Therapeutics for Hemophilia A
June, 2013
Roger L. Lundblad, Ph.D.
Consultant in Biotechnology
Chapel Hill, North Carolina
http://www.lundbladbiotech.com
And Adjunct Professor, Department of Pathology,
University of North Carolina, Chapel Hill, North Carolina
Hemophilia in 1960-1970
• University of North Carolina Experience
– 53 hemophilia A patients with 91 bleeding episodes;
13.3 units plasma/episode
– Roberts, H.R., Graham, J.B., Webster, W.P., and
Penick, G.D., Plasma transfusion therapy in
hemophlila, in The Hemophilias, ed. K.M.Brinkhous,
University of North Carolina, Chapel Hill, NC, USA,
1964
• Therapeutics Costs
– 10 to 20 cents a unit
– Green, D., Therapeutic materials, in Hemophilia, ed.
D.Green, C.C. Thomas, Springfield, IL, USA,. 1972
Hemophilia A – A Statement of the
Problem -2013
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Hemophilia A is a congenital disorder affecting 1 in 10,000 males associated with the absence of
factor VIII activity (severe, moderate, mild) – severe is less that 1% factor VIII with major problems
– raise level to 5% (moderate) and the clinical picture is markedly improved. With a world
population of 6 billion, there should be some 600,000 individuals with hemophilia A – my best
guess is that there are likely 150,000 individuals being treated with high purity or ultra-high purity
products.
The dominant cost in the treatment of hemophilia A is the cost of therapeutic product – in the US,
this is approximately $100,000 (US)/year – thus, despite a somewhat small consumer base, it is
business with a cash flow of approximately 2.0 billion U.S./year – and this does not consider
products such as FEIBA™ or NOVOSEVEN™ which are used to treat factor VIII inhibitors.
There are a number of recombinant and plasma-derived therapeutic products; All products
appear to be effective in providing hemostasis. Viral safety, while a risk factor with the plasmaderived products ( Modrow, S., Wenzel, J.J., Schimanski, S., et al., Prevalence of nucleic acid
sequences specific for human parvoviruses, hepatitis A and hepatitis E viruses in coagulation
factor concentrates, Vox Sang. 100, 351-238, 2011) does not appear to be a significant problem
with the use of plasma-derived products.
The development of inhibitors is the greatest risk factor in treatment of hemophilia A but there is
substantial progress in tolerization ( Auerswald, G.,Bidlingmaier, C., and Kurnik, K., Early
prophylaxis/FVIII tolerization regimen that avoids immunological danger signals is still effective in
minimizing FVIII inhibitor developments in previously untreated patients – long term follow-up and
continuing experience, Haemophilia 18, e18-e20, 2012).
Prophylaxis is still an emerging concept and value depends on metrics used.
The development of new factor VIII therapeutic products with extended half-life is proceeding
rapidly. It is not known as to how such products will affect treatment cost.
Gene therapy for hemophilia A is approaching reality.
Factor VIII Products -2013
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Treatment of hemophilia A is somewhat unique in that there are no
competing technologies other than replacement therapy with factor VIII
protein. The downside to this is that there are no “niche” markets for factor
VIII. Greater than 90% of hemophilia A care cost is therapeutic product.
Factor VIII protein may be obtained by recombinant DNA technology
(including fusion protein production) and subsequent modification of protein
(PEGylation).
Factor VIII can be obtained from plasma fractionation. Other products from
plasma such as IVIG and albumin support factor VIII manufacture from
plasma (see Grifols, V., Financing plasma proteins: unique challenges, in
Blood, Plasma and Plasma Proteins: A Unique Contribution to Modern
Healthcare, ed. J.L. Valverde, IOS Press, Amsterdam, Netherlands, 2006
(Pharmaceuticals Policy and Law 7, 185-198, 2005-2006). This is not
different from the petrochemical industry (see Curran, L.M., Waste
minimization practices in the petrochemical refining industry, J.Hazardous
Mater. 29, 189-197, 1992)
Concept in Hemophilia
Treatment -2013
• Home Treatment
– Pipe, S., Visions in haemophilia care, Thromb.Res. 124 (Suppl 2, S2-S5, 2009.
