VOLUME12
`
`JULY 2006
`
`EDITHDBY
`
`C.A.LEE
`
`SUPPLEMENT 3
`
`CSL1046
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`C.NLKESSLER
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`HAEMOPHILIA
`The Official Journal of the World Federation of Hemophilia
`Editors
`Christine A. Lee
`Craig M. Kessler
`Her/zopAi/ia Treatment Center and Coaginh/ion Laboratory
`Harman/9mg Cat/me mid Haemattm/J Unit
`Diririm Ly'Hemam/ygr/ Onto/0g]!
`ngl Fm Hot/)zlal
`Pom! fmel
`Cemgetaum [JIIII/flKr/ij', 3800 RermmirRoad
`Lam/mi [VIVJ ZQG, UK
`Nll” lffleJ/JIJIgI‘DIA DC 20057-2797, UJA
`
`
`Editorial Board
`1.. M. Aledort (Van) York, UM
`L. R. Battistella 81712:]!
`K. Beeton Habit/d. UK
`H. Herntorp [Md/”l0, Sit/eds”
`M. van den Berg Ulm'bt, The Neffitl'jflfll’l'
`I). Brettler Warrerter, UXA
`S. A. Brown Brit/7am, Aloha/ta
`A. Chuansumrit Baa/em, Tani/mid
`P. Collins Can/{fl UK
`D. DiMichele New York, USA
`A. B. Federici Ali/ml, [my
`P. L. F. Giangrandc Oafnrd, UK
`N. Goddard Lam/m, UK
`C. R. l\/l. Hay ilianrlimcr. UK
`L. Heijnen Hhizm, {/13 Netherlands
`M. Hcim Tel/1111a, [:rne/
`K. Hoots Harmon UXA
`J. Ingerslev Ca/mzhngm, Denmark
`R. Lassila Helm/ex) Fin/am!
`R. C. R. Liung (Ma/1110, fiver/ell
`
`
`Aims and scope. Haemfizz‘h’a is an international journal dedicated
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`

`HAEMOPHILIA
`
`VOLUME 12 SUPPLEMENT 3
`
`JULY 2006
`
`Edited by C. A. Lee and C. M. Kessler
`
`State of the Art
`
`XXVII International Congress of the
`World Federation of Hemophilia
`
`Vancouver, Canada, 21—25 May 2006
`
`’
`Guest Editors:
`P. Giangrande and G—E. Rivard
`
`if)...
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`HAEMOPHILIA Volume 12, Supplement 3, July 2006
`
`State of the Art
`XXVII International Congress of the World Federation of Hemophilia
`
`Contents
`
`The Management of Haemophilia
`The pharmacokinetics of coagulation factors
`M. Lee, M. Morfini. C. Negriur and V. Clmmouard
`
`Hacmophilia and ageing
`A. Street, K. Hill. B. Sussex, M. Warner and M.~F. Sadly
`
`The natural evolution of haemophilia care: developing and sustaining comprehensive care globally
`B. 1.. Hyatt
`
`Quality of life assessment in clinical practice in haemophilia treatment
`A. Cringcri, L. Mantuuam' and S. V. Markensen
`
`Ethical Issues
`
`Ethical issues in haemophilia
`D. DiMicbele, A. Ckuansumrit, A. ]. London, A. R. Thompson, C. C. Cooper. R. M. Killian, L. F. Ross, D. Lillicmp and
`]. Kimmelman
`
`Genetic Therapies and Novel Technologies
`Cellular and genetic therapies for haemophilia
`D. Lillicmp, T. VandenDriessche and K. High
`
`Strategies towards a longer acting fatter VIII
`E. L. Saw/20 and S. W. Pipe
`
`Inhibitors
`
`Why do inhibitors develop? Principles of and factors influencing the risk for inhibitor development in haemophilia
`j. Astermar/e
`
`Laboratory Aspects of Haemophilia Therapy
`Achieving and maintaining quality in the laboratory
