Download INOTROPIC AGENTS - Dr Ted Williams

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PHARMACY 753, WINTER-2006
MEDICINAL CHEMISTRY/PHARMACOLOGY
ANTICOAGULANTS AND
FIBRINOLYTIC/THROMBOLYTIC AGENTS
Dr. J.F. Stevens
-1-
Reading assignment: Wilson and Gisvold 11th ed., pp. 663 - 668
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ANTICOAGULANTS
BLOOD COAGULATION PROCESS
1) Platelet Aggregation:
Platelets – non-nucleated cells; contain RNA, clotting factors, serotonin, ADP, PAF, Thromboxane
etc. – normally do not adhere to smooth intact endothelial cells
They do stick to subendothelial tissue exposed by vessel damage
Triggers release of compounds stimulating more platelets to adhere
Serotonin release promotes local vasoconstriction to close off small, bleeding vessels
This aggregate of platelets form a large mass called primary hemostatic plug
alone can stop bleeding in areas of minor damage
Platelets also stimulate local activation of blood coagulation cofactors
leads to fibrin clot formation
primary plug also acts as a ‘net’ to catch the procoagulants and serves as a catalytic surface for
clot formation
2) Fibrin Clot Formation:
Formed by a series of zymogen activations
Each step involves a protease converting an inactive precursor protein (zymogen) into another
catalytically-active protease.
Non-enzyme protein cofactors and Ca2+ are also required at most steps
Also need organizing surface provided by platelet plug
One example of an enzyme cascade
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New bonds crosslinking
fibrin strands
soluble fibrin monomers
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Partial Pathway of Blood Coagulation and Clot Hydrolysis
The blood coagulation process can be activated by one of two pathways.
The tissue factor pathway (formerly known as the extrinsic pathway) and
the contact activation pathway (known as the intrinsic pathway).
Factor X can be activated
by either pathway
Factor X
(inactive)
Factor Xa (active)
Vitamin k
dependent
Ca2+, Va
Platelet Factor-3
Prothrombin
(inactive)
Thrombin (active)
Vitamin k
dependent
Fibrinogen
Fibrin (soluble)
Fibrin Stabilizing Factor
(Factor XIIIa)
Fibrin Clot
(insoluble)
Fibrinase-catalyzed formation of an -lysyl--glutamyl fibrin cross link
O
NH
H
H
N
O
H2N
H
N
H
HN
NH3
NH2
O
O
NH
N
H
O
4
O
H
H
N
H
HN
N
H
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Once the wound has healed, the clot is dissolved – FIBRINOLYSIS
Tissue
Plasminogen
Activator
Fibrin Clot
(insoluble)
Plasminogen
Plasmin
(inactive)
(active)
Fibrin Fragments
(soluble)
ORAL ANTICOAGULANTS
AGENTS AFFECTING VITAMIN K-DEPENDENT CLOTTING PROCESS
Vitamin K: fat soluble vitamin w/ crucial role in activation of coagulation factors II, VII, IX and X
–
each of these factors has 9-12 glutamate residues that are converted to -carboxyglutamates
–
Gla residues are required for these proteins to bind Ca2+
compounds such as warfarin block the fomation of Gla residues
the result is a protein factor that can’t bind Ca2+
if unable to bind Ca2+ these factors do not coordinate to platelets and are not brought into proximity
with other coagulation factors which is required for the enzyme cascade to function properly
BACKGROUND
1924: Cattle that had eaten spoiled sweet clover died of hemorrhagic disease
–
cause traced to  levels of prothrombin
OH
OH
CH 2
1939: Hemorrhagic agent identified as a bishydroxycoumarin:
O
1948: More potent synthetic agent developed as a rodenticide:
O
O
O
Dicouma rol
OH
5
O
O
CH 3
O
Warfarin
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1951: Person survives suicide attempt from warfarin O.D.
1955: Dwight D. Eisenhower receives warfarin after heart attack
Mechanism of Vitamin K-Dependent Coagulation Cofactor Activation
Inactive coagualtion factor
Active coagualtion factor
CH2
CH2
CH2
-O C
2
CO2-
CH
CO2-
-carboxyglutamate (Gla)
Glutamate (Glu)
Vitamin K-dependent
Carboxylase
H2O
O2, CO2
O
OH
CH3
CH3
O
R
R
O
OH
Vitamin K Epoxide
Vitamin K (reduced form)
NAD+
Vit. K Epoxide
Reductase
X-(SH)2
Vit. K Epoxide
Reductase
O
CH3
X-S2
R
NADH
O
Vitamin K
CH3
R=
CH3
CH3
6
CH3
CH3
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CHEMISTRY
Two structural classes used clinically
OH
O
R
4
1
O
R
2
O
O
4-Hydroxy Coum arins
1,3-Ind andi ones
Both block regeneration of reduced form of vitamin K.
