Association of circulating PLA2G7 levels with cancer cachexia and assessment of darapladib as a therapy.

BACKGROUND: Cancer cachexia (CCx) is a multifactorial wasting disorder characterized by involuntary loss of body weight that affects many cancer patients and implies a poor prognosis, reducing both tolerance to and efficiency of anticancer therapies. Actual challenges in management of CCx remain in the identification of tumour-derived and host-derived mediators involved in systemic inflammation and tissue wasting and in the discovery of biomarkers that would allow for an earlier and personalized care of cancer patients. The aim of this study was to identify new markers of CCx across different species and tumour entities. METHODS: Quantitative secretome analysis was performed to identify specific factors characteristic of cachexia-inducing cancer cell lines. To establish the subsequently identified phospholipase PLA2G7 as a marker of CCx, plasma PLA2G7 activity and/or protein levels were measured in well-established mouse models of CCx and in different cohorts of weight-stable and weight-losing cancer patients with different tumour entities. Genetic PLA2G7 knock-down in tumours and pharmacological treatment using the well-studied PLA2G7 inhibitor darapladib were performed to assess its implication in the pathogenesis of CCx in C26 tumour-bearing mice. RESULTS: High expression and secretion of PLA2G7 were hallmarks of cachexia-inducing cancer cell lines. Circulating PLA2G7 activity was increased in different mouse models of CCx with various tumour entities and was associated with the severity of body wasting. Circulating PLA2G7 levels gradually rose during cachexia development. Genetic PLA2G7 knock-down in C26 tumours only partially reduced plasma PLA2G7 levels, suggesting that the host is also an important contributor. Chronic treatment with darapladib was not sufficient to counteract inflammation and tissue wasting despite a strong inhibition of the circulating PLA2G7 activity. Importantly, PLA2G7 levels were also increased in colorectal and pancreatic cancer patients with CCx. CONCLUSIONS: Overall, our data show that despite no immediate pathogenic role, at least when targeted as a single entity, PLA2G7 is a consistent marker of CCx in both mice and humans. The early increase in circulating PLA2G7 levels in pre-cachectic mice supports future prospective studies to assess its potential as biomarker for cancer patients.

Lp-PLA2 inhibition prevents Ang II-induced cardiac inflammation and fibrosis by blocking macrophage NLRP3 inflammasome activation.

Macrophage-mediated inflammation plays an important role in hypertensive cardiac remodeling, whereas effective pharmacological treatments targeting cardiac inflammation remain unclear. Lipoprotein-associated phospholipase A2 (Lp-PLA2) contributes to vascular inflammation-related diseases by mediating macrophage migration and activation. Darapladib, the most advanced Lp-PLA2 inhibitor, has been evaluated in phase III trials in atherosclerosis patients. However, the role of darapladib in inhibiting hypertensive cardiac fibrosis remains unknown. Using a murine angiotensin II (Ang II) infusion-induced hypertension model, we found that Pla2g7 (the gene of Lp-PLA2) was the only upregulated PLA2 gene detected in hypertensive cardiac tissue, and it was primarily localized in heart-infiltrating macrophages. As expected, darapladib significantly prevented Ang II-induced cardiac fibrosis, ventricular hypertrophy, and cardiac dysfunction, with potent abatement of macrophage infiltration and inflammatory response. RNA sequencing revealed that darapladib strongly downregulated the expression of genes and signaling pathways related to inflammation, extracellular matrix, and proliferation. Moreover, darapladib substantially reduced the Ang II infusion-induced expression of nucleotide-binding oligomerization domain-like receptor with pyrin domain 3 (NLRP3) and interleukin (IL)-1ß and markedly attenuated caspase-1 activation in cardiac tissues. Furthermore, darapladib ameliorated Ang II-stimulated macrophage migration and IL-1ß secretion in macrophages by blocking NLRP3 inflammasome activation. Darapladib also effectively blocked macrophage-mediated transformation of fibroblasts into myofibroblasts by inhibiting the activation of the NLRP3 inflammasome in macrophages. Overall, our study identifies a novel anti-inflammatory and anti-cardiac fibrosis role of darapladib in Lp-PLA2 inhibition, elucidating the protective effects of suppressing NLRP3 inflammasome activation. Lp-PLA2 inhibition by darapladib represents a novel therapeutic strategy for hypertensive cardiac damage treatment.

