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

International Journal of Nanomedicine
Dovepress
open access to scientific and medical research
Open Access Full Text Article
R e v I e w
The use of PeGylated liposomes in the development
of drug delivery applications for the treatment
of hemophilia
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
5 August 2010
Number of times this article has been viewed
Rivka Yatuv
Abstract: Hemophilia A is a rare X-linked bleeding disorder caused by lack or dysfunction of
Micah Robinson
coagulation factor VIII (FVIII). Hemophilia A is treated with replacement therapy, but frequent
Inbal Dayan-Tarshish
injections of the missing FVIII often lead to the formation of inhibitory antibodies. Patients who
Moshe Baru
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
Omri Laboratories Ltd., Nes Ziona,
Israel
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, therapeutic proteins, pharmacodynamics, factor VIII,
factor VIIa
Introduction
Hemophilia
Hemophilia is an inherited bleeding disorder caused by the lack or dysfunction of
coagulation factors VIII (FVIII) or IX (FIX). The genes for FVIII and FIX are located
on the X chromosome, and the vast majority of individuals with hemophilia are males.
Hemophilia A accounts for approximately 75%–80% of all hemophilia cases with a
prevalence of approximately 1/10,000 in the male population, whereas the prevalence
of hemophilia B is 1/30,000.1,2 The severity of the bleeding tendency is determined by
the residual clotting factor activity. Approximately 40% of hemophilia patients have
a severe form of the disease, defined as having less than 1% (0.01 IU/ml) of normal
factor activity.3
Correspondence: Moshe Baru
Hemophilia patients experience spontaneous hemorrhages into the joints, particularly
Omri Laboratories Ltd, Bldg. 22, weizmann
Science Park, PO Box 4015, NesZiona
the weight bearing joints such as knees, ankles and hips. This bleeding is painful and leads
74140, Israel
to long term inflammation and deterioration of the joint, resulting in permanent deformi-
Tel +972 8930 2970
ties, misalignment and loss of mobility. This is the major complication of hemophilia.4,5
Fax +972 8930 2975
email moshe@omrilabs.co.il
Bleeding may also occur into the muscles, soft tissues and other organs.
submit your manuscript | www.dovepress.com
International Journal of Nanomedicine 2010:5 581–591
581
Dovepress
© 2010 Yatuv et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article
8603
which permits unrestricted noncommercial use, provided the original work is properly cited.

---

Yatuv et al
Dovepress
Hemophilia therapy
2%–3% of hemophilia B patients develop inhibitors.15,16
Hemophilia is treated by supplying patients with the missing
As expected, the more profound mutations resulting in the
coagulation factor (FVIII or FIX). Replacement therapy may
lack of circulating FVIII or FIX are associated with higher
be provided on-demand (to stop a bleed) or prophylactically
risk of inhibitor development.17 Neutralizing or inactivat-
(to prevent bleeds). When treatment is provided on-demand,
ing autoantibodies to FVIII may also develop in patients
injections must be initiated at the first onset of symptoms so
without a history of coagulation factor deficiency. This
as to limit both the amount of bleeding and the extent of the
condition, known as acquired hemophilia, affects 0.2–1 out
resulting tissue damage.6 Prophylactic treatment has been
of every million people per year.18 Despite the low incidence
shown to provide hemophilia patients with better overall
of acquired hemophilia, the disorder is often devastating
care than on-demand treatment as it reduces the frequency
and the costs of treatment are immense. There is therefore
of hemorrhages and slows the development of long term
considerable interest in improving and optimizing existing
arthropathy.7 However, prophylactic treatment is more
treatment regimens.18,19
expensive than on-demand treatment and is not accessible
to many patients.6
Bypassing agents
In this review, we focus on the treatment of hemophilia A,
Treatment of hemophilia patients with inhibitors is based
the most common form of the disease. The FVIII used to
primarily on controlling or preventing acute hemorrhages
treat hemophilia A may be produced in mammalian cell lines
and their sometimes life threatening complications. Patients
genetically engineered to synthesize human FVIII (recombi-
with low inhibitor titers may be given high doses of factor
nant FVIII, rFVIII) or may be purified from normal pooled
concentrate. While some of the factor is neutralized by the
plasma (plasma-derived FVIII, pdFVIII).8
inhibitors, enough remains in the circulation to induce hemo-
FVIII is a large glycoprotein (2332 amino acids) that
stasis. Patients with high inhibitor titers ($5 Bethasda units,
is synthesized as a 330 kDa precursor and undergoes mul-
BU) cannot be treated by replacement therapy because even
tiple processing steps to yield a heterodimer composed of
high doses of coagulation factor are rapidly inactivated by
a light chain (80 kDa) and a heterogeneous heavy chain
the circulating antibodies.20 These patients must be treated
(90–210 kDa)9 FVIII circulates in the plasma as an inactive
with agents capable of inducing hemostasis independent of
precursor, tightly complexed with von Willebrand factor
the presence of FVIII or FIX (bypassing agents). Two such
(vWF). Upon initiation of coagulation, FVIII is activated by
agents are in widespread use: activated prothrombin com-
thrombin. It dissociates from vWF, interacts with negatively
plex concentrate (aPCC, also known as factor eight inhibi-
charged phospholipids on the surface of activated platelets,
tor bypass activity, FEIBA, Baxter AG, Vienna, Austria)21
and acts as a cofactor for factor IXa in the activation of
and recombinant activated factor VII (rFVIIa; NovoSeven,
factor X.1 This in turn leads to the generation of substantial
Novo Nordisk AS, Bagsværd, Denmark).15,22 rFVIIa, given
amounts of thrombin, the key enzyme in the coagulation
at supraphysiological doses, has been approved in many
cascade, and to the generation of a hemostatic plug.
countries for the treatment of bleeding episodes in patients
The half-life of human FVIII is about 10–12 hours.10,11
with congenital hemophilia and inhibitors to FVIII or FIX
Effective prophylactic therapy therefore requires three
and patients with acquired hemophilia.23 The half-life of
weekly infusions of 20–40 IU/kg to maintain FVIII above
FVIIa in the circulation is approximately 2.3 hours.24,25 In
levels at which spontaneous bleeding occurs.7 The need for
most patients, bleeding episodes are therefore brought under
frequent infusions reduces quality of life and may lead to
control by administering two or three doses of 90 µg/kg given
problems with compliance and injection complications.12
at two hour intervals.26,27
Central venous access devices are frequently required, espe-
cially in children, but these devices are plagued by recurrent
The need for a long-acting therapy
infections and thrombosis.13
for hemophilia
A long-acting form of FVIII would provide extended protec-
Development of inhibitors
tion against bleeding and reduce gaps in protection caused
and acquired hemophilia
by drops in FVIII levels between injections. Therefore, fewer
Replacement treatment is complicated by the emergence of
injections would be required, resulting in fewer injection
antibodies that inhibit the activity of the injected protein.14
related complications, and better quality of life. Several
Approximately 20%–40% of hemophilia A patients and
attempts have been made to generate long-acting forms of
submit your manuscript | www.dovepress.com
582
International Journal of Nanomedicine 2010:5
Dovepress

