Platelet-activating factor (PAF) and PAF antagonists in asthma

PAF is produced by and activates inflammatory cells, such as monocytes/macrophages, mast cells, platelets, neutrophils, eosinophils and endothelial cells. Its ability to imitate anaphylaxis, inducing for instance bronchoconstriction (BC) in guinea-pigs, and its identification (and/or that of lyso-PAF) in exudates from shocked lungs, led to the hypothesis that PAF is involved in immediate hypersensitivity. Recent results of Bachelet et al. show that PAF reduces the increased cyclic AMP content of guinea-pig alveolar cell population exposed to PGE2, salbutamol or isoprenaline, which agrees with its hypothesized stimulating role in conditions where increased cyclic AMP may reduce mediator release. PAF antagonists are usually selected with in vitro platelet tests and their in vivo activity is characterized in normal animals. Recent data of Pretolani et al. demonstrate nevertheless that the antagonists may lose part of their ability to inhibit PAF itself if tested on lungs from actively sensitized guinea-pigs. These lungs differ from those of passively sensitized or of naive animals in that they become hyper-responsive to mediators (PAF, leukotriene D4 [LTD4], histamine, arachidonate [AA]): BC and formation of thromboxane A2 are enhanced, and histamine is released dose-dependently under conditions where it is absent from perfusates from LTD4, AA or PAF-stimulated naive lungs. Peripheral inflammatory cells (basophils, eosinophils, monocytes) are possibly recruited into the lungs of the actively sensitized animals sometime during the second and/or third week of sensitization, and provide a new target which may account for the enhanced lung responsiveness. Ultra-structural studies of Lellouch-Tubiana et al. (abstract in this meeting) support this concept. Neither the primary target nor the chemotactic substance responsible for the reported modifications are identified, but recent data of Bachelet et al, showing that alveolar populations from actively sensitized guinea-pigs are less responsive to the cyclic AMP stimulating effects of PGE2, salbutamol or isoprenaline suggest the existence of a cell defect which may be important for the triggering of allergen-induced BC and cell recruitment. Our present concept involves a "pre-inflamed" lung in actively sensitized guinea-pigs and in human asthmatics, a stand-by process following sensitization and which is revealed following the activation of a target cell. This may be the alveolar macrophage which releases substances (PAF, TXA2, IL1) likely to start BC and protracted cell recruitment and activation.     

Platelet-activating factor antagonists decrease the inflammatory nociceptive response in rats

Rationale: Platelet-activating factor (PAF) is a membrane-derived phospholipid mediator that has biological effects on a variety of cells and tissues. A variety of stimuli, including those producing inflammation, promote the synthesis and release of PAF from various cell types. Evidence suggests that PAF exerts cellular actions through a plasma membrane receptor as well as via intracellular (microsomal) PAF binding sites. Objective: The present study was designed to: 1) investigate the role of PAF in a model of inflammatory nociception in rats (i.e. the formalin test), and 2) localize PAF's site(s) of action in nociception. To do this, we assessed the effect of administering two PAF antagonists (BN 52021 and BN 50730, which are selective for cell surface and intracellular PAF binding sites, respectively) on formalin-induced nociceptive responses. Methods: Forty minutes prior to formalin injection into the rat hindpaw, male Sprague-Dawley rats received systemic injections of BN 52021 (10, 1, or 0.1 mg/kg), BN 50730 (10, 1, or 0.1 mg/kg), or vehicle (45% 2-hydroxypropyl-β-cyclodextrin in distilled water, HBC) and the effects of the drugs on nociceptive behavioral responses were measured. Results: Rats receiving systemic BN 52021 or BN 50730 displayed a significant reduction of nociceptive responses in the late, but not early, phase of formalin-induced nociception. Conclusions: These findings suggest a role for endogenous PAF in nociceptive transmission, especially for persistent pain such as that which occurs in the late phase of the formalin test. The findings also indicate that both intracellular and cell surface PAF binding sites are involved in nociceptive modulation in rats, and that PAF antagonists might be useful for treating some patients with acute or chronic pain. Electronic Publication     

