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.

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 IC(50) 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).

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 A(2) (sPLA(2)) in experimental atherosclerosis, the involvement of various sPLA(2) isozymes as mediators of pro-atherogenic lipoprotein remodeling and participants in vascular and systemic inflammatory responses, and the evidence that sPLA(2) 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 sPLA(2) 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.

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

BACKGROUND: The 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. METHODS: Twenty-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. RESULTS: Plasma 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%. CONCLUSION: The use of A-002 may have a beneficial effect in preventing diet-induced atherosclerosis in guinea pigs.

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.

The synergistic inhibition of atherogenesis in apoE-/- mice between pravastatin and the sPLA2 inhibitor varespladib (A-002).

Secretory phospholipase A2 (sPLA2) activity promotes foam cell formation, increases proinflammatory bioactive lipid levels, decreases HDL levels, increases atherosclerosis in transgenic mice, and is an independent marker of cardiovascular disease. The effects of the sPLA2 inhibitor A-002 (varespladib) and pravastatin as monotherapies and in combination on atherosclerosis, lipids, and paraoxonase (PON) activity in apoE(-/-) mice were investigated. Male apoE(-/-) mice were placed on a 12-week high-fat diet supplemented with A-002 alone or combined with pravastatin. Atherosclerotic lesions were examined for size and composition using en face analysis, Movat staining, anti-CD68, and anti-alpha actin antibodies. Plasma lipids and PON activity were measured. A-002 decreased atherosclerotic lesion area by approximately 75% while increasing fibrous cap size by over 200%. HDL levels increased 40% and plasma PON activity increased 80%. Pravastatin monotherapy had no effect on lesion size but when combined with A-002, decreased lesion area 50% and total cholesterol levels 18% more than A-002 alone. A-002, a sPLA2 inhibitor, acts synergistically with pravastatin to decrease atherosclerosis, possibly through decreased levels of systemic inflammation or decreased lipid levels. A-002 treatment also resulted in a profound increase in plasma PON activity and significantly larger fibrous caps, suggesting the formation of more stable plaque architecture.

Novel antibacterial azetidine lincosamides.

The synthesis and evaluation of novel azetidine lincosamides 1 are described. Eleven new (3-trans-alkyl)azetidine-2-carboxylic acids were synthesized via alkylation of N-TBS-4-oxo-azetidine-2-carboxylic acid and subsequent elaboration then coupled to 7-chloro-1-methylthio-lincosamine. The resulting lincosamides differ from the drug clindamycin in both the size of the ring and the position/structure of the alkyl side-chain. SAR within the series was explored with attention to alkyl variants in positions 1 and 3 of the azetidine ring.

Neutrophil chemotaxis by pathogen-associated molecular patterns--formylated peptides are crucial but not the sole neutrophil attractants produced by Staphylococcus aureus.

The chemotactic migration of phagocytes to sites of infection, guided by gradients of microbial molecules, plays a key role in the first line of host defence. Bacteria are distinguished from eukaryotes by initiation of protein synthesis with formyl methionine. Synthetic formylated peptides (FPs) have been shown to be chemotactic for phagocytes, leading to the concept of FPs as pathogen-associated molecular patterns (PAMPs). However, it remains unclear whether FPs are major chemoattractants released by bacteria and whether further chemoattractants are produced. A Staphylococcus aureus mutant whose formyltransferase gene was inactivated (Deltafmt) produced no FPs and the in vitro and in vivo ability of Deltafmt culture supernatants to recruit neutrophils was considerably reduced compared with those of the parental strain. However, some chemotactic activity was retained, indicating that bacteria produce also unknown, non-FP chemoattractants. The activity of these novel PAMPs was sensitive to pertussis toxin but insensitive to the formyl peptide receptor inhibitor CHIPS. Deltafmt culture supernatants caused reduced calcium ion fluxes and reduced CD11b upregulation in neutrophils compared with wild-type supernatants. These data demonstrate an important role of FPs in innate immunity against bacterial infections and indicate that host chemotaxis receptors recognize a larger set of bacterial molecules than previously thought.