The selective lipoprotein-associated phospholipase A2 inhibitor darapladib triggers irreversible actions on glioma cell apoptosis and mitochondrial dysfunction.

Although the development of a therapeutic strategy for glioblastoma multiforme (GBM), the most aggressive type of brain tumor in adults, is in progress, the prognosis is still limited. In this study, we evaluated the anti-glioma effects of darapladib, a selective reversible inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2) that is encoded by the PLA2G7 gene and serves as a predictive biomarker of sub-clinical inflammation in cardiovascular diseases. The three glioma cell lines (rat C6 glioma cell line, human U87MG, and human U251MG) and an ex vivo brain tissue slice-glioma cell co-culture system were used to validate the inhibitory effect of darapladib on the expansion of glioma cells. Exposure to darapladib at doses higher than 5 µM induced profound cytotoxicity in C6, U87MG, and U251MG. Moreover, the colony formation ability of the glioma cell lines was significantly repressed after the addition of darapladib. Although darapladib did not reduce the generation of the Lp-PLA2 downstream molecule, arachidonic acid (AA), in the glioma cells, this small compound triggered mitochondrial membrane depolarization and cell apoptosis in these glioma cells. In addition, transient exposure to darapladib induced the upregulation of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) levels, but reduced phosphorylation of AKT/PKB (protein kinase B). The results from an ex vivo brain slice culture system further confirmed the effective inhibition of darapladib on the expansion of glioma cells. In conclusion, darapladib acts as a potential anti-glioma compound via the induction of mitochondrial membrane depolarization and cell apoptosis, and the inhibition of AKT signaling in glioma cells.

Lp-PLA2 Selective Inhibitor (Darapladib) Effect In Lowering The Expression Level Of IL-1B And IL-6 In The Renal At Type 2 Diabetes Mellitus.

PURPOSE: The aim of this study is to prove that type 2 diabetes mellitus can induce increasing inflammation marker in renal and that the provision of darapladib as Lp-LA2 Inhibitor agents can inhibit inflammation that were measured from the expression of IL-1B and IL-6- type cytokine in renal. This study also discusses the correlation between IL-1B and IL-6- type cytokine expression in renal. METHODS: Thirty Sprague-Dawley (SD) rats were divided into three main groups; those are negative control group (NC), Type 2 Diabetes Mellitus group (T2DM) given high fat diet (HFD) with streptozotocin intraperitoneal injection (35mg/kg BW) and diabetes mellitus + darapladib group (DM + DP). Each group was treated within two serial treatment time: 8 weeks and 16 weeks. Expressions of IL-1B and IL-6- type cytokine in renal were the markers that we measured by immunofluorosense method. RESULTS: The administration of darapladib can significantly decrease the expression of IL-1B- type cytokine (p ANOVA = 0.029, p < 0.005) measured in rats' renal both at weeks 8 and 16 in the T2DM group. The Expression of IL-6- type cytokine also showed a significant difference after treated with darapladib both at weeks 8 and 16 in T2DM group with p-value of ANOVA = 0.033, p < 0.005. The Pearson correlation showed a strong correlation (linear regression value was r2 = 0.743). CONCLUSION: Our results show that atherosclerosis caused by inflammation in renal T2DM SD rats could be inhibited by the administration of darapladib.