---

Dovepress
PeGylated liposomes mediated drug delivery in hemophilia
FVIII.28 These included mutagenesis of the FVIII molecule
PEGylated liposome (PEGLip) technology is a new
to increase its stability29–31 and direct PEGylation of the
approach to improving the pharmacodynamic properties
FVIII protein.32–35 Modifying a large and complex protein
of therapeutic proteins. Instead of encapsulating the drug,
such as FVIII is a difficult undertaking, though. The integ-
PEGylated liposomes are used as carriers with the protein
rity of multiple active sites must be maintained and protein
bound non-covalently but with high specificity to the outer
conformation must be preserved. Development of modified
surface (Figure 1). Unlike approaches such as mutagenesis,
forms of FVIII is still in the preclinical stage and the safety
direct PEGylation, or fusion to carrier proteins, PEGLip
and efficacy of these approaches has yet to be demonstrated
technology does not involve changes to a protein’s amino
in humans.28
acid sequence and does not involve covalent attachment of
There is also a clinical need for a more potent and long-
stabilizing agents.
acting form of FVIIa. As in the case of FVIII, long-acting
The PEGLip that we typically produce are composed of a
FVIIa would require fewer injections, would reduce injection
97:3 molar ratio of 1-palmitoyl-2-oleoyl phosphatidylcholine
associated complications, and would provide patients with
(POPC) to 1,2 distearoyl-sn-glycero-3-phosphatidyletha-
better control of bleeding episodes with fewer dips in FVIIa
nolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-
levels. Several approaches have been employed in attempts
PEG 2000). The liposomes are prepared as follows. Lipids
to improve FVIIa. These include substitution of one or more
are dissolved in tert-butanol and lyophilized. The resulting dry
amino acids within the protein,36 PEGylation,37 and fusion
lipid powder is resuspended to 110 mM lipid in 50 mM sodium
of FVIIa to albumin.38 These proteins showed promising
citrate, pH 6.7 to form liposomes. The liposomes are then
results in-vitro and in-animal models, but as in the case of
down-sized by extrusion through sequentially smaller polycar-
FVIII, the efficacy and safety of these approaches has yet to
bonate filters until they reach a final diameter of 80–100 nm.
be demonstrated in humans.
The PEG molecules that extend outward from the liposome
surface mediate binding of FVIII, FVIIa and other proteins
PEGylated liposome (PEGLip)
to the liposome surface (Figure 1). As mentioned above, the
technology
PEG molecules also limit uptake of the liposomes by the RES,
Liposomes, artificial phospholipid vesicles, have proven to be
thereby extending half-life in the circulation.42–44
useful in stabilizing drugs and improving their pharmacological
The binding of PEGLip to FVIII and FVIIa was demon-
properties. In most cases, liposomes are used to encapsulate the
strated in real time using surface plasmon resonance (SPR).
therapeutic agent (usually a small molecule drug).39 Modify-
The protein being assayed was bound to the surface of a chip
ing the liposome surface with molecules such as polyethylene
and association of liposomes was measured as a liposome
glycol prevents adsorption of plasma proteins to the liposome
solution flowed over the chip. SPR measurements showed that
surface and interferes with recognition and uptake by the reti-
PEGLip bind both recombinant and plasma-derived FVIII45,46
coloendothelial system (RES). This results in the generation of
as well as FVIIa.47 PEGLip also bind additional proteins
liposomes with a dramatically extended circulation time.40,41
such as recombinant human granulocyte colony-stimulating
Consensus sequence
S/T-X-L/V-I/Q/S-S/T/Q-X-X-E
K (nM)
D
PC
FVIII 1787 S SLISYEE 1794 4.5
1635 T TLQSDQE 1660 1.9
FVIIa 378 T RVSQYIE 385
0.4
DSPE-PEG 2000
Protein
PEGLip-formulated protein
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 ) for FvIII (two binding sites) and FvIIa are shown above.
D
International Journal of Nanomedicine 2010:5
submit your manuscript | www.dovepress.com
583
Dovepress

