Lipidomics Screening of Celecoxib in ex Vivo Human Whole Blood Assay - Part B

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.

Inclusion Criteria:

- Age between 18 - 50

- Volunteers must be in good health as based on medical history

- All volunteers must be non-smoking and non-pregnant

Exclusion Criteria:

- Subjects with any medical condition, which according to the investigator, may
interfere with interpretation of the study results, be indicative of an underlying
disease state, or compromise the safety of a potential subject (cancer or history of
significant cardiovascular disease (including stroke or TIA), renal, hepatic,
gastrointestinal, respiratory, endocrine, metabolic, hematopoietic, or neurological
disorders).

- Subjects who have received an experimental drug within 30 days prior to the study

- Subjects who have taken medications at least two weeks prior to the study. Subjects
using hormonal birth control, however, will not be an exclusionary criterion.

- Subjects who have taken aspirin or aspirin containing products for at least two weeks
prior to the study.

- Subjects who are sensitive or allergic to celecoxib (Celebrex) or its components

- Subjects who have taken any formulation of celecoxib including but not limited to
Celebrex, Celebra, Onsenal for at least two weeks prior to the start of the study and
throughout the study

- Subjects who have taken acetaminophen, NSAIDs, COX-2 inhibitors (OTC or prescription)
for at least two weeks prior to the study.

- Subjects who are consuming any type of tobacco product(s).

- Subjects who consume high doses of antioxidant vitamins daily (vitamin C> 1000mg,
Vitamin E> 400IU, Beta Carotene> 1000IU, Vitamin A> 5000IU, Selenium> 200mcg, Folic
Acid> 1mg) for the two weeks prior to the start of the study and throughout the
study.

- Subjects who consume alcohol, caffeine or high fat food 24 hours prior to the study.