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NSAIDs
Thomas Grewal Ph.D.
thomas.grewal@sydney.edu.au Room N518
(with kind permission of A/Prof Alaina Ammit)
Lecture 1:
NSAIDs – Mechanism of Action
Lecture 2:
NSAIDs – COX-1/2 vs COX-2 inhibitors
Further reading
Title: Foye’s Principals of Medicinal Chemistry
Editors: D.A. Williams, T.L. Lemke
Publisher: Philadelphia, Lippincott Williams & Wilkins, 2008
Edition: 6th ed.
Lecture 1
NSAIDs – Mechanism of Action
Learning objectives:
• Nonsteroidal anti-inflammatory drugs: a definition
• Inflammation in health and disease
• Focus on prostanoids:
– Role in inflammation
– Structure and biosynthesis
• COX enzymes
– Role in production of prostanoids
– Similarities and differences between isozymes COX-1 and COX-2
– Physiological role of COX-1 and COX-2
NSAIDs: Best Sellers
• NSAIDs most commonly prescribed medication
– 70 million prescriptions/year (US), $ 6.8 billion worldwide,
– Use and cost is likely to increase with longer life expectancy
• Approximately 50 NSAIDs available, among the most commonly prescribed drugs
• UK - 18.5 million prescriptions are written for NSAIDs each year at a cost of £170
million (not including OTC)
• Aspirin is consumed in very high amounts (worldwide production of > 50,000 tons/year)
• US - COX-2 inhibitors, rofecoxib (Vioxx) and celecoxib (Celebrex), > US $4 billion
in 2000
NSAIDs
• NSAIDs do not reverse the course of disease, but treat the symptoms:
pain, fever and inflammation in many inflammatory disorders
• Heterogenous group of compounds to treat various conditions/diseases:
– analgesics, antipyretic and anti-inflammatory
• Focus on anti-inflammatory agents
• Prototype: acetylsalicyclic acid (Aspirin)
– Alternative: steroids as useful anti-inflammatory drugs:
- inhaled corticosteroid for long-term treatment of persistent
asthma
- glucocorticoids, metabolic side effects
- DMARDs (disease-modifying anti-rheumatic drugs)
Nonsteroidal anti-inflammatory drugs (NSAIDs): a
definition?
• Heterogenous groups of therapeutic drugs to relieve pain, fever and
inflammation
• Approx 50 NSAIDs:
- Similar pharmacological properties
- use often limited to approved indications by the regulatory agencies
• Considerable confusion in terminology, classification as NSAIDs is
somewhat misleading:
– Also antipyretic: Antipyretics cause the hypothalamus to override an
interleukin-induced increase in temperature to reduce fever.
– Also analgetic: Analgesic drugs act on the peripheral and central
nervous systems to relieve pain
• Should paracetamol be included in this group? Antipyretic and analgetic,
but essentially devoid of anti-inflammatory actions
Inflammation in Health and Disease
• In health, inflammation is a normal and essential response:
– Wound healing
– Infection
– Thrombosis
• In disease states, inflammation is excessive:
– Asthma
– Atherosclerosis
– Cancer
– Rheumatoid arthritis
Inflammation in Disease: Rheumatoid arthritis
Classical model for NSAIDs
Pathogenesis of Rheumatoid arthritis:
1. Unknown activation of cells in synovial joint
2. Causes production of immunoglobulins (IgG), forming immune complexes
3. Activates the complement system
4. Activates production of chemokines
5. Attracts Leucocytes which ingest the IgG complexes to become rheumatic arthritis
cells.
6. These cells secrete hydrolases into the extracellular space
7. Hydrolases degrade extracellular tissue, e.g. collagen, which provokes an inflammatory
response in rheumatoid joints (all of this induces cyclooxygenase expression)
8. Swelling of joints, low-grade fever, pain
9.