– Mondorf W, Siegmund B, Mahnel R, Richter H, Westfeld M, Galler A, Pollmann
H. Haemoassist--a hand-held electronic patient diary for haemophilia home care.
Haemophilia. 2009 Mar;15(2):464-72.
– Baker, J.R., Riske, B., Voutsis, M., et al., Insurance, home therapy, and
prophylaxis in U.S. youth with severe hemophilia, Am.J.Prev.Med. 41 (Suppl. 4),
S338-S345, 2011.
• Group Medical Treatment
– Lock, J., De Bruin, A., Scholten, M., et al., The group medical appointment
(GMA) in haemophilia and von Willebrand’s disease: a new development in
outpatient paediatric care, Haemophilia 12, 766-772, 2012.
• Future
– Franchini, M. and Mannucci, P.M., Past, present and future of hemophilia: a
narrative review, Orphanet J. Rare Dis.7:24, 2012.
– Knobe, K. and Berntorp, E., New treatments in hemophila: insights for the
clinician, Ther.Adv.Hematol. 3, 165-175, 2012.
– .Philippidis, A., Developing a balanced business model for gene therapy,
Hum.Gene Ther. 22, 645-646, 2011.
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Recent Advances in Factor VIII
• Development of products with extended circulatory half-life (see
Sheridan, C., Hemophilia market awaits next-generation therapies,
Nature Biotechnol. 29, 960, 2011).
– Pegylated Factor VIII
• Coupling to inserted surface cysteine
• Coupling to lysine residues
• Glycopegylation
– Fusion protein with Fc domain
– Factor VIII bound to PEGylated liposomes
– Engineered single chain factor VIII (Schulte, S., Pioneering
designs for recombinant coagulation factors, Thromb.Res. 128
(Suppl 1), S9-S12, 2011).
Pegylated Factor VIII
• There has been significant progress on the development
of pegylated factor VIII in the past two years.
– Ivens, I.A., Baumann, A., McDonald, T.A., et al., PEGylated
therapeutic proteins: a review for haemophilia caregivers,
Haemophilia 19, 11-20, 2013.
– Tang, L., Leong, L., Sim, D., et al., von Willebrand factor
contributes to longer half-life of PEGylated factor VIII in vivo,
Haemophilia , in press, 2013
– Kosloski, M.P., Pisai, D.S., Mager, D.E., and Balu-lyer, S.V.,
Allometry of factor VIII and informed scaling of next-generation
therapeutic proteins, J.Pharm.Sci., in press, 2013.
Factor VIII Engineered for
Extended Half-Life
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Eloctate™ (Factor VIII-Fc fusion protein), Biogen-Idec, BLA application
accepted by FDA
Concept of Fc fusion protein with prolonged half-life based on binding to
neonatal Fc receptor (Suzuki, T., Ishii-Watabe, A., Tada, M., Importance of
neonatal FcR in regulating the serum half-life of therapeutic proteins
contains the Fc domain of human IgG1: a comparative study of monoclonal
antibodies and Fc-fusion proteins to human neonatal FcR, J.Immunol. 184,
1968-1976, 2010.
Factor VIII-Fc fusion protein has demonstrated extended half-life in a clinical
trial (Powell, J.S., Josephson, N.C., Quon, D., et al., Blood, 119, 3031-3037,
2012.
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Inhibitor formation was not observed and there was an approximate 1.5 – fold extension of
therapeutic half-life
Peters, R.T., Toby, G., Lu, Q., et al., Biochemical and functional
characteristic of a recombinant monomeric factor VIII-Fc fusion protein,
J.Thromb.Haemost. 11, 132-141, 2013.