`M. S. l'iertzberg, j. Mam/nan, A. McCraul. S. C. Nair and A. Sriuustaua
`
`Laboratory issues in bleeding disorders
`D. Lillicrap, S. C. Nair, A. Srivastuuu. F. Rodeghz'era. I. Pabinger and A. B. Federicx'
`
`New approaches for measuring coagulation
`T. W. Barrtlwclifftr, M. Calumet), G. M. Podda, P. Bucctarelli, F. Lussana, A. Lea/ii, C. H. Tab, H. C. Hemker, S. Bégm'n,
`]. Ingerslev and B. Sarensen
`
`Genetic diagnosis of haemophilia and other inherited bleeding disorders
`F. I’eyuandi, G. jayaud/mmn. M. (.‘Imndy, A. Sriuastaua. S. M. Naknya, M. j. lolmson. A. R. Thompson, A. Gouda/e,
`I. Camgiola, S. Lauaretano, M. Menegatti, R. Palla. M. Spreafiw, I... firgi‘t‘abue, R. Asse/m. S. Bragg and P. M. Mmmuuci
`
`13
`
`22
`
`30
`
`36
`
`42
`
`52
`
`61
`
`68
`
`76
`
`82
`
`90
`
`Musculoskeletal Aspects of Haemophilia
`HIV and HCV eoinfected haemophilia patients: what are the best options of orthopaedic treatment?
`E. C. Rodriguez-Mercban
`
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`102
`
`108
`
`113
`
`117
`
`122
`
`128
`
`137
`
`143
`
`152
`
`159
`
`169
`
`Recent developments in elinimetric instruments
`K. Balaton, P. Kleijn, P. Hilliard, 5. Funk, N. Zourikizm, B.-M. Bergstrom, R. H. H. Engelbert, ]. ]. van der Net,
`M. ]. Manco-jo/mson, P. Petr-inf, M. van den Berg, A. Abad, B. M. Feldman, A. S. Doria, B. Lzmdin, P. M. Poovmoose,
`]. A. john, M. L. Kauitba. S. M. Padankatti, M. Devadurasini. D. Pazani, A. Srivastaua, F. R. Van Genderen and R. Vaclaa/atbz'ti
`
`Physiotherapy following elective orthopaedic procedures
`P. Kleifn, G. Blarney, N. Zourikizm, R. Dalzell and S. Lobet
`
`Total joint replacement in patients with inhibitors
`L. P. Solimmo, O. S. Perfcllo, G. Pasta and E. Santagoslino
`
`Pathogenesis of haemophilic arthropathy
`G. Rooscndaal and F. P. Lafcbcr
`
`Tissue engineering in musculoskeletal problems related to haemophilia
`H. Caviglia
`
`Rare Bleeding Disorders
`Congenital platelet disorders: overview of their mechanisms, diagnostic evaluation and treatment
`C. P. M. Hayward, A. K. Rat) and M. Cattaneo
`
`Rare bleeding disorders
`F. Peymmdi, R. ]. Kaufman, LI. Saligsalm, O. Salomon, P. H. B. Bolton-Maggs, M. Spreafico, M. Menegattz, R. Palla, S. Sibom‘
`and P. M. Marmucci
`
`Women and Bleeding Disorders
`Current understanding of von Willebrand’s disease in women — some answers, more questions
`P. A. Kouide‘s
`
`Von Willehrund‘s disease: clinical management
`A. B. Federici, G. Castaman, A. Thompson and E. Berntorp
`
`Prophylaxis
`Prevention and treatment of musculoskeletal disease in the hemophilia population: role of prophylaxis and synovectomy
`H. M. van den Berg, A. Dunn, K. Fischer and V. S. Blanchette
`
`World Federation of Hemophilia
`Treatment for all: a vision for the future
`M. W. Skinner
`
`5/15
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`

`

`Haemopbi/ia (2006), 12, (Suppl. 5), 42~51
`
`Strategies towards a longer acting factor VIII
`
`\X’. PIPE'i'
`E. L. SAENKO" and S.