Reduction in Vasa Vasorum Angiogenesis by Lp-PLA2 Selective Inhibitor Through The HIF-1α and VEGF Expression Under Dyslipidemic Conditions in Atherosclerosis Pathogenesis.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease which may lead to major cardiovascular events. The primary cause of atherosclerosis is Dyslipidemia. The increased level of lipid profile triggers endothelial dysfunction. This results in inflammation with the recruitment of monocyte, macrophage, T lymphocyte, and Mast cells secreted by an Lp-PLA2 enzyme which causes binding between macrophage and oxidized LDL. This binding results in the formation of foam cells and also the migration of smooth muscle cells. Following that, an Lp-PLA2 receptor hydrolizes OxPC which results in LysoPC and OxNEFA, bioactive compounds which stimulate the progression of atherosclerosis plaques. This process leads to cell hypoxia, which may result in the increase of HIF-1α and VEGF expressions and induction of vasa vasorum angiogenesis. Employing darapladib as an agent of Lp-PLA2 selective inhibitors, this study aimed to find out the effect of darapladib as an Lp- PLA2 selective inhibitor agent on the formation of vasa vasorum angiogenesis and the decrease of HIF-1α and VEGF expression in aortic tissue of rats with dyslipidemia. METHOD: A true laboratory experiment with a randomized post-test control group design used 30 male spraque dowley rats as animal models which were divided into 6 groups: Normal 8 weeks, Normal 16 weeks, Dyslipidemia (DL) 8 weeks, Dyslipidemia (DL) 16 weeks, Dyslipidemia with darapladib treatment (DLDP) 8 weeks and Dyslipidemia with darapladib treatment (DLDP) 16 weeks. The data measured in this study were the lipid profile (total cholesterol, HDL, and LDL). Using EnzyChrom TM kit, hematoxylin eosin, and double-labelling immunofluorescene, the levels of lipid profile, vasa vasorum, HIF-1α and VEGF were measured. RESULTS: The study results which were analyzed using NOVA test showed that with darapladib administration, there was a significant decrease in vasa vasorum angiogenesis (p=0.000), HIF-1α (p=0.005) and VEGF (p=0.009) expression in each time series. This result proves that Lp-PLA2 inhibitor reduces inflammatory process. CONCLUSION: Darapladib injection as an Lp-PLA2 selective inhibitor correlates with the decreasing vasa vasorum angiogenesis through alteration in HIF-1α and VEGF expressions in the aorta of high fat diet rats. We recommend further experiments to determine the effectiveness of darapladib with earlier time series in the atherosclerosis process.

Effect of lipoprotein-associated phospholipase A2 inhibitor on insulin resistance in streptozotocin-induced diabetic pregnant rats.

This paper aims to investigate the influence of lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibitor, darapladib, on insulin resistance (IR) in streptozotocin (STZ)-induced diabetic pregnant rats. The rat models were divided into Control (normal pregnancy), STZ + saline (STZ-induced diabetic pregnant rats), STZ + Low-dose and STZ + High-dose darapladib (STZ-induced diabetic pregnant rats treated with low-/high-dose darapladib) groups. Pathological changes were observed by Hematoxylin-eosin (HE) and Immunohistochemistry staining. Lp-PLA2 levels were determined by enzyme-linked immunosorbent assay (ELISA). An automatic biochemical analyzer was used to measure the serum levels of biochemical indicators, and homeostatic model assessment for insulin resistance (HOMA-IR) and insulin sensitivity index (ISI) were calculated. Western blot was applied to determine levels of inflammatory cytokines. Compared with Control group, rats in the STZ + saline group were significantly decreased in body weight, the number of embryo implantation, the number of insulin positive cells and pancreatic islet size as well as the islet endocrine cells, and high-density lipoprotein (HDL-C) level, but substantially increased in Lp-PLA2, low-density lipoprotein (LDL-C), fatty acids (FFA), serum total cholesterol (TC), triglyceride (TG) levels. Moreover, the increased fasting plasma glucose (FPG) and HOMA-IR and inflammatory cytokines but decreased fasting insulin (FINS) and ISI were also found in diabetic pregnant rats. On the contrary, rats in the darapladib-treated groups were just opposite to the STZ + saline group, and STZ + High-dose group improved better than STZ + Low-dose group. Thus, darapladib can improve lipid metabolism, and enhance insulin sensitivity of diabetic pregnant rats by regulating inflammatory cytokines.