Gene Therapy for Hemophilia A
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Johnson, J.M., Denning, G., Doering, C.B., and Spencer, C.T., Generation
of an optimized lentiviral vector encoding a high-expression factor VIII
transgene for gene therapy of hemophila A, Gene Ther., in press, 2013.
McIntosh, J., Lenting, P.J., Rosales, C., et al., Therapeutic levels of FVIII
following a single peripheral vein administration of rAAV vector encoding a
novel human factor varient, Blood 121, 3335-3344, 2013.
Zhu, F., Liu, Z., Wang, X., et al., Inter-chain disulfide bond improved protein
trans-splicing increases plasma coagulation activity in C57BL/6 mice
following portal vein FVIII gene therapy by dual vectors, Sci.China Life Sci.
56, 262-267, 2013.
The above studies show that a combination of vector development and
factor VIII engineering combine for progress in gene therapy for hemophilia
A.
Biomarkers for Factor VIII
• A biomarker is defined as “a characteristic that is objectively
measured and evaluated as an indicator of normal biological
processes, pathogenic processes, or pharmacological responses to
a therapeutic intervention.” Downing, D.O. for the Biomarkers
Definitions Working Group, NIH, Bethesda, MD, USA, Biomarkers
and surrogate endpoints: Preferred definitions and conceptual
framework, Clin.Pharmacol.Therapeutics 69, 89-95, 2001. See also
DeCaprio, A.P., Introduction to Toxicological Biomarkers, in
Toxicological Biomarkers, ed. A.P. DeCaprio, Taylor & Francis, New
York, NY, USA, 2006
• The major biomarker for hemophilia A is the activated partial
thromboplastin time
• Clinical endpoint = stopping of bleeding
• The availability of a documented clinical endpoint is a distinct
advantage for gene therapy.
Assay of Factor VIII
• Factor VIII is a cofactor (with apologies to hard-core
biochemists) for the activation of factor X by factor IXa in
a reaction which also requires calcium ions and platelets.
• The partial thromboplastin time (PTT) (developed in
Chapel Hill) is likely the most reliable assay.
• There are all sorts of chromogenic assays which
measure color and may or may not measure factor VIII.
• The assay is very important since factor VIII is “sold” by
the unit
• Lundblad RL, Kingdon HS, Mann KG, White GC. Issues
with the assay of factor VIII activity in plasma and factor
VIII concentrates. Thromb Haemost. 2000
Dec;84(6):942-8.
Development of Concept of Blood
Transfusion
• Development of transfusion medicine was critical to
hemophilia therapy
• Direct donor to recipient
– Pope Innocent vii (1406) donors and recipient expired, physician
left the country
• 1628 – William Harvey, Concept of Circulation
• 1665 – Richard Lower – Blood Transfusion in dogs
• 1818 – James Blundell – first successful blood
transfusion
– Transfusion is species specific
– Blood may be transfused directly from vessel to vessel
– Blood may be passed into an intermediate container and remain
for a short period of time before infusion into the recipient
First Treatment of Haemophilia A
• Lane, S., (1840), Lancet I, 185-188
– Treatment was reactive to a surgical
procedure.
– Use of Whole blood
– Blood collected from a donor into a
funnel/syringe device
– 4-6 ounces – 100 – 250 mL
– Within the scope of the article, the patient
recovered and did reasonably well
Technology Advances Required for
the Use of Blood for Haemophilia A
• J. Braxton Hicks – 1869 – use of sodium phosphate as
an anticoagulant – although mechanism not understood.
It was not very successful
– Hicks, J.Braxton, Cases of transfusion with some remarks on a
new method of performing the operation, Guys Hospital Reports,
Series 3, Volume 14, 1-14, 1869
• Compared phosphate with whipping to remove fibrine.
• Transfusion used basin to collect blood and funnels for transfer.
• Low success rate – use of phosphate was based on the
development of salt solutions for infusion.