In vitro antistaphylococcal activity of dalbavancin, a novel glycopeptide.

OBJECTIVES: Dalbavancin is a novel, semi-synthetic glycopeptide antibiotic. The aim of this study was to further explore its activity against staphylococci. METHODS: The bactericidal activity of dalbavancin was studied using MBC and time-kill methods. The potential for resistance to dalbavancin was examined using single-step and serial-passage experiments. RESULTS: Dalbavancin was bactericidal against methicillin-susceptible and -resistant Staphylococcus aureus, in both the presence and absence of human serum. No resistance was seen with any isolate tested. After serial passage, bacterial populations were more homogeneous in their susceptibility to dalbavancin than to vancomycin or teicoplanin. CONCLUSION: Dalbavancin is bactericidal for staphylococci. Resistance to this semi-synthetic glycopeptide is not readily developed in vitro.

Conformationally restricted analogs of deoxynegamycin.

Deoxynegamycin (1b) is a protein synthesis inhibitor with activity against Gram-negative (GN) bacteria. A series of conformationally restricted analogs were synthesized to probe its bioactive conformation. Indeed, some of the constrained analogs were found to be equal or better than deoxynegamycin in protein synthesis assay (1b, IC(50)=8.2 microM; 44, IC(50)=6.6 microM; 35e(2), IC(50)=1 microM). However, deoxynegamycin had the best in vitro whole cell antibacterial activity (Escherichia coli, MIC=4-16 microg/mL; Klebsiella pneumoniae, MIC=8 microg/mL) suggesting that other factors such as permeation may also be contributing to the overall whole cell activity. A new finding is that deoxynegamycin is efficacious in an E. coli murine septicemia model (ED(50)=4.8 mg/kg), providing further evidence of the favorable in vivo properties of this class of molecules.

New antibacterial tetrahydro-4(2H)-thiopyran and thiomorpholine S-oxide and S,S-dioxide phenyloxazolidinones.

Combinatorial libraries of N-acylated 5-(S)-aminomethyloxazolidinone derivatives of S-oxide and S,S-dioxide tetrahydro-4(2H)-thiopyranyl and thiomorpholine phenyloxazolidinone series have been synthesized on a solid phase and evaluated for antimicrobial activity. Several novel potent leads have been identified, including orally active oxazolidinones with enhanced activity against respiratory tract infection pathogens Haemophilus influenzae and Moraxella catarrhalis.

Novel oxazolidinone-quinolone hybrid antimicrobials.

Antimicrobial compounds incorporating oxazolidinone and quinolone pharmacophore substructures have been synthesized and evaluated. Representative analogues 2, 5, and 6 display an improved potency versus linezolid against gram-positive and fastidious gram-negative pathogens. The compounds are also active against linezolid- and ciprofloxacin-resistant Staphylococcus aureus and Enterococcus faecium strains. The MOA for these new antimicrobials is consistent with a combination of protein synthesis and gyrase A/topoisomerase IV inhibition, with a structure-dependent degree of the contribution from each inhibitory mechanism.

Targeting metalloenzymes: a strategy that works.

Faced with a wealth of antibacterial drug discovery targets as a result of bacterial genomics, we need to carefully select which ones to work with. Choosing bacterial metalloenzymes is one possible approach that can increase the probability of success. Metalloenzymes can be identified through specific motif searches of bacterial genomes. Current state-of-the-art medicinal chemistry allows for the design of inhibitor libraries targeting metalloenzymes and the efficient optimization of leads identified. This approach has been successfully applied to the discovery of in vivo active antibacterial agents that are inhibitors of bacterial peptide deformylase and UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase. Other bacterial metalloenzymes are open to the same approach.

Combinatorial modification of natural products: synthesis and in vitro analysis of derivatives of thiazole peptide antibiotic GE2270 A: A-ring modifications.

Thiazole peptide GE2270 A (1) possesses potent antimicrobial activity against many gram-positive pathogens, including methicillin resistant Staphylococcus aureus (S. aureus, MRSA; MIC(90)=0.06 microg/mL) and vancomycin resistant Enterococcus spp. (VRE; MIC(90)=0.03 microg/mL); however its poor aqueous solubility has prohibited its development for the clinical treatment of infections. An integrated combinatorial and medicinal chemistry program was employed to identify derivatives of 1 that retain activity but possess greatly enhanced aqueous solubility.