Darapladib inhibits atherosclerosis development in type 2 diabetes mellitus Sprague-Dawley rat model.

OBJECTIVE: Increase in the low-density lipoprotein (LDL) level in diabetes mellitus and atherosclerosis is related to lipoprotein associated phospholipase A2 (Lp-PLA2). Lp-PLA2 is an enzyme that produces lysophosphatidylcholine (LysoPC) and oxidized nonesterified fatty acids (oxNEFA). LysoPC regulates inflammation mediators, including intra-cellular adhesion molecule-1 (ICAM-1). Darapladib is known as a Lp-PLA2 specific inhibitor. The aim of this study was to reveal the effect of darapladib on the foam cell number, inducible nitric oxide synthase (iNOS), and ICAM-1 expression in aorta at early stages of the atherosclerosis in type 2 diabetes mellitus Sprague-Dawley rat model. METHODS: Thirty Sprague-Dawley male rats were divided into 3 main groups: control, rats with type 2 diabetes mellitus (T2DM), and T2DM rats treated with darapladib (T2DM-DP). Each group was divided into 2 subgroups according the time of treatment: 8-week and 16-week treatment group. Fasting blood glucose, insulin resistance, and lipid profile were measured and analyzed to ensure T2DM model. The foam cells number were detected using hematoxylin-eosin (HE) staining and the expression of iNOS and ICAM-1 was analyzed using double immunofluorescence staining. RESULTS: Induction of T2DM in male Sprague-Dawley rats after high fat diet and streptozotocin injection was confirmed by elevated levels of total cholesterol and LDL and increased fasting glucose and insulin levels compared to controls after both times of treatment. Moreover, T2DM in rats induced a significant increase (p<0.05) in the foam cells number and iNOS and ICAM-1 expression in aorta compared to controls after both treatment times. Darapladib treatment significantly reduced (p<0.05) foam cells number as well as iNOS expression in aorta in rats with T2DM after both treatment times. A significant decrease (p<0.05) in ICAM-1 expression in aorta was observed after darapladib treatment in rats with T2DM only after 8 weeks of treatment. CONCLUSION: Our data indicate that darapladib can decrease the foam cells number, iNOS, and ICAM-1 expression in aorta at the early stages of atherosclerosis in T2DM rat model.

Regioselectivity of thiouracil alkylation: Application to optimization of Darapladib synthesis.

Darapladib is one of the most potent Lp-PLA2 (Lipoprotein-associated phospholipase A2) inhibitor with an IC50 of 0.25 nM. We demonstrate that a crucial step of Darapladib synthesis was not correctly described in the literature, leading to the production of wrong regioisomers. Moreover we show that the inhibitory activity is directly linked to the position on N1 since compounds bearing alkylation on different sites have potentially less interaction within the active site of Lp-PLA2.

Lipoprotein-associated Phospholipase A2 and Coronary Heart Disease.

In the last decades, the role of inflammation in the pathogenesis of atherosclerosis has been the topic of intense research. Several markers of inflammation have shown predictive value for first and recurrent coronary events in patients without and with established Coronary Heart Disease (CHD). Among these markers, lipoprotein- associated phospholipase A2 (Lp-PLA2) has recently received considerable attention. In the present review, the potential role of Lp-PLA2 as a marker of CHD risk and as a therapeutic target is discussed. Elevated Lp- PLA2 mass and activity appears to be associated with increased risk for CHD, both in the general population and in patients with established CHD. However, it is unclear whether the measurement of Lp-PLA2 improves risk discrimination when incorporated in models that include traditional cardiovascular risk factors. Moreover, the lack of effect on CHD events of darapladib, a potent, selective Lp-PLA2 inhibitor, in two large, randomized, placebo-controlled trials and the mostly negative findings of genetic association studies suggest that Lp-PLA2 is unlikely to represent a causal factor in atherogenesis. Therefore, it is doubtful whether Lp-PLA2 will constitute a therapeutic target for the prevention of CHD.

