U.S. patent application number 14/394437 was filed with the patent office on 2015-03-26 for optimised subcutaneous therapeutic agents.
This patent application is currently assigned to CANTAB BIOPHARMACEUTICALS PATENTS LIMITED. The applicant listed for this patent is Cantab Biopharmaceuticals Patents Limited. Invention is credited to Michael James Earl, William Henry, John Charles Mayo, Richard Wolf-Garraway.
Application Number | 20150086524 14/394437 |
Document ID | / |
Family ID | 48190923 |
Filed Date | 2015-03-26 |
United States Patent
Application |
20150086524 |
Kind Code |
A1 |
Henry; William ; et
al. |
March 26, 2015 |
OPTIMISED SUBCUTANEOUS THERAPEUTIC AGENTS
Abstract
Methods and dosage formulations are provided for subcutaneous
administration in which therapeutic agents are modified to increase
the hydrophilicity and molecular dimensions in relation to the
native state of the therapeutic agent, in which the Cmax:Caverage
ratio is lower than the Cmax:Caverage ratio of the agent when
delivered intravenously.
Inventors: |
Henry; William; (London,
GB) ; Wolf-Garraway; Richard; (London, GB) ;
Mayo; John Charles; (London, GB) ; Earl; Michael
James; (London, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cantab Biopharmaceuticals Patents Limited |
Valetta |
|
MT |
|
|
Assignee: |
CANTAB BIOPHARMACEUTICALS PATENTS
LIMITED
Valetta
MT
|
Family ID: |
48190923 |
Appl. No.: |
14/394437 |
Filed: |
April 16, 2013 |
PCT Filed: |
April 16, 2013 |
PCT NO: |
PCT/EP2013/057928 |
371 Date: |
October 14, 2014 |
Current U.S.
Class: |
424/94.3 ;
435/226; 514/14.1; 530/383 |
Current CPC
Class: |
A61K 9/0019 20130101;
A61K 38/4846 20130101; A61P 31/00 20180101; A61K 47/60 20170801;
A61K 38/37 20130101; A61P 31/04 20180101; A61P 7/04 20180101; A61P
5/00 20180101 |
Class at
Publication: |
424/94.3 ;
514/14.1; 530/383; 435/226 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61K 47/48 20060101 A61K047/48; A61K 38/37 20060101
A61K038/37; A61K 38/48 20060101 A61K038/48 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 16, 2012 |
GB |
1206628.8 |
Aug 1, 2012 |
GB |
1213712.1 |
Aug 22, 2012 |
GB |
1214985.2 |
Claims
1. A method of administering a therapeutic agent to a patient,
comprising subcutaneously administering the therapeutic agent to
the patient, such that the C.sub.max:C.sub.average ratio is lower
than the C.sub.max:C.sub.average ratio of the agent when delivered
intravenously, and wherein the therapeutic agent is modified in
order to increase the hydrophilicity and modify the molecular
dimensions in relation to the native state of the therapeutic
agent.
2. A method of administering a therapeutic agent to the lymphatic
system of a patient, comprising the step of subcutaneously
administering the therapeutic agent, such that it does not directly
enter the circulatory system of the patient at the site of
injection, and wherein the therapeutic agent is modified in order
to increase the hydrophilicity and modify the molecular dimensions
in relation to the native state of the therapeutic agent.
3. A method of preventing entry of a therapeutic agent directly
into the local circulatory system of a patient upon subcutaneous
administration of the therapeutic agent to a patient, the method
comprising the step of subcutaneously administering the modified
agent to the patient and wherein the therapeutic agent is modified
in order to increase the hydrophilicity and modify the molecular
dimensions in relation to the native state of the therapeutic
agent, such that the modified therapeutic agent is unable to enter
the local vasculature directly from the site of administration.
4. A method of modulating the speed of delivery of a therapeutic
agent from a subcutaneous depot in a subject, comprising modifying
the therapeutic agent to alter the hydrophilicity of the agent,
wherein the level of hydrophilicity is proportional to the level of
bioavailability.