`Terrier for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, MD, USA; and
`JrDepartmrmzf of Pediatrics, University of Michigan, Ann Arbor, MI, USA
`
`improved joint
`Summary. The reduced mortality,
`outcomes and enhanced quality of life, which have
`been witnessed in the developed world for patients
`with haemophilia, have been an outstanding achieve-
`ment. Advancements in biotechnology contributed
`significantly through the development of improved
`pathogen screening, viral inactivation techniques and
`the development of recombinant clotting factors.
`These were partnered with enhanced delivery of care
`through comprehensive haemophilia centres, adop—
`tion of home therapy and most recently effective
`prophylaxis. This came at great costs to govern—
`ments, medical
`insurers and patients’ families.
`In
`addition, barriers persist limiting the adoption and
`adherence of effective prophylactic therapy. Biotech—
`nology has been successful at overcoming similar
`barriers in other disease states. Long-acting biologi—
`
`cal therapeutics are an incremental advance towards
`overcoming some of these barriers. Strategies that
`have been successful for other therapeutic proteins
`are now being applied to factor VIII
`(FVIII) and
`include modifications such as the addition of poly—
`ethylene glycol (PEG) polymers and polysialic acids
`and alternative formulation with PEG—modified lipo-
`somes, In addition, insight into FVIII structure and
`function has allowed targeted modifications of the
`protein to increase the duration of its cofactor
`activity and reduce its clearance in vii/o. The poten—
`tial advantages and disadvantages of these approa-
`ches will be discussed.
`
`factor VIII,
`Keywords: bioengineering, clearance,
`half-life, polyethylene glycol, polysialic acids
`
`Introduction
`
`The last three decades of the recombinant technology
`era witnessed the cloning of the factor VIII (FVIII)
`gene, the development of recombinant FVIII (rFVIII)
`for infusion and furthered the progress towards a
`genetic cure. The resultant
`increased capacity for
`FVIII concentrate production coupled with advances
`in
`haemophilia
`comprehensive
`care,
`pathogen
`screening and viral inactivation technology has led
`to both exciting and sobering observations within the
`developed and developing countries worldwide.
`Within the United States,
`life expectancy is now
`approximately 65 years; serious blood—borne infec—
`tions have not occurred since 1990 and joint disease
`has been eliminated for children under the age of 15
`
`Correspondence: Steven \W. Pipe, MD, Associate Professor of
`Pediatrics and Communicable Diseases, Hemophilia and Coagu—
`lation Disorders Program, University of
`iVIichigan, LZIIO
`\Women’s Hospital, 1500 E., Medical Center Drive, Ann Arbor,
`MI 4s109, usA.
`Tel.: +1-734-647-2893; fax: +1-734-936-7083;
`eemail: ummdswpflbmed.umichedu
`
`42
`
`[1]. Home treatment and early initiation of prophye
`laxis have been the most significant advances to
`impact on joint disease prevention and quality of life
`[2]. This has of course come at great economic cost.
`The average cost of the treatment for a person with
`haemophilia in the United States
`in 2001 was
`$139 000, 72% of which was due to costs for factor
`concentrates [3]. This cost is not dissimilar to those
`for patients in the developed countries with social-
`ized national health care systems such as the United
`Kingdom, Australia or Germany [4]. These kinds of
`costs are an insurmountable barrier
`for
`similar
`
`treatment in many developing countries. Although
`prophylaxis treatment strategies vary greatly,
`the
`typical regimen requires FVIII infused at a dose of
`20—40 IU kg’l
`three times per week. Despite the
`improved musculoskeletal outcomes and quality of
`life, enthusiasm for prophylaxis is hampered by
`suboptimal adherence to therapy [5] and frequent
`need for the placement of central venous access
`devices (CVAD) [6].
