is in line with the need of a low blood viscosity to the recruitment process that will improve leukocyte migration towards the endothelial wall. NO efflux from RBCs is also affected by the inflammatory response at the first hours of inflammation. This animal model demonstrates in vivo the association between an acute inflammatory response and the rheological properties of the blood, namely the RBCs deformability. For those reasons we consider this as an adequate model to study acute inflammatory responses as well as hemorheological parameters.

The responses of pulmonary and systemic circulation and airway to anaphylactic mediators in anesthetized BALB/c mice

AimsAnaphylactic shock sometimes accompanies pulmonary vaso- and broncho-constriction. We previously reported the hemodynamic features of mouse anaphylaxis (Life Sci. 2014; 116: 98–105). However, the effects of anaphylactic chemical mediators on the hemodynamics of in vivo mice are not well known. Furthermore, it is uncertain whether the mediators exert the same directional actions. Therefore, we determined their effects systematically on total peripheral resistance (TPR), pulmonary vascular resistance (PVR), or airway pressure (AWP) in anesthetized mice. Main methodsWe measured directly pulmonary arterial pressure, left atrial pressure, systemic arterial pressure, central venous pressure and aortic blood flow to determine PVR and TPR, as well as AWP, following injections of platelet-activating factor (PAF), histamine, serotonin, leukotriene (LT) C4, and prostaglandin (PG) D2 in anesthetized open-chest artificially ventilated BALB/c mice. Key findingsConsecutive administration of any agents increased PVR dose-dependently with the maximal responsiveness being PAF>LTC4>serotonin>>histamine=PGD2. Histamine caused a biphasic PVR response, an initial decrease, which was abolished by L-NAME, followed by an increase at high doses. PAF, serotonin, and histamine decreased TPR dose-dependently, while LTC4 or PGD2 yielded an increase or no change in TPR, respectively. Serotonin, but not the other agents, increased AWP. SignificanceAnaphylactic mediators exert non-uniform actions on the pulmonary and systemic circulation and airway in anesthetized BALB/c mice: PAF, LTC4 and serotonin cause substantial pulmonary vasoconstriction, while histamine biphasic responses of the initial nitric oxide dependent vasodilation followed by vasoconstriction; PAF, serotonin, and histamine, but not LTC4 or PGD2, evoke systemic vasodilatation; only serotonin induces airway constriction.

The Responses of Pulmonary and Systemic Circulation and Airway to Allergic Mediators in Anesthetized Rats.

Lung allergic diseases sometimes accompany pulmonary vaso- and broncho-constriction. Rats are currently used for the experimental study of lung allergies. However, their hemodynamic mechanisms are not fully understood. Therefore the effects of allergic mediators were determined systematically in vivo in rats in terms of pulmonary vascular resistance (PVR), airway pressure (AWP) and total peripheral resistance (TPR). We directly measured pulmonary arterial pressure, left atrial pressure, systemic arterial pressure, central venous pressure and aortic blood flow to determine PVR and TPR, as well as AWP, following injections of platelet-activating factor (PAF), histamine, serotonin, leukotriene (LT) C4, and prostaglandin (PG) D2 in anesthetized open-chest artificially ventilated Sprague-Dawley (SD) rats. PVR was dose-dependently increased by consecutive administration of PAF, LTC4, and PGD2, with the maximal responsiveness being PAF>LTC4>PGD2. However, neither histamine nor serotonin changed PVR. TPR was decreased by all agents except LTC4 which actually increased it. PAF and serotonin, but not the other agents, increased AWP. In conclusion, allergic mediators exert non-uniform actions on pulmonary and systemic circulation and airways in anesthetized SD rats: PAF, LTC4 and PGD2, but not histamine or serotonin, caused substantial pulmonary vasoconstriction; LTC4 yielded systemic vasoconstriction, while the others caused systemic vasodilatation; only two mediators, PAF and serotonin, induce airway constriction.

Steroid receptor coactivators 1 and 2 mediate fetal-to-maternal signaling that initiates parturition.