Termed indirect acting agents
SLOW ONSET: already functional coagulation cofactors are not affected by these compounds
depletion of circulating Vitamin K-dependent clotting factors must occur via normal protein
catabolism
clotting factor t1/2 range from 24-40 hrs
full ONSET of action can take 2-7 days.
DURATION after discontinuation can last 4-5 days
time required for new clotting factors to reach pre-drug levels
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INDIVIDUAL AGENTS
Warfarin (COUMADIN, ATHROMBIN-K)
O
OH
O
CH 3
O
Warfarin
can be adminstered orally, IV or IM
WARFARIN SAR
–
Need H+ at C-4 in order to form salts
–
S-(-) enatiomer is 5-8x more potent than R-(+) enantiomer
–
S-isomer is metabolized more rapidly
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Anisindione (MIRADON)
O
OCH3
O
anisindione
Oral
Pharmacologically, nearly identical to warfarin
Can discolor urine to reddish-orange
Non-Vitamin K Dependent Oral Anticoagulants
Ximelegatran (EXANTA)
Late phase III trials (AstraZeneca)
O
HO
N
H
H
N
O
N
HN
NH
O
OEt
Prodrug for melagatran
acts as a direct thrombin inhibitor
inhibits the free and clot bound enzyme
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ximelegatran has no effect on human thrombin in vitro
oral availability is ca. 20%, measured as melagatran in the plasma.
Applying for indications to treat DVT, pulmonary embolism and stroke due to atrial fibrillation
in a phase III trial vs warfarin for DVT, ximelegatran was equally effective and did not require routine
coagulation monitoring or dose adjustment .
IV ANTICOAGULANTS
Heparin
•
Large polysaccharide chain
–
synthesized as chains of alternating D-glucuronic acid and N-Ac-D-glucosamine units
–
heavily O- and N-sulfated which gives heparin a high number of anionic groups
–
very heterogeneous mixture
Antithrombin-binding structure of heparin
CH2OSO3-
(or –H)
CH2OSO3-
CO2-
O
O
O
O
OH
OH
HO CO2-
OSO3-
NHCOCH3
(or –SO3-)
N-acetyl
glucosamine
6-O-sulfate
OH
glucuronic acid
NHSO3N-sulfated
glucosamine
3,6-O-disulfate
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O
O
O
O
O
CH2OSO3-
O
OSO3
-
O
OSO3iduronic
acid
2-O-sulfate
NHSO3N-sulfated
glucosamine
6-O-sulfate
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–
commercial heparin is usually isolated from bovine lung or porcine intestinal mucosa
o the unmodified polysaccharide is degraded to 5 – 30 kDa fragments during isolation
–
unstable at low pH and must be given IV
–
onset of action is immediate
Mechanism of Action
Heparin alone has no anticoagulant action
•
It serves as a catalyst to increase the rate at which antithrombin III neutralizes thrombin and factor Xa
–
heparin binds to antithrombin III
– induces a conformational change that promotes binding to protease clotting factors
–
the clotting factor (protease) then cleaves a specific peptide bond in antithrombin III
–
a conformational change occurs that results in a very stable clotting factor/antithrombin III
complex
–
heparin is released from complex and can go an bind to another thrombin or factor Xa molecule
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LOW MOLECULAR WEIGHT HEPARIN
•
Lower proportion of the critical pentasaccharide sequence required for antithrombin III bindings
–
increases action of antithrombin III on factor Xa but has less effect on thrombin
o only 25-50% of LMWH molecules bind and inactivate thrombin

referred to as the Factor Xa:IIa ratio
–
average MW is 4000 – 5000
–
more predictable and higher bioavailability than heparin and longer t1/2
o HMWH is only 30% bioavailable, but up to 90% of a dose of LMWH is absorbed
LMWH Marketed in U.S.
