Lipidomics Screening of Anti-inflammatory Drugs and Drug Candidates in Vitro - Part A

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). Our laboratory 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). We 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). We 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). We have 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). We 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, we 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). Healthy, non-smoking, male and female volunteers will
be asked to donate blood. Human whole blood assays will include (i) determination of the
baseline lipid levels at various time points in stimulated whole blood, (ii) measurement of
lipids in pre-stimulated whole blood treated with a single intervention compound, (iii)
quantitation of lipids in pre-stimulated whole blood treated with a combination of
intervention compounds. We expect that the compounds at focus will affect various
inflammatory pathways resulting in new patterns of substrate rediversion and measurement of
previously unknown lipid products.

Inclusion Criteria:

- age between 18-50

- non-pregnant females

- non-smoking males and females

- in good health as based on medical history

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.

- 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 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.

- Pregnant female subjects