The precise mechanisms that lead to parturition are incompletely defined. Surfactant protein-A (SP-A), which is secreted by fetal lungs into amniotic fluid (AF) near term, likely provides a signal for parturition; however, SP-A-deficient mice have only a relatively modest delay (~12 hours) in parturition, suggesting additional factors. Here, we evaluated the contribution of steroid receptor coactivators 1 and 2 (SRC-1 and SRC-2), which upregulate SP-A transcription, to the parturition process. As mice lacking both SRC-1 and SRC-2 die at birth due to respiratory distress, we crossed double-heterozygous males and females. Parturition was severely delayed (~38 hours) in heterozygous dams harboring SRC-1/-2-deficient embryos. These mothers exhibited decreased myometrial NF-κB activation, PGF2α, and expression of contraction-associated genes; impaired luteolysis; and elevated circulating progesterone. These manifestations also occurred in WT females bearing SRC-1/-2 double-deficient embryos, indicating that a fetal-specific defect delayed labor. SP-A, as well as the enzyme lysophosphatidylcholine acyltransferase-1 (LPCAT1), required for synthesis of surfactant dipalmitoylphosphatidylcholine, and the proinflammatory glycerophospholipid platelet-activating factor (PAF) were markedly reduced in SRC-1/-2-deficient fetal lungs near term. Injection of PAF or SP-A into AF at 17.5 days post coitum enhanced uterine NF-κB activation and contractile gene expression, promoted luteolysis, and rescued delayed parturition in SRC-1/-2-deficient embryo-bearing dams. These findings reveal that fetal lungs produce signals to initiate labor when mature and that SRC-1/-2-dependent production of SP-A and PAF is crucial for this process.

Fetal-to-maternal signaling to initiate parturition.

Multiple processes are capable of activating the onset of parturition; however, the specific contributions of the mother and the fetus to this process are not fully understood. In this issue of the JCI, Gao and colleagues present evidence that steroid receptor coactivators 1 and 2 (SRC-1 and SRC-2) regulate surfactant protein-A (SP-A) and platelet-activating factor (PAF) expression, which increases in the developing fetal lung. WT dams crossed with males deficient for both SRC-1 and SRC-2 had suppressed myometrial inflammation, increased serum progesterone, and delayed parturition, which could be reconciled by injection of either SP-A or PAF into the amnion. Together, the results of this study demonstrate that the fetal lungs produce signals to initiate labor in the mouse. This work underscores the importance of the fetus as a contributor to the onset of murine, and potentially human, parturition.

Sphingosine-1-phosphate receptor 2 protects against anaphylactic shock through suppression of endothelial nitric oxide synthase in mice

Sphingosine-1-phosphate receptor 2 (S1P(2)) is expressed in vascular endothelial cells (ECs). However, the role of S1P(2) in vascular barrier integrity and anaphylaxis is not well understood. Endothelial nitric oxide synthase (eNOS) generates nitric oxide to mediate vascular leakage, compromising survival in patients with anaphylaxis. We recently observed that endothelial S1P(2) inhibits Akt, an activating kinase of eNOS. We tested the hypothesis that endothelial S1P(2) might suppress eNOS, exerting a protective effect against endothelial barrier disruption and anaphylaxis. Mice deficient in S1P(2) and eNOS underwent antigen challenge or platelet-activating factor (PAF) injection. Analyses were performed to examine vascular permeability and the underlying mechanisms. S1pr2 deletion augmented vascular leakage and lethality after either antigen challenge or PAF injection. PAF injection induced activation of Akt and eNOS in the aortas and lungs of S1pr2-null mice, which were augmented compared with values seen in wild-type mice. Consistently, PAF-induced increase in cyclic guanosine monophosphate levels in the aorta was enhanced in S1pr-null mice. Genetic Nos3 deletion or pharmacologic eNOS blockade protected S1pr2-null mice from aggravation of barrier disruption after antigen challenge and PAF injection. ECs isolated from S1pr2-null mice exhibited greater stimulation of Akt and eNOS, with enhanced nitric oxide production in response to sphingosine-1-phosphate or PAF, compared with that seen in wild-type ECs. Moreover, S1pr2-deficient ECs showed more severe disassembly of adherens junctions with augmented S-nitrosylation of β-catenin in response to PAF, which was restored by pharmacologic eNOS blockade. S1P(2) diminishes harmful robust eNOS stimulation and thereby attenuates vascular barrier disruption, suggesting potential usefulness of S1P(2) agonists as novel therapeutic agents for anaphylaxis.