`While we wait on an ultimate genetic cure, perhaps
`through gene
`therapy,
`the question is whether
`innovations
`in
`drug
`delivery
`or
`recombinant
`
`© 2006 The Authors
`Journal compilation © 2006 Blackwell Publishing Ltd
`
`6/15
`
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`

`

`technology will be able to overcome these barriers
`(cost, compliance, need for CVAD)
`to continued
`effective prophylaxis. Certainly, the development of
`clotting factor delivery systems that did not rely on
`needle and syringe would be a major breakthrough,
`but even strategies to extend the functional half—life
`of FVIII could have a significant impact. There have
`been considerable advances in biological therapy in
`recent years in other disease states that have included
`nonvinvasive methods of delivering proteins and
`increasing their biological half—life. This review will
`explore successful strategies for other therapeutic
`proteins, the advantages and disadvantages of such
`strategies as applied to rFVlll delivery as well as new
`innovations in rFVIlI therapy towards a longer acting
`FVIII protein.
`
`Polyethylene glycol conjugation
`
`in vivo factors can reduce the efficacy of
`Several
`biological
`therapeutic agents. These include poor
`solubility at physiological pH, neutralization by host
`antibodies, rapid renal clearance, cellular clearance
`and proteolysis [7]. Modification of proteins through
`conjugation with polyethylene glycol
`(PEG) poly
`mers, or PEGylation, has been a successful strategy
`to overcome some of these efficacy limitations. PEG
`polymers are amphiphilic, non—toxic and immuno—
`logically inert. They consist of ethylene oxide
`subunits linked in a chain with a hydroxyl group at
`each terminal and may be linear
`(5—30 kDa) or
`branched (40—60 kDa)
`through connections pro-
`duced by chemical linkers [7]. PEGylation was first
`described in the 1970s [8,9] and has resulted recently
`in several commercialized therapeutics. These modi—
`fied biologics can have better efficacy, provided the
`parent protein’s biological activity is retained.
`Polyethylene glycol polymers incorporate many
`water molecules within their hydrophilic structure,
`so that their volume is disproportionately larger than
`that predicted by their molecular weight
`[10].
`PEGylation increases the resultant size of the conju—
`gated therapeutic protein abovc the limit for kidney
`filtration, thereby extending the circulating half-life
`[7]. The hydrated PEG chains are also highly mobile
`in aqueous medium potentially shielding antigenic
`determinants on the protein from access to immune-
`mediating cells
`[7]. PEGylation can reduce pro—
`teolysis by proteascs through steric hindrance or
`sitespecific conjugation that eliminates cleavage sites
`[10]. Some proteins are relatively insoluble with a
`tendency to aggregate. PEGylation increases the
`solubility of several proteins making it easier to
`formulate
`for administration [7]. All of
`these
`
`© 2006 The Authors
`Journal compilation © 2006 Blackwell Publishing Ltd
`
`LONGER ACTING FACTOR VI“
`
`43
`
`improved features could result in improved efficacy
`in m'uo but may also contribute to greater patient
`adherence by reducing the frequency of administra—
`tion and improving quality of life [7].
`Commercialized PEGylated protein therapeutics
`have included PEG—asparaginase for the treatment
`of acute lymphoblastic leukaemia, PEGiadenosine
`deaminase for severe combined immunodeficiency
`disease, PEG-interferon for hepatitis C, PEG—gra-
`nulocyte colony stimulating factor (G7CSF) for che»
`motherapy—induced neutropenia and PEG-growth
`hormone for
`the treatment of acromegaly. The
`PEGylation of GiCSF results in a molecule whose
`mode of elimination is almost entirely neutrophil—
`mediated clearance, thus the drug‘s elimination from
`plasma is dependent on the recovery of the patient’s
`neutrophil count. UnPEGylated interferons require
`dosing three times per week, not too dissimilar from
`prophylactic FVIII administration regimens. PEGy—
`lation of interferon resulted in efficacious dosing
`schedules of once per week [11]. With regard to
`potential reduced immunogenicity from PEGylation,
`one study observed that some children who experi-
`enced anaphylaxis to unmodified asparaginase toler—
`ated the PEGylated version without an allergic
`reaction [12].
