Lovaza's Effect on the Activation of Platelets

Cardiovascular disease remains a leading cause of death in North America (1). Uncontrolled
platelet activation, adhesion and aggregation initiated by vessel wall plaque rupture are
thought to be responsible for acute vascular occlusion in many situations (2-5). Although
many platelet inhibition drugs are available, all currently available drugs have undesirable
toxicity profiles (6-8). Thus, reduction in toxicity and improved management of patients
with thrombotic diseases remains an unmet medical need.

Platelet activation plays a pivotal role in the pathogenesis of acute coronary syndromes,
strokes and other thrombophilic diseases. Atheromatous plaque rupture changes the shear
forces of blood flowing over the injured vessel surface and also exposes collagen as well as
other prothrombotic factors (9-11). As the initial hemostatic event, platelets become
activated and cover the injured surface. Following platelet activation highly active
substances like adenosine diphosphate (ADP) and thromboxane A2 (TxA2) are released from the
platelet to promote and recruit further platelet aggregation to the injury site (12). If
this process proceeds unabated, as it often does in atherosclerotic diseases, the vessel
becomes occluded and infarction may follow.

Lovaza® (Reliant Pharmaceutical Inc., Liberty Corner, NJ), a commercially available
formulation that contains 90 % omega-3-acid ethyl esters (46% eicosapentaenoic acid -EPA-
and 38% docosohexaenoic acid -DHA-), has the potential ability to modify the recruitment of
additional platelets to the growing thrombus by promoting synthesis of thromboxane A3
(TxA3), a poor platelet activator, instead of thromboxane A2, a potent platelet activator.
Agents used to inhibit platelet function such as aspirin and clopidogrel are not always
effective (13-16). Unfortunately, some patients do not respond to these therapeutics
(17-24). Realistic numbers for patient resistance to these drugs are probably 10-15% for
ASA and 20-30% for clopidogrel. Almost all resistant patients have less favorable outcomes
and are unaware of this potentially life-threatening problem until a severe cardiac adverse
event occurs. Lovaza® may add additional therapeutic benefit to these patients.(25,26)
Beyond the occasional patient with complaints of eructation or a "fishy" taste in their
mouth, Lovaza® has a benign toxicity profile. If Lovaza® can be shown to have a clinically
relevant anti-platelet effect, it may have a use to either replace or reduce the dose of
more toxic anti-platelet agents.

The proposed biochemical mechanism for the anti-platelet effect of omega n3 fatty acids is
based on modifications in platelet prostaglandin metabolism (27-31). Cellular membranes are
primarily composed of phospholipids (PL). The backbone of PL's is glycerol. The glycerol
hydroxyl groups in position 1 and 2 bind two fatty acid molecules through formation of ester
bonds (31). The third hydroxyl binds the so-called head group, which may be choline,
inositol, ethanolamine or serine. At least in the case of platelets the fatty acid at the
C-2 position is often the unsaturated arachidonic fatty acid (an omega n6 fatty acid). When
Lovaza® is ingested (an omega n3 fatty acid), the unsaturated fatty acid at the C2 position
can be DHA or EPA. Several important differences result from this substitution including an
important effect on platelet function. As part of the platelet activation process,
phospholipase A2 clips the fatty acid at the C-2 position, either arachidonic acid or
DHA/EPA (31). In the case of the platelet, the fatty acid is then metabolized through an
enzyme called COX-1 to a thromboxane (32-35). When the fatty acid is arachidonic acid,
thromboxane A2 is synthesized (TxA2). TxA2 is a very potent platelet activator and
vasoconstrictor. In the case of DHA or EPA, a series 3 TxA3 is synthesized, a poor platelet
activator and vasoconstrictor (32-35). Production of TxA3 underlies the potential
anti-platelet effect of Lovaza®.

The second effect of DHA inclusion in PL's is a newly discovered alteration in the cell
membrane structure. It is now well established that DHA promotes "lipid raft" formation in
cellular membranes (36-38). These rafts, primarily composed of sphingomyelin and
cholesterol, form the sites where some transmembrane proteins can be inserted into the
membrane. These transmembrane proteins may be sites for ion channels or receptors that
define important cellular functions and can be a means to activate cells. Thus, DHA's
ability to promote raft formation may have a profound beneficial effect on platelet
function.