---

Yatuv et al
Dovepress
factor (G-CSF), granulocyte macrophage colony-stimulating
PeGLip-formulated FvIII
factor (GM-CSF), and glucagon-like peptide 1 (GLP-1).48
FVIII binds PEGLip specifically and with high affinity (two
Affinity constants (K ) for all of these interactions were
D
binding sites, K of 4.5 and 1.9 nM), but this interaction
D
in the low nM range (0.4–12 nM). All of the proteins that
does not alter FVIII activity, as shown by maintenance of full
bind PEGLip share a consensus sequence of 8 amino acids
activity after formulation.45,46 The association of FVIII with
(S/T–X–L/V–I/Q/S–S/T/Q–X–X–E) (Figure 1). A synthetic
PEGLip does not affect the FVIII protein’s structure. This was
peptide derived from one of the two consensus sequences in
demonstrated by binding of several anti-FVIII antibodies to
FVIII (amino acids 1783–1796 of FVIII) also binds PEGLip
FVIII following PEGLip formulation45 Moreover, PEGLip
with high affinity (K of 2.3 nM). PEGLip do not bind sev-
D
formulation allows FVIII to interact with its natural binding
eral proteins that lack the consensus sequence, highlighting
partners. This was shown by in-vitro binding of PEGLip-
its significance for binding. These proteins include human
formulated FVIII to vWF.45 Thus binding of PEGLip to FVIII
serum albumin (HSA), human IgG, insulin, interferon
does not change the protein’s biological properties.
alpha 2a, interferon alpha 2b, human growth hormone, and
PEGLip formulation of both recombinant and plasma–
erythropoietin.48 Together, our data show that the consensus
derived forms of FVIII extends hemostatic efficacy in vivo.
sequence is sufficient to mediate the binding of the proteins/
This was demonstrated by better survival of hemophilic mice
peptides to PEGLip.
following tail-vein transection. Standard FVIII, PEGLip-
We also sought to identify the molecules or structure on
FVIII, or saline were administered to hemophilic mice
PEGLip responsible for protein/peptide binding. We found
24 hours prior to transection of the left lateral tail vein. Mice
that non-PEGylated POPC liposomes do not bind FVIII,45,46
that received PEGLip-FVIII bled less and survived signifi-
FVIIa,47 or the FVIII-derived synthetic peptide.48 Analy-
cantly longer (P , 0.05) than mice that received standard
ses performed with liposomes composed of various types
FVIII or saline (Figure 2A).45,46 This significantly increased
of lipids and lipid polymers revealed that the interaction
survival of tail-vein transected hemophilic mice following
between PEGLip and proteins is mediated primarily by the
the injection of PEGip formulated recombinant FVIII was
PEG molecule and the carbamate group adjacent to the PEG
also demonstrated by others.50 The increased survival rate
molecule within DSPE-PEG 2000.48 When PEG molecules
was dependent on the pre-formation of a complex of FVIII
were not present on the liposome surface (as in the case of
and PEGLip. Clotting times of whole blood samples from
POPC liposomes) no binding occurred. Likewise, when
hemophilic mice injected with PEGLip-FVIII were much
proteins lacking the consensus sequence were assayed, no
shorter than the clotting times of blood samples from mice
binding was observed. Liposome-protein binding is therefore
injected with free FVIII. This faster clotting was detected
highly specific.
shortly after injection and at various time points up to
72 hours post injection.50
Formulation of FVIII and FVIIa
PEGLip-FVIII provides human subjects with extended
with PEGLip
protection from bleeding.49 Several clinical trials tested the
Formulation of a protein with PEGylated liposomes is very
safety and the efficacy of PEGLip rFVIII (also referred to as
straightforward. The lyophilized protein powder of FVIII
BAY 79-4980).51 A single arm, subject blinded clinical trial
or FVIIa is simply reconstituted in liposome solution and
was performed to assess the efficacy and safety of two levels
allowed to fully dissolve. Even large and sensitive proteins
of FVIII (25 or 35 IU/kg) with a fixed dose of liposomes
such as FVIII may be formulated without any change in
(22 mg lipids/kg). Twenty-three severe hemophilia A patients
the production process or the purification procedure.45–47,49
were treated in three study segments. Standard FVIII was

Formulation is very gentle and it does not involve any cova-
administered in the first study segment whereas PEGLip-FVIII
lent modification of the protein. The protein’s native structure
was provided in the second and third segments. Segments were
is not changed, and the likelihood of antibody production
separated by 4-day washout periods and each prophylactic
does not increase. The protein is fully active immediately
infusion was administered while subjects were in a non-
after formulation45–47 and it is free to interact with its normal
bleeding state. As a measure of efficacy, the time between
binding partners.45 Administration to patients remains for
each prophylactic infusion and the next spontaneous bleed
the most part unchanged, though dosage and frequency of
was recorded. Prophylactic treatment with PEGLip-FVIII
injections may need to be adjusted to account for extended
nearly doubled the length of time subjects were protected
therapeutic activity.
from spontaneous bleeding (Figure 2B). A single prophylactic
submit your manuscript | www.dovepress.com
584
International Journal of Nanomedicine 2010:5
Dovepress