5. A method according to any one of claims 1 to 4, wherein the
subcutaneous administration of the modified therapeutic agent
enables a higher dose of the modified agent to be administered to
the patient than can be safely delivered by a single intravenous
bolus injection.
6. A method according to any one of claims 1 to 5, wherein the
subcutaneous administration of the modified therapeutic agent
enables the patient to be re-dosed earlier than if the modified
agent is administered intravenously.
7. A method according to any one of claims 1 to 6, wherein the
subcutaneous administration of the modified therapeutic agent
produces a lesser or equivalent immunogenic response than the
intravenous administration of the agent in its modified or native
form.
8. A method according to any one of claims 1 to 7, wherein the
hydrophilicity of the therapeutic agent is increased by at least
50% in relation to the native state of the therapeutic agent.
9. A method according to any one of claims 1 to 8, wherein the
therapeutic agent is an antibiotic, a blood clotting factor, a
hormone, another therapeutic peptide or protein, a small molecule
or a monoclonal antibody.
10. The method according to claim 9, wherein the blood clotting
factor is selected from the group consisting of Factor VII, Factor
VIIa, Factor VIII, Factor IX, Factor X, Factor Xa, Factor XI,
Factor XIII, Factor V, von Willebrand's Factor, and Protein C.
11. A method according to any one of claims 1 to 10, wherein the
modification is conjugation of the therapeutic agent to a
biocompatible polymer.
12. The method according to claim 11, wherein the biocompatible
polymer is polyethylene glycol (PEG), poly-phosphatidyl choline
(PC), polypropylene glycol (PPG), copolymers of ethylene glycol and
propylene glycol, polyethylene oxide (PEO), polyoxyethylated
polyol, polyolefinic alcohol, polyhydroxyalkylmethacrylate,
polysaccharides, poly .alpha.-hydroxy acid, polyvinyl alcohol,
polyphosphosphasphazene, poly N-acryloylmorpholine, polyalkyene
oxide polymers, polymaleic acid, poly DL-alanine,
carboxymethylcellulose, dextran, starch or starch derivatives,
hyaluronic acid chitin, polymethacrylates, polysialic acid (PSA),
polyhydroxy alkanoates, poly amino acids and combinations
thereof.
13. The method according to claim 11 or 12, wherein the
biocompatible polymer is PEG
14. A method according to any one of claims 1 to 13, wherein the
modification is a fusion to a polypeptide to produce a fusion
protein; incorporation into vesicular delivery vehicles such as
liposomes, transfersomes or micelles; incorporation into or
attachment to dendrimers or the formation of oligomer complexes of
the therapeutic agent.
15. A method according to any one of claims 1 to 14, wherein the
subcutaneous delivery volume of the therapeutic agent is no more
than 2 ml.
16. A method according to any one of claims 1 to 15, wherein the
modified therapeutic agent is deliverable at a concentration higher
than the concentration of the modified agent than can be safely
delivered intravenously.
17. A method according to any one of claims 1 to 16, wherein
subcutaneous delivery of the modified therapeutic agent provides a
therapeutic benefit to the patient for a duration of at least 12
hours longer than the therapeutic benefit of the modified agent
when administered intravenously.
18. A method according to any one of claims 1 to 17, wherein the
subcutaneous delivery is by subcutaneous injection, topical
application, transdermal patch, microdermal abrasion, or high
pressure dry powder delivery.
19. A method according to any one of claims 1 to 18, wherein the
subcutaneous delivery is at least once per day, at least twice per
day, at least once per week, at least twice per week, at least once
per two weeks or at least once per month.
20. A modified agent comprising a therapeutic agent and a
modification, wherein the modification increases the hydrophilicity
and modifies the molecular dimensions of the agent in relation to
the native state of the therapeutic agent for use in a method
according to any one of claims 1 to 19.
21. A modified agent for use according to claim 19, wherein the
modification increases the hydrophilicity of the agent by at least
50% in relation to the native state of the therapeutic agent.
22. A modified agent as claimed in claim 20 or in claim 21, in
which the modification is a biocompatible polymer fused to the
therapeutic agent.
