AN UPDATE ON CYCLOOXYGENASE ENZYME AS THERAPEUTIC AGENTS

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AN UPDATE ON CYCLOOXYGENASE ENZYME AS THERAPEUTIC
AGENTS

• Dr. Kripa Shanker Gupta
• Post Grad Student 2nd Yr
• MAMC, N. Delhi

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History

• 1930 Goldbatt extraction of PGs from semen
  
• Von Euler BP smooth muscle
  contraction
• 1950s Purification of Prostaglandins
• 1971 John Vane Aspirin acts through COX
• 1980s COX constitutively expressed and
  unregulated.
• Late 80s COX induced by Inflammatory cytokines
• 1991 Second isoform of COX COX 2
• 2002 3rd variant COX 3 ?

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EICOSANOID FACTS

• 20-carbon compounds
• Include prostaglandins, prostacyclins,
  thromboxanes, leukotrienes
• Physiological effects at very low concentrations
• Many of their effects mediated by cyclic AMP or
  calcium second messengers
• Unlike hormones, not transported in the blood
• Local mediators that act where synthesized or in
  adjacent cells

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Phospholipase A2 (PLA2)
GC induce lipocortin that inhibits PLA2
Prostaglandins and thromboxanes (Cyclic/ring
product)
Leukotrienes (Linear product)
Aspirin inhibits irreversibly Indomethacin forms
a salt bridge in the binding site Ibuprofen
competes for substrate binding
Zyflo competes with AA for binding
Figure 1. Liberation of arachidonic acid and its
metabolism to prostaglandins/ thromboxanes or to
leukotrienes
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Arachidonic Acid (?6) derived from membrane
phospholipids
X
Prostaglandin endoperoxide synthase
PGH2 central intermediate (Head of pathway)
Figure 3. Conversion of arachidonic acid to PGH2
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PGH2 central intermediate (Head of pathway)
PGF2? labor induction (Fetus)
Figure 3. Conversion of PGH2 to prostaglandins
and thromboxane of the 2-series
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Cyclooxygenase Enzyme

• Earlier tissue homogenates used as source of
  enzymatic activity
• Present in all cells
• Purified enzyme by amide gel electrophoresis
• Classified as integral microsomal membrane
  protein
• Different PG synthesis rate and activity reported

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Evidence of COX isoforms

• Autoinactivation rates of COX, inhibition of
  NSAIDS and time course profile of PGE2 and
  PGF2alpha synthesis
• 
  Activating platelets
• within minutes
• PG Synthesis
• Mitogen stimulated fibroblasts
• hours
• Steroids inhibited the IL-1 induced COX activity
  but not basal COX activity
• Cell Growth Studies genes products inducible
  in-vitro exhibiting COX similarity
• Western blot hybridization c DNA probe 2
  different mRNA 4 kbps and 2.8 kbps

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Discovery of COX - 2

• Studies on cell division
• Mitogen activated early genes activity in
  fibroblasts
• Swiss 3T3 cells sequence encoded new inducible
  gene
• Mouse TIS10 cDNA expression increased
  prostaglandin E2 activity of which suppressed by
  NSAIDS

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Structural Details

• Both are dimer bound to microsomal membrane
• 4 domains
• Dimerization domain
• Membrane binding domain
• Catalytic domain differ in structure
• Terminal signal peptide domain differ in length
• Post/co translational glycosylation

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Figure 8. Actions of two known isoforms of
cyclooxygenase (COX).
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Newer isoforms ( Variants )

• COX 1 Variants
• COX -1 V 1 (COX 3)
• PCOX 1A
• PCOX 1B
• COX 1 SNPs
• Other

• COX 2 Variants
• COX -2 V 1
• PCOX 2 B
• COX 2 SNPs
• Other

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Isoforms
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COX 3 a theory without evidence?