Performance characteristics of lipoprotein-associated phospholipase A2 activity assay on the Dimension Vista analyser and preliminary study of a healthy Italian population.

INTRODUCTION: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a marker of vascular inflammation associated with coronary heart disease and stroke. We evaluated analytical performance of the PLAC® Activity Test on Siemens Dimension Vista® 1500 analyzer and measured Lp-PLA2 activity in Italian adults to establish reference intervals (RIs) and evaluate correlation with circulating lipids and age. MATERIALS AND METHODS: The evaluation protocol consisted of precision, linearity, sensitivity, method comparison, substrate depletion ("hook") effect and interference assessment. Inhibitor (Darapladib) effect was also evaluated. Lp-PLA2 activity was measured in 250 healthy donors (123 males, 127 females, aged 18-70 years). Central 95% RIs were established using nonparametric statistics. RESULTS: Intra-assay and inter-assay precision showed CVs of 0.6% - 1.4% and 0.9% - 2.0%, respectively. Linearity replicates showed R2 > 0.98. Limit of quantitation was 5.8 U/L (CV = 9.4%). Bland Altman plot showed bias - 0.9, 95% limits of agreement -6.5 - 4.72. Passing-Bablok regression showed excellent correlation (Slope = 1.02, 95% CI: 1.01 to 1.03; Intercept = - 1.86, 95% CI: - 3.08 to - 1.26; R2 = 0.999). No "hook effect" was observed at Lp-PLA2 activities ≤ 1000 U/L. Average Lp-PLA2 activity in 250 healthy donors was 182 ± 44 U/L (mean ± SD). Males showed statistically significant higher activities than females (P < 0.001). RIs were 107 - 265 U/L for males and 84 - 225 U/L for females. Moderate significant correlation (r = 0.29, P < 0.001) was found between Lp-PLA2 activity and total cholesterol. CONCLUSIONS: The PLAC® Activity Test shows very good performance characteristics on Dimension Vista® 1500.

Investigation of a Bicyclo[1.1.1]pentane as a Phenyl Replacement within an LpPLA2 Inhibitor.

We describe the incorporation of a bicyclo[1.1.1]pentane moiety within two known LpPLA2 inhibitors to act as bioisosteric phenyl replacements. An efficient synthesis to the target compounds was enabled with a dichlorocarbene insertion into a bicyclo[1.1.0]butane system being the key transformation. Potency, physicochemical, and X-ray crystallographic data were obtained to compare the known inhibitors to their bioisosteric counterparts, which showed the isostere was well tolerated and positively impacted on the physicochemical profile.

Genetic invalidation of Lp-PLA2 as a therapeutic target: Large-scale study of five functional Lp-PLA2-lowering alleles.

Aims Darapladib, a potent inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2), has not reduced risk of cardiovascular disease outcomes in recent randomized trials. We aimed to test whether Lp-PLA2 enzyme activity is causally relevant to coronary heart disease. Methods In 72,657 patients with coronary heart disease and 110,218 controls in 23 epidemiological studies, we genotyped five functional variants: four rare loss-of-function mutations (c.109+2T > C (rs142974898), Arg82His (rs144983904), Val279Phe (rs76863441), Gln287Ter (rs140020965)) and one common modest-impact variant (Val379Ala (rs1051931)) in PLA2G7, the gene encoding Lp-PLA2. We supplemented de-novo genotyping with information on a further 45,823 coronary heart disease patients and 88,680 controls in publicly available databases and other previous studies. We conducted a systematic review of randomized trials to compare effects of darapladib treatment on soluble Lp-PLA2 activity, conventional cardiovascular risk factors, and coronary heart disease risk with corresponding effects of Lp-PLA2-lowering alleles. Results Lp-PLA2 activity was decreased by 64% ( p = 2.4 × 10-25) with carriage of any of the four loss-of-function variants, by 45% ( p < 10-300) for every allele inherited at Val279Phe, and by 2.7% ( p = 1.9 × 10-12) for every allele inherited at Val379Ala. Darapladib 160 mg once-daily reduced Lp-PLA2 activity by 65% ( p < 10-300). Causal risk ratios for coronary heart disease per 65% lower Lp-PLA2 activity were: 0.95 (0.88-1.03) with Val279Phe; 0.92 (0.74-1.16) with carriage of any loss-of-function variant; 1.01 (0.68-1.51) with Val379Ala; and 0.95 (0.89-1.02) with darapladib treatment. Conclusions In a large-scale human genetic study, none of a series of Lp-PLA2-lowering alleles was related to coronary heart disease risk, suggesting that Lp-PLA2 is unlikely to be a causal risk factor.