Treatment strategy: inhibit production of inflammatory
mediators (step 4, 7: e.g. histamine, serotonin,
eicosanoids, prostanoids)
NSAIDs: Mechanism of Action
Cell membrane phospholipids
NSAIDs
X
Prostanoids
Prostanoids
• Regulate/increase inflammatory response
• Naturally occurring 20-carbon cyclopentano-fatty acid derivatives
• Produced in mammalian tissue from polyunsaturated fatty acids in cell membrane
phospholipids
• The term "prostanoids" relates strictly to the products of the cyclooxygenase
pathway:
– Prostaglandin
– Thromboxanes
– Prostacyclin (also known as PGI2)
• Found in essentially every compartment of the body
• Many prostanoids exist, with diverse pharmacological properties
General Structure
Classified by the capital letters A, B, C, D, E, F, G, H, I, J, K depending on
the nature and stereochemistry of oxygen substituents at the 9- and 11positions
http://www.cyberlipid.org/prost1/pros0001.htm
Subscripts
• The number of double bonds in the side chain connected to the
cyclopentane ring is designated by subscripts 1, 2 or 3
• Reflects the nature of the biosynthetic precursors:
– Subscript 1, derived from 8,11,14-eicosatrienoic acid
– Subscript 2, derived from arachidonic acid (5,8,11,14-eicosatetraenoic
acid) plus additional cis bond at C-5, C-6 positions
– Subscript 3, derived from 5,8,11,14,17-eicosapentaenoic acid plus
additional cis bond at C-17, C-18
• Most common fatty acid in humans is arachidonic acid (AA),
hence PGs of the 2 series play an important biological role
PGs of the 2 Series are Derived from AA
Arachidonic acid is derived from linoleic acid, diet, phospholipids
(phosphatidylethanolamine, phosphatidylcholine) in cell membranes
Phospholipase A2
Foye’s Principals of Medicinal Chemistry, 2002
Foye’s Principals of Medicinal Chemistry, 2002
Foye’s Principals of Medicinal Chemistry, 2002
The complexity of prostanoids: distinct prostaglandin receptors
(GPCR’s) in different tissues
Grosser T. Thromb Haemost. 2006 Oct;96(4):393-400.
http://www.hosppract.com/issues/2001/03/ceper.htm
COX-1/COX-2: Similarities
• COX-1 and COX-2 are isoenzymes
• COX-1 gene is located on chromosome 9, COX-2 gene is on chromosome 1
• Molecular weight is similar: COX-1 67 kDa (599 aa), COX-2 72 kDa (604 aa)
• Both consist of a long narrow channel open at one end and with a hairpin bend at
the other
• In amino acid sequence, the isoforms are 65% identical
• They are almost completely identical at their catalytic site to carry out similar
enzymatic functions
• AA is pulled into the COX's open end and fully formed prostaglandins leave the cell
via facilitated diffusion
European Journal of Medicinal Chemistry (2001), 36 (2) , 109-126
COX-1/COX-2: Differences
• The differences between COX-1 and COX-2 are genetically determined and cause
several amino acid changes
• Position 523: valine in COX-2, bulky isoleucine in COX-1, causes a structural
difference in the active site of the enzyme
• Valine 523 in COX-2 creates an additional side pocket, which is a pre-requisite for
COX-2 drug selectivity. Access to this side pocket is restricted in COX-1
• Valine 434 in COX-2 and Isoleucine 434 in COX-1, wider entry into the additional
side pocket of COX-2, helps to establish COX-2 drug selectivity
European Journal of Medicinal Chemistry (2001), 36 (2) , 109-126
NSAIDs: COX-1/COX-2 selectivity
• Both COX-1 and COX-2 have arginine at position 120 of their amino-acid
sequence
• Non-selective NSAIDs bind Arginine 120 to block arachidonic acid uptake
• Structural differences at Pos 523 (Val/Isoleucine)
pharmacological targeting of either COX-1 or COX-2
permit
selective
• Valine, a smaller amino acid in COX-2 creates a side pocket that allows binding of
COX-2-selective inhibitors (too bulky for COX-1)
http://www.hosppract.com/issues/2001/03/ceper.htm
NSAIDs: COX-1/COX-2 selectivity
http://www.hosppract.com/issues/2001/03/ceper.htm
COX-1 (“Good”) vs. COX-2 (“Bad”):
An oversimplification…
COX-1 (“Good”) vs. COX-2 (“Bad”):
• The thinking behind development of COX-2 selective
NSAIDs…
– COX-1 is constitutive, maintaining normal cell function within
the mucosa of many organs
– COX-2 is an inducible enzyme, it becomes abundant in activated
macrophages and other cells at sites of inflammation
– Underlying paradigm - selective COX-2 inhibitors would
primarily target inflammation, with minimal disturbance of
homeostatic COX-1 function
Summary
• Nonsteroidal anti-inflammatory drugs (NSAIDs) are antiinflammatory, analgesic
and antipyretic
• Inflammation plays a role in health and disease
• Prostanoids, especially those derived from arachidonic acid, induce inflammation
• NSAIDs act on COX enzymes to inhibit prostanoid synthesis
• COX-1 and COX-2 are isozymes
• Differences in COX enzyme structure can be exploited to make selective COX-2
inhibitors
NSAIDs Lecture 2
Selective vs. Non-selective COX Inhibitors
Learning objectives:
• Review the drug discovery timeline for NSAIDs
• role and structure of COX isozymes
• structural basis of COX inhibition for commonly used NSAID drug classes:
– Salicylates (e.g. aspirin)
– Arylalkanoic acids
» Aryl- and Heteropropionic acids (e.g. ibuprofen)
» Aryl- and Heteroarylacetic acids (e.g. indomethacin, sulindac, diclofenac and
nabumetone)
– Selective COX-2 inhibitors (e.g. celecoxib and rofecoxib)
• What went wrong?