---

Dovepress
PeGylated liposomes mediated drug delivery in hemophilia
A 100
PEGLip- FVIII
90
FVIII
80
Saline
70
60
n = 20
50
40
p = 0.02
30
n = 20
p < 0.01
20
n = 30
Survival of hemophilic mice (%)
p = 0.06
10
0
0
10
20
30
40
50
Hours post tail vein transection
Standard FVIII
Clinical results
B
PEGLip-FVIII
p < 0.01
16
p < 0.01
14
s
12
10
8
13.3
6
10.9
bleed-free days 4
Post-prophylaxi
5.9
7.2
2
0
25 IU/kg
35 IU/kg
n = 12
n = 11
FVIII dose
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.
injection of 25 IU/kg PEGLip-FVIII resulted in a mean
the efficacy and safety of prophylactic infusions of various

bleed-free interval of 10.9 days compared to 5.9 days with
PEGLip doses (4.2, 12.6, or 22.1 mg/kg) with a fixed FVIII
standard FVIII. Similarly, injection of 35 IU/kg PEGLip-FVIII
dose (35 IU/kg). Mean number of bleeding-free days after
resulted in a mean bleed-free interval of 13.3 days compared to
each infusion increased from 7.8 days for 35 IU/ml of
7.2 days with standard FVIII. This difference was significant
standard FVIII to 8.7, 10.8, and 10.9 days for 35 IU/ml of
(P , 0.05) for both dose levels (Figure 2B).49
FVIII formulated in 4.2, 12.6, and 22.1 mg/kg of PEGLip,
A subsequent randomized, subject blinded, four-way
respectively.52 The study showed a dose response to PEGLip
crossover study involving 16 hemophilia A patients evaluated
that reached saturation at the highest dose level.
International Journal of Nanomedicine 2010:5
submit your manuscript | www.dovepress.com
585
Dovepress

---

Yatuv et al
Dovepress
An additional study tested the safety of PEGLip-FVIII
with an affinity (K ) of 0.4 nM.47 Formulation of FVIIa with
D
and compared its pharmacokinetic profile to that of standard
PEGLip did not affect its in vitro activity.47 Most importantly,
FVIII.53 In this randomized double-blind study, 26 severe
formulation of FVIIa with PEGLip improved survival of hemo-
hemophilia A patients received a single injection of standard
philic mice following tail vein transection. Hemophilic mice
FVIII (35 IU/kg) followed by 12 observation days and a 4-day
were injected with standard FVIIa, PEGLip-FVIIa, or saline.
Washout period. Patients then received a single injection of
Fifteen minutes after injection, tail veins were transected and
PEGLip-FVIII (35 IU/kg FVIII, 13 or 22 mg/kg PEGLip) fol-
survival was scored up to 24 hours. We found that mice injected
lowed once again by 12 days of observation. Pharmacokinetic
with PEGLip-FVIIa survived significantly longer (P , 0.05)
(PK) analysis based on samples taken from patients during the
than mice injected with standard FVIIa (Figure 3A).
trial showed no significant difference between standard FVIII
The safety and efficacy of PEGLip-FVIIa were tested in
and PEGLip-FVIII. This suggests that the increased protection
humans in an open label, exploratory, cross over, phase I/II
from bleeding observed with PEGLip-FVIII did not result from
clinical study in six adult subjects with severe hemophilia
a simple prolongation of FVIII half-life in the blood stream.
A and inhibitory FVIII antibodies.56 Each subject received
Safety and tolerability of PEGLip-FVIII were assessed
two infusions: one infusion of 90 µg/kg standard FVIIa and
in another clinical trial involving 18 severe hemophilia A
one infusion of 90 µg/kg PEGLip-formulated FVIIa. The
patients. This study was directed primarily at determining the
two infusions were separated by a 10-day washout period.
optimal infusion rate for PEGLip-FVIII. The study showed
Injection volume was kept constant while injection order was
that PEGLip-FVIII may be administered at an infusion rate
randomized. Blood samples were collected from patients at
similar to that of standard FVIII.54
various time points both before and after each infusion of
In all four of the studies described above, no production
FVIIa or PEGLip-FVIIa. Whole blood samples were then
of inhibitory antibodies was detected, and no serious adverse
analyzed by rotational thrombelastography, a method record-
events were reported. However, a few subjects experienced
ing the kinetics of clot formation and the firmness of the clots.
increased breathing frequency and flushing.53,54 At the first
PEGLip-formulated FVIIa produced significantly (P , 0.05)
sign of symptoms, infusions were stopped and the patients
shorter clotting times and higher clot firmness than standard
recovered fully without further medical intervention. This
FVIIa up to 5 hours post injection. Clotting time of standard
type of hypersensitivity reaction is known as complement
FVIIa 30 min post injection was the same as clotting time of
activation related pseudoallergy (CARPA) and has been
PEGLip-FVIIa 2 hours post injection. Clotting time of standard
described following administration of radiocontrast media,
FVIIa 1 hour post injection was the same as clotting time of
liposomal drugs, and micellar solvents. Unlike IgE medi-
PEGLip-FVIIa 3.2 hours post injection (Figure 3B). Maximal
ated reactions, CARPA reactions arise at first treatment and
clot firmness induced by standard FVIIa 0.5 hour post injection
become milder or disappear upon repeated exposure.54,55
was as high as that induced by PEGLip-FVIIa ∼2.7 hours post
The combined results of the phase I and II clinical studies
injection. Thrombin generation assays showed that thrombin
described above show that PEGLip-FVIII is well tolerated
was produced faster and more efficiently following infusion
and provides extended protection from bleeding following
of PEGLip-FVIIa than following infusion of standard FVIIa.
prophylactic treatment.
No significant differences were detected between the PK of
PEGLip-FVIIa and standard FVIIa.
PeGLip-formulated FvIIa
One of the subjects in the trial experienced transient
PEGLip formulation is desirable for FVIIa because this thera-
hyperemia, an increase in blood pressure and anxiety during
peutic protein has an extremely short half-life in the circula-
the infusion of the first 1 mL of PEGLip-FVIIa. This non-IgE
tion (approximately 2.3 hours).24,25 In most patients treated
mediated reaction (CARPA) is associated with PEGLip.55 and
with FVIIa, effective hemostasis is achieved only after two or
is not related to FVIIa. The reaction subsided within one hour.
three doses of 90 µg/kg26,27 given at two hour intervals.
In all other subjects, PEGLip-FVIIa was well tolerated and
SPR analyses showed that PEGLip bind specifically to
there were no serious adverse events nor were there any sig-
FVIIa. As in the case of FVIII, binding of liposomes to FVIIa
nificant changes in vital signs, clinical chemistry or hemato-
was dependant on the presence of the DSPE-PEG lipopolymer.
logical parameters. Measurements of coagulation parameters
POPC liposomes lacking DSPE-PEG bound FVIIa to a much
indicated that there was no increase in thrombotic risk.
lower level than PEGLip. Kinetic analysis of the PEGLip-
An analysis of the results of the clinical trial indicates
FVIIa interaction indicated that the PEGLip bind to FVIIa
that PEGLip-FVIIa provides about two more “efficacy hours”
submit your manuscript | www.dovepress.com
586
International Journal of Nanomedicine 2010:5
Dovepress

