Susanne Fries

Drug Resistance and Pseudoresistance An Unintended Consequence of Enteric Coating Aspirin

Background— Low dose aspirin reduces the secondary incidence of myocardial infarction and stroke. Drug resistance to aspirin might result in treatment failure. Despite this concern, no clear definition of aspirin resistance has emerged, and estimates of its incidence have varied remarkably. We aimed to determine the commonality of a mechanistically consistent, stable, and specific phenotype of true pharmacological resistance to aspirin—such as might be explained by genetic causes. Methods and Results— Healthy volunteers (n=400) were screened for their response to a single oral dose of 325-mg immediate release or enteric coated aspirin. Response parameters reflected the activity of the molecular target of aspirin, cyclooxygenase-1. Individuals who appeared aspirin resistant on 1 occasion underwent repeat testing, and if still resistant were exposed to low-dose enteric coated aspirin (81 mg) and clopidogrel (75 mg) for 1 week each. Variable absorption caused a high frequency of apparent resistance to a single dose of 325-mg enteric coated aspirin (up to 49%) but not to immediate release aspirin (0%). All individuals responded to aspirin on repeated exposure, extension of the postdosing interval, or addition of aspirin to their platelets ex vivo. Conclusions— Pharmacological resistance to aspirin is rare; this study failed to identify a single case of true drug resistance. Pseudoresistance, reflecting delayed and reduced drug absorption, complicates enteric coated but not immediate release aspirin administration. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00948987.

COX-2–Derived Prostacyclin Modulates Vascular Remodeling

Suppression of prostacyclin (PGI2) biosynthesis may explain the increased incidence of myocardial infarction and stroke which has been observed in placebo controlled trials of cyclooxygenase (COX)-2 inhibitors. Herein, we examine if COX-2–derived PGI2 might condition the response of the vasculature to sustained physiologic stress in experimental models that retain endothelial integrity. Deletion of the PGI2 receptor (IP) or suppression of PGI2 with the selective COX-2 inhibitor, nimesulide, both augment intimal hyperplasia while preserving luminal geometry in mouse models of transplant arteriosclerosis or flow-induced vascular remodeling. Moreover, nimesulide or IP deletion augments the reduction in blood flow caused by common carotid artery ligation in wild-type mice. Generation of both thromboxane (Tx)A2 and the isoprostane, 8, 12 –iso iPF2α-VI, are increased in the setting of flow reduction and the latter increases further on administration of nimesulide. Deletion of the TxA2 receptor (TP) reduces the hyperplastic response to nimesulide and carotid ligation, despite further augmentation of TP ligand production. Suppression of COX-2–derived PGI2 or deletion of IP profoundly influences the architectural response of the vasculature to hemodynamic stress. Mechanism based vascular remodeling may interact with a predisposition to hypertension and atherosclerosis in contributing to the gradual transformation of cardiovascular risk during extended periods of treatment with selective inhibitors of COX-2.

Tetranor PGDM, an Abundant Urinary Metabolite Reflects Biosynthesis of Prostaglandin D2 in Mice and Humans