Platelet-activating factor antagonists protect amyloid-beta damaged neurons from microglia-mediated death

Neurons treated with sub-lethal concentrations of amyloid-beta1-42 developed phenotypic changes and selectively bound a CD14-IgG chimera; in co-cultures, microglia recognised and killed these amyloid-beta1-42 -damaged neurons. Pre-treatment with the platelet-activating factor (PAF) antagonists (Hexa-PAF, CV6209 or ginkgolide B) reduced CD14-IgG binding to amyloid-beta1-42 -damaged neurons, and the presence of PAF antagonists in co-cultures increased neuronal survival in a dose-dependant manner. PAF antagonists also protected neurons treated with HuPrP82-146, a peptide found in prion diseases. Second messenger studies demonstrated that the addition of PAF mimicked some of the effects of amyloid-beta1-42 on neurons. PAF-damaged neurons bound CD14-IgG, and PAF-damaged neurons were killed by microglia in a CD14-dependent process. Neuronal death was inversely related to both the concentration of PAF, and the number of microglia added. The effects of PAF were reduced by an antagonist of the prostanoid D receptor (BWA868C) indicating that neuronal damage induced by PAF is partly mediated by prostaglandins. These observations are compatible with the hypothesis that sub-lethal concentrations of amyloid-beta1-42 stimulate a cascade of second messengers including PAF and the prostaglandins. At nanomolar concentrations PAF induces a change in neuronal phenotype that activates microglia via the CD14 molecule, these activated microglia then kill the amyloid-beta1-42 damaged neurons.     

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.     

The mechanism of action of opiate antagonists in experimental traumatic shock

Acute experiments on 86 adult rabbits have shown that in the early period of Cannon's traumatic shock nalophine and naloxone relieve torpidity, restore motor activity and responses to standard stimuli. This prolongs the animal life (nalorphine) and increases the survivability (naloxone). However in some rabbits a premature relief of torpidity (0.5 mg/kg of nalorphine) precipitates the animal death. It is concluded that in the late period the drugs exert no effect on the shock outcome and that inactivation of opioid suppression system can either trigger the mechanism of shock withdrawal or promote a reduction of its severity.     

Preclinical studies with platelet-activating factor antagonists in models of septic shock

Since the isolation and elucidation of the structure of platelet-activating factor (PAF) in the late 1970's, several preclinical studies have suggested that PAF is a key mediator of septic shock induced in animals by either endotoxin or by Gram-negative bacteria. A number of PAF antagonists have been sythesized that protect animals from the lethal effects of endotoxin. Some of these antagonists are in early stages of clinical development. The most advanced cadidate is BN 52021, a ginkgolide, that is in Phase II/III clinical trials in patients with septic shock. Preliminary results with BN 52021 indicate that it is efficacious and significantly reduces mortality associated with Gram-negative sepsis. Pivotal trials with BN 52021 aer ongoing. The present review summarizes the biological effects of PAF and the effect of PAF antagonists in animal models of septic shock. The interrelationship of PAF and tumor necrosis factor (another key mediator of septic shock) is also discussed.     

Patent Update: Pulmonary-Allergy,Dermatological, Gastrointestinal & Arthritis: Platelet-activating factor receptor antagonists

Platelet-activating factor (PAF) has been implicated in a range of inflammatory conditions and an appropriate antagonist should prove to be a useful therapeutic agent. Although PAF antagonists have yet to be commercialised, a number are undergoing clinical trials. In disease states such as asthma there is evidence for the involvement of a variety of inflammatory mediators and the recent literature describes a variety of approaches to incorporating more than one mode of action in a single compound. This review is primarily an update on PAF antagonist patent publications for the period July 1993 to February 1995. Reference has been made to earlier compounds to provide an update on clinical progress and for comparison with newer antagonists.     

Platelet-activating factor increases the expression of metalloproteinase-9 in human bronchial epithelial cells.