---

Dovepress
PeGylated liposomes mediated drug delivery in hemophilia
A
n = 18
100
90
80
p < 0.01
70
n = 18
60
50
p < 0.01
40
n = 16
30
PEGLip-FVIIa
p < 0.01
20
FVIIa
Survival of hemophilic mice (%)
10
Saline
0 0
5
10
15
20
25
Hours post tail vein transection
B
Clinical results
6000
FVIIa
*
5500
PEGLip-FVIIa
5000
4500
*
4000
*
3500
**
3000
*
2500
Total clotting time (s) 2000
1500
1000
0
1
2
3
4
5
Time post injection (h)
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-FVI a,
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).
than standard FVIIa. In treatment of bleeding episodes in
clinical experiments showed that there was no difference
hemophilia patients with inhibitors, one 90 µg/kg dose of between the pharmacokinetic behavior of standard FVIII and
PEGLip-FVIIa may be roughly equivalent to two infusions
PEGLip-formulated FVIII53 and between standard FVIIa and
of standard FVIIa given at 2-hour intervals.
PEGLip-formulated FVIIa.56 This suggests that extension of

circulation half-life is not the primary mechanism responsible
Mechanism of action
for increased hemostatic efficacy.
In preclinical models, formulation of FVIII and FVIIa
Extensive in vitro experiments measuring clot forma-
with PEGLip improved pharmacokinetic properties in vivo
tion and lysis by rotational thrombelastography, indicate that
and increased circulation half-life.45–47 However, phase I
platelets must be present in order for PEGLip formulation to
International Journal of Nanomedicine 2010:5
submit your manuscript | www.dovepress.com
587
Dovepress

---

Yatuv et al
Dovepress
improve the hemostatic properties of FVIII and FVIIa.47,57
between liposomes of various compositions, including PEG
When PEGLip-FVIII and PEGLip-FVIIa were added to severe
liposomes, and blood cells have been reported previously by
hemophilic whole blood or platelet rich plasma, clots formed
others.50,58
much faster than when similar concentrations of standard FVIII
Having shown that PEGLip bind FVIII and FVIIa45,47
and FVIIa were used. Clots were also firmer and more resistant
and that PEGLip bind platelets, we next tested whether
to fibrinolysis. Similarly, ex-vivo rotational thrombelastometry
PEGLip are capable of delivering proteins to platelets.
experiments in whole blood drawn from hemophilic mice indi-
Fluorescently labeled FVIII and FVIIa were formulated with
cated that clotting times were much faster in mice injected with
non-fluorescent PEGLip and shown by flow cytometry to
PEGLip-FVIII rather than with free FVIII.50 Such improve-
bind human platelets in-vitro. These results were also found
ments in kinetics, clot firmness, and resistance to fibrinolysis
in-vivo as PEGLip-formulated fluorescent proteins interacted
depended on platelets as they were not observed when experi-
with the platelets of hemophilic mice following injection.57
ments were performed in platelet poor plasma.57
Fluorescently labeled human serum albumin (HSA), which
In order to gain a deeper understanding of the mechanisms
does not bind to PEGLip, did not show increased binding to
responsible for these improvements in efficacy, we tested
platelets following formulation with PEGLip. This indicates
whether PEGLip themselves bind platelets. Flow cytometry
that PEGLip-mediated association of proteins with platelets
analysis indicated that fluorescent PEGLip associate with
is dependent on prior binding of the proteins to PEGLip.
platelets in-vitro in a dose related manner. This associa-
The combined results indicate the following mechanism
tion was further confirmed in-vivo following the injection
(Figure 4): Formulation of FVIII or FVIIa with PEGLip leads
of fluorescent PEGLip into hemophilic mice.57 Interactions
to non-covalent binding of the protein to the outer surface of
Activation of coagulation cascade
Blood vessel
FVIIIa + FIXa
FVIIa
FX
FXa
Vessel injury
Pro-thrombin
Thrombin
4. Faster clotting
and stronger clot
Fibrinogen
Fibrin
Platelet
activation
3. Platelets are
recruited to the
wound site and
activated
2. Non-activated
platelets carrying
PEGLip-FVIIa/FVIII
1. Injected material:
PEGLip-FVIIa/FVIII
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.
submit your manuscript | www.dovepress.com
588
International Journal of Nanomedicine 2010:5
Dovepress