Prostaglandin D2 (PGD2) is a cyclooxygenase (COX) product of arachidonic acid that activates D prostanoid receptors to modulate vascular, platelet, and leukocyte function in vitro. However, little is known about its enzymatic origin or its formation in vivo in cardiovascular or inflammatory disease. 11,15-Dioxo-9α-hydroxy-2,3,4,5-tetranorprostan-1,20-dioic acid (tetranor PGDM) was identified by mass spectrometry as a metabolite of infused PGD2 that is detectable in mouse and human urine. Using liquid chromatography-tandem mass spectrometry, tetranor PGDM was much more abundant than the PGD2 metabolites, 11β-PGF2α and 2,3-dinor-11β-PGF2α, in human urine and was the only endogenous metabolite detectable in mouse urine. Infusion of PGD2 dose dependently increased urinary tetranor PGDM > 2,3-dinor-11β-PGF2α > 11β-PGF2α in mice. Deletion of either lipocalin-type or hemopoietic PGD synthase enzymes decreased urinary tetranor PGDM. Deletion or knockdown of COX-1, but not deletion of COX-2, decreased urinary tetranor PGDM in mice. Correspondingly, both PGDM and 2,3-dinor-11β-PGF2α were suppressed by inhibition of COX-1 and COX-2, but not by selective inhibition of COX-2 in humans. PGD2 has been implicated in both the development and resolution of inflammation. Administration of bacterial lipopolysaccharide coordinately elevated tetranor PGDM and 2,3-dinor-11β-PGF2α in volunteers, coincident with a pyrexial and systemic inflammatory response, but both metabolites fell during the resolution phase. Niacin increased tetranor PGDM and 2,3-dinor-11β-PGF2α in humans coincident with facial flushing. Tetranor PGDM is an abundant metabolite in urine that reflects modulated biosynthesis of PGD2 in humans and mice.

Novel Eicosapentaenoic Acid-derived F3-isoprostanes as Biomarkers of Lipid Peroxidation

Isoprostanes (iPs) are prostaglandin (PG) isomers generated by free radical-catalyzed peroxidation of polyunsaturated fatty acids (PUFAs). Urinary F2-iPs, PGF2α isomers derived from arachidonic acid (AA) are used as indices of lipid peroxidation in vivo. We now report the characterization of two major F3-iPs, 5-epi-8,12-iso-iPF3α-VI and 8,12-iso-iPF3α-VI, derived from the ω-3 fatty acid, eicosapentaenoic acid (EPA). Although the potential therapeutic benefits of EPA receive much attention, a shift toward a diet rich in ω-3 PUFAs may also predispose to enhanced lipid peroxidation. Urinary 5-epi-8,12-iso-iPF3α-VI and 8,12-iso-iPF3α-VI are highly correlated and unaltered by cyclooxygenase inhibition in humans. Fish oil dose-dependently elevates urinary F3-iPs in mice and a shift in dietary ω-3/ω-6 PUFAs is reflected by an increasing slope [m] of the line relating urinary 8, 12-iso-iPF3α-VI and 8,12-iso-iPF2α-VI. Administration of bacterial lipopolysaccharide evokes a reversible increase in both urinary 8,12-iso-iPF3α-VI and 8,12-iso-iPF2α-VI in humans on an ad lib diet. However, while excretion of the iPs is highly correlated (R2 median = 0.8), [m] varies by an order of magnitude, reflecting marked inter-individual variability in the relative peroxidation of ω-3 versus ω-6 substrates. Clustered analysis of F2- and F3-iPs refines assessment of the oxidant stress response to an inflammatory stimulus in vivo by integrating variability in dietary intake of ω-3/ω-6 PUFAs.

Differential impairment of aspirin-dependent platelet cyclooxygenase acetylation by nonsteroidal antiinflammatory drugs

Significance Painkillers classified as nonsteroidal antiinflammatory drugs (NSAIDs) are among the most commonly consumed drugs. Although they ameliorate pain effectively by inhibiting the enzyme cyclooxygenase, they can cause serious cardiovascular complications, including heart attack and stroke. Additionally, NSAIDs have the potential to render low-dose aspirin taken to reduce the risk of heart attack and stroke ineffective through a drug–drug interaction, and there is great uncertainty in how to manage pain in patients with cardiovascular disease. We developed a MS assay to quantitate precisely the interaction of aspirin with NSAIDs. Exposure of volunteers to aspirin revealed a potent drug–drug interaction with ibuprofen and naproxen but not celecoxib. This observation has relevance to the interpretability of ongoing randomized clinical trials comparing the safety of NSAIDs. The cardiovascular safety of nonsteroidal antiinflammatory drugs (NSAIDs) may be influenced by interactions with antiplatelet doses of aspirin. We sought to quantitate precisely the propensity of commonly consumed NSAIDs—ibuprofen, naproxen, and celecoxib—to cause a drug–drug interaction with aspirin in vivo by measuring the target engagement of aspirin directly by MS. We developed a novel assay of cyclooxygenase-1 (COX-1) acetylation in platelets isolated from volunteers who were administered aspirin and used conventional and microfluidic assays to evaluate platelet function. Although ibuprofen, naproxen, and celecoxib all had the potential to compete with the access of aspirin to the substrate binding channel of COX-1 in vitro, exposure of volunteers to a single therapeutic dose of each NSAID followed by 325 mg aspirin revealed a potent drug–drug interaction between ibuprofen and aspirin and between naproxen and aspirin but not between celecoxib and aspirin. The imprecision of estimates of aspirin consumption and the differential impact on the ability of aspirin to inactivate platelet COX-1 will confound head-to-head comparisons of distinct NSAIDs in ongoing clinical studies designed to measure their cardiovascular risk.