Platelet-activating factor (PAF) plays an important role in the pathogenesis of bronchial asthma. To investigate the role of PAF in the maintenance and remodeling of the extracellular matrix, we evaluated the expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 from human bronchial epithelial cells after PAF treatment. Gelatin zymography of human bronchial epithelial cell-conditioned media showed major pro-matrix metalloproteinase-9 and minor pro-matrix metalloproteinase-2 expression and these expressions were totally inhibited by the metalloproteinase inhibitor EDTA. The identification of matrix metalloproteinase-9 was confirmed by Western blot analysis. Northern blotting and zymography demonstrated that PAF induced the mRNA of matrix metalloproteinase-9 from human bronchial epithelial cells and an increase in the gelatinolytic activity of pro-matrix metalloproteinase-9 but not in that of pro-matrix metalloproteinase-2. Lyso-PAF did not induce matrix metalloproteinase-9 mRNA or the gelatinolytic activity of pro-matrix metalloproteinase-9. CV6209, an receptor antagonist of PAF, reduced the increases of pro-matrix metalloproteinase-9 mRNA and gelatinolytic activity induced by PAF. Another receptor antagonist of PAF, hexanolamine PAF, did not inhibit the increases in the synthesis or release of pro-matrix metalloproteinase induced by PAF. Based on these results, we propose that matrix metalloproteinase-9 may be actively involved in the PAF-induced physiopathological remodeling in human bronchial epithelial cells.     

Platelet-activating factor drives eotaxin production in an allergic pleurisy in mice

1. The activation of eosinophils via G-protein-coupled seven transmembrane receptors play a necessary role in the recruitment of these cells into tissue. The present study investigates a role for PAF in driving eotaxin production and eosinophil recruitment in an allergic pleurisy model in mice. 2. The intrapleural injection of increasing doses of PAF (10(-11) to 10(-9) moles per cavity) induced a dose- and PAF receptor-dependent recruitment of eosinophils 48 h after stimulation. 3. Intrapleural injection of PAF induced the rapid (within 1 h) release of eotaxin into the pleural cavity of mice and an anti-eotaxin antibody effectively inhibited PAF-induced recruitment of eosinophils. 4. Eosinophil recruitment in the allergic pleurisy was markedly inhibited by the PAF receptor antagonist UK-74,505 (modipafant, 1 mg kg(-1)). Moreover, recruitment of eosinophils in sensitized and challenged PAF receptor-deficient animals was lower than that observed in wild-type animals. 5. Blockade of PAF receptors with UK-74,505 suppressed by 85% the release of eotaxin in the allergic pleurisy. 6. Finally, the injection of a sub-threshold dose of PAF and eotaxin cooperated to induce eosinophil recruitment in vivo. 7. In conclusion, the production of PAF in an allergic reaction could function in multiple ways to facilitate the recruitment of eosinophils -- by facilitating eotaxin release and by cooperating with eotaxin to induce greater recruitment of eosinophils.     

Platelet-activating factor relaxes ferret tracheal smooth muscle and reduces transepithelial potential difference in vitro.

1. The effects of platelet activating factor (PAF) were examined on the smooth muscle tone, mucus volume, lysozyme and albumin outputs and potential difference (PD) across the ferret tracheal wall. 2. PAF (0.1-10 microM) had no direct effect on mucus volume, lysozyme or albumin output from the ferret trachea. PAF produced concentration-dependent relaxations of the tracheal smooth muscle and reductions in PD across the tracheal wall. There was no change in the histological appearance of the trachea after exposure to PAF. 3. The PAF-induced smooth muscle relaxation was not affected by FPL55712, a combination of mepyramine and cimetidine, or by a combination of the oxygen free-radical scavengers catalase and superoxide dismutase (SOD); but was abolished by indomethacin or the PAF-receptor antagonist WEB2086. 4. The PAF-induced reduction in PD was not affected by indomethacin, FPL55712 or mepyramine and cimetidine, but was prevented by catalase and SOD, and by WEB2086. 5. We conclude that PAF relaxes ferret tracheal smooth muscle in vitro by receptor-mediated release of a bronchodilator prostaglandin, possibly PGE2. PAF also reduces PD across the trachea suggesting changes in epithelial function; however, there is no histological epithelial damage after PAF. The reduction in PD with PAF is probably produced by receptor-mediated release of oxygen free-radicals. The cellular source of these free-radicals and of the dilator prostaglandin is unclear.     