---

Dovepress
PeGylated liposomes mediated drug delivery in hemophilia
the PEGylated liposomes. After injection of the formulated
immunogenicity. Extensive experience with PEGLip-FVIII
proteins to the bloodstream the liposomes associate with
in several clinical studies has shown no increase in antibody
non-activated platelets and a complex of platelet-PEGLip-
generation. PEGLip formulation thus avoids one of the major
protein (FVIII/FVIIa) is formed in-vivo. Following injury,
pitfalls of the drug development process.49,52,53
platelets are recruited to sites of injury where they adhere to
PEGLip-FVIII/FVIIa are well tolerated and suitable for
the damaged vessel wall. Due to their prior association with
long-term treatment of hemophilia. This was demonstrated
PEGLip, they carry FVIII and FVIIa with them. Platelets are
in toxicology studies, which included injecting high doses
activated at the wound site and coagulation complexes form
of PEGLip-FVIII and PEGip-FVIIa into rats and rabbits
on the surface of the activated platelets. Since FVIII and
(acute toxicology) and repeated injections (up to nine months
FVIIa are already present on the platelets prior to activation,
of weekly injections) of PEGLip into rats and rabbits.45
the coagulation cascade is more efficient. Clots form faster
PEGylated liposomes were also shown to be non-toxic in
and the clots are more stable.
mice and dogs.62
The association of PEGLip-FVIII or PEGLip-FVIIa with
In the years to come, we expect PEGLip-FVIII and PEG-
platelets may also lead to increased concentration of FVIII
Lip-FVIIa to become an attractive treatment for hemophilia
or FVIIa at the wound site, even when the overall concentra-
A patients and hemophilia patients with inhibitors.
tion of FVIII or FVIIa in the circulation is low. Accordingly,
expression of FVIII in platelets was shown to be effective
Disclosure
at inducing hemostasis even when FVIII protein expression
The authors are employees of Omri Laboratories Ltd. The
levels did not exceed 1% of normal levels. This was probably
authors report no conflicts of interest in this work.
due to recruitment of platelets to the sites of injury and local
release of FVIII from activated platelets at wound sites.59
References
1. Dahlback B. Blood coagulation and its regulation by anticoagulant
Summary
pathways: genetic pathogenesis of bleeding and thrombotic diseases.
J Intern Med. 2005;257:209–223.
PEGLip technology improves the pharmacodynamic prop-
2. Hedner U, Ginsburg D, Lusher JM, High KA. Congenital Hemorrhagic
Disorders: New Insights into the Pathophysiology and Treatment
erties of both coagulation factors VIII and VIIa. This was
of Hemophilia. Hematology Am Soc Hematol Educ Program. 2000:
demonstrated in preclinical experiments and in five differ-
241–265.
ent clinical trials with both FVIII and FVIIa. The combined
3. Soucie JM, Evatt B, Jackson D. Occurrence of hemophilia in the United
States. The Hemophilia Surveillance System Project Investigators. Am
results indicate that PEGLip-FVIII prevents bleeding for a
J Hematol. 1998;59:288–294.
significantly longer period than standard FVIII, and that one
4. Kaufman RJ, Anthonorakis SE, Fay PJ. Hemostasis and Thrombosis:
Basic Principles and Clinical Practice. 4th ed. Philadelphia: Lippincott
dose of PEGLip-FVIIa may be roughly equivalent to two
Williams and Wilkins, 2001.
infusions of standard FVIIa given at 2 hour intervals when
5. Roosendaal G, Mauser-Bunschoten EP, De Kleijn P, et al. Synovium
treating a bleed in hemophilia patients with inhibitors.
in haemophilic arthropathy. Haemophilia. 1998;4:502–505.
6. Arun B, Kessler C. Inherited hemorrhagic disorders. In: Colman W, Hirsh J,
Of the approaches currently available to generate long-
Marder V, Clowes A, George J, editors. Hemostasis and thrombosis. 4th
acting forms of FVIII and FVIIa, PEGLip formulation has
ed. Philadelphia: Lippincott Williams & Willkins, 2001:815–825.
7. Hoots WK, Nugent DJ. Evidence for the benefits of prophylaxis in the
the advantage of being the only approach that has been tested
management of hemophilia A. Thromb Haemost. 2006;96:433–440.
extensively in clinical trials. PEGLip-FVIII and PEGLip-
8. Kessler CM. New perspectives in hemophilia treatment. Hematology
FVIIa have been shown to be safe in trials involving more
Am Soc Hematol Educ Program. 2005:429–435.
9. Jankowski MA, Patel H, Rouse JC, Marzilli LA, Weston SB, Sharpe PJ.
than 90 hemophilia patients. In general, treatments with
Defining ‘full-length’ recombinant factor VIII: a comparative structural
PEGLip-formulated FVIII and FVIIa were well tolerated.
analysis. Haemophilia. 2007;13:30–37.

