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PEGylation of a Factor VIII–Phosphatidylinositol Complex: Pharmacokinetics and Immunogenicity in Hemophilia A Mice

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Abstract

Hemophilia A is an X-linked bleeding disorder caused by the deficiency of factor VIII (FVIII). Exogenous FVIII is administered therapeutically, and due to a short half-life, frequent infusions are often required. Fifteen to thirty-five percent of severe hemophilia A patients develop inhibitory antibodies toward FVIII that complicate clinical management of the disease. Previously, we used phosphatidylinositol (PI) containing lipidic nanoparticles to improve the therapeutic efficacy of recombinant FVIII by reducing immunogenicity and prolonging the circulating half-life. The objective of this study is to investigate further improvements in the FVIII–PI formulation resulting from the addition of polyethylene glycol (PEG) to the particle. PEGylation was achieved by passive transfer of PEG conjugated lipid into the FVIII–PI complex. PEGylated FVIII–PI (FVIII–PI/PEG) was generated with high association efficiency. Reduced activity in vitro and improved retention of activity in the presence of antibodies suggested strong shielding of FVIII by the particle; thus, in vivo studies were conducted in hemophilia A mice. Following intravenous administration, the apparent terminal half-life was improved versus both free FVIII and FVIII–PI, but exposure determined by area under the curve was reduced. The formation of inhibitory antibodies after subcutaneous immunization with FVIII–PI/PEG was lower than free FVIII but resulted in a significant increase in inhibitors following intravenous administration. Passive transfer of PEG onto the FVIII–PI complex does not provide any therapeutic benefit.

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Abbreviations

aPTT:

Activated partial thromboplastin time

DMPC:

Dimyristoylphosphatidylcholine

DMPE-PEG2000 :

1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000]

DSPE-PEG2000 :

1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy (polyethylene glycol)-2000]

FVIII:

Factor VIII

LDLR:

Low-density lipoprotein receptor

LRP:

Low-density lipoprotein receptor-related protein

PEG:

Polyethylene glycol

PI:

Phosphatidylinositol

PS:

Phosphatidylserine

RES:

Reticuloendothelial system

rFVIII:

Recombinant factor VIII

i.v.:

Intravenous

s.c.:

Subcutaneous

vWf:

von Willebrand factor

APCs:

Antigen presenting cells

References

  1. Foster PA, Zimmerman TS. Factor VIII structure and function. Blood Rev. 1989;3(3):180–91.

    Article  PubMed  CAS  Google Scholar 

  2. Mannucci PM, Tuddenham EG. The hemophilias—from royal genes to gene therapy. N Engl J Med. 2001;344(23):1773–9. doi:10.1056/NEJM200106073442307.

    Article  PubMed  CAS  Google Scholar 

  3. Klinge J, Ananyeva NM, Hauser CA, Saenko EL. Hemophilia A—from basic science to clinical practice. Semin Thromb Hemost. 2002;28(3):309–22. doi:10.1055/s-2002-32667.

    Article  PubMed  CAS  Google Scholar 

  4. Brettler DB. Inhibitors in congenital haemophilia. Baillieres Clin Haematol. 1996;9(2):319–29.

    Article  PubMed  CAS  Google Scholar 

  5. Healey JF, Lubin IM, Nakai H, Saenko EL, Hoyer LW, Scandella D, et al. Residues 484–508 contain a major determinant of the inhibitory epitope in the A2 domain of human factor VIII. J Biol Chem. 1995;270(24):14505–9.

    Article  PubMed  CAS  Google Scholar 

  6. Lubin IM, Healey JF, Barrow RT, Scandella D, Lollar P. Analysis of the human factor VIII A2 inhibitor epitope by alanine scanning mutagenesis. J Biol Chem. 1997;272(48):30191–5.

    Article  PubMed  CAS  Google Scholar 

  7. Zhong D, Saenko EL, Shima M, Felch M, Scandella D. Some human inhibitor antibodies interfere with factor VIII binding to factor IX. Blood. 1998;92(1):136–42.