• acetaminophen as a "COX-3" inhibitor or even a
  "selective COX-3 inhibitor" in rat studies
• induced enzyme appearing 48 hours after the start
  of the inflammation
• COX 1 gene with intron -1 changing protein
  folding and active site confirmation
• Expressed more in brain cells

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Nonsteroidal Anti-inflammatory Drugs(NSAIDs)

• Common therapeutic indications
• Common adverse effects
• Different pharmacokinetics and potency
• Different chemical families
• Common mechanism of action (cyclooxygenase
  inhibition)
• Different selectivities to COX I and II
• Similarities more striking than Differences

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Common Therapeutic Uses

• Analgesic (CNS and peripheral effect) may involve
  non-PG related effects
• Antipyretic (CNS effect)
• Anti-inflammatory (except acetaminophen)
  rheumatic fever, rheumatoid arthritis, other
  rheumatological diseases due mainly to PG
  inhibition.
• Dysmenorrhoea
• Prophylaxis of diseases due to platelet
  aggregation (CAD, post-op DVT)
• PDA Closure
• Pre-eclampsia and hypertension of pregnancy
  (?excess TXA2)
• Some are Uricosuric

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COX inhibitors

• Non Selective COX inhibitors
• Non competitive
• Aspirin
• Competitive
• Phenylbutazone
• Ibuprofen
• Naproxen
• Diclofenac
• Piroxicam
• Ketorolac
• Analgesic with Antipyretic without anti
  inflammatory action
• Paracetamol
• Metamizol
• Nefopam

• Preferential COX 2 inhibitors
• Nimesulide
• Meloxicam
• Nabumetone
• Selective COX -2 inhibitors
• Celcoxib
• Rofecoxib
• Valdecoxib
• Etoricoxib
• Parecoxib
• Lumoracoxib

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Figure 4. Structure and mechanism of action of
aspirin
CH2
OH
Ser
?
Cyclooxygenase (active)
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Need of Selective COX -2 Inhibitors

• inhibition of COX-2 - anti-inflammatory effects
• inhibition of COX-1 - recognized toxicities of
  NSAIDs,
• a) peptic ulcers and the associated risks of
  bleeding, perforation and obstruction

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NSAID
Loss of PGI2 induced inhibition of LTB4 mediated
endothelial adhesion and activation of
neutrophils
Loss of PGE2 and PGI2 mediated inhibition of acid
secretion and cytoprotective effect
? Leukocyte-Endothelial Interactions
Capillary Obstruction
Proteases Oxygen Radicals
Ischemic Cell Injury
Endo/Epithelial Cell Injury
Mucosal Ulceration
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Endoscopic Picture of Gastric Ulcer10
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Need of Selective COX -2 Inhibitors

• inhibition of COX-2 - anti-inflammatory effects
• inhibition of COX-1 - recognized toxicities of
  NSAIDs,
• a) peptic ulcers and the associated risks of
  bleeding, perforation and obstruction
• b) prolonged bleeding time and,
• c) renal insufficiency .
• inflamed tissues target without disturbing the
  homeostatic functions of prostaglandins in
  noninflamed organs.
• Theoretically, selective COX-2 inhibition should
  preserve the anti-inflammatory efficacy

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What is Selectivity

• Ratio of COX 1 / COX 2 inhibition activity
• COX -1 activity ability to inhibit TXB 2
  production from platelets
• COX 2 activity ability to inhibit PGI 2
  production from monocytes in response to stimuli

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Rise of COXIBS

• Large scale trials showed equal efficacy and
  lower GI side effects
• Market taken by a storm
• Celecoxib and then Rofecoxib became billion
  dollar drugs
• Extensive chronic usage in Inflammatory disorders
  like RA, Osteoarthritis and other Inflammatory
  disorders
• Newer Applications Adenomas , AD

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Comparison of GI Benefits
Adapted from Cardiovascular Safety of Celecoxib
Risk-Benefit Assessment - Celebrex, Pfizer,
2/16/05
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Big Questions

• Does COX-2 serve a physiological function(s)?
• COX -2 in macula densa response to Na
  restriction
• Role in ovulation and fertility
• Brain temperature control
• Does COX-1 serve an inflammatory function(s)?
• No clear cut evidence dubious gene knock out
  studies

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Correlation with GI symptoms

• Theoretically COX 2 inhibitors should be free
  of side effects
• Increase in selectivity ratio should decrease the
  GI side effects.