10. Fijnvandraat K, Berntorp E, ten Cate JW, et al. Recombinant, B-domain
One of the most problematic complications of hemo-
deleted factor VIII (r-VIII SQ): pharmacokinetics and initial safety aspects
philia treatment is the induction of inhibitory antibodies.
in hemophilia A patients. Thromb Haemost. 1997;77:298–302.
PEGLip formulation does not change the structure of FVIII

11. Weiss HJ, Sussman, II, Hoyer LW. Stabilization of factor VIII in plasma
by the von Willebrand factor. Studies on posttransfusion and dissociated
and FVIIa. It thereby avoids one of the biggest obstacles to
factor VIII and in patients with von Willebrand’s disease. J Clin Invest.
producing improved coagulation factors: changes in structure
1977;60:390–404.

12. Santagostino E, Mancuso ME. Barriers to primary prophylaxis in
that induce antibody formation. Because of the large size
haemophilic children: the issue of the venous access. Blood Transfus.
and complexity of the FVIII protein and the sensitivity of
2008;6:S12–S16.
hemophilia A patients to even small changes in the protein,

13. Saenko EL, Ananyeva NM, Moayeri M, Ramezani A, Hawley RG.
60,61
Development of improved factor VIII molecules and new gene transfer
FVIII can be considered a worst case scenario for increased
approaches for hemophilia A. Curr Gene Ther. 2003;3:27–41.
International Journal of Nanomedicine 2010:5
submit your manuscript | www.dovepress.com
589
Dovepress

---

Yatuv et al
Dovepress

14. Mathew P. Current opinion on inhibitor treatment options. Semin

36. Allen GA, Persson E, Campbell RA, Ezban M, Hedner U, Wolberg AS.
Hematol. 2006;43:S8–S13.
A variant of recombinant factor VIIa with enhanced procoagulant and

15. Lusher JM, Roberts HR, Davignon G, et al. A randomized, double-blind
antifibrinolytic activities in an in vitro model of hemophilia. Arterioscler
comparison of two dosage levels of recombinant factor VIIa in the
Thromb Vasc Biol. 2007;27:683–689.
treatment of joint, muscle and mucocutaneous haemorrhages in persons

37. Stennicke HR, Ostergaard H, Bayer RJ, et al. Generation and bio-
with haemophilia A and B, with and without inhibitors. rFVIIa Study
chemical characterization of glycoPEGylated factor VIIa derivatives.
Group. Haemophilia. 1998;4:790–798.
J Thromb Haemost. 2008;100:920–928.

16. Shapiro A. Inhibitor treatment: state of the art. Dis Mon. 2003;49:

38. Schulte S. Use of albumin fusion technology to prolong the half-life of
22–38.
recombinant factor VIIa. Thromb Res. 2008;122:S14–S19.

17. Rick ME, Walsh CE, Key NS. Congenital bleeding disorders. Hematol-

39. Zamboni WC. Concept and clinical evaluation of carrier-mediated
ogy Am Soc Hematol Educ Program. 2003:559–574.
anticancer agents. Oncologist. 2008;13:248–260.

18. Zeitler H, Ulrich-Merzenich G, Hess L, et al. Treatment of acquired

40. Elbayoumi TA, Torchilin VP. Liposomes for targeted delivery of
hemophilia by the Bonn-Malmo Protocol: documentation of an in vivo
antithrombotic drugs. Expert Opin Drug Deliv. 2008;5:1185–1198.
immunomodulating concept. Blood. 2005;105:2287–2293.

41. Gabizon A, Shmeeda H, Barenholz Y. Pharmacokinetics of pegylated

19. Ma AD, Carrizosa D. Acquired factor VIII inhibitors: pathophysiology
liposomal Doxorubicin: review of animal and human studies. Clin
and treatment. Hematology Am Soc Hematol Educ Program. 2006:
Pharmacokinet. 2003;42:419–436.
432–437.

42. Gabizon A, Catane R, Uziely B, et al. Prolonged circulation time and

20. Kempton CL, White GC 2nd. How we treat a hemophilia A patient
enhanced accumulation in malignant exudates of doxorubicin encap-
with a factor VIII inhibitor. Blood. 2009;113:11–17.
sulated in polyethylene-glycol coated liposomes. Cancer Res. 1994;

21. Tjonnfjord GE, Holme PA. Factor eight inhibitor bypass activity
54:987–992.
(FEIBA) in the management of bleeds in hemophilia patients with

43. Gabizon AA, Barenholz Y, Bialer M. Prolongation of the circulation
high-titer inhibitors. Vasc Health Risk Manag. 2007;3:527–531.
time of doxorubicin encapsulated in liposomes containing a polyethyl-

22. Hedner U, Glazer S, Pingel K, et al. Successful use of recombinant
ene glycol-derivatized phospholipid: pharmacokinetic studies in rodents
factor VIIa in patient with severe haemophilia A during synovectomy.
and dogs. Pharm Res. 1993;10:703–708.
Lancet. 1988;2:1193.

44. Klibanov AL, Maruyama K, Torchilin VP, Huang L. Amphipathic poly-

23. Ingerslev J. Efficacy and safety of recombinant factor VIIa in the
ethyleneglycols effectively prolong the circulation time of liposomes.
prophylaxis of bleeding in various surgical procedures in hemophilic
FEBS Lett. 1990;268:235–237.
patients with factor VIII and factor IX inhibitors. Semin Thromb Hemost.