    PubMed  CAS  Google Scholar 

  8. Healey JF, Barrow RT, Tamim HM, Lubin IM, Shima M, Scandella D, et al. Residues Glu2181-Val2243 contain a major determinant of the inhibitory epitope in the C2 domain of human factor VIII. Blood. 1998;92(10):3701–9.

    PubMed  CAS  Google Scholar 

  9. Scandella D, Gilbert GE, Shima M, Nakai H, Eagleson C, Felch M, et al. Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site. Blood. 1995;86(5):1811–9.

    PubMed  CAS  Google Scholar 

  10. Fay PJ, Scandella D. Human inhibitor antibodies specific for the factor VIII A2 domain disrupt the interaction between the subunit and factor IXa. J Biol Chem. 1999;274(42):29826–30.

    Article  PubMed  CAS  Google Scholar 

  11. Fijnvandraat K, Celie PH, Turenhout EA, ten Cate JW, van Mourik JA, Mertens K, et al. A human alloantibody interferes with binding of factor IXa to the factor VIII light chain. Blood. 1998;91(7):2347–52.

    PubMed  CAS  Google Scholar 

  12. Reding MT, Okita DK, Diethelm-Okita BM, Anderson TA, Conti-Fine BM. Human CD4+ T-cell epitope repertoire on the C2 domain of coagulation factor VIII. J Thromb Haemost. 2003;1(8):1777–84.

    Article  PubMed  CAS  Google Scholar 

  13. Reding MT, Okita DK, Diethelm-Okita BM, Anderson TA, Conti-Fine BM. Epitope repertoire of human CD4(+) T cells on the A3 domain of coagulation factor VIII. J Thromb Haemost. 2004;2(8):1385–94.

    Article  PubMed  CAS  Google Scholar 

  14. Hu GL, Okita DK, Conti-Fine BM. T cell recognition of the A2 domain of coagulation factor VIII in hemophilia patients and healthy subjects. J Thromb Haemost. 2004;2(11):1908–17.

    Article  PubMed  CAS  Google Scholar 

  15. Morfini M. Pharmacokinetics of factor VIII and factor IX. Haemophilia. 2003;9 Suppl 1:94–9. discussion 100.

    Article  PubMed  CAS  Google Scholar 

  16. Eaton D, Rodriguez H, Vehar GA. Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry. 1986;25(2):505–12.

    Article  PubMed  CAS  Google Scholar 

  17. Fay PJ, Anderson MT, Chavin SI, Marder VJ. The size of human factor VIII heterodimers and the effects produced by thrombin. Biochim Biophys Acta. 1986;871(3):268–78.

    Article  PubMed  CAS  Google Scholar 

  18. Lenting PJ, Neels JG, van den Berg BM, Clijsters PP, Meijerman DW, Pannekoek H, et al. The light chain of factor VIII comprises a binding site for low density lipoprotein receptor-related protein. J Biol Chem. 1999;274(34):23734–9.

    Article  PubMed  CAS  Google Scholar 

  19. Saenko EL, Yakhyaev AV, Mikhailenko I, Strickland DK, Sarafanov AG. Role of the low density lipoprotein-related protein receptor in mediation of factor VIII catabolism. J Biol Chem. 1999;274(53):37685–92.

    Article  PubMed  CAS  Google Scholar 

  20. Ljung R. Prophylactic therapy in haemophilia. Blood Rev. 2009;23(6):267–74. doi:10.1016/j.blre.2009.08.001.

    Article  PubMed  CAS  Google Scholar 

  21. Carlsson M, Berntorp E, Bjorkman S, Lindvall K. Pharmacokinetic dosing in prophylactic treatment of hemophilia A. Eur J Haematol. 1993;51(4):247–52.

    Article  PubMed  CAS  Google Scholar 

  22. Sharathkumar A, Lillicrap D, Blanchette VS, Kern M, Leggo J, Stain AM, et al. Intensive exposure to factor VIII is a risk factor for inhibitor development in mild hemophilia A. J Thromb Haemost. 2003;1(6):1228–36.