Platelet‐activating factor (PAF) induces wheal and flare skin reactions independent of mast cell degranulation

Platelet-activating factor (PAF) causes wheal and flare responses which are abrogated by H1-antihistamines giving rise to the hypothesis that PAF-induced wheal development is secondary to histamine release from dermal mast cells. But is this hypothesis correct? Wheal and flare responses were induced by intradermal injection of PAF, codeine and histamine in 14 healthy volunteers. Dermal histamine and PGD2 contractions were measured using microdialysis. PAF, unlike histamine and codeine, did not cause a statistically significant rise in mean histamine levels with ten persons showing negligible histamine release. Codeine caused a significant but variable histamine release, ranging from 29 to 282 ng/ml. Codeine, but not PAF or histamine, caused a small but statistically significant release of PGD2. Wheal and flare reactions in human skin induced by PAF are not associated with histamine release and, therefore, appear to be independent of mast cell degranulation.

The role of PAF/PAFR signaling in zymosan-induced articular inflammatory hyperalgesia

Platelet-activating factor (PAF) and its receptor (PAFR) have been shown to be involved in several inflammatory events, including neutrophil chemoattraction and nociception. The present study addressed the role of PAF in the genesis of articular hyperalgesia in a model of joint inflammation. Zymosan-induced articular hyperalgesia, oedema and neutrophil migration were dose-dependently reduced following pretreatment with selective PAFR antagonists, UK74505 (5, 10 and 20mg/kg) and PCA4248 (3, 10, 30mg/kg). These parameters were also reduced in PAF receptor-deficient mice (PAFR(-/-)). The hyperalgesic action of PAF was further confirmed by the demonstration that joint injection of PAF induces a dose- (0.3, 1 and 3μg/joint), time- and PAFR-dependent articular hyperalgesia and oedema. The PAF hyperalgesic mechanisms were dependent on prostaglandins, leukotrienes and neutrophils, as PAF-induced articular hyperalgesia was inhibited by indomethacin (COX inhibitor), MK886 (leukotrienes synthesis inhibitor) or fucoidan (leukocyte rolling inhibitor). Furthermore, PAF-induced hyperalgesia was reduced in 5-lypoxigenase-null mice. In corroboration of these findings, intra-articular injection of PAF promotes the production of LTB(4) as well as the recruitment of neutrophils to the joint. These results suggest that PAF may participate in the cascade of events involved in the genesis of articular inflammatory hyperalgesia via stimulation of prostaglandins, leukotrienes and neutrophil migration. Finally, targeting PAF action (e.g., with a PAFR antagonist) might provide a useful therapeutic approach to inhibit articular inflammatory hyperalgesia.

Glutamine inhibits platelet-activating factor-mediated pulmonary tumour metastasis

Inflammation has been increasingly recognised as an important component of tumourigenesis. Platelet-activating factor (PAF), a potent inflammatory mediator, has the ability to enhance tumour growth and metastasis. In this study, we have investigated (i) the role of mitogen-activated protein kinases (MAPKs) and (ii) the therapeutic efficacy of the non-essential amino acid, l-glutamine (Gln), which evidences MAPKs inhibition activity in PAF-mediated B16F10 melanoma metastasis to the lungs. Mice were given intraperitoneal injection of PAF. ERK, JNK, and p38 MAPKs were activated rapidly by PAF in the lungs, and the PAF-induced metastasis of B16F10 was inhibited in a dose-dependent manner by pretreatment with either U0126 (ERK inhibitor), SP600125 (JNK inhibitor), or SB202190 (p38 inhibitor). Intraperitoneal administration of Gln after, but not before, PAF injection deactivated ERK, JNK, and p38 by dephosphorylating them. Gln inhibited PAF-induced metastasis when Gln was administered either intraperitoneally or orally. PAF induced pronounced angiogenic activity in an in vivo mouse Matrigel implantation model. MAPK inhibitors as well as Gln significantly inhibited PAF-induced angiogenesis. These data indicate that Gln exerts a beneficial effect against inflammation-associated enhanced tumour metastasis via the deactivation of MAPKs.