45. Baru M, Carmel-Goren L, Barenholz Y, et al. Factor VIII efficient
2000;26:425–432.
and specific non-covalent binding to PEGylated liposomes enables

24. Fridberg MJ, Hedner U, Roberts HR, Erhardtsen E. A study of the
prolongation of its circulation time and haemostatic efficacy. J Thromb
pharmacokinetics and safety of recombinant activated factor VII in
Haemost. 2005;93:1061–1068.
healthy Caucasian and Japanese subjects. Blood Coagul Fibrinolysis.

46. Dayan I, Robinson M, Baru M. Enhancement of haemostatic efficacy
2005;16:259–266.
of plasma-derived FVIII by formulation with PEGylated liposomes.

25. Lindley CM, Sawyer WT, Macik BG, et al. Pharmacokinetics and
Haemophilia. 2009;15:1006–1013.
pharmacodynamics of recombinant factor VIIa. Clin Pharmacol Ther.

47. Yatuv R, Dayan I, Carmel-Goren L, et al. Enhancement of factor
1994;55:638–648.
VIIa haemostatic efficacy by formulation with PEGylated liposomes.

26. Ingerslev J, Thykjaer H, Kudsk Jensen O, Fredberg U. Home treatment

Haemophilia. 2008;14:476–483.
with recombinant activated factor VII: results from one centre. Blood

48. Yatuv R, Carmel-Goren L, Dayan I, Robinson M, Baru M. Binding
Coagul Fibrinolysis. 1998;9:S107–S110.
of proteins to PEGylated liposomes and improvement of G-CSF effi-

27. Key NS, Aledort LM, Beardsley D, et al. Home treatment of mild to
cacy in mobilization of hematopoietic stem cells. J Control Release.
moderate bleeding episodes using recombinant factor VIIa (Novo-
2009;135:44–50.
seven) in haemophiliacs with inhibitors. J Thromb Haemost. 1998;80:

49. Spira J, Plyushch OP, Andreeva TA, Andreev Y. Prolonged bleeding-
912–918.
free period following prophylactic infusion of recombinant factor VIII

28. Saenko EL, Pipe SW. Strategies towards a longer acting factor VIII.
reconstituted with pegylated liposomes. Blood. 2006;108:3668–3673.
Haemophilia. 2006;12 Suppl 3:42–51.

50. Pan J, Liu T, Kim JY, et al. Enhanced efficacy of recombinant FVIII in

29. Gale AJ, Pellequer JL. An engineered interdomain disulfide bond
noncovalent complex with PEGylated liposome in hemophilia A mice.
stabilizes human blood coagulation factor VIIIa. J Thromb Haemost.
Blood. 2009;114:2802–2811.
2003;1:1966–1971.

51. Powell JS. Liposomal approach towards the development of a longer-

30. Gale AJ, Radtke KP, Cunningham MA, Chamberlain D, Pellequer JL,
acting factor VIII. Haemophilia. 2007;13:23–28.
Griffin JH. Intrinsic stability and functional properties of disulfide

52. Spira J, Plyushch OP, Andreeva TA, Khametova RN. Evaluation of
bond-stabilized coagulation factor VIIIa variants. J Thromb Haemost.
liposomal dose in recombinant factor VIII reconstituted with pegylated
2006;4:1315–2132.
liposomes for the treatment of patients with severe haemophilia A.

31. Pipe SW, Kaufman RJ. Characterization of a genetically engineered
J Thromb Haemost. 2008;100:429–434.
inactivation-resistant coagulation factor VIIIa. Proc Natl Acad Sci U S A.

53. Powell JS, Nugent DJ, Harrison JA, et al. Safety and pharmacokinet-
1997;94:11851–11856.
ics of a recombinant factor VIII with pegylated liposomes in severe

32. Mei B, Pan C, Jiang H, et al. Rational design of a fully active, long-
hemophilia A. J Thromb Haemost. 2008;6:277–283.
acting PEGylated factor VIII for hemophilia A treatment. Blood.

54. Martinowitz U, Lalezari S, Luboshitz J, Lubetsky A, Spira J. Infusion

33. Murphy JE, Pan C, Barnett T, et al. Site-specific PEGylation of rFVIII
rates of recombinant FVIII-FS with PEGylated liposomes in haemo-
results in prolonged in vivo efficacy. J Thromb Haemost. 2007;5:
philia A. Haemophilia. 2008;14:1122–1124.
P-T-022.

55. Szebeni J. Complement activation-related pseudoallergy: a new class of

34. Regan LM, Jiang X, Ramsey P, et al. Biological activity of PEGylated
drug-induced acute immune toxicity. Toxicology. 2005;216:106–121.
factor VIII. J Thromb Haemost. 2007;5:P-T-026.

56. Spira J, Plyushch OP, Zozulya N, et al. Safety, pharmacokinetics and

35. Tang L, Pan C, Atwal H, et al. PEGylation protects factor VIII from
efficacy of factor VIIa formulated with PEGylated liposomes in haemo-
the inhibition of antibody inhibitors. J Thromb Haemost. 2007;5:
philia A patients with inhibitors to factor VIII. An open label, explor-
P-T-036.
atory, cross over, phase I/II study. Haemophilia. 2010; In Press.
submit your manuscript | www.dovepress.com
590
International Journal of Nanomedicine 2010:5
Dovepress

---