Recombinant factor VIII SQ--stability of VIII: C in homogenates from porcine, monkey and human subcutaneous tissue.
The aim of this paper was to investigate whether a formulation-based approach to understanding and addressing stability could generate a subcutaneous factor VIII preparation for patients as an alternative to the existing intravenous products. The low bioavailability of subcutaneously administered factor VIII could have several causes: proteolytic degradation of the protein in the interstitium; adsorption to tissue, in particular to acidic phospholipids such as L-alpha phosphatidyl-L-serine (phosphatidylserine); the absence of free von Willebrand factor in the interstitium; phagocytosis by macrophages in the interstitium or in the lymph nodes; and coagulation could be initiated upon injection. This study was undertaken to investigate the first three factors in-vitro (i.e., proteolytic degradation, adsorption to tissue and the protective effect of von Willebrand factor). The influence of some other macromolecular stabilisers and protease inhibitors was also investigated. The stability of factor VIII activity (VIII: C) was investigated in homogenates from porcine, monkey and human subcutaneous tissue. Possible coagulation was prevented in these studies by the presence of both citrate and antithrombin. An exploratory in-vivo study was performed in the pig; plasma samples were assayed with a factor VIII:Ag (90kDa) ELISA. The decrease in VIII:C appeared to be more pronounced in homogenates from monkey and human tissues than in porcine homogenate. The results from human tissue homogenate resembled the degradation profile seen in monkey homogenate. Both the von Willebrand factor and phosphatidylserine/phosphatidylcholine (PS/PC) liposomes showed a significant stabilising effect on VIII:C in the tissue homogenates. The qualitative pattern was similar in porcine, monkey and human tissue. A combination of several protease inhibitors seemed to have a protective effect on the stability of VIII: C albeit at high concentrations of inhibitors and the effect was less than that of PS/PC. An exploratory in-vivo study was performed in the pig with phosphatidylserine in two formulations; either in the form of PS/PC liposomes or together with Polysorbate 80 in the form of mixed micelles (phosphatidylserine/P80). Including phosphatidylserine in the formulations appeared to increase the availability, of subcutaneously administered r-VIII SQ in the pig. However, further studies are necessary, preferably in the monkey where in-vitro studies indicate a closer resemblance to the human. In conclusion, a proposed inactivation mechanism for r-VIII SQ in subcutaneous tissue could be adsorption to phospholipid surfaces followed by proteolytic degradation. However, additional studies are required due to the multitude of factors influencing the subcutaneous absorption route. A combination of protease inhibitor(s) together with phosphatidylserine-containing liposomes are suggested for further investigation, preferably in a monkey animal model.
Department of Preformulation Sciences, Pharmacia & Upjohn AB, Stockholm, Sweden. angelica@everyday.com
A
A
Fatouros
Y
Y
Lidén
B
B
Sjöström
D000818:Animals; D004337:Drug Carriers; D016503:Drug Delivery Systems; D004355:Drug Stability; D010450:Endopeptidases / Q000378:metabolism; D005169:Factor VIII / Q000008:administration & dosage / Q000378:metabolism*; D000882:Haplorhini; D006801:Humans; D008081:Liposomes; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000378:metabolism*; D012867:Skin / Q000378:metabolism*; D013552:Swine; D014841:von Willebrand Factor / Q000378:metabolism
D004337:Drug Carriers; D008081:Liposomes; D011994:Recombinant Proteins; D014841:von Willebrand Factor; C078147:F8 protein, human; D005169:Factor VIII; D010450:Endopeptidases