Recombinant factor VIIISQ - Stability of VII : C in homogenates from porcine, monkey and human subcutaneous tissue

The aim of this paper was to investigate whether a formulation-based approa ch to understanding and addressing stability could generate a subcutaneous factor VIII preparation for patients as an alternative to the existing intr avenous products. The low bioavailability of subcutaneously administered fa ctor VIII could have several causes: proteolytic degradation of the protein in the interstitium; adsorption to tissue, in particular to acidic phospho lipids such as L-alpha phosphatidyl-L-serine (phosphatidylserine); the abse nce of free von Willebrand factor in the interstitum; phagocytosis by macro phages in the interstitium or in the lymph nodes; and coagulation could be initiated upon injection. This study was undertaken to investigate the firs t three factors in-vitro (i.e., proteolytic degradation, adsorption to tiss ue and the protective effect of von Willebrand factor). The influence of so me other macromolecular stabilisers and protease inhibitors was also invest igated. The stability of factor VIII activity (VIII:C) was investigated in homogenates from porcine, monkey and human subcutaneous tissue. Possible co agulation was prevented in these studies by the presence of both citrate an d antithrombin. An exploratory in-vivo study was performed in the pig; plas ma samples were assayed with a factor VIII:Ag (90 kDa) ELISA. The decrease in VIII:C appeared to be more pronounced in homogenates from m onkey and human tissues than in porcine homogenate. The results from human tissue homogenate resembled the degradation profile seen in monkey homogena te. Both the von Willebrand factor and phosphatidylserine/phosphatidylcholi ne (PS/PC) liposomes showed a significant stabilising effect on VIII:C in t he tissue homogenates. The qualitative pattern was similar in porcine, monk ey and human tissue. A combination of several protease inhibitors seemed to have a protective effect on the stability of VIII: C albeit at high concen trations of inhibitors and the effect was less than that of PS/PC. An explo ratory in-vivo study was performed in the pig with phosphatidylserine in tw o formulations; either in the form of PS/PC liposomes or together with Poly sorbate 80 in the form of mixed micelles (phosphatidylserine/P80). Includin g phosphatidylserine in the formulations appeared to increase the availabil ity of subcutaneously administered r-VIII SQ in the pig. However, further s tudies are necessary, preferably in the monkey where in-vitro studies indic ate a closer resemblance to the human. In conclusion, a proposed inactivation mechanism for r-VIII SQ in subcutane ous tissue could be adsorption to phospholipid surfaces followed by proteol ytic degradation. However, additional studies are required due to the multi tude of factors influencing the subcutaneous absorption route; A combinatio n of protease inhibitor(s) together with phosphatidylserine-containing lipo somes are suggested for further investigation, preferably in a monkey anima l model.