Name
Trade
name
Manufacturer
Enoxaparin
Lovenox
Dalteparin
Fragmin
Tinzaparin
Innohep
Rhone-Poulenc
Rorer
Pharmacia and
Upjohn
Novo
Ardeparin
Normiflo
Wyeth-Ayerst
Fractionation
method
LMWH Used
Worldwide
Nadroparin
Sanofi
Parnaparin
Opocrin
Reviparin
Knoll
Benzylation and alkaline
depolymerization
Nitrous acid
depolymerization
Enzymatic
degradation
Peroxidative
depolymerization
Nitrous acid
depolymerization
Cupric acid and
hydrogen
peroxide
degradation
Nitrous acid
depolymerization
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Halflife
(h)
Factor Xa:IIa
ratio (daltons)
Average
molecular
4.5
2.7:1
weight
4,500
2-4
2.0:1
4,000-6,000
1.3
2:1
4,900
1.2-3.3
2.0:1
5,600-6,500
3.5
2-4.1
4,500
4
3:1
4,500-5,000
NA
3-5:1
4,300
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Fondaparinux sodium (ARIXTRA®)
•
Synthetic sulfated pentasaccharide, MW = 1728
–
•
contains only the critical region of heparin required to bind to antithrombin III
Fondaparinux is more selective for the inhibition of factor Xa than heparins and LMWH
–
Selectivity increases the neutralization of factor Xa by antithrombin
More effective than unfractionated heparins and LMWH in preventing venous thromboembolism after
orthopedic surgery for hip fracture, hip replacement, and knee replacement
•
well-absorbed when given by subcutaneous injection
–
very predictable effect and patients do not require monitoring as frequently as patients receiving other
agents,
Heparin Side Effects
Most serious is Heparin Induced Thrombocytopenia (HIT): typically occurs 7-14 days after initiation of
therapy. Results in heparin-induced platelet aggregation and the production of antibiodies directed
towards platelets with heparin bound to surface platelet factor 4 (PF4).
Requires terminating heparin and adminstration of antiplatelet agents and oral anticoagulants or
defibrination agents
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Ancrod –glycosylated serine protease (234 amino acid, 35 kDa) from snake venom
high specificity for fibrinogen. Rapidly removes normal fibrinogen from the blood stream w/o
affecting other clotting factors.
Lepirudin (REFLUDAN) – recombinant hirudin produced in yeast cells. Hirudin is a direct thrombin
inhibitor, does not require antithrombin III. Lepirudin is Leu1-Thr2-63-desulfohirudin. The polypeptide is
composed of 65 amino acids and has a molecular weight of 7.0 kDa. Natural hirudin is produced in trace
amounts as a family of highly homologous polypeptides by the leech Hirudo medicinalis. For patients
who cannot tolerate heparin.
Argatroban: a new reversible direct thrombin inhibitor for
treatment of HIT and for preventing HIT in patients
receiving heparin.
NH
H2N
O
N
H
CO2H
N
NH
O
S
CH3
O
CH3
Hemorrhaging from heparin use is managed with protamine sulfate – highly cationic proteins (mostly
arginine residues) from salmon sperm. Fairly specific for binding the anionic heparin and rendering it
inactive.
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ANTIPLATELET AGENTS
Coagulation process is slowed if platelets are prevented from aggregating
Several classes of agents that affect platelet aggregation
•
Aspirin and other agents that block thromboxane A2 synthesis
e.g., sulfinpyrazone (ANTURANE)
O
O
aspirin
•
O
O
N
N
OH
S
O
O
sulfinpyrazone
Inhibit cAMP phoshodiesterase > increasing platelet cAMP > decreasing AMP and ADP.
e.g., dipyridamole (PERSANTINE). Use: prevent thrombus formation on prosthetic heart valves.
OH
N
N
N
HO
N
N
OH
N
N
N
OH

ADP receptor antagonists. Clopidrogel/Clopidogrel (Plavix)
causes irreversible inhibition of platelet function. It is a prodrug
that is activated by cytochrome P450 in the liver. The active
metabolite binds covalently to ADP receptor subtype P2Y2 on
platelets, causing inhibition of ADP-mediated activation of GP
IIb/IIIa receptors on platelet surface (no binding with fibrinogen).
Use: atherosclerotic vascular disease.
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Cl
O
O
N
S
clopidrogel
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GP IIb/IIIa Receptor Antagonists
•
Platelet aggregation is a key step in thrombus formation
–
•
can lead to myocardial infarction and acute coronary syndrome, including unstable angina.
Glycoprotein IIb/IIIa receptors are found on platelets and, when activated, bind fibrinogen which leads to
the final step in platelet aggregation.
Several inhibitors of GP IIb/IIIa activation have proven useful as antithrombotics and to treat unstable angina
Abciximab (REOPRO®)
first agent of this class to demonstrate clinical effectiveness. Used during percutaneous coronary intervention
chimeric human-murine Fab fragment of a monoclonal antibody against the GP IIb/IIIa receptor
–
lacks high receptor-blocking specificity
–
can be antigenic
–
very expensive
Need for agents with greater specificity, reduced tendency for allergic reaction and shorter half-life led to
search for peptdomimetics. Numerous snake venoms known to inhibit platelet aggregation. Venom peptides
and proteins provided the leads for drug development.