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Disturbing Reports from the long term trials of
the Coxibs

• CLASS AND VIGOR TRIALS
• RISE IN CVS EVENTS

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Celecoxib clinical trials

• Four Main Trials
• CLASS
• APC
• PreSAP
• ADAPT

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CLASS Trial

• Purpose
• GI toxicity
• Pain alleviation efficacy
• Patients
• 8000 patients
• Average age of 60 y.o.
• With Osteoarthritis or Rheumatoid Arthritis
• Without history of GI disease

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CLASS TrialCelecoxib vs. NSAIDs
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CLASS TrialSerious CV Events
Adapted from COX-2 CV Safety - Celecoxib, Dr.
James Witter, MD, PhD. 2/16/05
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CLASS TrialConclusions

• GI risk is lower than NSAIDs
• No significant difference between NSAIDs and
  Celecoxib for CV risk

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APC Trial

• Purpose
• Reduce probability of adenomatous polyps
  (reduction of colon cancer)
• Patients
• 2000 patients
• Average age of 60 y.o.
• Prior adenomas

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APC Trial Baseline Characteristics
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0
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Adapted from a New England Journal of Medicine
article Cardiovascular Risk Associated with
Celecoxib in a Clinical Trial for Colorectal
Adenoma Prevention, March 17, 2005.
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APC Trial Death Rates
Adapted from Celecoxib in Adenoma Prevention -
The APC Trial, Dr. Ernest Hawk, MD, MPH, NIH,
2/15/2005
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0
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APC TrialConclusions

• Early trial suspension
• Planned duration of 3-5 years
• Stopped early but followed patients for about 3
  years
• Fair trial results
• Significant difference between Celebrex CV risk
  and placebo CV risk

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PreSAP Trial

• Purpose
• Reduce risk of cancerous polyps
• Patients
• 1500 patients
• Average age of 61 y.o.
• Had growths surgically removed

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PreSAP Trial CV Death Rates
Relative to placebo
Adapted from Celecoxib in Adenoma Prevention -
The PreSAP Trial, Dr. Bernard Levin, MD FDA,
2/16/2005
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PreSAP TrialData
Adverse CV event risk in Celecoxib vs. Placebo
Estimated probability of CV death, heart attack,
stroke, or CHF (log)
Months since first dose
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PreSAP TrialConclusions

• Trial suspended early
• Planned duration of 3-5 years
• Stopped because of APC trial
• No significant difference between placebo CV risk
  and Celecoxib CV risk

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ADAPT Trial

• Purpose
• Reduce risk of Alzheimers Disease
• Patients
• 2625 patients
• Average age of 70 y.o.
• At risk of Alzheimers

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ADAPT TrialData

• Naproxen showed CV risk after 1.5 years
• Planned duration of 7 years
• Began 2001
• Stopped 2004, 3 days after APC Trial was stopped

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ADAPT TrialConclusions

• Significant difference in data shows Naproxen as
  worse than Celecoxib
• Celecoxib is still significantly worse than
  Placebo
• Data contradicts previous Naproxen trials
• Results are unexplainable