Timeline of Drug Discovery: NSAIDs
• 1763 Edward Stone described efficacy of salicylate-containing plant extracts for the
treatment of malarian fever and rheumatic complaints.
• 1860 - salicylic acid was chemically synthesized by Kolbe
• 1898 - acetylsalicylic acid used medicinally (aspirin - a from acetyl and spirin, an older
name given to salicyclic acid (spiric acid))
• 1899 - acetylsalicylic acid was registered and introduced commercially as "aspirin" by
Bayer (Germany).
• 1940s – phenylbutazone (Arylalkanoic Acid; largest group of NSAIDs; 1950 - arthritis
therapy)
• 1950s - fenamates
• 1960s – indomethacin by Shen (Merck, most potent Arylalkanoic Acid)
• 1970s and 1980s - number of chemical abstracts dealing with potential inhibitors of the
COX enzyme rose markedly ~ 2,500 per year 1970s – including the proprionates and
the oxicams
• 1980s – COX-2 discovered
• 1990s – selective COX-2 inhibitors (1997 – 2000: celecoxib, rofecoxib, valicoxib)
• 2002 – COX-3 discovered
• 2004 – selective COX-2 inhibitors withdrawn from market
NSAIDs: Common Structural Features
• Acidic centre
• Aromatic/heteroaromatic ring
• Additional centre of lipophilicity
(either an alkyl chain or additional
aromatic ring)
• Corresponds with carboxylic acids
and double bonds of arachidonic acid
5,8,11,14-eicosatetraenoic acid
Salicylates (e.g. aspirin)
•Acidic centre
•Aromatic ring
Aspirin (Acetylsalicyclic acid)
Salicyclic acid
•Acetyl group
Aspirin: Interaction with COX (I)
•
•
•
•
Irreversible inhibitor of COX-1 and COX-2
Aspirin acetylates Ser-530 so that AA cannot reach the catalytic center
Arg-120 in COX stabilizes the carboxylate group of aspirin
The aromatic ring structure accommodated within the largely
hydrophobic binding channel (Tyr-385)
Aspirin: Interaction with COX (II)
hydrophobic
hydrophobic
acetylates
acetylates
stabilizes carboxyl
stabilizes carboxyl
Nature Reviews Drug Discovery 2, 179-191 (2003)
Aspirin (and other salicylic acids):
Mechanism of action:
inhibition of histamine release
inhibition of hydrolase release
inhibition of leucocyte accumulation
inhibition of prostaglandin synthesis
Aspirin is the only NSAID that covalently modifies COX-1/2
10-100 more potent to inhibit COX-1 than COX-2
rapid absorption upon oral administration,
absorption highly dependent on gastric pH,
aspirin binds to albumin (drug-drug interactions)
Side effects: peptic ulcers in GI tract
(lesion in the mucous membranes of the digestive
tract, due to excessive damage to the mucosal cells
that line the stomach, can become malignant)
Salicylates (aspirin) inhibit prostaglandin synthesis, in particular PGE, which normally
inhibits gastric acid secretion---gastric mucosa cell damage
plus inhibition of platelet aggregation---bleeding
Non-selective NSAIDs: Dual Insult
Arylalkanoic Acids
– Largest group of NSAIDs
– All non-selective
– Center of acidity: carboxylic, enolic or hydroxamide function,sulfonamide
or tetrazole ring; relates to carboxylic acid of arachidonic acid
– Consist of:
» Aryl- and Heteroarylacetic acids, e.g.