• Duncan, J., On re-infusion of blood on primary and other
amputations, Brit.Med.J. I, 192, 1886
• Cotterill, J.M., Severe injury from dynamite, tranfusion of
blood, four times, recovery, Brit.Med.J. ii, 630, 1886
Technology Advances Required for
the Use of Blood in the Treatment
of Haemophilia A
• The development of anticoagulants for blood
– As noted by P.L. Mollison in an excellent review, the
use of citrate allowed the separation of donor and
recipient in space while the use of glucose enabled
separation in time.
– Mollison, P.L., The introduction of citrate as an
anticoagulant for transfusion and of glucose as a red
cell preservative, Brit.J.Haematol. 108, 13-18, 2000.
– Boulton, F., A hundred years of cascading – started
by Paul Morowitz (1879-1936), a pioneer of
haemostasis and transfusion, Transfusion Medicine
16, 1-10, 2006.
Factor VIII Therapy – 1936 Status
• 1840 – Lane (London) – Successful transfusion of whole
blood for hemophilia
• 1886 – Contrill (Edinburgh)-Phosphate anticoagulant
• 1900 – Landsteiner - ABO blood grouping
• 1911 – Addis – Defective prothrombin conversion in
hemophilia
• 1911 – Todd and White – Citrated blood for transfusion
• 1915 – Ottenberg – Citrated blood for transfusion in
hemophilia – correction for several days
• 1916 – Addis – blood (phosphate) and serum corrected
hemophilia for 24 hrs
• 1916 – Rous and Turner – sucrose and citrate
• 1936 – Blood bank at Cook County Hospital
Cohn Fractionation
• Developed by E.J. Cohn and others at Harvard
University in the late 1930’s
• Major emphasis during WWII
• Fractionation of plasma by organic solvents, pH,
and ionic strength
• Still forms the basis for most human plasma
fractionation
• Blombäcks developed derivatives of Cohn
Fraction I such as I-0
• Pavlovsky used tannic acid precipitation to
prepare derivatives of Cohn Fraction I
Early Development of Plasma
Protein Therapeutics
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Dried Plasma – 1941
Formalin-stabilized plasma – 1942
Products from human placenta - 1952
Stable plasma protein fraction (PPF) –1952
Cohn Fraction I – 1958
Commercial IVIG – 1962 (although there were some
studies in the 1940’s
Cryoprecipitate – 1963
Commercial Factor VIII Preparations-1965
Rh IgG – 1968
Factor IX Concentrates – 1970 (also inhibitor products)
Economic “Drivers” for Plasma
Fractionation
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1940 – 1960 - Albumin
1960 – 1990 - Factor VIII
1900 – Present – IVIG
See Grifols, V., Financing plasma proteins:
Unique challenges, in Blood, Plasma and
Plasma Proteins: A Unique Contribution to
Modern Healthcare, ed. J.L. Valverde, IOS
Press, Amsterdam, Netherlands, 2006
Development of Modern Factor VIII
Therapeutics
• Despite the advances in chromatography, plasma
fractionation was (and for that matter with one exception)
is still dominated by precipitation technologies.
• Chromatography did have an effect on plasma-derived
therapeutic products until 1985 and that was
immunoaffinity chromatography; immunoaffinity
chromatography was developed in 1975 and successful
in this context required the production of monoclonal
antibodies.
• Introducing chromatography into a plasma fractionation
facility is a disruptive innovation – see Govindarajan, V.
and Kopalle, P.K., The Usefulness of Measuring
Disruptiveness of Innovations Ex Post in Making Ex Ante
Predictions, J.Prod.Innov.Manag. 23, 12-18, 2006.
Development of Modern Factor VIII
Therapeutics
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Various groups continued to attempt make more effective derivatives of Cohn Fraction I.