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Comparison of Risk of Death, Heart Attack, and
Stroke
Relative Risk
Celecoxib vs.
Placebo
NSAID
Naproxen
Diclofenac
Ibuprofen
0.1
0.1 0.3 1.0 3.0 10.0
Favors celecoxib
Favors comparator
Adapted from Cardiovascular Safety of Celecoxib
Risk-Benefit Assessment - Celebrex, Pfizer , Feb
15, 2005
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Summary of Trials
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VIGOR - Summary of GI Endpoints
Rofecoxib
RR 0.46 (0.33, 0.64)
Naproxen
5
RR 0.38 (0.25, 0.57)
4
RR 0.43 (0.24, 0.78)
3
Rates per 100 Patient-Years
2
1
0
Confirmed Clinical Upper GI Events
ConfirmedComplicated Upper GI Events
All Clinical GI Bleeding
p 0.005.
( ) 95 CI.
p lt 0.001.
Source Bombardier, et al. N Engl J Med. 2000.
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VIGOR - Confirmed Thrombotic Cardiovascular Events
Patients with Events (Rates per 100 Patient-Years)
Rofecoxib N4047
Naproxen N4029
Relative Risk (95 CI)
Event Category
45 (1.7)
19 (0.7)
0.42 (0.25, 0.72)
Confirmed CV events
28 (1.0)
10 (0.4)
0.36 (0.17, 0.74)
Cardiac events
8 (0.3)
0.73 (0.29, 1.80)
Cerebrovascular events
11 (0.4)
0.17 (0.00, 1.37)
Peripheral vascular events
6 (0.2)
1 (0.04)
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Investigator-Reported Thrombotic Cardiovascular
Events in the VIGOR Study Compared with Phase
II/III OA Study
3.5
3.0
2.5
Ibuprofen, Diclofenac, Nabumetone (OA)
2.0
Rofecoxib (OA)
Cumulative Incidence
1.5
1.0
0.5
0.0
0
2
4
6
8
10
12
14
Months of Follow-up
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Effect of Celecoxib Rofecoxib on PGIM
Urinary 2,3 dinor-6-keto-PGF1a (PGIM)
Two Weeks Rx
Single Dose Rx
200
200
160
160
120
120
Urinary PGI-M (pg/mg creatinine) (Mean SE)

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80



40
40
0
0
Placebo N7
Celecoxib 400 mg N7
Ibuprofen 800 mg N7
Placebo N12
Rofecoxib50 mg QDN12
Indomethacin50 mg TIDN10
plt0.05 vs. placebo.
Proc. Natl. Acad Sci. USA 199996272-277.
plt0.01 vs. placebo.
J. Pharmacol. Exp. Ther. 1999289735-741.
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APPROVe trial

• Adenomatous Polyp Prevention on VIOXX
• 2,586 Patients of adenomatous polyp randomized to
  25 mg of rofecoxib or placebo
• trial was stopped early
• increased risk for serious cardiovascular events,
  such as heart attacks and strokes, first observed
  after 18 months

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Blood Vessel Wall Endothelial Cell
Platelet
?cAMP ? aggregation
? Ca2/vessel smooth muscle constricts
Figure 5. Normal physiologic interaction between
PGI2 and TXA2 in platelet and endothelial cell
biology
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Endothelial Cell
Blood Vessel Wall
Platelet
Smooth Muscle Cell
TXA2
PGI2
Injury
Normal Physiology
HEALTHY
Figure 6. Reendothelialization of the artery
wall following injury and the key role of
platelet aggregation stimulated by TXA2 in
response to injury.
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Figure 6. Reendothelialization of the artery
wall following injury and the key role of
platelet aggregation stimulated by TXA2 in
response to injury.
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Figure 6. Reendothelialization of the artery
wall following injury and the key role of
platelet aggregation stimulated by TXA2 in
response to injury.
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PGI3
Platelets lack nucleic cannot replace COX TXA
preferentially ?
Figure 7. Control of platelet aggregation.
Suppression of platelet aggregation by drug or
dietary intervention
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What FDA says

• FDA 1st release Feb 18 , 2005
• Supported the marketing of vioxx in market
• Committee recommend black box warning on
  cardiovascular risk, dose should be only 12.5 mg
  instead of 25/50 mg
• FDA 2nd release Feb 18 , 2005
• The effect is evident in every drug of this class
  and is dependant on both time period and the dose
  of the drug

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What the FDA Says

• Neutral Celecoxib requires further study to
  estimate longer-term CV risks
• Positive There does not appear to be any
  clinically or statistically significant trend
  with celecoxib to suggest additional
  cardiovascular risks over the comparator drugs.
• Negative (almost) Some studies appear to show
  an increased risk of CV events, but the findings
  are not consistent across the COX-2 selective
  NSAIDs.