    Article  PubMed  CAS  Google Scholar 

  23. Koestenberger M, Raith W, Muntean W. High titre inhibitor after continuous factor VIII administration for surgery in a young infant. Haemophilia. 2000;6(2):120.

    Article  PubMed  CAS  Google Scholar 

  24. Claxton AJ, Cramer J, Pierce C. A systematic review of the associations between dose regimens and medication compliance. Clin Ther. 2001;23(8):1296–310.

    Article  PubMed  CAS  Google Scholar 

  25. Ramani K, Miclea RD, Purohit VS, Mager DE, Straubinger RM, Balu-Iyer SV. Phosphatidylserine containing liposomes reduce immunogenicity of recombinant human factor VIII (rFVIII) in a murine model of hemophilia A. J Pharm Sci. 2008;97(4):1386–98. doi:10.1002/jps.21102.

    Article  PubMed  CAS  Google Scholar 

  26. Levchenko TS, Rammohan R, Lukyanov AN, Whiteman KR, Torchilin VP. Liposome clearance in mice: the effect of a separate and combined presence of surface charge and polymer coating. Int J Pharm. 2002;240(1–2):95–102.

    Article  PubMed  CAS  Google Scholar 

  27. Awasthi VD, Garcia D, Goins BA, Phillips WT. Circulation and biodistribution profiles of long-circulating PEG-liposomes of various sizes in rabbits. Int J Pharm. 2003;253(1–2):121–32.

    Article  PubMed  CAS  Google Scholar 

  28. Allen TM, Hansen C, Martin F, Redemann C, Yau-Young A. Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochim Biophys Acta. 1991;1066(1):29–36.

    Article  PubMed  CAS  Google Scholar 

  29. Zeisig R, Shimada K, Hirota S, Arndt D. Effect of sterical stabilization on macrophage uptake in vitro and on thickness of the fixed aqueous layer of liposomes made from alkylphosphocholines. Biochim Biophys Acta. 1996;1285(2):237–45.

    Article  PubMed  CAS  Google Scholar 

  30. Ramani K, Purohit V, Miclea R, Gaitonde P, Straubinger RM, Balu-Iyer SV. Passive transfer of polyethylene glycol to liposomal-recombinant human FVIII enhances its efficacy in a murine model for hemophilia A. J Pharm Sci. 2008;97(9):3753–64. doi:10.1002/jps.21266.

    Article  PubMed  CAS  Google Scholar 

  31. Peng A, Straubinger RM, Balu-Iyer SV. Phosphatidylinositol containing lipidic particles reduces immunogenicity and catabolism of factor VIII in hemophilia a mice. AAPS J. 2010;12(3):473–81. doi:10.1208/s12248-010-9207-z.

    Article  PubMed  CAS  Google Scholar 

  32. Stoilova-McPhie S, Villoutreix BO, Mertens K, Kemball-Cook G, Holzenburg A. 3-Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography. Blood. 2002;99(4):1215–23.

    Article  PubMed  CAS  Google Scholar 

  33. Pratt KP, Shen BW, Takeshima K, Davie EW, Fujikawa K, Stoddard BL. Structure of the C2 domain of human factor VIII at 1.5 A resolution. Nature. 1999;402(6760):439–42.

    Article  PubMed  CAS  Google Scholar 

  34. Purohit VS, Ramani K, Kashi RS, Durrani MJ, Kreiger TJ, Balasubramanian SV. Topology of factor VIII bound to phosphatidylserine-containing model membranes. Biochim Biophys Acta. 2003;1617(1–2):31–8.

    PubMed  CAS  Google Scholar 

  35. Bartlett GR. Phosphorus assay in column chromatography. J Biol Chem. 1959;234(3):466–8.

    PubMed  CAS  Google Scholar 

  36. Heath TD, Macher BA, Papahadjopoulos D. Covalent attachment of immunoglobulins to liposomes via glycosphingolipids. Biochim Biophys Acta. 1981;640(1):66–81.