Platelet-activating factor enhanced the pressor response of endothelin-1

Both endothelin-1 (ET-1) and platelet-activating factor (PAF) have been suggested to play a role in the regulation of the cardiovascular system. In view of the limited data regarding the interaction between ET-1 and PAF, the hemodynamic effects of ET-1 and PAF, either alone or in combination, were investigated in the current study. Anesthetized male Sprague Dawley rats received bolus intravenous injections of ET-1 (1 and 2 nmol/kg) and/or PAF (0.075, 0.15 and 0.3 nmol/kg). In some experiments, the ET receptor antagonist, FR-139317 (2.5 or 5 mg/kg), were injected 5 min before the administration of ET-1 or PAF. ET-1 caused a biphasic response consisting of an initial depressor followed by a delayed but sustained pressor response. Injection of PAF to anesthetized rats resulted only in a decrease in arterial blood pressure. Interestingly, the pressor effect of ET-1 was significantly enhanced in the concomitant presence of PAF. Pretreatment with FR-139317 inhibited the magnitude of ET-1-induced hypertension and increased the duration of the depressor action of ET-1. The time-course of PAF-induced decrease of arterial blood pressure was also prolonged in rats pretreated with FR-139317. These data therefore suggested that ET receptors were activated, either directly or indirectly, by PAF, possibly to facilitate the return of blood pressure to resting level following a depressor response. Thus the activation of ET receptors by PAF might result in the enhancement of the pressor response of ET-1 observed in the current study.     

ACTH fragments reverse experimental haemorrhagic shock in rats.

Hypovolemic shock was produced in rats by withdrawing about 50% of estimated total blood volume. Following mean arterial pressure stabilization in the range of 25-27 mmHg, the rats were given intravenous ACTH (1-4) (4-9) (4-10) (5-10) (1-10) in comparison with ACTH (1-24). The ACTH fragments, administered within 5 min after shock induction, promptly and dose-dependently improved mean arterial pressure and survival of rats; the ACTH (1-10) showed an activity similar to that of ACTH (1-24) whereas the other fragments were less active.     

Corticotropin-releasing factor receptor antagonists

Corticotropin-releasing factor (CRF) coordinates the neural, endocrine and immune responses of the body to stress. Therefore, CRF receptors are important targets for the design of drugs for depression, anxiety and stress-related disorders. Several laboratories have published extensive preclinical and limited clinical research into the role of CRF in human disease. This review covers developments in the patent literature during 2002 – 2006 and outlines the prospects for CRF-based therapy for mental illness.     

Platelet-activating factor acetylhydrolase: selective inhibition by potent n-alkyl methylphosphonofluoridates