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Jorpes, J.E., Blomback, B.,Blomback, M., and Magnusson, S., A pilot plant for the preparation of a human plasma fraction
containing the human antihemophilic factor A (factor VIII) and v. Willebrand’s factor, Acta Med. Scand. Suppl. 379, 7-21, 1962
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Steinbuch, M., Precipitation methods in plasma fractionation, Vox Sang. 23, 92-106, 1972
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Newman, J., Johnson, A.J., Karpatkin, M.H., and Puszkin, S., Methods for the production of clinically effective intermediate- and
high-purity factor VIII concentrates, Brit.J.Haematol. 21, 1-20, 1971
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Brinkhous, K.M., Wagner, R.H., Roberts, H.R., and Webster, W.P., Use of aliphatic amino acid precipitated antihemophilic
factor in therapy of hemophilia, Bibl.Haemtol. 29, 1104-1108, 1968
Judith Pool - 1965 – Cryoprecipitate
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Pool, J.G. and Robinson, J., Observations in plasma banking and transfusion procedures for haemophilic patients using a
quantitative assay fo antihemophilic globulin (AHG), Brit.J.Haematol. 5. 24-30, 1959.
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Pool, J.G., Hershgold, E.J, and Pappenhaggen, A.R., High-potency antihemophilic factor concentrate prepared from cryoglobulin
precipitate, Nature 203, 312, 1964.
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Bennett, E., Dormandy, K.M., Churchill, W.G.L., Coward, A.R., Smith, M., and Cleghorn, T.E., Cryoprecipitate and the plastic
blood-bag system: Provision of adquequate replacement therapy for routine treatment of haemophilia, Brit.Med.J. 2, 88-91, 1967.
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This enabled every blood bank to produce an effective therapeutic for hemophilia A.
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It was not possible to scale up the cryoprecipitate step for commercial fractionation, it was possible to include a “cold
drop” at the start of the conventional plasma fractionation process as the cryosupernatant fraction could be taken
through the Cohn Fractionation process.
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Still a useful approach (Kasper, C.K., Products for clotting factor replacement in developing countries, Semin.Thromb.Haemost.
31, 507-512, 2005)
Immunoaffinity Chromatography
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Pedro Cuatrecasas – 1960’s
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Livingston, D.M., Immunoaffinity chromatography of proteins, Methods Enzymol. 34, 723-731, 1974
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Application to Factor VIII – 1982 – Baxter and Aventis
Recombinant DNA technology
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Stanford recombinant DNA – Cohen-Boyer patent
• Hughes, S.S., Making dollars out of DNA. The first major patent in biotechnology and the commercialization of molecular
biology, 1974-1980, Isis 92, 541-575, 2001
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Cutter/Genentech and Baxter/Genetics Institute
Factor VIII Concentrates
1960-1983
• Kenneth Brinkhous, Harold Roberts, Gilbert White set
the standards for hemophilia A care for several decades.
– Hemophilia dog colony
• Robert Wagner and colleagues at UNC-CH
– Amino Acid Precipitation – Method IV Factor VIII
• Alan Johnson at NYU
– PEG precipitation
• Virus Problems
• Hougie, C., Thrombosis & Bleeding: an era of discovery,
Trafford Publications, Victoria, BC, 2004
Development of Ultrahigh Purity
Concentrates
• Drivers for something better than high
purity concentrates
– Product safety –”non-A,non-B hepatitis” –was
not solved by heat treatment
– Fibrinogen overload – German regulatory
agency – The circulatory half-life of factor VIII
is approximately 14 hours; fibrinogen half-life
is 4 days!
Development of Ultrahigh Purity
Concentrates
• Immunoaffinity Chromatography
– Isocratic method – best fit to manufacturing
– Antibodies to factor VIII
– Antibodies to von Willebrand Factor
– Concomitant development of
solvent/detergent technology at New York
Blood Center.
Manufacture of Monoclonal-Purified
Factor VIII
Frozen Plasma
Thaw at 2-4oC/centrifuge
Cryoprecipitate
Supernatant Fraction to Cohn
Cold Precipitate
Fibrinogen and Other Stuff
Supernatant Fraction to
MAB-Affinity step
The highly purified factor VIII preparations no longer are effective for the
treatment of von Willebrand Disease – the high-molecular weight vWF is
found in the cold precipitate.
