The use of PEGylated liposomes in the development of drug delivery applications for the treatment of hemophilia

Abstract

Hemophilia A is a rare X-linked bleeding disorder caused by lack or dysfunction of coagulation factor VIII (FVIII). Hemophilia A is treated with replacement therapy, but frequent injections of the missing FVIII often lead to the formation of inhibitory antibodies. Patients who develop high levels of inhibitors must be treated with bypassing agents such as activated FVII (FVIIa). Both FVIII and FVIIa have short half-lives and require multiple injections. Long-acting forms of these proteins would therefore reduce the frequency of injections, improve patient compliance and reduce complications. In this article we present a new platform technology that produces long-acting forms of FVIII and FVIIa and improves the efficacy of hemophilia treatment. This technology is based on the binding of proteins/peptides to the outer surface of PEGylated liposomes (PEGLip). Binding is dependent on an amino acid consensus sequence within the proteins and is highly specific. At the same time, binding is non-covalent and does not require any modification of the therapeutic agent or its production process. Association of proteins with PEGLip results in substantial enhancements in their pharmacodynamic properties following administration. These improvements seem to arise from the association of formulated proteins with platelets prior to induction of coagulation.

Keywords: PEGylated liposomes; factor VIII; factor VIIa; pharmacodynamics; therapeutic proteins.

Figure 1

A schematic diagram showing a PEGLip formulated coagulation factor VIII (FVIII) or activated factor VII (FVIIa). The protein is non-covalently bound to a polyethylene glycol moiety on the outer surface of a PEGylated liposome. Binding is mediated by an amino acid consensus sequence within the protein (boxed in red). The actual consensus sequences, locations within the proteins’ sequences and affinity constants (K_D) for FVIII (two binding sites) and FVIIa are shown above.

Figure 2

Efficacy of PEGLip-formulated FVIII in preclinical experiments and a clinical trial. A) Efficacy in an animal model. Hemophilic mice were injected into the tail vein with PEGLip-formulated FVIII, standard FVIII (both 0.1 IU/mouse), or saline. Twenty-four hours after injection, the left lateral tail vein of each mouse was cut and survival was scored. B) Efficacy in a clinical trial. Hemophilia A patients were given 25 IU/kg or 35 IU/kg of standard or PEGLip-formulated FVIII and the time between the prophylactic infusion and the next spontaneous bleed was recorded. The number of bleeding-free days following each treatment is shown. Results are average ± SEM.

Figure 3

Efficacy of PEGLip-formulated FVIIa in preclinical experiments and a clinical trial. A) Efficacy in an animal model. Hemophilic mice were injected with PEGLip-FVIIa, standard FVIIa (both 10 μg/mouse), or saline. The right and left lateral tail veins of each mouse were cut 15 min post injection and survival was scored. B) Efficacy in a clinical trial. Hemophilia patients with inhibitors were injected prophylactically with 90 μg/kg FVIIa (○) or 90 μg/kg PEGLip-FVIIa (●). Total clotting times at 0.5–5 hours post injection were analyzed by thrombelastography. No clotting (total clotting time >3600 sec) was detected in any of the subjects in the hour preceding infusions. The dashed lines compare clotting times induced by PEGLip-FVIIa to those induced by standard FVIIa. Results are average ± SEM (n = 6). ^*P < 0.05, ^**P = 0.08 (FVIIa vs PEGLip-FVIIa, paired t-test).

Figure 4

Mechanism of action of PEGLip-formulated FVIII and FVIIa. 1. Formulation of FVIII or FVIIa with PEGLip leads to non-covalent binding of the protein to the outer surface of the PEGylated liposomes. 2. The liposomes are then injected into the bloodstream where they associate with non-activated platelets. 3. When injury occurs, platelets are recruited to the wound where they adhere to the damaged vessel wall. They carry FVIII and FVIIa with them. Platelet activation and initiation of the coagulation cascade occur simultaneously. 4. Coagulation complexes form on the surface of the activated platelets. Since FVIII and FVIIa are already present on the platelets prior to activation, the coagulation cascade is more efficient. Clots form faster and the clots are more stable.