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Clinical Trials of New Cox-2 Inhibitors
Etoricoxib
Parecoxib
Valdecoxib
Lumiracoxib
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Valdecoxib, selective Cox-2 inhibitor

• Approved by the FDA in November 2001, but now
  under close watch
• Risk-Benefit Analysis
• Efficacy is comparable to nonselective NSAIDs
• Better GI safety profile
• Generally similar cardiovascular risks
• Serious skin reactions (Stevens-Johnson)
• Less promising than the new investigational drugs
• New clinical testing in progress

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CV Risks in CABG I Surgery Setting
Study 071 Higher risk in almost all CV categories
adjudicated
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CV Risks in CABG II Surgery Setting
Study 071 Higher risk in almost all CV categories
adjudicated
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Black box warning Valdecoxib

• Contraindicated in CABG patients
• Serious skin reaction Steven johnson syndrome
  and erythema multiforme
• Discontinue after first skin rash or mucosal
  lesion

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Etoricoxib

• Etoricoxib, selective COX-2 inhibitor
• Developed to diversify existing treatment options
  for inflammatory conditions
• Currently approved in 60 countries
• New Drug Application (NDA)
• Recent Phase III clinical trials
• Chronic Exposure Studies 4087 patients, 13
  trials
• EDGE Study diclofenac as active comparator

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Clinical Efficacy

• Osteoarthritis (OA), Rheumatoid Arthritis (RA),
  chronic low back pain, acute gouty arthritis,
  acute pain, and ankylosing spondylitis
• Efficacy demonstrated by statistically
  significant improvements in 3 areas
• 1. Physical Function
• 2. Pain Subscale
• 3. Patient Evaluation

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Gastrointestinal Safety and Tolerability

• Primary endpoints gastroduodenal perforations,
  symptomatic gastroduodenal ulcers, and upper GI
  bleeding)
• Overall superior GI safety and tolerability
  compared to traditional NSAIDs (COX-1 expression,
  fewer discontinuations)
• Support for etoricoxib as an alternate therapy
  for patients experiencing significant GI problems
  with nonselective NSAIDs

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Cumulative Rate of GI Ulcers
Etoricoxib Naproxen
Ibuprofen
86
Renovascular Safety

• Inhibition of renal prostaglandin synthesis
  complicates renal blood flow -gt impaired kidney
  function
• 1. fluid retention (edema)
• 2. hypertension
• 3. Congestive Heart Failure
• Edema, hypertension, and CHF were mild and
  infrequently led to discontinuations
• Generally consistent with rates observed for
  nonselective NSAIDs

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Incidence of Hypertension

• Elevation in blood pressure

Mean Incidence (Etoricoxib)
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Cardiovascular Safety

• Primary Endpoint Confirmed Thrombotic Serious
  Adverse Experiences
• - unstable angina, myocardial infarction,
  ischemic stroke, and transient ischemic attacks
• Pooled CV Phase IIb/III data Over 6700 patients

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Thrombotic Cardiovascular Incidence
Placebo-Controlled
Naproxen-Controlled
Non-Naproxen-Controlled
Higher Incidence
Lower Incidence
Higher Incidence
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Incidence of Edema-Related Events

• One-year continuous exposure (Etoricoxib vs.
  Naproxen)
• no dose-related effect
• consistent with rates observed for NSAIDs

Mean Incidence (Etoricoxib)
Lower Extremity Edema
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Etoricoxib Conclusions

• Great clinical efficacy for OA, RA, and other
  inflammatory conditions
• Two unique treatment options ankylosing
  spondylitis and acute gouty arthritis
• No major CV risk pattern with current evidence
• Significantly improved GI safety (ulcer incidence
  and upper GI complications)
• Further studies needed EDGE, MEDAL, EDGE II
  (gt34500 patients, largest NSAID analysis)

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Lumiracoxib, selective COX-2 inhibitor

• Developed as a new treatment option with lower GI
  side effects
• Approved in 21 other countries, including the
  U.K.
• Phase III clinical trials and NDA in progress

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Clinical Efficacy

• Efficacy demonstrated for
• 1. Osteoarthritis and Rheumatoid Arthritis
• 2. Other chronic pain conditions
• 3. Acute pain (post-surgical)
• 50 clinical and clinical pharmacology trials -
  13000 patients worldwide
• As effective as celecoxib in pain relief
• FDA approval and market viability dependent on
  side effect profile