Platelet-Activating Factor and Pain

Platelet-activating factor (PAF) is a phospholipid mediator that regulates the functions of a variety of cells in the peripheral tissues and in the nervous system. Findings that injection of PAF exogenously at the skin or in the spinal cord induced pain hypersensitivity gave us much attention to its role in pain signaling. Studies using pharmacological and genetic tools to control the functions of the PAF receptor (PAFR) revealed that the PAF/PAFR system plays a role in tissue injury-induced pain, but not in the acute physiological pain evoked by thermal and mechanical stimuli. Recent investigations have focused on the roles of PAFR in pathological chronic pain such as the neuropathic pain that occurs after nerve injury for which there is currently no effective therapy. Nerve injury upregulated PAFRs in dorsal root ganglion (DRG) neurons. Studies using PAFR antagonists and PAFR-deficient mice indicated a crucial role of PAFR in production of tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) in the DRG and in developing and maintaining neuropathic pain. Thus, blocking PAFRs may be a viable therapeutic strategy for treating neuropathic pain.

Pharmacology of AM803, a novel selective five-lipoxygenase-activating protein (FLAP) inhibitor in rodent models of acute inflammation

We evaluated the in vivo pharmacological properties of AM803 3-[3- tert-butylsulfanyl-1-[4-(6-ethoxy-pyridin-3-yl)-benzyl]-5-(5-methyl-pyridin-2-ylmethoxy)-1 H-indol-2-yl]-2,2-dimethyl-propionic acid, a selective five-lipoxygenase-activating protein (FLAP) inhibitor, using rat and mouse models of acute inflammation. Oral administration of AM803 (1 mg/kg) resulted in sustained inhibition of ex vivo ionophore-challenged whole blood LTB 4 biosynthesis with > 90% inhibition for up to 12 h and an EC 50 of ∼ 7 nM. When rat lungs were challenged in vivo with calcium-ionophore, AM803 inhibited LTB 4 and cysteinyl leukotriene (CysLT) production with ED 50s of 0.12 mg/kg and 0.37 mg/kg, respectively. The inhibition measured 16 h following a single oral dose of 3 mg/kg was 86% and 41% for LTB 4 and CysLTs, respectively. In an acute inflammation setting, AM803 dose-dependently reduced LTB 4, CysLTs, plasma protein extravasation and neutrophil influx induced by peritoneal zymosan injection. Finally, AM803 increased survival time in mice exposed to a lethal intravenous injection of platelet activating factor (PAF). The magnitude of effect was similar to that of an inhibitor of five-lipoxygenase (5-LO) and LTA 4 hydrolase but superior to a leukotriene CysLT 1 receptor antagonist. In summary, AM803 is a novel, potent and selective FLAP inhibitor that has excellent pharmacodynamic properties in vivo and is effective in animal models of acute inflammation and in a model of lethal shock.

Role of PAF Receptor in Proinflammatory Cytokine Expression in the Dorsal Root Ganglion and Tactile Allodynia in a Rodent Model of Neuropathic Pain