    Article  PubMed  CAS  Google Scholar 

  37. Bi L, Lawler AM, Antonarakis SE, High KA, Gearhart JD, Kazazian Jr HH. Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A. Nat Genet. 1995;10(1):119–21. doi:10.1038/ng0595-119.

    Article  PubMed  CAS  Google Scholar 

  38. Verbruggen B, Novakova I, Wessels H, Boezeman J, van den Berg M, Mauser-Bunschoten E. The Nijmegen modification of the Bethesda assay for factor VIII:C inhibitors: improved specificity and reliability. Thromb Haemost. 1995;73(2):247–51.

    PubMed  CAS  Google Scholar 

  39. Purohit VS, Ramani K, Sarkar R, Kazazian Jr HH, Balasubramanian SV. Lower inhibitor development in hemophilia A mice following administration of recombinant factor VIII-O-phospho-L-serine complex. J Biol Chem. 2005;280(18):17593–600.

    Article  PubMed  CAS  Google Scholar 

  40. Nedelman JR, Gibiansky E, Lau DT. Applying Bailer’s method for AUC confidence intervals to sparse sampling. Pharm Res. 1995;12(1):124–8.

    Article  PubMed  CAS  Google Scholar 

  41. Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry. 1991;30(43):10363–70.

    Article  PubMed  CAS  Google Scholar 

  42. Lollar P, Fay PJ, Fass DN. Factor VIII and factor VIIIa. Methods Enzymol. 1993;222:128–43.

    Article  PubMed  CAS  Google Scholar 

  43. Reipert BM, Ahmad RU, Turecek PL, Schwarz HP. Characterization of antibodies induced by human factor VIII in a murine knockout model of hemophilia A. Thromb Haemost. 2000;84(5):826–32.

    PubMed  CAS  Google Scholar 

  44. Peng A, Gaitonde P, Kosloski MP, Miclea RD, Varma P, Balu-Iyer SV. Effect of route of administration of human recombinant factor VIII on its immunogenicity in hemophilia A mice. J Pharm Sci. 2009;98(12):4480–4. doi:10.1002/jps.21765.

    Article  PubMed  CAS  Google Scholar 

  45. Pisal DS, Balu-Iyer SV. Phospholipid binding improves plasma survival of factor VIII. Thromb Haemost. 2010;104(5):1073–5. doi:10.1160/TH10-06-0422.

    Article  PubMed  CAS  Google Scholar 

  46. Pisal DS, Kosloski MP, Balu-Iyer SV. Delivery of therapeutic proteins. J Pharm Sci. 2010;99(6):2557–75. doi:10.1002/jps.22054.

    Article  PubMed  CAS  Google Scholar 

  47. Abuchowski A, van Es T, Palczuk NC, Davis FF. Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol. J Biol Chem. 1977;252(11):3578–81.

    PubMed  CAS  Google Scholar 

  48. Abuchowski A, McCoy JR, Palczuk NC, van Es T, Davis FF. Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase. J Biol Chem. 1977;252(11):3582–6.

    PubMed  CAS  Google Scholar 

  49. Mei B, Pan C, Jiang H, Tjandra H, Strauss J, Chen Y, et al. Rational design of a fully active, long-acting PEGylated factor VIII for hemophilia A treatment. Blood. 2010;116(2):270–9. doi:10.1182/blood-2009-11-254755.

    Article  PubMed  CAS  Google Scholar 

  50. Baru M, Carmel-Goren L, Barenholz Y, Dayan I, Ostropolets S, Slepoy I, et al. Factor VIII efficient and specific non-covalent binding to PEGylated liposomes enables prolongation of its circulation time and haemostatic efficacy. Thromb Haemost. 2005;93(6):1061–8. doi:10.1267/THRO05061061.

    PubMed  CAS  Google Scholar 

  51. Ananyeva NM, Kouiavskaia DV, Shima M, Saenko EL. Catabolism of the coagulation factor VIII: can we prolong lifetime of f VIII in circulation? Trends Cardiovasc Med. 2001;11(6):251–7.

