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Therapeutic Uses for Blood Products and
Enzyme Therapeutics
BIT 230
Walsh Chapter 9
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Blood and Blood Products
Source of traditional biologics Blood composed of
red blood cells white blood cells platelets plasma (contain cellular elements) - therapeutic
proteins come from plasma
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Therapeutic Blood Products
Clotting factors Factor VIIa VIII, IX and XIII
Platelet concentrate Hemoglobin whole blood (hemorrhage) Red blood cells
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Risk with Blood Product Use
Transmission of infectious diseases Hepatitis B and C HIV Cytomegalovirus (CMV) (cold symptoms or more
serious in people with HIV) Treponema pallidum (syphilis) Trypanosomes
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Strict GMP guidelines for using blood products
Careful screening of all donors and donations use of pathogen removing methods or
inactivation during processing steps stringent screening of all finished products TRACEABILITY important! - no test 100%
accurate (1/42,000 HIV negative blood unit donated is actually positive)
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Treatment of Blood Products
Can’t heat inactivate or chemically treat blood product to remove pathogens
Rely on careful screening procedures Chromatography steps effective in separation
of pathogens from the blood product
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Use of Platelets
Key role in blood clotting process Administered either prophylactically or
therapeutically to prevent or minimize blood loss- people suffering from thrombocytopenia (low number of thrombocytes, alternate word for platelets)
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Human Serum Albumin (HSA)
Most abundant blood protein (Table 9.4 page 356 table of known plasma proteins)
65.5 kDa protein 60% of total plasma protein Responsible for most of blood osmotic
pressure (retaining sufficient fluid in blood vessels)
makes blood “thicker than water”
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Therapeutic HSA
Available in aqueous form or concentrated form
plasma extender in hemorrhage, shock, burns and edema
used after surgery market value about $1 billion
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Hemostasis
Maintain constant blood volume; Mechanisms for hemostasis:
congregation and clumping of platelets at site of vascular injury
localized constriction of blood vessel at damage site
induction of the blood coagulation cascade
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Coagulation Cascade
Fibrinogen converts into fibrin Fibrin aggregate at injury site, causing
thrombus (clot) formation seals off damages area to prevent further
blood loss Process: 12+ blood clotting factors involved
in cascade
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Coagulation CascadeCoagulation pathway
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Factor Name
I Fibrinogen
II Prothrombin
III Tissue factor or thromboplastin
IV Calcium ions
V Proaccelerin (Labile factor)
VII Proconvertin (Stable factor)
VIII Antihemophilic factor A, Antihemophilic globulin
IX Antihemophilic factor B, Plasma thromboplastin component, Christmas factor
X Stuart-Prower factor
XI Plasma thromboplastin antecedent, Hemophilia C, Rosenthal syndrome
XII Hageman factor
XIII Fibrin stabilizing factor, Laki-Lorand factor
Coagulation Factors
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Coagulation Factors cont’d
Roman numeral designation (see factors in Table 9.5 page 359 and next slide)
Intrinsic and extrinsic pathways Intrinsic - cascade that utilizes only factors
that are soluble in the plasma Extrinsic - some factors that are insoluble in
the plasma, e.g., membrane-bound factors (factor VII)
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coagulation lecture
See this PowerPoint Presentation
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RJ GilliesBC801
Overview of blood coagulation
VesselInjury
PlateletActivation
TissueFactor
CoagulationCascade
PlateletAggregation
PlateletPlug
Thrombin
Clot
Vasocon-striction
Adhesion to vWF
Release of Prostaglandins
Release of ATP, factor V, fibrinogen, HMWK , Ca2+
www.biochem.arizona.edu
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Genetic disorders
Lack of gene expression of clotting factor altered amino acid sequence of clotting factor both intrinsic and extrinsic pathways must
work for proper clotting results of mutations bruising or prolonged
bleeding
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Disorders cont’d
90% of characterized disorders deficiency in factor VIII, rest due to factor IX
treatment administration of whole blood or relevant coagulation factor (purified from whole blood)
recombinant factors important to minimize risk of exposure to blood pathogens
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Factor VIII - Hemophilia