Microfluidic Assay of Platelet Deposition on Collagen by Perfusion of Whole Blood from Healthy Individuals Taking Aspirin

BACKGROUND Microfluidic devices can create hemodynamic conditions for platelet assays. We validated an 8-channel device in a study of interdonor response to acetylsalicylic acid (ASA, aspirin) with whole blood from 28 healthy individuals. METHODS Platelet deposition was assessed before treatment or 24 h after ingestion of 325 mg ASA. Whole blood (plus 100 μmol/L H-d-Phe-Pro-Arg-chloromethylketone to inhibit thrombin) was further treated ex vivo with ASA (0-500 μmol/L) and perfused over fibrillar collagen for 300 s at a venous wall shear rate (200 s(-1)). RESULTS Ex vivo ASA addition to blood drawn before aspirin ingestion caused a reduction in platelet deposition [half-maximal inhibitory concentration (IC50) approximately 10-20 μmol/L], especially between 150 and 300 s of perfusion, when secondary aggregation mediated by thromboxane was expected. Twenty-seven of 28 individuals displayed smaller deposits (45% mean reduction; range 10%-90%; P < 0.001) from blood obtained 24 h after ASA ingestion (no ASA added ex vivo). In replicate tests, an R value to score secondary aggregation [deposition rate from 150 to 300 s normalized by rate from 60 to 150 s] showed R < 1 in only 2 of 28 individuals without ASA ingestion, with R > 1 in only 3 of 28 individuals after 500 μmol/L ASA addition ex vivo. At 24 h after ASA ingestion, 21 of 28 individuals displayed poor secondary aggregation (R < 1) without ex vivo ASA addition, whereas the 7 individuals with residual secondary aggregation (R > 1) displayed insensitivity to ex vivo ASA addition. Platelet deposition was not correlated with platelet count. Ex vivo ASA addition caused similar inhibition at venous and arterial wall shear rates. CONCLUSIONS Microfluidic devices quantified platelet deposition after ingestion or ex vivo addition of aspirin.

Cyclooxygenase inhibitors repress vascular hyaluronan-synthesis in murine atherosclerosis and neointimal thickening

Hyaluronan (HA) is a key molecule of the extracellular matrix that is thought to be critically involved in both atherosclerosis and restenosis. Recently, it has been demonstrated that the cyclooxygenase (COX) products, prostacyclin and prostaglandin E2, induce HA synthesis in vitro by transcriptional up‐regulation of HA‐synthase 2 (HAS2) and HAS1. The relative roles in atherosclerotic and restenotic artery disease of tissue specifically expressed COX‐1 and COX‐2 are still under debate. Thus, the present study aimed to investigate the effect of COX isoform inhibition on HA‐accumulation and regulation of HAS isoform expression in two models of pathologic artery remodelling in vivo. Firstly, ApoE‐deficient mice were treated with a prototypic isoform non‐selective inhibitor, indomethacin or with a prototypic COX‐2 selective inhibitor, rofecoxib, for 8 weeks. Aortic HAS mRNA expression and HA‐accumulation in atherosclerotic aortic root lesions were analyzed. Secondly, neointimal hyperplasia was induced by carotid artery ligation in ApoE‐deficient mice on a high fat diet and the effects of the COX inhibitors were determined after 4 weeks of treatment. Intimal HA‐accumulation was markedly reduced in both models by indomethacin and rofecoxib. This coincided with a strong inhibition of HAS1 mRNA expression in both models and with decreased HAS2 mRNA in the aorta of ApoE‐deficient mice. HAS3 was not affected. The repression of HA‐accumulation by both COX‐2 selective and non‐selective COX inhibition implicates COX‐2 in the regulation of HA synthesis via stimulation of HAS1 and HAS2 expression in vivo. Modulation of vascular HA‐accumulation might play a role in chronic effects of COX inhibitors on the progression of atherosclerosis.