`Polyethylene glycolylation of FVIII would seem to
`be a natural progression for the advancement of
`haemophilia
`therapeutics. However,
`there
`are
`important drawbacks to consider with PEGylation
`as well as unique elimination properties for FVIII,
`which may limit this application. The main disad—
`vantage of PEGylated protein formulations is that an
`improved pharmacokinetic profile can be accompani
`ied by decreased specific activity [7]. Cross-linking
`PEG to proteins
`typically involves coupling of
`activated PEG molecules to amino groups of lysine
`or the protein’s aminoterminal group [7,13]. With-
`out
`the ability tO discriminate where the PEG
`polymers conjugate to the protein,
`there may be
`decreased accessibility for key activating proteases or
`other protein—protein interactions integral
`to the
`therapeutic protein’s biological activity. For exam—
`ple, PEGylated winterferon retains only 7% of the
`antiviral activity of the native protein. However,
`in viva, by virtue of
`its dramatically improved
`pharmacokinetics it still shows improved efficacy
`[14]. This degree of reduced biological activity would
`be detrimental to the efficacy of PEGylatcd FVIII for
`several reasons. FVIII is inactive in its native form,
`requiring proteolytic cleavage by thrombin for acti-
`vation and interaction with the phospholipid (PL)
`surface, factor IXa (FIXa) and factor X (PX) in order
`to exert its cofactor function. PEGylation, depending
`
`Haemopbilza (2006), 12, (Suppl. 3), 4275]
`
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`

`44 E. L. SAENKO and S. W. PIPE
`
`on the number of PEG conjugates, would potentially
`interfere with all of these macromolecular interac-
`
`tions. Although reduced biological activity could be
`compensated by a dramatic improvement in phar—
`macokinetic properties, this may not be the case for a
`PEGylated form of FVIII. FVIII is already a large
`molecule and is not subject to renal elimination. lts
`plasma half—life
`is already significantly extended
`through its interaction with von W'illebrand factor
`(VWF) in viz/o. Whereas the plasma half-life of FVIII
`in the absence of VWF is N2 h,
`it is ~12 h when
`associated with VWF [15]. PEGylation of FVIII risks
`interfering with VWF—FVIII affinity and actually
`compromising its plasma half—life. Although this may
`in turn be compensated by reduced interaction with
`cellular clearance mechanisms [16],
`in vivo studies
`are required to address this issue. Reduced biological
`activity of a PEGylated FVIII could be tolerated if the
`extended half-life resulted in a sustained biological
`action. However,
`the instability of activated FVIII
`(FVIIIa) may limit its utility in a PEGylated form.
`FVIII (domain structure A1—A2—B—A3-Cl—C2) exists
`in plasma as an inactive heterodimer of a variably
`sized heavy chain (HC, subunits A1~A2~B, ~90—
`200 kDa) and a light chain (LC, subunits A3-C1—C2,
`N80 kDa) associated via a copper (Cu+)—dependent
`interaction between the A1 and A3 subunits [17719].
`Upon activation with thrombin, proteolysis removes
`the B-domain and bisects the HC resulting in an
`FVIIIa heterotrimer
`(subunits A1/A2/A3VC1»C2)
`[20]. However, the EVllla heterotrimer is unstable
`and subject to spontaneous decay of its procoagulant
`activity attributable to firsteorder dissociation of its
`free A2 subunit, which occurs at physiological pH
`[21,22]. This
`type of proteolytic activation and
`inactivation does not occur with other commercial—
`
`ized PEGylated protein therapeutics. Merely improv—
`ing the pharmacokinetics of
`the inactive FVIII
`heterodimer by PEGylation may have limited effect
`on in Uil/O efficacy, if the active form has significantly
`reduced the biological activity secondary to interfer-
`ence by the PEG conjugates as well as the inherent
`instability of FVIII-a.