Hemophilia A X-linked recessive disease Intact VIII- made up of 2 products, factor VIII
and von Willebrand factor (vWF) 1-2 million Da fully intact VIII needed to enhance activation
rate of factor IX of intrinsic system
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Hemophilia
Level of intact factor VIII affects severity of disease (need at least 5% expression)
rarer von Willibrand’s disease- lack both components of factor VIII complex
Administer weekly factor VIII complex for life
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rVIII
Expressed VII:C cDNA in CHO and BHK cells
clinical studies show equivalent effects of recombinant and native isolated factor VIII
some problems due to immunogenic response
same production issues as with any biologic
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Factor IX
Hemophilia B rarer disease treatment is administration of concentrated
factor IX produced recombinantly in CHO cells also X-linked recessive
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Anticoagulants
Inappropriate clotting dangerous Occurs in blood vessels, impedes blood flow results in heart attack (coronary thrombosis
resulting in m.i.) or stroke (blood vessel that supplies the brain)
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Major Anticoagulants
Heparin Warfarin Hirudin Ancrod Protein C Dicoumerol
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Heparin
Proteoglycan - make up of large carbohydrate component, so more like a poly-sugar than a protein
injected drug found in mast cells in endothelium of blood vessels activates antithrombin (plasma protein)- inactivates
clotting factor One of few carbohydrate product with therapeutic
application
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Vitamin K Antimetabolites
Dicoumarol and warfarin- given orally prevent vitamin K-dependent carboxylation of
factors II, VII, IX and X hinders effective functioning of the coag
cascade Side effect: prolonged bleeding need to choose dosage carefully
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Hirudin
Directly inhibits thrombin by binding to it isolated from leech saliva in Hirudo medicinalis short 65 aa polypeptide does not require a co-factor weak immunogen not as much bleeding side effects Trade name: Refludan (purifying scheme in Figure
9.15, page 378)
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Thrombolytic Agents
Thrombosis function to plug damaged blood vessel
after repair, blood clot is removed via enzyme degradation (fibrinolysis)
when inappropriate clot forms, damage minimized if you can speed up removal of clot
Agents developed to do so
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Thrombolytic Agents
tPAs (see diagram of action Figure 9.18 Page 382) Activase (Genetech) Ecokinase (Galenus Mannheim) Retavase (Boehringer-Mannheim/Centocor) Rapilsyn (BM)
TNKase Streptokinase Urokinase
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Tissue plasminogen activator
Plasmin catalyzes degradation of fibrin in clots (dissolving clot)
plasmin derived from plasminogen plasminogen synthesized and released from
kidneys; 90 kDa protein tPA potently activates plasminogen
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Early tPA
Studied in 40’s; normal low level made it difficult to characterize
Bowes melanoma cell line secretes large amounts of tPA
cloned in “83 and produced in CHO cells Genentech got first tPA license in 1987
(Activase) need to treat within hours of m.i.
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Recombinant tPA
Several modified forms to improve efficacy (faster- acting, prolonged half-life)
produced in E. coli (Ekokinase, Retavase and Rapilysin)
no glycosylation leads to prolonged serum half-life
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Enzymes as Therapies
Table 9.12, page 389 Enzymes given intravenously encapsulate or covalently modify enzymes to
counteract antigenicity or short half-lives capsule needs to be non-immunogenic
material Liposomes- facilitate entry into cell (used in
transfection protocols)
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Glucocerebrosidase
Used to treat Gaucher’s disease disease affects lipid metabolism - these are
lipids such as sphingosine, found in brain and neural tissue, associated with myelin sheath of nerves
symptoms- enlargement of liver and spleen, mental retardation
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Glucocerebrosidase cont’d
Genzyme Corp. in Boston- marked in 1991- brand name Ceredase -taken from placenta
administration to Gaucher’s patients relieve symptoms of disease
Cerezyme- recombinant version produced in CHO cells
$200 million market
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Digestive aids
Used in compromised digestive functions, such as cystic fibrosis
another e.g. - lactase, aids in lactose intolerance
pepsin- degradation of dietary protein pancreatin - degradation of dietary
carbohydrate, fat and protein