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GI Safety and Tolerability

• Multifold-fold reduction in upper GI ulcer
  complications compared to NSAIDs

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Renovascular Safety

• De Novo Hypertension
• Kaplan-Meier Estimate ()
• Lower rates for Lumiracoxib than Ibuprofen
• Same trend for aggravation and severe
• Clinically significant
• Edema (fluid retention)
• Lower rates for Lumiracoxib than Ibuprofen
• Few discontinuations

lumiracoxib
gt95 CL
lumiracoxib
gt95 CL
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Cardiovascular Safety

• Primary Endpoint Anti-Platelet Trialist
  Collaboration
• myocardial infarction, stroke, or vascular death
• 33,933 patients in these trials
• Overall Results APTC endpoint

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Cardiovascular Safety Conclusions

• No statistically significant difference
• between lumiracoxib and NSAIDs for
• MI, ischemic stroke, and APTC
• events
• Slightly better CV profile than Vioxx
• Smaller changes in mean BP
• Smaller relative risk compared to Naproxen
• Less inhibition of vasoactive PGI-2 that
  suppresses platelet aggregation
• (15 vs. 73)

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Lumiracoxib Conclusions

• No new patient populations less likely to see
  major market success
• Improved GI and renovascular safety profile
  compared to nonselective NSAIDs
• Phase III possibly suggests fewer cardiovascular
  risks than Vioxx
• More clinical trials needed for approval

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Parecoxib, selective COX-2 inhibitor

• Developed to provide significant analgesia
  without GI profile
• Medical need inadequate treatment options for
  postoperative pain have prolong hospitalization
  and allow progression to chronic status
• Not yet approved by the FDA (original NDA
  submitted Sept. 2001), but sold in 45 other
  countries
• First injectable COX-2 inhibitor
• Total of 65 studies completed, including 24
  analgesia studies

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Clinical Efficacy
Conventional treatments for post-operative pain

• 3 Major Data Sets
• Placebo-Controlled
• Morphine-Controlled (opioid analgesic)
• Ketorolac-Controlled (conventional NSAID)
• Efficacy in controlling post operative pain
  (Phase III)

• Scale ranged from 0-11
• High number indicates impaired function

2.0
1.5
Mean Scorea
1.0
Placebo (n519)
Parecoxib / Valdecoxib (n520)
0.5
Days
0
2
3
4
5
6
7
8
9
10
101
Reduction in Need for Opioids
Placebo (n519)
Parecoxib / Valdecoxib (n520)
Significant reduction in need for opiod
analgesics with same levels of pain control
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Side Effect Profile

• Normal therapeutic dose does not appear to affect
  COX-1 enzyme
• Generally lower incidence of gastrointestinal
  ulceration and bleeding compared to nonselective
  NSAIDs
• Avoids respiratory depression and other common
  opioid side affects
• Often combined with Bextra for clinical
  administration

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Cardiovascular Safety

• Study 069 General Surgery Study
• No significant cardiovascular risk relative to
  placebo
• CV risk only found in CAGB surgery setting

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Parecoxib Conclusions

• Medical need
• Unique advantages over current analgesics
• 1. better side effect profile
• 2. comparable or better efficacy
• Minimal cardiovascular risk under normal surgical
  settings - only observed risk for CAGB surgery
• Opens COX-2 inhibitors to a new subgroup of
  patients great market potential

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Overall Conclusions

• Greatly improved GI profile because of
  selectivity
• Lumiracoxib and Parecoxib offer the most
  promising cardiovascular profile
• Important to evaluate blood pressure effects and
  platelet aggregation independently
• R D to expand treatment groups and continue
  minimizing side effects
• More clinical evaluation and analysis needed
  statistically significant trends

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Further Research

• Other emerging investigational drugs that have
  not yet been approved by the FDA
• Further analysis of the relative CV profiles of
  all four drugs compared to Rofecoxib and
  Celecoxib
• Possible alternate uses (chemopreventive effects
  and Alzheimers)
• Status of each NDA
• New Phase III clinical trials

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Current Status

• Merck Has withdrawn Vioxx
• USFDA says Rofecoxib should come with a warning
• Researchers say Its a CLASS Effect ?
• India Has banned Rofecoxib and Valdecoxib
• Celecoxib has been approved in Adenomatous polyp
  by US FDA
• Some European countries have banned the drug ,
  others follow suit of US FDA rulings