Lipoxygenase and prostaglandin G/H synthase cascades in cardiovascular disease

The 12/15-lipoxygenase (LO) cascade governs the generation of 12-hydroperoxy-eicosatetraenoic acid (HPETE) and 15-HPETE from arachidonic acid. The 5-LO pathway plays a fundamental role in the biosynthesis of leukotrienes, essential inflammatory lipid mediators. Cyclooxygenase (COX)-1 and -2 biosynthetic pathways are responsible for prostaglandin and thromboxane formation. Experimental investigations in animal models using 12/15-LO deficient mice, 12/15-LO or 15-LO transgenic mice, or pharmacological 15-LO inhibition have all demonstrated the essential role of 12/15-LO in atherogenesis. The underlying mechanisms are linked to low-density lipoprotein oxidation, pro-inflammatory Th1 cytokine production and enhanced monocyte–endothelial cell interaction. Human genetic studies as well as disruption of the 5-LO gene in mouse models of hyperlipidemia revealed that 5-LO and 5-LO-activating protein are associated with risks of human cardiovascular disease, and that this cascade plays an important role in aortic aneurysm pathogenesis through leukotriene-mediated inflammatory chemokine production. COX-1 plays an active role in atherogenesis via thromboxane A2, while COX-2-derived prostaglandin (PGI2) protects against atherosclerosis in murine models. Recent data demonstrated that selective inhibition of COX-2 augments the risk of cardiovascular events in patients. Selective inhibition or blockade of selective components in these two enzymatic pathways through systemic drug delivery or medical device approaches (e.g., drug-eluting stents) may have therapeutic benefit against certain cardiovascular diseases.

CHAPTER 31 – Thromboxane Generation

A short-lived lipid mediator, thromboxane (Tx) A2, is synthesized by activated platelets and released as a local signal, which amplifies activation and recruits additional platelets to the site of clot formation1,2 TxA2 is also a potent vasoconstrictor and stimulates mitogenesis, accelerating hemostasis and the proliferative response to vascular injury.3-6 Derived from arachidonic acid (AA) by the sequential action of prostaglandin G/H synthase (PGHS)-1 and thromboxane synthase (TxAS) (Fig. 31-1), TxA2 targets a specific G protein-coupled transmembrane thromboxane receptor (TP) that signals to facilitate dense granule secretion and integrin αIIbβ3 activation in platelets. Patients deficient in TxA2 formation have a mild bleeding disorder,7-16 as have those with a defective response to TxA2, 17-19 such as an arginine60 to leucine mutation of the TP which disrupts receptor-G protein interactions. 20

Platelet Prostanoids and their receptors

The prostanoids, which include the prostaglandins (PGs) and thromboxane (Tx)A2, are a family of locally acting lipid mediators. Two isozymes, cyclooxygenase (COX)-1 and COX-2, of which only COX-1 is expressed in mature platelets, form an unstable intermediate product, PGH2, from arachidonic acid released from membrane phospholipids. At least nine PG synthases metabolize PGH2 further to five biologically active prostanoids, which each act through one or more specific G-protein coupled receptor.