BackgroundNeuropathic pain is a highly debilitating chronic pain following damage to peripheral sensory neurons and is often resistant to all treatments currently available, including opioids. We have previously shown that peripheral nerve injury induces activation of cytosolic phospholipase A2 (cPLA2) in injured dorsal root ganglion (DRG) neurons that contribute to tactile allodynia, a hallmark of neuropathic pain. However, lipid mediators downstream of cPLA2 activation to produce tactile allodynia remain to be determined.Principal FindingsHere we provide evidence that platelet-activating factor (PAF) is a potential candidate. Pharmacological blockade of PAF receptors (PAFRs) reduced the development and expression of tactile allodynia following nerve injury. The expression of PAFR mRNA was increased in the DRG ipsilateral to nerve injury, which was seen mainly in macrophages. Furthermore, mice lacking PAFRs showed a reduction of nerve injury-induced tactile allodynia and, interestingly, a marked suppression of upregulation of tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) expression in the injured DRG, crucial proinflammatory cytokines involved in pain hypersensitivity. Conversely, a single injection of PAF near the DRG of naïve rats caused a decrease in the paw withdrawal threshold to mechanical stimulation in a dose-dependent manner and an increase in the expression of mRNAs for TNFα and IL-1β, both of which were inhibited by pretreatment with a PAFR antagonist.ConclusionsOur results indicate that the PAF/PAFR system has an important role in production of TNFα and IL-1β in the DRG and tactile allodynia following peripheral nerve injury and suggest that blocking PAFRs may be a viable therapeutic strategy for treating neuropathic pain.

Pharmacological Characterization of 3-[3-tert-Butylsulfanyl-1-[4-(6-methoxy-pyridin-3-yl)-benzyl]-5-(pyridin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethyl-propionic Acid (AM103), a Novel Selective 5-Lipoxygenase-Activating Protein Inhibitor That Reduces Acute and Chronic Inflammation

Leukotrienes (LTs) are proinflammatory lipid mediators synthesized by the conversion of arachidonic acid (AA) to LTA(4) by the enzyme 5-lipoxygenase (5-LO) in the presence of 5-LO-activating protein (FLAP). 3-[3-tert-Butylsulfanyl-1-[4-(6-methoxy-pyridin-3-yl)-benzyl]-5-(pyridin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethyl-propionic acid (AM103) is a novel selective FLAP inhibitor in development for the treatment of respiratory conditions such as asthma. In a rat ex vivo whole-blood calcium ionophore-induced LTB(4) assay, AM103 (administered orally at 1 mg/kg) displayed >50% inhibition for up to 6 h with a calculated EC(50) of approximately 60 nM. When rat lung was challenged in vivo with calcium ionophore, AM103 inhibited LTB(4) and cysteinyl leukotriene (CysLT) production with ED(50) values of 0.8 and 1 mg/kg, respectively. In this model, the EC(50) derived from plasma AM103 was approximately 330 nM for inhibition of both LTB(4) and CysLT. In an acute inflammation setting, AM103 displayed dose-dependent inhibition of LTB(4), CysLT, and plasma protein extravasation induced by peritoneal zymosan injection. In a model of chronic lung inflammation using ovalbumin-primed and challenged BALB/c mice, AM103 reduced the concentrations of eosinophil peroxidase, CysLTs, and interleukin-5 in the bronchoalveolar lavage fluid. Finally, AM103 increased survival time in mice exposed to a lethal intravenous injection of platelet-activating factor. In summary, AM103 is a novel, potent and selective FLAP inhibitor that has excellent pharmacodynamic properties in vivo and is effective in animal models of acute and chronic inflammation and in a model of lethal shock.

Mechanisms underlying the nociceptive responses induced by platelet-activating factor (PAF) in the rat paw