Aronson (Aronson, D.L. and Chang, P., Ultracentrifugal analysis
of factor VIII and von Willebrand factor in therapeutic preparations,
Vox.Sang.69, 8-13, 1995) suggests that the interaction is complex with data
suggesting while infused factor VIII associated randomly with vWF while material
purified from plasma using vWF affinity is unique
Recombinant DNA Technology and
Factor VIII
Cutter/Genentech
– Kogenate®
• Baxter/Genetics Institute
– Recombinate®
– Advate®
• Pharmacia/Wyeth(Genetics Institute)
– ReFacto® (B-domainless factor VIII)
Future Directions for Factor VIII
Therapeutics
• Oldenburg, J., Dolan, G., and Lemm, G.,
Haemophilia care then, now and in the
future, Haemophilia 15 (Suppl 1, 2-7, 2009
– Life expectancy have moved from 11 years in
1939 to 68 years in 1980
– Prophylactic use to prevent joint bleeding
– Delivery of therapy
• Gene therapy
• Improved product
Future Directions for Factor VIII
Therapeutics
• Current market is almost commodity in nature
– Growth through product enhancement
– Factor VIII as a biopharmaceutical
• Growth into developing markets is constrained
by infrastructure
• Market needs
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Product Safety
Product efficacy
Therapeutic effect driven by clinical endpoint
While a great product requires great science,
great science does not make a great product
Future Directions for Hemophilia A
Therapy Delivery
• “Cure”
– Gene Therapy
• Gene Fixing
• Gene Augmentation
• Delivery of current product (avoid venipuncture)
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Transdermal
Oral
Pulmonary
Nasal
Continuous infusion
• Implantable pumps
– Formulation issues
– Stability issues
• Modified Product (fraught with technical challenges)
– Engineered
– Chemically Modified
Gene Therapy for Hemophilia A
• Why has this not happened??
– Incredibly attractive commercial target for “proof of
principle” for a technology
– However, given the above, what is the “bottle?” – Is
gene therapy a product or a service?
– Despite all of our knowledge, we still don’t understand
gene expression??
– Addition cis-factors and trans-factors??
– Unknown epigenetic factors??
– Is there a specific cell type such that, for example,
you cannot get sustained expression in muscle.
– Is the expression of factor VIII and von Willebrand
Factor linked??
Gene Therapy for Hemophilia A
• Linkage of vWF and Factor VIII – Is this
important for design of gene therapy?
– Fricke, W.A. and Yu, M.Y., Characterization of von
Willebrand factor in factor VIII concentrates,
Am.J.Hematol. 31, 41-45, 1989.
• Highly purified factor VIII from plasma associated with a
small vWF multimer
• Highly purified factor VIII preparations do not correct von
Willebrand disease.
– Montgomery, R.R. and Gill, J.C., Interactions between
von Willebrand factor and Factor VIII: were did they
first meet?, J.Pediatr.Hematol.Oncol. 22, 269-275,
2000.
Gene Therapy for Hemophilia A
• Co-expression of VIII and vWF required?
• Need different approach
• Gene Therapy needs a business model – is it a
service, a drug, a combination product – in some
ways, has the same business issues as stem
cells.
• Someone will make it work because
– Great target – clear clinical endpoint and several
animal model systems
– Great “proof-of-principle” such that company does not
have to make money off success as it would validate
a platform technology for a larger clinical target.
Future Directions for Hemophilia A
Therapeutics
• Drug Delivery
– Needle-less delivery (See Prabhu, S., et al.,
Needles and needleless devices for infusion
of anti-hemophilic factor concentrate: impact
on protein structure and function, Haemophilia
12, 58-61, 2006)
– Infusion pumps – problems with long-term
product stability – if this was easy, it would
have happened for insulin.