23. A modified agent as claimed in any one of claims 20 to 22,
wherein subcutaneous delivery of the modified agent provides a
therapeutic benefit to the patient for a duration of at least 12
hours longer than the therapeutic benefit of the modified agent
when administered intravenously.
24. A dosage form of a pharmaceutical composition of a modified
therapeutic agent, wherein subcutaneous delivery of the modified
agent provides a therapeutic benefit to the patient for a duration
of at least 12 hours longer than the therapeutic benefit of the
modified agent when administered intravenously.
25. A dosage form of a pharmaceutical composition of a modified
blood coagulation factor for subcutaneous administration which when
formulated for subcutaneous administration to a patient provides a
no more than once per month dosage form sufficient to maintain a
whole blood clotting time in said patient of no more than 20
minutes.
26. A liquid dosage form of a PEGylated blood coagulation factor
for subcutaneous administration no more than once per month wherein
the dosage form has a C.sub.max of at least 10% and no more than
90% compared to an equivalent reference dosage form when
administered intravenously, for use in the treatment of a blood
clotting disorder.
27. A dosage form according to any one of claims 24 to 26, wherein
the dosage form provides a no more than once per fortnight, no more
than once per week, no more than once per half week, no more than
once per two days or no more than once per day dosage.
28. A dosage form according to any one of claims 24 to 26, wherein
the dosage form is sufficient to maintain a whole blood clotting
time in said patient of less than 15 minutes.
29. A dosage form according to any one of claims 24 to 26, wherein
the dosage form is sufficient to maintain a whole blood clotting
time in said patient of less than 12 minutes.
30. A dosage form as claimed in any one of claims 24 to 29, in
which the blood clotting factor is selected from the group
consisting of Factor VIIa, Factor VII, Factor VIII, Factor IX,
Factor X, Factor Xa, Factor XI, Factor XIII, Factor V, von
Willebrand's Factor and Protein C.
31. A dosage form as claimed in any one of claims 24 to 30, wherein
the modification is PEGylation.
32. The dosage form as claimed in any one of claims 24 to 31 in
which the dosage form has a C.sub.max of at least 10% and no more
than 90% compared to an equivalent reference dosage form when
administered intravenously.
33. The dosage form as claimed in claim 32 in which the formulation
has a C.sub.max of from 10% to 20% compared to an equivalent
reference dosage form when administered intravenously.
34. The dosage form as claimed in claim 33, wherein the agent is
Factor VIII.
35. The dosage form as claimed in claim 32 in which the formulation
has a C.sub.max of from 40% to 60% compared to an equivalent
reference dosage form when administered intravenously.
36. The dosage form as claimed in claim 35, wherein the agent is
Factor IX.
37. The dosage form as claimed in claim 32 in which the formulation
has a C.sub.max of from 75% to 80% compared to an equivalent
reference dosage form when administered intravenously.
38. The dosage form according to claim 32 in which the formulation
has a C.sub.max of 75% compared to an equivalent reference dosage
form when administered intravenously.
39. The dosage form as claimed in claim 37 or claim 38, wherein the
agent is Factor VII.
40. A dosage formulation according to any one of claims 24 to 39,
in which the dosage is of from 1 to 1000 IU/kg.
41. A dosage formulation according to any one of claims 24 to 39 in
which the dosage is of from 5 to 500 IU/kg.
42. A dosage formulation according to any one of claims 24 to 39,
in which the dosage is of from 100 to 250 IU/kg.
43. A dosage formulation according to any one of claims 24 to 39,
in which the dosage is of from 50 to 200 IU/kg.
44. A dosage formulation according to any one of claims 24 to 39,
in which the dosage is of from 25 to 50 IU/kg.
45. A dosage formulation according to any one of claims 24 to 39,
wherein the dosage form is for administration at least once per
day, at least twice per day, about once per week, about twice per
week, about once per two weeks, or about once per month.
46. A dosage formulation of a therapeutic agent wherein the dosage
form is for subcutaneous administration and wherein the therapeutic
agent is modified to alter the hydrophilicity of the agent, wherein
the level of hydrophilicity is proportional to the level of
bioavailability.