Platelet-activating factor (PAF) is an inflammatory mediator widely known to exert relevant pathophysiological functions. However, the relevance of PAF in nociception has received much less attention. Herein, we have investigated the mechanisms underlying PAF-induced spontaneous nociception and mechanical hypersensitivity in the rat paw. PAF injection (1–30 nmol/paw) resulted in a dose-related overt nociception, whilst only the dose of 10 nmol/paw produced a significant and time-related mechanical hypersensitivity. Local coinjection of PAF antagonist WEB2086 significantly inhibited both spontaneous nociception and mechanical hypersensitivity. Moreover, the coinjection of the natural IL-1β receptor antagonist (IRA) notably prevented both PAF-induced nociceptive responses, whilst these responses were not altered by anti-TNFα coinjection. Interestingly, pretreatment with the ultrapotent vaniloid agonist resiniferotoxin, coinjection of the TRPV1 receptor antagonist SB366791, or mast cell depletion with compound 48/80 markedly prevented PAF-induced spontaneous nociception. Conversely, PAF-elicited mechanical hypersensitivity was strikingly susceptible to distinct antineutrophil-related strategies, namely the antineutrophil antibody, the selectin blocker fucoidin, the chemokine CXCR2 receptor antagonist SB225002, and the C5a receptor antibody anti-CD88. Notably, the same antineutrophil migration strategies significantly prevented the increase of myeloperoxidase activity induced by PAF. The mechanical hypersensitivity caused by PAF was also prevented by the cyclooxygenase inhibitors indomethacin or celecoxib, and by the selective β 1 adrenergic receptor antagonist atenolol. Collectively, the present results provide consistent evidence indicating that distinct mechanisms are involved in the spontaneous nociception and mechanical hypersensitivity caused by PAF. They also support the concept that selective PAF receptor antagonists might constitute interesting targets for the development of new analgesic drugs.

Involvement of Platelet-Activating Factor in Ultraviolet B-Induced Hyperalgesia

Ultraviolet B (UVB) radiation causes cutaneous inflammation. One important clinical consequence of UVB-induced inflammation is increased pain or hyperalgesia, which is likely mediated by enhanced sensitivity of cutaneous sensory neurons. Previous studies have demonstrated that UVB radiation generates the lipid mediator, platelet-activating factor (PAF), as well as oxidized phospholipids that act as PAF-mimetics. These substances exert effects through the PAF receptor (PAF-R). This study was designed to assess whether PAF-R is involved in UVB-induced hyperalgesia. Intradermal injection of carbamoyl PAF (CPAF; 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine) resulted in an enhanced response to mechanical stimuli in wild-type mice but not in PAF-R knockout (KO) mice. There was no significant change in paw withdrawal to noxious thermal stimuli in either genotype after intradermal injection of CPAF. Exposure of the hind paw to 1,500 J m(-2) UVB radiation caused an increased sensitivity to both mechanical and thermal stimulation in wild-type mice but not in PAF-R KO mice. The thermal hyperalgesia caused by UVB irradiation was inhibited in mice that lacked PAF-R in bone marrow-derived cells. These data demonstrate that the PAF-R is important for UVB-induced hyperalgesia. Further investigation of the role of PAF-R signaling in UVB-induced hyperalgesia could provide better understanding of the pathological processes initiated by UVB-induced skin damage.

Involvement of platelet-activating factor and tumour necrosis factor in the pathogenesis of joint inflammation in rabbits.

We have studied the participation of platelet-activating factor (PAF) in antigen-induced arthritis in rabbits, as well as the possible co-operation between PAF and tumour necrosis factor (TNF) in their ability to induce joint inflammation when injected into the knees of healthy rabbits. The administration of two structurally different PAF receptor antagonists, BN52021 and Alprazolam, from 4 h before the intra-articular injection of ovalbumin in preimmunized rabbits, induced an important reduction in the synovial fluid volume, in the amount of cells infiltrating the articular cavity and the synovial membrane, as well as in the prostaglandin E2 (PGE2) concentration. Furthermore, proteoglycans of the articular cartilage, which were found diminished in animals with non-treated arthritis, were well preserved in rabbits treated with PAF antagonists. All the synovial fluids from joints with arthritis had detectable amounts of PAF. The injection of either TNF or PAF into the joints of normal rabbits induced a mild inflammation. When TNF was administered 1 h before PAF, a synergistic response was noted in the synovial fluid volume, in the accumulation of leucocytes, and in the amount of PGE2. The administration of BN50726, a hetrazepine with a potent PAF-receptor antagonist effect, induced a diminution in those parameters. Our results suggest that PAF may be an early and important mediator of joint damage, and that TNF can amplify the inflammatory response induced by PAF. PAF receptor antagonists could play some role in the treatment of inflammatory joint diseases.