– Need a new paradigm for this problem
Future Directions for Hemophilia A
Therapeutics – Modified Proteins
• Saenko, E.L. and Pipe, S.W., Strategies towards a longer acting
factor VIII, Haemophilia 12(suppl. 3), 42-51, 2006
– PEG Modification (Baxter and Nektar)
• Kingdon and Lundblad worked on this at UNC in the 1970’s with a total lack
of success
• Modification of lysine with small reagents results in factor VIII inactivation
and the one published study on PEG modification does not provide optimism
– Manning, F., et al., Effects of chemical modification on recombinant factor VIII
activity, Thromb.Res. 80, 247-254, 1995
– Röstin, J., et al., B-domain deleted recombinant coagulation factor VIII modified
with monomethoxy polyethylene glycol, Bioconjug.Chem. 11, 387-386, 2000
– PEGylated lipsomes (Bayer/Zilip (Amsterdam)/UC-Davis)
– Engineering to Improve Stability
• Pipe (Michigan)
• Lollar (Emory)
Future Direction for Hemophilia A
Therapeutics
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Peptide Analogues – I was enthusiastic about this at one time but no longer
– too many issues with regulation of activity expression.
• Lenting, P.J., et al., Ca2+ binding to the first epidermal growth factorlike domain of human blood coagulation factor IX promotes enzyme
activity and factor VIII light chain binding, J.Biol.Chem. 271, 2533225337, 1996
• Fay, P.J., et al., The A1 and A2 subunits of Factor VIIIa
synergistically stimulate factor IXa catalytic activity, J.Biol.Chem.
274, 15401-15406, 1999
• Kolkman, J.A., et al., Regions 301-303 and 333-339 in the catalytic
domain of blood coagulation factor IX are factor VIII-interactive sites
involved in stimulation of enzyme activity, Biochem.J. 339, 217-221,
1999
• Bajaj, S.P., Factor IXa:Factor VIIIa interaction. Helix 330-338 of
factor IXa interacts with residues 558-565 and spatially adjacent
regions of the A2 subunit of Factor VIIIa, J.Biol.Chem. 276, 1630216309, 2001
Future Directions for Hemophila A
Therapeutics
• Carbohydrate Modification
– Is carbohydrate heterogeneity a problem?
• Carbohydrate Engineering
– Dwek, R.A. et al., Targeting glycosylation as a
therapeutic approach, Nat.Rev.Drug Disc. 1,
65-75, 2002
– Jones, J., Controlling N-linked glycan site
occupancy, Biochim.Biophys.Acta 1726,121137, 2005
Some Basic Questions about
Factor VIII
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Why is proteolysis associated with secretion?
Why is factor VIII so big?
Why is circulatory half-life dependent on von Willebrand factor?
Why is half-life so short ?
– Hours instead of days?
Why is there is a free sulfhydryl group?
Why is copper bound? – does bound copper have anything to do with the free
sulfhydryl group?
What is the role of factor VIII in the activation of factor X by factor IXa?
– Factor IXa as a regulatory protease – what are factors involved in the increase in
the increase in Vmax ? The decrease in Km is easier to manage.
Is binding to platelets unique? Walsh PN, Camp E, Dende D. Different requirements
for intrinsic factor-Xa forming activity and platelet factor 3 activity and their
relationship to platelet aggregation and secretion. Br J Haematol. 1978
Oct;40(2):311-31.
Why is secretion of factor VIII
associated with proteolysis?
• Activity (or lack thereof) of single chain material
– Would single-chain material be a better product?
• ReFacto™
• Why is thrombin activation required for normal
factor VIII function?
• Is inactivation by activated protein C necessary?
• You need the answers to these questions when
you consider engineered/modified factor VIII
• Factor VIII as a thrombotic risk factor
– Pabinger I, Ay C. Biomarkers and venous
thromboembolism. Arterioscler Thromb Vasc Biol.
2009 Mar;29(3):332-6.