Mechanisms of Action of Acetaminophen

Acetaminophen has antipyretic and moderate analgesic properties, but largely lacks
anti-inflammatory activity. While its mechanism of action is not entirely understood, it is
probably both an isoform nonspecific and partial cyclooxygenase (COX) inhibitor in humans at
doses commonly taken for mild pain and pyrexia, such as 1000 mg. Although no inhibition of
platelet aggregation is observed at this dosage, platelet thromboxane formation by COX is
depressed by roughly 40%. Epidemiological studies suggest that at higher doses, 2000 mg and
above, acetaminophen exhibits a gastrointestinal adverse effect profile indistinguishable
from traditional, nonspecific NSAIDs. Thus, it is possible that maximal COX inhibition is
achieved at higher doses. Interestingly, complete COX inhibition by non-selective COX
inhibitors has the potential to antagonize the irreversible platelet inhibition induced by
aspirin. In contrast to reversible inhibitors, aspirin acts by acetylation of a serine
residue in the substrate binding channel of COX. For example, ibuprofen, a reversible and
non-selective COX inhibitor, is thought to prevent aspirin from gaining access to this target
site. This study investigates, whether COX inhibition by acetaminophen is dose dependent in
humans and whether acetaminophen interacts with the irreversible COX inhibition by low dose
aspirin. It addresses the dose-related effect of acetaminophen on COX activity and assesses
potential pharmacological interactions with low dose aspirin in normal healthy volunteers.
The primary hypothesis is that administrating acetaminophen before aspirin would antagonize
the irreversible effects of aspirin, as assessed by the measurement of serum thromboxane B2
and platelet aggregation 24 hrs after the administration of the first study drug on day 6 of
combination therapy.

The second aim will determine the effects of acetaminophen on oxidant stress and
cyclooxygenase activity in patients who smoke. While the structural interaction of
acetaminophen with COX is unknown, it may inactivate the enzyme by a molecular mechanism
different from other NSAIDs. Thus, acetaminophen, which is a good reducing agent, might act
to reduce COX from its active, oxidized form. When uninhibited, the peroxidase component of
this bifunctional enzyme oxidizes its catalytic center to generate a tyrosyl radical that is
required for its activity. Indeed, some reducing agents have the capacity to prevent COX
activation in vitro. If reduction were the basis for COX inhibition by acetaminophen in vivo,
it would be expected to be less pronounced under conditions of high peroxide tone, as occurs
in inflammation. Indeed, acetaminophen, which is a phenol derivative, may act as a free
radical scavenging antioxidant like other phenolic compounds, such as vitamin E and has been
shown to alleviate oxidative damage in model systems. This study explores the potential
antioxidant effect of acetaminophen in smokers. Such individuals represent a human model of
oxidant stress. Novel approaches to the quantitative assessment of free radical induced
damage to lipids are applied, which are elevated in smokers. Additionally, it is determined
whether COX inhibition by acetaminophen is conditioned by oxidant tone in vivo.

Part B:

To develop and validate of a method to measure platelet COX-1 acetylation by aspirin.

Inclusion Criteria:

- Age between 18 - 55

- Subjects recruited for the "non-smoker group" must be in good health as based on
medical history, physical examination, vital signs, and laboratory tests.

- Subjects recruited for the "smokers group" will be chronic smokers of at least 4 years
duration, but no longer than 20 years duration, who smoke 11-20 cigarettes per day.
Smokers must be otherwise healthy as described above.

- Female subjects of child bearing potential must be using a medically acceptable method
of contraception (oral contraception, depo-provera injection, IUD, condom with
spermicide, diaphragm, cervical cap, progestin implant, abstinence, tubal ligation,
oophorectomy, TAH) throughout the entire study period. All female subjects must
consent to a urine pregnancy test at screening and just prior to the start of each
treatment phase of the study, which must be negative at all time points.

- Subjects must be within 30% of their ideal body weight.

Exclusion Criteria:

- Female subjects who are pregnant or nursing a child.

- Subjects, who have received an experimental drug, used an experimental medical device
within 30 days prior to screening, or who gave a blood donation of ≥ one pint within 8
weeks prior to screening.

- Subjects with any coagulation, bleeding or blood disorders.

- Subjects who are sensitive or allergic to acetaminophen and/or aspirin, as well as any
of their components.

- Subjects with documented history of any gastrointestinal disorders, including bleeding
ulcers.

- Subjects with any evidence of cancer.

- Subjects with a history of heart disease, including myocardial infarction, angina,
coronary artery disease, any evidence of coronary artery stenosis, arrhythmias, heart
failure, having had a CABG or significant irregularities in the EKG.

- Subjects with history of peripheral artery disease (claudication, bypass surgery or
stent placement in extremity.)

- Subjects with a history of stroke or transitory ischemic attacks (TIA).

- Subjects with renal, hepatic, respiratory, endocrine, metabolic, hematopoietic or
neurological disorder.

- Subjects with a history of liver disease or abnormal LFTs (>2x upper limit normal).

- Subjects with any abnormal laboratory value or physical finding that 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 had a history of drug or alcohol abuse within the last 6 months.