`Rostin et al. [23] published their experience with
`PEGylation of B»domain—delctcd rFVIII (r-VIH SQ).
`They used several strategies for PEG conjugation.
`Using random PEG coupling at amino groups of
`lysines, they observed a significant decrease in the
`FVIII activity that was proportional to the degree of
`modification but was significant at even low degrees
`of modification; 1 PEG/r—VIII SQ reduced the specific
`activity to 50%. Using a less reactive PEG polymer, a
`larger excess of polymer was required to modify
`the protein. The reduced reactivity is
`thought
`to
`
`Haemopbz/in (2006), 12, (Suppl. 3), 42—51
`
`correlate with higher selectivity for conjugation. This
`resulted in some preservation of the FVIII activity;
`with 3 PEG/reVIH SQ, the specific activity was 50%.
`Finally,
`they adsorbed the FVIII
`to an anionic
`exchange column in order to protect reactive lysines,
`presumably at the acidic regions of FVIII, from PEG
`conjugation. This had been successful in protecting
`the functional sites on target proteins in previous
`studies. This allowed the preservation of 50% of the
`FVIII activity with 4 PEG/r—VHI SQ. Western blot
`analyses
`suggested that modification sites were
`located on both the HC5 and the LCs of FVIII. Their
`studies also suggested that the VWF—binding site was
`disturbed by PEGylation, as only 26—43% of the
`protein content was able to bind to VWF. The in vivo
`consequences of this perturbation on plasma half—life
`were not studied.
`
`The success of PEGylation as a strategy to extend
`the half—life of FVIII may depend on new targeted
`methodologies for conjugation sites of PEG poly-
`mers. These strategies include specifically targeting
`non-essential functional groups on the surface of the
`protein such as free cysteines or oligosaccharides or
`even employing chemical strategies towards site,
`directed PEGylation [24]. One can also employ
`standard l’EGylation while protecting active sites
`with an inhibitor or a substrate specific to the active
`site. This could be possible for FVIII but would
`require protection of multiple key sites; sites for
`thrombin recognition/cleavage, VWF interaction as
`well as interactive sites for FlXa and FX and Pls.
`
`Alternatively, one could employ chromatographic
`separation of isomers of PEGylated FVIII, selecting
`those with the most biological activity for evaluation.
`The B-domain of FVlll would make an attractive
`
`for site»directed selective PEGylation. This
`target
`large domain has a significant role in intracellular
`trafficking of FVIII within the secretion pathway, but
`when deleted FVIII still retains high specific activity
`and is efficacious therapeutically [25]. The B-domain
`does not play a significant role for interaction with
`VWF, FIXa or FX or the PL surface. Therefore,
`PEGylation in this region would be less likely to have
`a negative effect on VWF affinity or biological
`activity. In addition, upon thrombin activation the
`B—domain is ptoteolytically removed. Therefore, any
`potential steric hindrance of active sites by PEG
`would be abrogated after activation. Recent studies
`have demonstrated that
`the Bedomain protein
`sequence can be modified significantly without
`impairing FVIII secretion [26]. Therefore, a strategy
`designed to introduce targeted sites for PEG modi—
`fication (such as introducing cysteines to create thiol—
`reactive sites) has a high likelihood of success. Recent
`
`© 2006 The Authors
`Journal compilation © 2006 Blackwell Publishing Ltd
`
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`

`

`partnerships between technology leaders in targeted
`PEGylation strategies
`(Nektar Therapeutics, San
`Carlos, CA, USA) and manufacturers of
`rFVIII
`(Baxter International, Deertield,
`IL, USA) suggest
`that
`there is ongoing investigation in this area
`towards a novel FVIII product [27],
`There is some concern as to the ultimate clearance
`mechanism for PEG as it is considered non—biode-
`gradable. Although some potential pathways for
`PEG degradation have been described, they appear to
`occur at a low rate and are not considered normal
`detoxification mechanisms [28]. PEG would then end
`up in tissues participating in the uptake of PEGylated
`constructs where it would accumulate intralysoso—
`mally [29]. The potential adverse effects of long—term
`exposure to large amounts of PEG over an extended
`period of time (i.e. lifelong as would be predicted for
`haemophilia applications) are unknown.