    Article  PubMed  CAS  Google Scholar 

  52. Bieri S, Djordjevic JT, Daly NL, Smith R, Kroon PA. Disulfide bridges of a cysteine-rich repeat of the LDL receptor ligand-binding domain. Biochemistry. 1995;34(40):13059–65.

    Article  PubMed  CAS  Google Scholar 

  53. Bovenschen N, Boertjes RC, van Stempvoort G, Voorberg J, Lenting PJ, Meijer AB, et al. Low density lipoprotein receptor-related protein and factor IXa share structural requirements for binding to the A3 domain of coagulation factor VIII. J Biol Chem. 2003;278(11):9370–7.

    Article  PubMed  CAS  Google Scholar 

  54. Sarafanov AG, Ananyeva NM, Shima M, Saenko EL. Cell surface heparan sulfate proteoglycans participate in factor VIII catabolism mediated by low density lipoprotein receptor-related protein. J Biol Chem. 2001;276(15):11970–9. doi:10.1074/jbc.M008046200.

    Article  PubMed  CAS  Google Scholar 

  55. Bovenschen N, Rijken DC, Havekes LM, van Vlijmen BJ, Mertens K. The B domain of coagulation factor VIII interacts with the asialoglycoprotein receptor. J Thromb Haemost. 2005;3(6):1257–65. doi:10.1111/j.1538-7836.2005.01389.x.

    Article  PubMed  CAS  Google Scholar 

  56. Klibanov AL, Maruyama K, Torchilin VP, Huang L. Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes. FEBS Lett. 1990;268(1):235–7.

    Article  PubMed  CAS  Google Scholar 

  57. Gaitonde P, Peng A, Straubinger RM, Bankert RB, Balu-Iyer SV. Downregulation of CD40 signal and induction of TGF-beta by phosphatidylinositol mediates reduction in immunogenicity against recombinant human factor VIII. J Pharm Sci. 2011; doi:10.1002/jps.22746.

  58. Ishida T, Atobe K, Wang X, Kiwada H. Accelerated blood clearance of PEGylated liposomes upon repeated injections: effect of doxorubicin-encapsulation and high-dose first injection. J Control Release. 2006;115(3):251–8. doi:10.1016/j.jconrel.2006.08.017.

    Article  PubMed  CAS  Google Scholar 

  59. Ishida T, Ichihara M, Wang X, Kiwada H. Spleen plays an important role in the induction of accelerated blood clearance of PEGylated liposomes. J Control Release. 2006;115(3):243–50. doi:10.1016/j.jconrel.2006.08.001.

    Article  PubMed  CAS  Google Scholar 

  60. Koide H, Asai T, Hatanaka K, Akai S, Ishii T, Kenjo E, et al. T cell-independent B cell response is responsible for ABC phenomenon induced by repeated injection of PEGylated liposomes. Int J Pharm. 2010;392(1–2):218–23. doi:10.1016/j.ijpharm.2010.03.022.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by a grant from the National Institutes of Health (R01 HL-70227) to SVB. We are grateful to Dr. ZP Bernstein of Western New York Hemophilia Foundation, for providing albumin-free recombinant factor VIII (Advate). The authors thank Dr. K Huang from the Department of Chemistry at University at Buffalo for performing the NMR study.

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, 521 Hochstetter Hall, Amherst, New York, 14260, USA

    Aaron Peng, Matthew P. Kosloski, Genki Nakamura, Hong Ding & Sathy V. Balu-Iyer

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  1. Aaron Peng
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  2. Matthew P. Kosloski
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  3. Genki Nakamura
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  4. Hong Ding
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  5. Sathy V. Balu-Iyer
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Correspondence to Sathy V. Balu-Iyer.

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Peng, A., Kosloski, M.P., Nakamura, G. et al. PEGylation of a Factor VIII–Phosphatidylinositol Complex: Pharmacokinetics and Immunogenicity in Hemophilia A Mice. AAPS J 14, 35–42 (2012). https://doi.org/10.1208/s12248-011-9309-2

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