Platelet-activating factor (PAF) is a potent endogenous phospholipid modulator of diverse biological activities, including inflammation and shock. PAF levels are primarily regulated by PAF acetylhydrolases (PAF-AHs). These enzymes are candidate secondary targets of organophosphorus (OP) pesticides and related toxicants. Previously known OP inhibitors of other serine hydrolases were tested with PAF-AH from mouse brain and testes of established functional importance compared with the structurally different human plasma enzyme. Several key OP pesticides and their oxon metabolites were very poor inhibitors of mouse brain and human plasma PAF-AH in vitro but moderately active for mouse brain and blood PAF-AH in vivo (e.g., tribufos defoliant and profenofos insecticide, presumably following oxidative bioactivation). OP compounds were then designed for maximum in vitro potency and selectivity for mouse brain PAF-AH vs. acetylcholinesterase (AChE). Lead compounds were found in a series of benzodioxaphosphorin 2-oxides. Ultrahigh potency and selectivity were achieved with n-alkyl methylphosphonofluoridates (long-chain sarin analogs): mouse brain and testes IC50 < or = 5 nM for C(8)-C(18) analogs and 0.1-0.6 nM for C(13) and C(14) compounds; human plasma IC50 < or = 2 nM for C(13)-C(18) analogs. AChE inhibitory potency decreased as chain length increased with maximum brain PAF-AH/AChE selectivity (>3000-fold) for C(13)-C(18) compounds. The toxicity of i.p.-administered PAF (LD50 ca. 0.5 mg/kg) was increased less than 2-fold by pretreatment with tribufos or the C(13)n-alkyl methylphosphonofluoridate. These studies with a mouse model indicate that PAF-AH is not a major secondary target of OP pesticide poisoning. The optimized PAF-AH inhibitors may facilitate investigations on other aspects of PAF metabolism and action.     

Corticotropin-releasing factor antagonists in affective disorders

Following a search lasting nearly three decades, corticotropin-releasing factor (CRF), a 41 amino acid-containing peptide, was isolated and characterised in 1981. In the preceding 18 years, a concatenation was developed that appears to show that CRF integrates not only the endocrine, but also the autonomic, immunologic and behavioural responses of mammalian organisms to stress. Direct CNS administration of CRF to laboratory animals produces actions similar to those observed after exposure to stress. Moreover, CNS administration of peptidergic CRF antagonists blocks many of the behavioural responses to stress. Since both early untoward life events as well as recently experienced stress have been implicated in the pathophysiology of affective disorders, and because there is substantial evidence for CRF neuronal hyperactivity in patients with affective disorders, small molecule, lipophilic CRF antagonists have been hypothesised to possess antidepressant and/or anxiolytic activity. Within the last few years, a number of pharmaceutical companies have developed selective, small molecule CRF(1) receptor antagonists. These compounds block the effects of CRF both in vitro and in vivo. There is also evidence that these agents possess anxiolytic and antidepressant activity in animal behavioural models. Compounds that act upon the CRF system have been hypothesised to be of value not only for certain psychiatric disorders, but also in neurodegenerative and inflammatory disorders. Some of these CRF(1) receptor antagonists are currently undergoing clinical trials to determine their efficacy and tolerability in patients with mood and anxiety disorders.     

Platelet-activating factor (PAF) involvement in acetaminophen-induced liver toxicity and regeneration

Acetaminophen-induced toxicity has been attributed to cytochrome P-450-generated metabolites, which covalently modify target proteins. However, the mechanism of liver injury pathogenesis needs to be further elucidated. Platelet-activating factor (PAF) is one of the mediators involved in inflammatory tissue alterations associated with acute liver failure. In this study, alterations in blood PAF levels and the serum activity of PAF-acetylhydrolase (PAF-AH) were investigated over the time course of liver injury and regeneration induced by acetaminophen treatment in rats. The administration of a toxic dose of acetaminophen (3.5 g/kg) in rats caused acute hepatic injury, as evident by alterations of biochemical (serum enzymes: ALT, AST and ALP) and liver histopathological (degree of inflammation and apoptosis) indices between 20 and 40 h post-treatment. The hepatic damage was followed by liver regeneration, made evident by three independent indices ([3H]thymidine incorporation into hepatic DNA, liver thymidine kinase activity and hepatocyte mitotic index), presenting a peak at 72 h. The PAF levels were elevated at 24 and 28 h, presenting a remarkable peak at 32 h post-treatment. PAF-AH activity presented different kinetics to that of PAF. The enzyme activity was relatively low at all time points examined before the rise in PAF activity, peaking later, at 72, 84 and 96 h. Our data demonstrate that PAF is involved in the pathogenesis of acute liver failure and in augmented compensatory liver tissue repair post-acetaminophen treatment. However, the putative role of PAF during liver toxicity and regeneration remains to be established.