`
`LONGER ACTING FACTOR VIII 45
`
`assays) to FVIII formulated in the absence of SSL.
`l’harmacokinetic studies of l’EGLip—PVHI compared
`with standard formulation FVIII in haeinophilic mice
`(exon 16 FVIII knockout) demonstrated similar peak
`recovery after injection. Average half—life, area under
`the curve and mean residence time were 1.5—1.6
`times higher for PEGLip-FVHI compared with the
`free FVIII. Significantly, the haemostatic efficacy of
`PEGLip»FVHI in haemophilia mice was prolonged
`following tail vein injection compared with standard
`formulation FVHI. This new formulation of FVIII is
`
`presently beginning Phase I trials at the University of
`California — Davis Medical Center in Sacramento
`
`and the Children’s Hospital of Orange County in
`California through a partnership between Zilip—
`Pharma (Amsterdam, The Netherlands) and Bayer
`Biological Products (Research Triangle Park, NC,
`USA) [34].
`
`PEGylated liposomes
`
`Polysialic acid polymers
`
`Liposomes (lipid vesicles) are capable of encapsula-
`ting drugs either within their aqueous phase or
`within their lipid bilayer and have been utilized in a
`broad range of drug delivery applications
`[30].
`Unfortunately, they are rapidly cleared by phagocytic
`cells of the reticuloendothelial system (RES). By
`incorporating PEGylated lipids onto the liposome
`surface,
`there is reduced RES clearance and pro—
`longed circulation tirne. These modified liposomes
`are referred to as sterically stabilized liposomes (SSL)
`and have been successfully formulated with the
`chemotherapy drug doxorubicin and lL—Z [31,32].
`Doxorubicin formulated with SSL resulted in pro-
`longed circulation time and enhanced accumulation
`of the drug in the malignant exudates of cancer
`patients.
`the disadvantages of
`Acknowledging some of
`direct modification of the FVIII protein by PEGyla—
`tion, formulation of unmodified FVIII with SSI. has
`garnered recent
`interest. Baru er
`a1.
`[33] have
`published their preclinical characterization of a full—
`length rFVHI with SSL. In this formulation strategy,
`SSL were prepared containing distearoylphosphati-
`dylethanolamine (DSPE) conjugated with PEG. They
`report that for this preparation (designated PEGLip—
`FVIII) FVIII is not encapsulated within the intralipo—
`some aqueous phase or inserted into the lipid bilayer
`but rather is associated with the liposomes through
`high—affinity interaction with the PEG-DSPE. Binding
`studies using surface plasmon resonance
`(SPR)
`measurements demonstrated no hindrance to VWF
`
`binding and similar FVIII activity (measured by
`one-stage clotting assays and two—stage chromogenic
`
`© 2006 The Authors
`Journal compilation © 2006 Blackwell Publishing Ltd
`
`Polysialic acids (PSA) are considered nature’s ‘stealth’
`technology. PSA are linear polymers of N—acetylneu—
`raminic acid (sialic acid) and are abundantly present
`on the surface of cells and many proteins [29]. PSA
`protect against invading bacteria by interfering with
`host complement activation and phagocytic activity.
