WO2005012354A1

WO2005012354A1 - Method for extending the half-life of fviii

Info

Publication number: WO2005012354A1
Application number: PCT/EP2004/008484
Authority: WO
Inventors: Olaf Walter; Tobias Suiter
Original assignee: Zlb Behring Gmbh
Priority date: 2003-07-31
Filing date: 2004-07-29
Publication date: 2005-02-10

Abstract

The patent describes a stabilized factor VIll preparation and a method for increasing the stability of factor VIll in which the factor VIll is stabilized by adding a sufficient quantity of a von Willebrand factor whose A2 domain has been completely or partially deleted.

Description

AVENTIS BEHRING GMBH

Method for extending the half-life of FVIII

The invention relates to a method for extending the half-life of blood coagulation factor VIll and to a pharmaceutical preparation which comprises a factor FVIII which has an extended half-life.

Classical hemophilia, or hemophilia A, is the most frequently occurring cause of blood coagulation disturbances. It is due to a deficiency of blood coagulation factor FVIII, which is in turn brought about by an error in chromosome X, and, at a frequency of between 1 and 2 cases per 10 000 individuals, affects males almost exclusively. The defect in the X chromosome is transmitted by females who are themselves not hemophiliacs, however. Hemophilia A is clinically manifested by an abnormal tendency to bleed and, before treatment with factor VIll concentrates was introduced medically, reduced the average life expectancy of individuals suffering from severe hemophilia to less than 20 years. The use of factor VIll concentrates derived from plasma has substantially improved the situation for hemophilia patients. It has been possible to decisively extend average life expectancy and to enable most patients to nowadays lead a more or less normal life, particularly when a prophylactic injection is administered 2 - 3 times per week.

In the plasma, factor VIll circulates as a noncovalently bound complex with von Willebrand factor (VWF). In this complex, the molecular mass of the von Willebrand factor is about 98% of the molecular mass of the factor VIll. The von Willebrand factor controls the synthesis and activity of the factor VIll. The binding of factor VIll to von Willebrand factor is of crucial importance for the stability of factor VIll against breakdown in vivo. A deficiency of VWF, or indeed the complete absence of VWF, leads to an accelerated breakdown of factor VIll and to an increase in the tendency to bleed, which increase is known as von Willebrand disease.

It has already been shown that factor VIll which is bound to VWF is protected both against phospholipid-dependent proteolysis and direct proteolysis by activated protein C (APC). The two crucial sites at which the VWF binds to the factor VIll molecule have by now been located, with the first site being between amino acids 1 ,670 and 1 ,689 in the aminoterminal segment of the light chain within the A3 domain and with the second binding site being between amino acids 2,303 and 2,332 in the carboxy- terminal segment of the C2 domain. Both segments are evidently of crucial importance for the high-affinity binding of factor VIll to the D' and D3 domains of VWF.

The APC-binding site in the factor VIll molecule has been located between amino acids 2,009 and 2,018 in the A3 domain. Nogami et al. showed that the binding sites for APC and VWF on factor VIll overlap and that the protective effect of VWF in regard to the APC-catalyzed inactivation of factor VIll has therefore to be explained not only by factor VIll being prevented from binding to the phospholipid but also by VWF directly preventing APC from binding to factor VIll (Nogami et al., Blood, Vol. 99, 3993-3998).

Another mechanism for degrading factor VIll is controlled by LRP (low density lipoprotein receptor-related protein), which regulates the breakdown in the liver. The binding of factor VIll to LRP takes place at two sites, which are separated from each other, within the A2 and C2 domains of factor VIll. The C2 domain overlaps the VWF binding site. For this reason, only the A2 region remains accessible in the factor VIII/VWF complex whereas the C2 region is masked by VWF. Saenko et al. showed that an isolated factor VIll is broken down about twice as rapidly by LRP-expressing cells as is factor VIll which is complexed with VWF (Saenko et al., TCM, Vol. 11 , No. 6, 2001). These observations demonstrate that, apart from the binding of LRP to the A2 region, which is identified by amino acids 717 - 909, the binding to the C2 domain also contributes substantially to the LRP-controlled mechanism for degrading factor VIll and that this mechanism is dependent on VWF.

All the previously known experimentally supported attempts to extend the biological half-life of factor VIll have been based on altering the factor VIll molecule. Surprisingly, it has now been found that extending the biological half-life of vWF, or of a mutant of vWF, leads to the biological half-life of factor VIll being extended. The biological half-life of vWF can be extended, for example, by deleting the A2 domain since this thereby impedes the proteolytic degradation of the vWF.

An example of such a mutant is a variant of VWF which has recently become available and which completely lacks the A2 domain (A2DDrVWF). This vWF has been found to be resistant to proteolysis, in the plasma, by ADAMTS13 at the Tyr⁸⁴²-Meth⁸⁴³ bond within the VWF A2 domain. Koppelman et al. (Blood, Vol. 87, No. 6, 1996, pp 2292 - 2300) have shown that this deletion does not impair the ability to bind to factor VIll.

In principle, it is possible, within the meaning of the invention, to use all vWF variants, mutants and fragments which extend the biological half-life of vWF and whose ability to bind to F VIll is not less, or only insignificantly less, than that of the wild-type vWF.

While, in principle, it is possible to add such an altered vWF as a single substance for supporting the endogenous F VIll, preference is given to adding F VIll and vWF as a complex.

The invention therefore relates to a stabilized factor VIll preparation which comprises a complex between blood coagulation factor VIll and von Willebrand factor whose biological half-life is extended, in particular a vWF whose A2 domain is entirely or partially lacking.

In such a complex, the increase in stability of the VWF also at the same time increases the stability of the factor VIll.

If a factor VIll preparation which has been stabilized in this way is employed for treating hemophilia A patients, the concentration of unactivated factor VIll in the plasma is then increased quite substantially above the previously known half-life of 14 hours. It is then possible to protect the patient from severe hemorrhages even when the factor VIll preparation which is present in the complex with the VWF whose A2 region has been deleted is administered much less frequently.

The invention also relates to a method for increasing the stability of blood coagulation factor VIll, which method comprises adding, to the factor VIll, a sufficient quantity of a VWF whose A2 domain has been completely or partially deleted.

Patients in whom the tendency to bleed has to be attributed to a deficiency of VWF (von Willebrand disease) can be successfully treated by administering VWF whose A2 domain has been entirely or partially removed since the factor VIll, which is present in these patients in a quantity which is per se adequate, is so effectively protected against biological degradation by the VWF which has been stabilized in the described manner, and is therefore particularly long-lived, that it is available in a quantity which is adequate for the blood coagulation mechanism.

Claims

AVENTIS BEHRING GMBH 2003/M013(A74)Patent claims:

1. A stabilized factor VIll preparation which comprises a complex composed of the factor VIll and of von Willebrand factor (vWF) whose biological half-life is extended as compared with that of native vWF.

2. The stabilized factor VIll preparation as claimed in claim 1, wherein the vWF whose biological half-life is extended as compared with that of native vWF contains a complete or partial deletion of the A2 domain.

3. A method for increasing the stability of factor VIll which comprises adding, to the factor VIll, a sufficient quantity of von Willebrand factor whose A2 domain has been completely or partially deleted.