Nonsteroidal anti-inflammatory drugs (NSAIDs), selective for inhibition of cyclooxygenase (COX)-2, alleviate pain and inflammation by suppressing COX-2-derived prostacyclin (PGI2) and prostaglandin (PG) E2 (1). However, eight placebo-controlled clinical trials have revealed that NSAIDs, designed to inhibit specifically COX-2, predispose patients to increased cardiovascular risks including myocardial infarction, stroke, systemic and pulmonary hypertension, congestive heart failure, and sudden cardiac death (1-3). The cardiovascular adverse effects are attributable to the suppression of COX-2-derived PGI2, a potent vasodilator and inhibitor of platelet activation (4; 5). The research team has shown that global deletion, selective inhibition or mutation of COX-2, or deletion of the receptor for PGI2 elevate blood pressure and accelerate thrombogenesis in mouse models (6). The investigators have further demonstrated that vascular COX-2 deletion predisposes mice to thrombosis and hypertension (7), and that selective deletion of COX-2 in cardiomyocytes leads to cardiac dysfunction and enhanced susceptibility to induced arrhythmogenesis (8) that may contribute to the heart failure and cardiac arrhythmias reported in patients taking NSAIDs specific for inhibition of COX-2.
This cardiovascular hazard from NSAIDs prompted interest in the microsomal prostaglandin E synthase-1 (mPGES-1) as an alternative drug target. mPGES-1 is the inducible PG terminal synthase that acts downstream of COX-2 and catalyzes the conversion of the intermediate COX endoperoxide product PGH2 to PGE2 (9). The investigators have previously reported that similar to the interference with COX-2 expression or function, global or cell-specific deletion of mPGES-1 suppresses PGE2 production; but unlike with COX-2, global mPGES-1 deficiency augments biosynthesis of PGI2 and does not predispose normo- or hyperlipidemic mice to thrombogenic or hypertensive events (9-11). Both suppression of PGE2 and augmentation of PGI2 in mPGES-1-/- mice result from the rediversion of the accumulated PGH2 substrate to PGI2 synthase (10). Furthermore, global deletion of mPGES-1 limits the vascular proliferative response to wire injury (12), retards atherogenesis and suppresses angiotensin II-induced abdominal aortic aneurysm formation in hyperlipidemic mice (10; 13). The research team has also shown that mPGES-1-deficiency does not affect ozone-induced airway inflammation or airway hyper-responsiveness suggesting that pharmacological inhibition of mPGES-1 and endoperoxide rediversion to PGD2 may not predispose patients at risk to airway dysfunction (14). In addition, studies by others indicate that global deletion of mPGES-1 reduces the post-ischemic brain infarction and neurological dysfunction in cerebral ischemia/reperfusion in mice (15). mPGES-1 deficiency also renders mice less susceptible to excessive inflammation and hypersensitivity in rodent models of analgesia (16; 17). Taken together, these findings suggest that pharmacological inhibition of mPGES-1 may retain anti-inflammatory effects from PGE2 suppression, but due to PGI2 augmentation, targeting of mPGES-1 might avoid the cardiovascular risks associated with selective COX-2 inhibitors.
PGH2 substrate rediversion consequent to mPGES-1 deletion is a ubiquitous event observed at the cellular level and systemically (urinary prostaglandin metabolites); the profile of the rediversion products, however, varies by cell and tissue type, the disease model, and the extent of system perturbation (6; 10-14; 18-21). The investigators have shown that in mice deficient in mPGES-1 in endothelial cells (EC) or vascular smooth muscle cells (VSMC), PGI2 is the predominant substrate rediversion product, whereas deletion of mPGES-1 in myeloid cells results in shunting of PGH2 mostly towards TxA2(11). Functionally, mice lacking mPGES-1 in myeloid cells, exhibited a poor response to vascular injury implicating myeloid mPGES-1 as a cardiovascular drug target. Therefore, cell-specific mPGES-1 deletion leads to a differential pattern of substrate rediversion and may affect biological function of the system, thus complicating drug development. What is unknown is whether genetic deletion or pharmacological inhibition of mPGES-1 can directly (through substrate rediversion) or indirectly (by effects of prostaglandin rediversion products on enzyme expression or their further metabolism to transcellular products (22)) influence the lipidome beyond the prostaglandin pathway with functional consequence. For example, disruption of AA-PGE2 metabolism might influence arachidonate product formation by the cytochrome P450 (23; 24), leukotriene, anandamide, 2-arachidonylglycerol (2-AG) and other cascades (25). At the cellular level, mPGES-1-/- macrophages, pretreated with LPS and stimulated with arachidonic acid (AA), exhibit a 5-fold increase in 12-HHT (12-hydroxyheptadecatrienoic acid), indicating substrate rediversion towards thromboxane A synthase (18). Inhibition and deletion of COX-2 have been reported to augment metabolites of 5-lipoxygenase (5-LO) pathway 5-HETE (5-hydroxyeicosatetraenoic acid) and leukotrienes LTB4, LTC4, LTD4 (26-28), and metabolites of CYP450 cascade 14,15-DHET/EET (dihydroxyeicosatrienoic/epoxyeicosatrienoic acid) (26). Therefore, the substrate AA may be shunted from one pathway to the other when a particular branch of the cascade is pharmacologically inhibited or genetically ablated.
Here, the research team will conduct a broad-spectrum lipidomics screening of anti-inflammatory drugs and drug candidates that antagonize receptors (LTC4, LTB4, EP4 receptors) or inhibit specific components (COX-1, COX-2, mPGES-1, 5-KO, FLAP, LTA4A) of arachidonic acid pathway in an in vitro human whole-blood assay (hWBA).
Preliminary in vitro results from Part A demonstrated that targeting of COX-2 with a selective COX-2 inhibitor celecoxib affected not only cyclooxygenase pathway but also lipoxygenase cascade. Celecoxib inhibited COX-derived products PGE2, PGF2a and TxB2 and significantly reduced levels of 15-HETE, a product of 15-LOX cascade.
In Part B, the investigators propose to study the effect of celecoxib on plasma lipids ex vivo. Healthy, non-smoking, male and female volunteers will be asked to take a single, therapeutic dose of 200 mg of celecoxib or a placebo pill and provide blood and urine samples before and after the drug administration. Experiments will include (i) the ex vivo whole blood assay, in which lipids will be measured in blood collected before and 3 hours (Tmax) after administration of celecoxib and stimulated with LPS, (ii) lipid metabolites will be measured in pre- and post-celecoxib urine samples, (iii) celecoxib plasma and urine concentrations will be measured to evaluate the pharmacokinetic profile of the study drug.
The investigators expect that lipid profile from ex vivo hWBA done on celecoxib-treated subjects will recapitulate findings from the in vitro hWBA received with celecoxib-treated human blood.
