Joaquim Trias

Varespladib (A-002), a secretory phospholipase A2 inhibitor, reduces atherosclerosis and aneurysm formation in ApoE-/- mice.

The family of secretory phospholipase A2 (sPLA2) enzymes has been associated with inflammatory diseases and tissue injury including atherosclerosis. A-001 is a novel inhibitor of sPLA2 enzymes discovered by structure-based drug design, and A-002 is the orally bioavailable prodrug currently in clinical development. A-001 inhibited human and mouse sPLA2 group IIA, V, and X enzymes with IC50 values in the low nM range. A-002 (1 mg/kg) led to high serum levels of A-001 and inhibited PLA2 activity in transgenic mice overexpressing human sPLA2 group IIA in C57BL/6J background. In addition, the effects of A-002 on atherosclerosis in 2 ApoE−/− mouse models were evaluated using en face analysis. (1) In a high-fat diet model, A-002 (30 and 90 mg/kg twice a day for 16 weeks) reduced aortic atherosclerosis by 50% (P < 0.05). Plasma total cholesterol was decreased (P < 0.05) by 1 month and remained lowered throughout the study. (2) In an accelerated atherosclerosis model, with angiotensin II-induced aortic lesions and aneurysms, A-002 (30 mg/kg twice a day) reduced aortic atherosclerosis by approximately 40% (P < 0.05) and attenuated aneurysm formation (P = 0.0096). Thus, A-002 was effective at significantly decreasing total cholesterol, atherogenesis, and aneurysm formation in these 2 ApoE−/− mouse models.

A-002 (Varespladib), a phospholipase A2 inhibitor, reduces atherosclerosis in guinea pigs

BackgroundThe association of elevated serum levels of secretory phospholipase A2 (sPLA2) in patients with cardiovascular disease and their presence in atherosclerotic lesions suggest the participation of sPLA2 enzymes in this disease. The presence of more advanced atherosclerotic lesions in mice that overexpress sPLA2 enzymes suggest their involvement in the atherosclerotic process. Therefore, the sPLA2 family of enzymes could provide reasonable targets for the prevention and treatment of atherosclerosis. Thus, A-002 (varespladib), an inhibitor of sPLA2enzymes, is proposed to modulate the development of atherosclerosis.MethodsTwenty-four guinea pigs were fed a high saturated fat, high cholesterol diet (0.25%) for twelve weeks. Animals were treated daily with A-002 (n = 12) or vehicle (10% aqueous acacia; n = 12) by oral gavage. After twelve weeks, animals were sacrificed and plasma, heart and aorta were collected. Plasma lipids were measured by enzymatic methods, lipoprotein particles size by nuclear magnetic resonance, aortic cytokines by a colorimetric method, and aortic sinus by histological analyses.ResultsPlasma total cholesterol, HDL cholesterol and triglycerides were not different among groups. However, the levels of inflammatory cytokines interleukin (IL)-10, IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were significantly reduced in the treatment group. This group also had a significant 27% reduction in cholesterol accumulation in aorta compared with placebo group. Morphological analysis of aortic sinus revealed that the group treated with A-002 reduced atherosclerotic lesions by 24%.ConclusionThe use of A-002 may have a beneficial effect in preventing diet-induced atherosclerosis in guinea pigs.

Varespladib methyl in cardiovascular disease.

Importance of the field: The high risk of recurrent cardiovascular events amongst patients with cardiovascular disease receiving evidence-based therapies has prompted investigations into complimentary treatments that may reduce residual risk. Analyses of clinical trials in statin-treated patients demonstrate that elevated lipid levels and an activated systemic inflammatory state are associated with a higher risk of recurrent cardiovascular events. Areas covered in this review: This article reviews evidence supporting the causal role for secretory phospholipase A2 (sPLA2) in experimental atherosclerosis, the involvement of various sPLA2 isozymes as mediators of pro-atherogenic lipoprotein remodeling and participants in vascular and systemic inflammatory responses, and the evidence that sPLA2 inhibition reduces atherosclerosis in experimental models and biomarkers associated with cardiovascular events in coronary heart disease (CHD) patients. What the reader will gain: The experimental basis for sPLA2 inhibition with varespladib methyl as a potential candidate for lowering recurrent cardiovascular events particularly in acute coronary syndrome patients is discussed. Take home message: Varespladib methyl therapy reduces atherogenic lipoprotein concentrations and systemic inflammatory markers in CHD patients. The future role of varespladib methyl in CHD patients awaits the results of ongoing clinical trials.

Varespladib Inhibits Secretory Phospholipase A2 in Bronchoalveolar Lavage of Different Types of Neonatal Lung Injury

Secretory phospholipase A2 (sPLA2), which links surfactant catabolism and lung inflammation, is associated with lung stiffness, surfactant dysfunction, and degree of respiratory support in acute respiratory distress syndrome and in some forms of neonatal lung injury. Varespladib potently inhibits sPLA2 in animal models. The authors investigate varespladib ex vivo efficacy in different forms of neonatal lung injury. Bronchoalveolar lavage fluid was obtained from 40 neonates affected by hyaline membrane disease, infections, or meconium aspiration and divided in 4 aliquots added with increasing varespladib or saline. sPLA2 activity, proteins, and albumin were measured. Dilution was corrected with the urea ratio. Varespladib was also tested in vitro against pancreatic sPLA2 mixed with different albumin concentration. Varespladib was able to inhibit sPLA2 in the types of neonatal lung injury investigated. sPLA2 activity was reduced in hyaline membrane disease (P <.0001), infections (P = .003), and meconium aspiration (P = .04) using 40 μM varespladib; 10 μM was able to lower enzyme activity (P = .001), with an IC50 of 87 μM. An inverse relationship existed between protein level and activity reduction (r = 0.5; P = .029). The activity reduction/protein ratio tended to be higher in hyaline membrane disease. Varespladib efficacy was higher in vitro than in lavage fluids obtained from neonates (P <.001).