47. A method of treatment of a disease in a patient, comprising
administering subcutaneously a dosage form of a modified
therapeutic agent according to claim 24.
48. A method of treatment of a blood clotting disease or trauma in
a patient comprising administering subcutaneously a dosage form of
a blood clotting factor according to any one of claims 24 to 46 to
a patient in need thereof.
49. A method of treatment according to claim 47 or claim 48,
wherein the dosage form is administered at least once per day, at
least twice per day, at least about twice per week, at least about
once per week, at least once per two weeks, or at least about once
per month.
50. A method of treatment according to claim 47 or claim 48,
wherein the dosage form is administered at least once per day.
51. A method of treatment according to claim 50, wherein the dosage
form is administered twice per day, in which the patient receives a
first dosage form in a first administration and a second dosage
form in a second administration.
52. A method of treatment according to claim 51, wherein the second
dosage form is administered separately, simultaneously or
sequentially to the first dosage form.
53. A kit of parts comprising a subcutaneous dosage form according
to any one of claims 24 to 46, and an administration vehicle.
Description
[0001] The present invention relates to the subcutaneous delivery
of therapeutic agents, as well as the modifications of such agents
to render them suitable for subcutaneous delivery.
[0002] Many hydrophobic (lipophilic) molecules are used in the
treatment of infection, disease and disorders. Lipophilic molecules
are generally administered directly into the bloodstream of a
patient, in order to ensure rapid delivery to the site of the
infection, disease etc. However, the half life and/or
bioavailability of such molecules may be sub-optimised.
Disadvantages of intravenous administration include local and
general reactions to the delivery of relatively large amounts of
agent into a patient and the inconvenience of intravenous
administration.
[0003] The present inventors have surprisingly found that modifying
a therapeutic agent, and thereby increasing the hydrophilicity and
the molecular dimensions of the agent, results in the inability of
such an agent to directly enter the vascular system. However, the
modified agent still becomes bioavailable due to its ability to
enter the circulatory system of a patient via the aqueous lymphatic
system. The modification is chosen in order to reduce surface
adherence of the therapeutic agent to the connective tissues and to
increase its solubility in tissue fluid. The modified therapeutic
agents of the present invention are particularly useful when they
are delivered to the subcutaneous space, since they are too large
to enter the vascular system directly from the subcutaneous space
and therefore are transported around the body by the lymphatic
system, entering the circulatory system via the thoracic duct
(right lymphatic duct and subclavian veins). This surprisingly
results in a predictable, steady infusion of the agent into the
circulatory system of the patient. Accordingly, the present
invention is concerned with the subcutaneous delivery of a modified
agent, in order to render the effect of the modified agent more
predictable in its longevity, infusion rate and elimination rate
and thus duration of effect. This is achieved by causing the agent
to be more hydrophilic and modifying its molecular dimensions such
that upon subcutaneous delivery to the patient, the modified agent
is unable to pass through the blood vessel walls to enter the blood
stream but is transported by interstitial fluid such that it enters
the lymphatic system. This results in a controlled, predictable
release into the vascular system, from the lymphatic system. It
removes the need to consider the level of vascularisation around a
site of delivery as discussed below.
[0004] The invention can be applied to peptides, biomolecules,
including all blood factors, hormones, antibiotics, monoclonal
antibodies and some small molecules. Any suitable modification can
be used that does not interfere with the therapeutic effect of the
molecule, and that increases the hydrophilicity and, modifies its
molecular dimensions (which may include molecular weight, or the
physical size of the modified agent) to ensure that it cannot
directly enter the vasculature without first passing into the
subclavian vein via the lymphatic system at the thoracic duct. The
chosen modification may have the concomitant effect of regulating
the elimination of the agent from the body (by excretion,
digestion, immunologic attack or other means) such that the rate of
infusion and rate of elimination of the agent are "balanced" for an
optimal therapeutic effect.