Tirofiban (AGGRASTAT®). Used in patients undergoing PTCA (percutaneous transluminal coronary
angioplasty)
CO2H
Developed from an anticlotting protein isolated from
venom of saw-scaled viper
O
H
HN
Injectable, used to treat unstable angina
Can cause bleeding
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(CH2)4 O
N
H
S
O
(CH2)3
CH3
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Eptifibatide (INTEGRILIN®)
Based on barbourin – a peptide from the southeastern pygmy rattlesnake
showed high specificity for GP IIb/IIIa
Injectable, used to treat unstable angina and during
percutaneous transluminal angioplasty
Can cause bleeding
Prostacyclin (PGI2) the natural antiplatelet aggregation agent
stable, orally active analogs
COOH
O
O
HO
COOH
HO
OH
PGI2 = prostacyclin = Epoprostenol
administered by continuous infusion
17
OH
Beraprost (PGI2 analog, oral)
approved in Japan
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FIBRINOLYTICS AND THROMBOLYTIC AGENTS
used to dissolve or break up unwanted thrombi
Tissue
Plasminogen
Activator
Fibrin Clot
(insoluble)
Plasminogen
Plasmin
(inactive)
(active)
Fibrin Fragments
(soluble)
Normally, Tissue Plasminogen Activator (t-PA) is released from endothelial cells in response to various
signals
t-PA binds to the fibrin clot and cleaves and activates plasminogen to plasmin
t-PA cleaves fibrin-bound plasminogen 100x faster than circulating plasminogen
** shows the importance of proximity and orientation/alignment
Streptokinase (KABIKINASE, Streptase): Acute pulmonary embolism, DVT, acute MI
non-enzyme protein isolated from a streptococci bacterium
Indirect plasminogen activator
Forms a 1:1 complex with plasminogen and induces a conformational change
new conformation gives the complex plasmin-like catalytic activity
can cleave other plasminogen molecules to plasmin
PROBLEMS: SK is a foreign protein and antibodies are produced.
requires a loading dose to overcome antibodies present in system from previous streptococcal
infections
can not reuse SK for several months
t1/2 after Ab depletion is ~80 min
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Major side effect is bleeding
allergic response
other disadvantage is poor selectivity for clot-bound vs. circulating (free) plasminogen
Ideally want to localize plasminogen activation to the site of the clot
Anistreplase = Anisoylated Plasminogen Streptokinase Activator Complex (APSAC) (EMINASE®), acute MI
complex of streptokinase and plasminogen
O
Active site serine is acylated with a p-anisoyl group, which gets
O
Ser O
hydrolyzed in vivo
essentially SK prodrug has a longer t1/2
slightly higher fibrin specificity
Urokinase (ABBOKINASE): Urinary Plasminogen Activator. Pulmonary embolism and DVT
Originally isolated form urine, now produced by kidney cell culture
Urokinase is a protease cleaves a peptide bond in plasminogen to form plasmin
Advantage over streptokinase is that it is non-antigenic
t1/2 = 15 min
very expensive
Recombinant TPA (rTPA, alteplase) (ACTIVASE) Acute MI
Human gene for TPA is inserted into CHO cells and over expressed
first recombinant protein approved for clinical use
endogenous factor – highest fibrin specificity and non-antigenic
t1/2 = 3 min
some controversy regarding efficacy
two early trials (40K and 22K patients) found no difference between the two drugs
Genentech claimed protocol was flawed - not given soon enough
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Subsequent trial w/ 41K patients showed a statistically significant 1% difference in favor of rTPA
study also included aspirin
Tenecteplase: variant of alteplase (six point mutations) > significant increase in plasma half life (20 min).
Administered by IV bolus injection.
Reteplase: deletion variant of t-PA, lacks fibrin-binding sites and oligosaccharide side chains (expressed in
prokaryotic E. coli.). Eliminated slower than alteplase. Given in two bolus injections 30 minutes apart.
INVESTIGATIONAL THROMBOLYTICS
Cost of rTPA driving search for source of low cost PA with low immunogenicity and high fibrin specificity
Staphylokinase from S.aureus
indirect PA like streptokinase
more clot specific than SK
less induction of neutralizing Abs
Bat-PA
recombinant form of PA isolated from vampire bat (Desmodus rotundus salivary plasminogen activator,
DSPA or desmoteplase)
low immunogenicity
sequence is 80% homologous w/ human t-PA
higher fibrin specificity than human t-PA
(Developed by Professor Schleuning at PAION/Schering Berlin)
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