Surfactant and Varespladib Co-Administration in Stimulated Rat Alveolar Macrophages Culture

Acute lung injury is a life-threatening condition characterized by surfactant dysfunction and raised secretory phospholipase A2 (sPLA2) activity. Varespladib is a sPLA2 inhibitor shown to be effective in animal models of acute lung injury. We aimed at investigating the effect of co-administration of surfactant and varespladib on sPLA2 activity. Alveolar macrophages were cultured and stimulated with lipopolysaccharide and then treated with either varespladib, surfactant, varespladib followed by surfactant or nothing. sPLA2 activity, free fatty acids, tumour necrosis factor-α (TNF-α) and protein concentrations were measured in culture supernatants. Treatment with varespladib (p=0.019) and varespladib + surfactant (p=0.013), reduced the enzyme activity by approximately 15% from the basal level measured in the untreated cultures. Surfactant, varespladib and varespladib + surfactant, respectively decreased free fatty acids by -45% (p=0.045), - 62% (p=0.009) and -48% (p=0.015), from the baseline concentration of the untreated cultures. Varespladib and poractant- α co-administration reduces sPLA2 activity and free fatty acids release in cultured rat alveolar macrophages, although a clear drug synergy was not evident. Since co-administration may be useful to reduce inflammation and surfactant inactivation in acute lung injury, further in vivo studies are warranted to verify its clinical usefulness.

58 Effect of Varespladib-Protected Surfactant in Cultured Rat Alveolar Macrophages Stimulated with LPS

Background Secretory phospholipase A2 (sPLA2) is a crucial enzyme for inflammatory response and surfactant catabolism. Acute lung injury (ALI) is a life-threating syndrome characterized by surfactant dysfunction and raised levels of sPLA2. Varespladib is a potent selective sPLA2 inhibitor that is effective in animal models of ALI. Nothing is known about the joint administration surfactant+varespladib and we aimed at studying the effect on the sPLA2 pathway. Methods 1x106 normal alveolar macrophages (from Rattus Norvegicus) were cultured in Ham’sF12 medium + 2% fetal bovine serum. Cultures were incubated with 15 ng/mL LPS for 24h, then treated with 200 µg poractant-α, 90 µM varespladib, both or nothing. These concentrations were those achieving 50% sPLA2 activity reduction in previous experiments. After 24h, culture supernatants were assayed for sPLA2 activity, free fatty acids (FFA) and total proteins concentrations. Results sPLA2 activity corrected for the protein level is 0.26±0.02, 0.24±0.02, 0.24±0.02 and 0.28±0.02 IU/µg in cultures treated with surfactant, varespladib, both or nothing (overall p=0.016; Dunnett post-hoc between cultures treated with varespladib and varespladib+surfactant against untreated cultures p=0.01). FFA are higher in untreated cultures (394±82 µM), than in surfactant- (219±70 µM) and in varespladib-treated ones (148±51 µM). Combined treatment reduced FFA to 206±47 µM (overall p=0.017; Sidak post-hoc p=0.036 and p=0.023 for the varespladib and combined treatment against control cultures). Conclusions The joined administration of varespladib and poractant-α significantly reduce sPLA2 activity and FFA production. Surfactant+varespladib affect sPLA2 pathway significantly more than the surfactant alone.

22 New Anti-Inflammatory Therapy for Neonatal Acute Lung Injury: Varespladib |[lpar]|A-001|[rpar]| Inhibits Ex-Vivo Secretory Phospholipase

Background: Secretory phospholipase A2(sPLA2) is crucial for surfactant catabolism and inflammation. We demonstrated high sPLA2 activity during infection related respiratory failure (IRRF), hyaline membrane disease (iRDS), RSV-triggered ARDS. sPLA2 is also responsible for lung damage during meconium aspiration syndrome (MAS). Varespladib is the first new anti-inflammatory drug able to directly inhibit sPLA2 and it resulted efficacious in animals and promising in adults. Aim: To test varespladib efficacy in an ex-vivo model of direct endotracheal administration in various types of neonatal acute lung injury. Methods: 24 neonates affected by iRDS, 12 with IRRF, 5 with MAS have been subjected to non-bronchoscopic broncho-alveolar lavage according to European Respiratory Society advices. Supernatant has been subdivided in aliquots which have been added with various concentrations of varespladib or equivalent volumes of saline. Real time sPLA2 activity and total protein content have been measured. Enzyme activity has been corrected for dilutional factors and varespladib absorbance. Clinical data were registered up to the intensive care unit discharge. Results: Varespladib reduces sPLA2 during iRDS(-20%; p< 0.0001), MAS(-23%; p=0.04), IRRF(- 15%; p=0.003). Varespladib efficacy is higher when a lower capillary extravasation is present (ratio sPLA2 activity reduction/total proteins = 2.17 in iRDS; 0.59 in IRRF, 0.53 in MAS). IC50 has been calculated and varespladib efficacy correlates with the basal enzyme activity (rho= 0.375; p=0.017). No correlations with gestational age, birth weight, surfactant cumulative need, mechanical ventilation and oxygen therapy duration were found. Conclusions: Varespladib efficaciously inhibits sPLA2 in an ex-vivo model of direct administration. This might have important anti-inflammatory effects and spare surfactant from its catabolism.