[0005] Examples of suitable modifications include the conjugation
of the agent with a polymer, suitably a biocompatible polymer, such
as polyethylene glycol (PEG), poly-phosphatidyl choline (PC),
polypropylene glycol (PPG), copolymers of ethylene glycol and
propylene glycol, polyethylene oxide (PEO), polyoxyethylated
polyol, polyolefinic alcohol, polyhydroxyalkylmethacrylate,
polysaccharides, poly .alpha.-hydroxy acid, polyvinyl alcohol,
polyphosphosphasphazene, poly N-acryloylmorpholine, polyalkyene
oxide polymers, polymaleic acid, poly DL-alanine,
carboxymethylcellulose, dextran, starch or starch derivatives,
hyaluronic acid, chitin, polymethacrylates, polysialic acid (PSA),
polyhydroxy alkanoates, poly amino acids and combinations thereof.
The biocompatible polymer may have a linear or branched
structure.
[0006] Other examples of biocompatible polymers are a protein
selected from, but not limited to, the group consisting of albumin,
transferrin, immunoglobulins including monoclonal antibodies,
antibody fragments for example; single-domain antibodies, V.sub.L,
V.sub.H, Fab, F(ab').sub.2, Fab', Fab3, scFv, di-scFv, sdAb, Fc and
combinations thereof.
[0007] Other methods of modifying the therapeutic agent might be
through the use of fusion proteins; incorporation into vesicular
delivery vehicles such as liposomes, transfersomes or micelles;
incorporation into/attachment to dendrimers; formation of oligomer
complexes of the agent. The chosen modification may have the
concomitant effect of regulating the elimination of the agent from
the body (by excretion, digestion, immunologic attack or other
means) such that the rate of infusion and rate of elimination of
the agent are "balanced" for an optimal therapeutic effect.
[0008] Once delivered to the subcutaneous space the modified agent
thus located is able to be transported via the lymphatic system to
infuse into the vascular system via the subclavian veins, after
which such modifications also control the elimination of the agent
from the body in such a way that the ratio of infusion rate from
the subcutaneous space into the circulation to elimination rate of
the drug product from the body may be balanced and controlled in a
manner to optimise the therapeutic efficiency and effectiveness of
the modified agent.
[0009] An example of therapeutic agents that may be modified for
subcutaneous delivery in this way include blood coagulation
factors. The blood coagulation cascade involves a number of
different proteins which variously serve to activate each other and
promote the formation of a blood clot and maintain healthy
haemostasis. In some embodiments, the blood coagulation factor to
be modified in accordance with the invention is selected from the
group consisting of Factor VII, Factor VIIa, Factor VIII, Factor
IX, Factor X, Factor Xa, Factor XI, Factor VIIa, Factor V, Factor
XIII, von Willebrand's Factor and Protein C. In some embodiments
the blood coagulation factor is suitably Factor VII, Factor VIII or
Factor IX.
[0010] An example of therapeutic agents to which the invention
relates includes, blood coagulation Factor VII (herein referred to
as FVII), which is a 53,000 Dalton (Da), glycosylated, Vitamin K
dependent, single-chain zymogen, containing 12 native disulphide
bonds (O'Hara et al., Proc. Nat'l Acad. Sci. USA, 84: 5158-5162
(1987)). The protein is predominantly produced in the liver. FVII
is involved in the extrinsic blood clotting cascade (FIG. 1). The
protein is organised into four discrete domains: an N-terminal
.gamma.-carboxyglutamate (Gla) domain, two epidermal growth
factor-like (EGF) domains and a C-terminal serine protease domain.
The circulating zymogen shows very little protease activity in the
absence of its cofactor tissue factor (TF) which is found in the
vascular subendothelium. Following vascular damage, FVII binds to
TF with high affinity and is converted to the active, two-chain
enzyme FVIIa by specific cleavage of the peptide bond between
arginine 152 and isoleucine 153. The FVIIa light-chain is composed
of the N-terminal Gla and EGF-like domains and the heavy-chain is
composed of the serine protease domain. The heavy and light chains
are held together by a single disulphide bond between cysteine 135
and cysteine 262. Once activated, FVIIa rapidly catalyses the
conversion of FX to FXa and FIX to FIXa. FXa then forms a complex
with FVa to cleave prothrombin, resulting in the generation of
small amounts of thrombin (Aitken, M. G. EMA, 16: 446-455 (2004)).