Darapladib, a Lipoprotein-Associated Phospholipase A2 Inhibitor, Reduces Rho Kinase Activity in Atherosclerosis.

PURPOSE: Increased lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and Rho kinase activity may be associated with atherosclerosis. The principal aim of this study was to examine whether darapladib (a selective Lp-PLA2 inhibitor) could reduce the elevated Lp-PLA2 and Rho kinase activity in atherosclerosis. MATERIALS AND METHODS: Studies were performed in male Sprague-Dawley rats. The atherosclerosis rats were prepared by feeding them with a high-cholesterol diet for 10 weeks. Low-dose darapladib (25 mg·kg⁻¹·d⁻¹) and high-dose darapladib (50 mg·kg⁻¹·d⁻¹) interventions were then administered over the course of 2 weeks. RESULTS: The serum levels of triglycerides, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), high-sensitivity C-reactive protein (hs-CRP), and Lp-PLA2, significantly increased in atherosclerosis model groups, as did Rho kinase activity and cardiomyocyte apoptosis (p<0.05 vs. sham group), whereas nitric oxide (NO) production was reduced. Levels of TC, LDL-C, CRP, Lp-PLA2, and Rho kinase activity were respectively reduced in darapladib groups, whereas NO production was enhanced. When compared to the low-dose darapladib group, the reduction of the levels of TC, LDL-C, CRP, and Lp-PLA2 was more prominent in the high-dose darapladib group (p<0.05), and the increase of NO production was more prominent (p<0.05). Cardiomyocyte apoptosis of the high-dose darapladib group was also significantly reduced compared to the low-dose darapladib group (p<0.05). However, there was no significant difference in Rho kinase activity between the low-dose darapladib group and the high-dose darapladib group (p>0.05). CONCLUSION: Darapladib, a Lp-PLA2 inhibitor, leads to cardiovascular protection that might be mediated by its inhibition of both Rho kinase and Lp-PLA2 in atherosclerosis.

Pharmacological inhibition of eicosanoids and platelet-activating factor signaling impairs zymosan-induced release of IL-23 by dendritic cells.

The engagement of the receptors for fungal patterns induces the expression of cytokines, the release of arachidonic acid, and the production of PGE2 in human dendritic cells (DC), but few data are available about other lipid mediators that may modulate DC function. The combined antagonism of leukotriene (LT) B4, cysteinyl-LT, and platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) inhibited IL23A mRNA expression in response to the fungal surrogate zymosan and to a lower extent TNFA (tumor necrosis factor-α) and CSF2 (granulocyte macrophage colony-stimulating factor) mRNA. The combination of lipid mediators and the lipid extract of zymosan-conditioned medium increased the induction of IL23A by LPS (bacterial lipopolysaccharide), thus suggesting that unlike LPS, zymosan elicits the production of mediators at a concentration enough for optimal response. Zymosan induced the release of LTB4, LTE4, 12-hydroxyeicosatetraenoic acid (12-HETE), and PAF C16:0. DC showed a high expression and detectable Ser663 phosphorylation of 5-lipoxygenase in response to zymosan, and a high expression and activity of LPCAT1/2 (lysophosphatidylcholine acyltransferase 1 and 2), the enzymes that incorporate acetate from acetyl-CoA into choline-containing lysophospholipids to produce PAF. Pharmacological modulation of the arachidonic acid cascade and the PAF receptor inhibited the binding of P-71Thr-ATF2 (activating transcription factor 2) to the IL23A promoter, thus mirroring their effects on the expression of IL23A mRNA and IL-23 protein. These results indicate that LTB4, cysteinyl-LT, and PAF, acting through their cognate G protein-coupled receptors, contribute to the phosphorylation of ATF2 and play a central role in IL23A promoter trans-activation and the cytokine signature induced by fungal patterns.