D-Ala-D-lac binding is not required for the high activity of vancomycin dimers against vancomycin resistant enterococci.

Covalent dimerization and oligomerization of vancomycin is an important and extensively used strategy to develop analogues active against vancomycin resistant enteroccoci (VRE). Here, we have carried out investigations to probe the role of peptide binding (Lys-d-Ala-d-Lac) in the high anti-VRE activities of covalently linked vancomycin dimers. Covalent dimers of damaged vancomycin (desleucyl) were prepared, and their anti-VRE activities and binding affinities toward various model peptides were measured. Despite the dramatic loss in affinity toward several model peptides in comparison to the corresponding intact vancomycin dimers, these damaged dimers maintained good activity against VRE. These results strongly suggest that the high anti-VRE activities of covalent vancomycin dimers are conferred from mechanisms other than Lys-d-Ala-d-Lac binding.

N- and C-terminal modifications of negamycin.

Negamycin 1 is a bactericidal antibiotic with activity against Gram-negative bacteria, and served as a template in an antibiotic discovery program. An orthogonally protected beta-amino acid derivative 3a was synthesized and used in parallel synthesis of negamycin derivatives on solid support. This advanced intermediate was also used for N- and C-terminal modifications using solution-phase methodologies. The N-terminal modifications have resulted in the identification of active analogues, whereas the C-terminal modifications resulted in complete loss of antibacterial activity. The N-methyl negamycin analogue, 19a, inhibits protein synthesis (IC(50)=2.3 microM), has antibacterial activity (Escherichia coli, MIC=16 microgram/mL), and is efficacious in an E. coli murine septicemia model (ED(50)=16.3mg/kg).

Identification of potent and broad-spectrum antibiotics from SAR studies of a synthetic vancomycin analogue.

Dimeric vancomycin analogues based on a lead compound identified from a library of synthetic analogues of vancomycin have up to 60-fold greater activity than vancomycin against vancomycin-resistant Enterococcus faecium (VRE, VanA phenotype). Simplified analogues have also been prepared and found to maintain activity against VRE and have broad-spectrum antibiotic activity.

N-alkyl urea hydroxamic acids as a new class of peptide deformylase inhibitors with antibacterial activity.

Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth and is a new target for the development of antibacterial agents. All previously reported PDF inhibitors with sufficient antibacterial activity share the structural feature of a 2-substituted alkanoyl at the P(1)' site. Using a combination of iterative parallel synthesis and traditional medicinal chemistry, we have identified a new class of PDF inhibitors with N-alkyl urea at the P(1)' site. Compounds with MICs of 200 micro M for matrilysin and other mammalian metalloproteases. Structure-activity relationship analysis identified preferred substitutions resulting in improved potency and decreased cytotoxity. One of the compounds (VRC4307) was cocrystallized with PDF, and the enzyme-inhibitor structure was determined at a resolution of 1.7 A. This structural information indicated that the urea compounds adopt a binding position similar to that previously determined for succinate hydroxamates. Two compounds, VRC4232 and VRC4307, displayed in vivo efficacy in a mouse protection assay, with 50% protective doses of 30.8 and 17.9 mg/kg of body weight, respectively. These N-alkyl urea hydroxamic acids provide a starting point for identifying new PDF inhibitors that can serve as antimicrobial agents.

Mining bacterial cell wall biosynthesis with new tools: multitarget screens.

The cytoplasmic steps of peptidoglycan synthesis remain underexplored for the discovery of novel antibiotics. Pathway screens are well suited to screen for novel inhibitors because several targets are tested at once. Whole-cell based assays are easier to set up but they tend to favor identification of compounds that inhibit enzymes that are at the limiting rate in the pathway. Screens that use purified enzymes are optimized to detect with similar probability inhibitors of any of the targets in the pathway. This approach is being used to identify novel promising molecules. Copyright 1999 Harcourt Publishers Ltd.