`In addition, PSA modulate cell—to—cell
`inhibition,
`reducing the adherence of cancer cells and facilitating
`metastatic migration. Because of this insulating fea—
`ture of PSA, it has been proposed that polysialylation
`of therapeutic molecules could improve their phar—
`macokinetics. The rationale would be
`that
`the
`
`extreme hydrophilicity of the PSA would form a
`‘watery cloud’ around the therapeutic molecule pro—
`tecting it from proteolytic enzymes, clearance recep
`tors and even antibodies. Investigators have used PSA
`based on polysaccharides 0n Escherichia coli that are
`immunologically identical to PSA in the host; thus
`they are non—immunogenic even when conjugated to
`therapeutic proteins [29]. PSA are biodegradable
`giving them an advantage over PEGylation when
`applied to a therapeutic protein that will be delivered
`in large doses and over an extended period of time (as
`would be the case for haemophilia applications).
`Polysialylation has been applied to a wide range of
`therapeutics
`and
`has
`demonstrated
`important
`improvements over the parent molecule. For exam—
`ple, polysialylation of asparaginase protected it from
`proteolytic degradation in the presence of serum
`while its function was preserved [35]. Fully preserved
`function has also been observed for polysialylated
`forms of cab—interferon and insulin even while their
`half-lives in circulation and areas under the curve
`
`Haemopbi/ia (2006), 12, (Suppl. 3), 42—51
`
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`

`

`46 E. L. SAENKO and S. \V. PIPE
`
`were significantly increased [29]. There is also
`evidence of reduced immunological reactivity for
`polysialylated forms of asparaginase and insulin
`[36,37].
`As applied to FVIII, polysialylation holds promise
`for extending the half-life of FVIII without compro—
`mising its functional activity. It may even reduce the
`immunogenicity of FVIII or protect it from neutral-
`izing antibodies
`in patients with inhibitors.
`It
`remains
`to be demonstrated that polysialylated
`forms of FVlll can retain VWF affinity and still
`be
`effectively activated by
`thrombin. Recently
`announced collaborations between Lipoxen Techno-
`logies (London, UK) and Baxter International is the
`evidence of research interest in this area [27].
`
`Stabilized forms of FVllla
`
`Successful progress has been made in overcoming the
`inherent limitations of FVlll expression and biologi—
`cal function through techniques to improve rFVIII
`biosynthesis and secretion, functional activity, half—
`life and antigenicity/itnmunogenicity [38]. With
`regard to producing a longer acting FVIII molecule,
`FVIII bioengineered for resistance to inactivation
`holds promise.
`is
`(A2/A2/A3—C1-C2)
`The FVIIIa heterotrimer
`unstable and susceptible to proteolytic inactivation
`by activated protein C (APC), FIXa or factor Xa.
`However, studies have also demonstrated that loss of
`procoagulant
`activity after
`thrombin activation
`results from a
`reversible dissociation of
`the A2
`subunit from the heterotrimer and is another factor
`
`limiting the procoagulant activity of FVIIIa in viva.
`In order to address this limitation, an inactivation-
`resistant FVlll
`(1R8) was genetically engineered
`which is not susceptible to the dissociation of the
`A2—domain subunit and proteolytic inactivation by
`APC and therefore has a prolonged cofactor activity
`[39]. In designing IRS, B-domain residues 794—1689
`were deleted and Arg740 was replaced by alanine,
`eliminating the thrombin cleavage sites at Arg740
`and Arg1689. As a result, FVIII activation by
`thrombin occurs via a single cleavage after Arg372.
`This leads to the generation of an FVllIa dimer that
`retains the A2-domain covalently attached to the LC,
`thus preventing its spontaneous dissociation. Addi
`tionally, missense mutations at APC inactivation
`Cleavage sites provided resistance to further proteo-
`lysis of FVIlIa. The specific activity of IRS proved to
`be markedly higher than that of wild-type T‘VIII
`(FVIII WT),
`and significant peak activity was
`retained for hours in vitro, whereas FVIII WT, under
`similar conditions was inactivated by thrombin after
`
`Hartmopbilin (2006), 12, (Suppl. 3), 42751
`
`10 min. The LC ac