Plasma PAFAH/PLA2G7 Genetic Variability, Cardiovascular Disease, and Clinical Trials.

PAFAH is specific for short acyl groups esterified at the sn-2 position of glycerol in phospholipids, and apart from PAF, it hydrolyzes oxidized phospholipids produced during LDL oxidation. As the majority of the plasma PAFAH activity is bound in humans to LDL, it is also called the lipoprotein-associated phospholipase A2 (Lp-PLA2), and it was associated with the proinflammatory processes in atherosclerosis. The epidemiological studies in Caucasian populations demonstrated that high PAFAH levels might be a risk factor for cardiovascular disease through generation of proinflammatory lysoPC/lysoPAF and oxidized fatty free acids and led to the development of darapladib, a reversible PAFAH inhibitor. In the preclinical study in diabetic and hypercholesterolemic pigs, darapladib decreased both plasma and in situ lesion PAFAH/Lp-PLA2 activity, reduced lesion lysoPC content, and also reduced the complex coronary lesion development by reducing the necrotic core. In the recently published double-blind trial with darapladib (STABILITY study), it was shown that darapladib did not affect the time to cardiovascular death, myocardial infarction, or stroke. Similarly, in the SOLID-TIMI 52 study, darapladib did not reduce the risk of major coronary events; for those reasons, the clinical trials with darapladib will probably definitely stop in this pathology. Finally, in the absence of a tangible effect of V279F loss-of-function mutation on the cardiovascular risk in Asiatic populations and no effect of A379V polymorphism which modifies PAFAH activity in Caucasians, combined with no effect of the anti-PAFAH/Lp-PLA2 drug darapladib in clinical trials, let us conclude that it is unlikely that PAFAH could be implicated in atherosclerosis per se. We rather believe that PAFAH/Lp-PLA2 is a biomarker of atherosclerosis.

Deciphering the Causal Role of sPLA2s and Lp-PLA2 in Coronary Heart Disease.

Over the last 10 to 15 years, animal and human observational studies have identified elevated levels of both proinflammatory secretory phospholipase A2-IIA and lipoprotein-associated phospholipase A2 as potential risk factors for coronary heart disease. However, Mendelian randomization, a genetic tool to test causality of a biomarker, and phase III randomized controlled trials of inhibitors of theses enzymes (varespladib and darapladib) converged to indicate that elevated levels are unlikely to be themselves causal of coronary heart disease and that inhibition had little or no clinical utility. The concordance of findings from Mendelian randomization and clinical trials suggests that for these 2 drugs, and for other novel biomarkers in future, validation of potential therapeutic targets by genetic studies (such as Mendelian randomization) before embarking on costly phase III randomized controlled trials could increase efficiency and offset the high risk of drug development, thereby facilitating discovery of new therapeutics and mitigating against the exuberant costs of drug development.

The pharmacokinetics and safety of darapladib in subjects with severe renal impairment.