Since it is the Lovaza®-alteration of the platelet membrane that leads to its clinical
benefit, assays to determine how the lipid composition of the platelet membrane changes
after ingestion of Lovaza® will be carried out. The concept of these experiments is fairly
simple. A standard well-established 1H NMR method will be used to detect changes in the
lipid composition of the platelet membrane as a function of the Lovaza® dose (39-41). From
these experiments we will be able to prove that DHA or EPA from Lovaza® is actually directly
incorporated into a platelet membrane

Inclusion Criteria:

- Males or females older than 18 years old who are able to ingest omega n3 fatty acids
are eligible for this trial and are:

- On no antiplatelet and anticoagulation therapy, OR

- On chronic therapy with warfarin or aspirin alone (< or =325 mg/day)or combination
therapy with clopidogrel and aspirin (< or =325 mg/day).

- The subject must be able to read, understand, and sign an informed consent form and
follow protocol.

- To be enrolled in the study, subjects must be clinically stable on stable medical
therapy throughout the duration of the study and meet the following criteria:

- Healthy volunteers

- Volunteers with stable coronary artery disease are those with:

- Prior MI (>1 month) OR

- Prior revascularization: angioplasty ± stenting (> 1 month) OR

- Coronary artery bypass grafting (>3 months) OR

- Documented disease on coronary angiography.

- No planned no planned procedures or changes in medical therapies over the 24-week
duration of the study

- Volunteers with stable atrial fibrillation are those with:

- Rate-controlled or paroxysmal atrial fibrillation on stable antiarrhythmic therapy.

- On a stable dose of warfarin and regular follow-up in an anticoagulation ("coumadin")
clinic.

- No planned changes in antiarrhythmic therapies or cardioversion during the duration
of the study.

- No recent admissions for atrial fibrillation (> 3 months)

- Subjects may not ingest other drugs known to cause a significant platelet abnormality
while participating in this trial. (See list of prohibited medications, as outlined
in Section 9)

- Patients must be assessable to the investigator for scheduled clinic visits during
the duration of the trial.

- All female subjects of child bearing potential must have a negative serum pregnancy
test prior to randomization and not plan on getting pregnant for the duration of the
study.

Exclusion Criteria:

- Any medical condition that would preclude ingestion of omega n3 fatty acids
(Lovaza®).

- Subjects taking nutritional supplements of fish oil or flaxseed oil. These patients
may become eligible if they are willing to discontinue these nutritional supplements
for a 2-week washout period.

- Any other medical condition that would adversely affect the study objectives.

- Chronic medical conditions known to be associated with abnormal platelet function
including:

- Liver dysfunction including abnormal liver function tests (AST, ALT, or alkaline
phosphatase > upper limit of normal), known cirrhosis or chronic hepatitis.

- Chronic kidney disease with a calculated creatinine clearance < 60 ml/min (MDRD)
and/or a serum creatinine > 2.0 mg/dl.

- History of significant anemia, or baseline hemoglobin < 11.0 g/dl.

- Baseline PT>ULN, INR>1.3, and aPTT>ULN in subjects who are not on chronic warfarin
therapy.

- History of thrombocytopenia, or baseline platelet count of < 100,000

- History of thrombocytosis, or baseline platelet count of > 600,000

- Known bleeding diathesis and/or congenital hemostasis disorder and/or congenital
platelet abnormalities.

- Any history of stroke in the past 12 months.

- History of peptic ulcer disease in the past year or gastrointestinal bleeding in the
last 3 months.

- Genitourinary bleeding in the last 3 months.

- HIV or other infectious diseases that would expose laboratory personnel to
unacceptable risks.

- Treatment within 30 days with an antiplatelet agent other than aspirin or clopidogrel
such as eptifibatide, tirofiban or abciximab.

- Treatment within the past 7 days with unfractionated or low-molecular- weight
heparin.

- Allergy to iodine, fish, or other components of the study drug.

- Alcohol or substance abuse.

- Emotionally or psychiatrically unstable.

- Use of any investigational drug or device within the past 30 days

- Any other factor that the investigator feels would put the patient at increased risk
if participating in the study.

- Any Terminal illness or illness that may cause mortality that could obscure the
results of the test in any way for them to appear inaccurate.