• indomethacin
• sulindac
• diclofenac
• nabumetone
» Aryl- and Heteropropionic acids, e.g.
• ibuprofen
• flurbiprofen
Indomethacin: Interaction with COX (I)
• Non-selective COX inhibitor, causes
slow, time-dependent inhibition of
COX-1 and COX-2
• Chlorobenzoyl group is stabilized by
hydrophobic interactions
• The acidic function of indomethacin
associates with Arg-120 of COX-1/2
• One of the most potent NSAIDs
• More potent antipyretic and analgetic
effects compared to aspirin
• Potent inhibitor of prostaglandin
synthesis
Sulindac
• Sulindac is a prodrug, aimed to reduce GI side effects of
indomethacin
• Converted to a metabolite ~ 8X more effective than aspirin
Sulindac
active sulphide metabolite
8-fold more potent than aspirin
Diclofenac
• Unique amongst NSAIDs:
1. Potency (3-1000 x more potent
than other NSAIDs – strong
decrease
in
prostaglandins,
thromboxanes)
2. Inhibits lipoxygenase –decreased
leukotrienes (e.g. LKB4)
3. Inhibits AA release and stimulates
AA re-uptake – reduced AA
availability
4. Most widely used NSAID
Nabumetone: a non-acidic NSAID
• Non-acidic - no primary insult;
• poor ability to inhibit COX in gastric mucosa - minimum secondary
insult
• Nabumetone is metabolized to 6MNA (6-methoxy-naphthalene-2acetic acid)
• 6MNA is an effective inhibitor of COX in joints
• Classic pro-drug approach in drug design
Aryl- and Heteropropionic Acids
• Class name “profens”
• Substitution of a methyl group on the carbon atoms separating the
acid centre from the aromatic ring (increased anti-inflammatory
activity)
• Examples include:
» ibuprofen
» flurbiprofen
• Reversible, competitive inhibitors of COX-1 and COX-2
(S) (+)-isomer is biologically active
COX-1 (“Good”) vs. COX-2 (“Bad”): An oversimplification…
• The thinking behind development of COX-2 selective
NSAIDs…
– COX-1 is constitutive, maintaining normal cell
function within the mucosa of many organs
– COX-2 is an inducible enzyme, it becomes abundant in
activated macrophages and other cells at sites of
inflammation
• Underlying paradigm - selective COX-2 inhibitors would
primarily target inflammation, with minimal disturbance
of homeostatic COX-1 function
• An oversimplification…
Selective COX-2 Inhibitors:
celecoxib (Celebrex) and rofecoxib (Vioxx)
COX-1 (“Good”) vs. COX-2 (“Bad”): An oversimplification…
COX2-hypothesis
Selective COX2 inhibitors (‘coxibs’) are molecules with side
chains which fit into the hydrophobic pocket, but are too large
to block COX1 with equal affinity
Hypothesis
1) GI-toxicity of NSAIDs is due to inhibition of COX1-mediated
production of PGE2 and TXA2 in gastric epithelium and
platelets
2) COX2 is induced at inflammatory sites and the major source of
pain and inflammation mediating PGs
But:
COX1 is also induced with inflammation
COX2 is constitutively expressed in many non-inflamed tissues
COX-1 (“Good”) vs. COX-2 (“Bad”): An oversimplification…
Increased COX2 selectivity increases cardiovascular risk: withdrawn worldwide after
detection of increased CVD events of risk patients
Grosser T, Thromb Haemost, 2006, 96, 393-400
COX2 inhibitors: marked reduction in PGI2, a potent inhibitor of platelet function and
vasorelaxing agent
- Loss of a local inhibitor of prothrombotic stimuli; patients with pre-existing risk factors
for thrombosis are at risk; even partial inhibition of PGI2 increases thrombotic process
COX-2 inhibitors depressed the biosynthesis of PGI2 without a
concomitant effect on thromboxane or platelet aggregation