AIM: Darapladib is a potent and reversible orally active inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2 ). The aim of the study was to assess the effects of severe renal impairment on the pharmacokinetics and safety/tolerability of darapladib compared with normal renal function. METHODS: This was an open label, parallel group study of darapladib following 10 day once daily 160 mg oral dosing in subjects with normal (n = 8) and severe renal impairment (estimated glomerular filtration rate <30 ml min(-1) 1.73 m(-2) , n = 8). Plasma concentrations of total and unbound darapladib as well as total darapladib metabolites were determined in samples obtained over 24 h on day 10. RESULTS: Plasma concentrations of total and unbound darapladib as well as all three metabolites were higher in subjects with severe renal impairment. Area under the plasma concentration vs. time curve between time zero and 24 h (AUC(0,24 h) and maximum plasma concentration (Cmax ) of total darapladib in severely renally impaired subjects were 52% and 59% higher than those in the matched healthy subjects, respectively. Similar results were found with the darapladib metabolites. Darapladib was highly plasma protein bound with 0.047% and 0.034% unbound circulating in plasma in severely renally impaired and healthy subjects, respectively. Unbound plasma darapladib exposures were more than two-fold higher in severely renally impaired subjects than in healthy controls. Adverse events (AE) were reported in 38% of healthy subjects and 75% of severely renally impaired subjects, most of which were mild or moderate in intensity. CONCLUSIONS: The results of this study showed that darapladib exposure was increased in subjects with severe renal impairment compared with healthy controls. However, darapladib was generally well tolerated in both groups.

Atherosclerotic plaque inflammation varies between vascular sites and correlates with response to inhibition of lipoprotein-associated phospholipase A2.

BACKGROUND: Despite systemic exposure to risk factors, the circulatory system develops varying patterns of atherosclerosis for unclear reasons. In a porcine model, we investigated the relationship between site-specific lesion development and inflammatory pathways involved in the coronary arteries (CORs) and distal abdominal aortas (AAs). METHODS AND RESULTS: Diabetes mellitus (DM) and hypercholesterolemia (HC) were induced in 37 pigs with 3 healthy controls. Site-specific plaque development was studied by comparing plaque severity, macrophage infiltration, and inflammatory gene expression between CORs and AAs of 17 DM/HC pigs. To assess the role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in plaque development, 20 DM/HC pigs were treated with the Lp-PLA2 inhibitor darapladib and compared with the 17 DM/HC untreated pigs. DM/HC caused site-specific differences in plaque severity. In the AAs, normalized plaque area was 4.4-fold higher (P<0.001) and there were more fibroatheromas (9 of the 17 animals had a fibroatheroma in the AA and not the COR, P=0.004), while normalized macrophage staining area was 1.5-fold higher (P=0.011) compared with CORs. DM/HC caused differential expression of 8 of 87 atherosclerotic genes studied, including 3 important in inflammation with higher expression in the CORs. Darapladib-induced attenuation of normalized plaque area was site-specific, as CORs responded 2.9-fold more than AAs (P=0.045). CONCLUSIONS: While plaque severity was worse in the AAs, inflammatory genes and inflammatory pathways that use Lp-PLA2 were more important in the CORs. Our results suggest fundamental differences in inflammation between vascular sites, an important finding for the development of novel anti-inflammatory therapeutics.

Darapladib for the treatment of cardiovascular disease.

Elevated levels of phospholipase A2 have been linked to atherosclerotic plaque progression, instability via promoting inflammation and subsequent acute coronary events. Epidemiological studies have demonstrated the correlation between elevated levels associated phospholipase A2 and cardiovascular events. Therefore, specific inhibition of lipoprotein-associated phospholipase A2 with darapladib has been tested as a therapeutic option for atherosclerosis. The aim of this profile is to review the physiologic aspects of lipoprotein-associated phospholipase A2 and to revisit the clinical evidence of darapladib as therapeutic option for atherosclerosis.

A report from the European Society of Cardiology Congress 2014 (August 30-September 3 - Barcelona, Spain).

The European Society of Cardiology (ESC) Annual Congress is the largest cardiology conference in the world and this year ran in Barcelona from August 30 to September 3. During the meeting, more than 30,000 cardiologists from over 100 countries met to share their knowledge in all cardiovascular fields, from basic science to management and prevention of cardiovascular diseases. Apart from more than 4,500 interesting abstracts presented in posters and oral sessions, five new ESC Clinical Practice Guidelines were presented among the latest clinical trial results, updates and registries.