This thrombin generation activates platelets and cofactors V, VIII
and XI on the platelet surface. The activation leads to the
formation of a thrombin burst which causes fibrin polymerisation
and the formation of a haemostatic plug.
[0011] Human recombinant FVIIa has been developed and
commercialised by Novo Nordisk as NovoSeven.RTM. (eptacog alfa
[activated], ATC code B02BD08). NovoSeven.RTM. is licensed for the
treatment of bleeding episodes in haemophilia A or B patients who
have developed inhibitory antibodies against FVIII or IX,
respectively (Jurlander et al., Seminars in Thrombosis and
Hemostasis, 27: 373-383 (2001); Roberts et al., Blood, 15:
3858-3864 (2004)). The treatment has proved to be safe and
effective since its launch in 1996. However, due to the proteins
relatively short in vivo half-life (2.3 hours; Summary Basis for
Approval NovoSeven.RTM., FDA reference number 96-0597) multiple
infusions of high doses of the product (90 .mu.g kg.sup.-1) may be
required over time during a single bleeding episode in order to
attain haemostasis. The short half-life of the product and the high
dose required to render the desired therapeutic effect preclude the
common use of NovoSeven.RTM. for prophylactic treatment of
haemophiliacs with inhibitors. Clearly, therefore, there is a need
for the development of FVIIa molecules which have an increased
half-life, producing improvements in pharmacokinetics (PK) and
pharmacodynamics (PD).
[0012] Factor VIII (FVIII) is an essential blood clotting factor
also known as anti-haemophilic factor (AHF). In humans, Factor VIII
is encoded by the F8 gene. Defects in this gene results in
haemophilia A, a well-known recessive X-linked coagulation disorder
effecting approximately 1 in 5,000 males.
[0013] The X-linked F8 gene encodes a polypeptide of 2351 amino
acids from 26 exons which after signal peptide cleavage renders a
mature FVIII molecule of 2332 amino acids (Wang et al. Int. J.
Pharmaceutics, 259: 1-15 (2003)). FVIII has been found to be
synthesized and released into the bloodstream by the vascular,
glomerular, and tubular endothelium, and the sinusoidal cells of
the liver though there is still considerable ambiguity as to what
the primary site of release in humans is. The FVIII molecule is
organised into six protein domains; NH.sub.2-A1-A2-B-A3-C1-C2-COOH.
The mature molecule contains a number of post-translational
modifications including N-linked and O-linked glycosylation,
sulphonation and disulphide bond formation. FVIII contains a total
of 23 cysteine residues, 16 of these form 8 disulphide bonds in the
A and C domains of the protein (McMullen et al. Protein Science, 4:
740-746 (1995)). Due to the post-translational modification of the
protein, its circulation molecular weight can be up to 330 kDa
depending on the level and type of glycosylation. FVIII is also
proteolytically processed so that the circulating species is a
heterodimer composed of a heavy chain (A1-A2-B) and light chain
(A3-C1-C2). When FVIII is secreted into the circulation it binds to
von Willebrand Factor (vWF) in a non-covalent manner. The binding
of the two molecules involves the A3 and C2 domains of the light
chain of FVIII (Lacroix-Desmazes et al. Blood, 112: 240-249
(2008)). Binding to vWF increases the stability and circulating
half-life of FVIII. Although binding to vWF increases the
circulating half-life of FVIII, its native half-life is 15-19
hours.
[0014] Factor VIII is an essential cofactor participating in the
intrinsic blood coagulation pathway. Its role in the coagulation
cascade is to act as a "nucleation template" to organise the
components of the FXase complex in the correct spatial orientation
on the surface of activated platelets (Shen et al. Blood, 111:
1240-1247 (2008)). FVIII is initially activated by thrombin (Factor
Ila) or FXa and it then dissociates from vWF in the form of FVIIIa.
FVIIIa then binds to activated platelets at the site of vascular
injury and binds FIXa through an A2 and A3 mediated interaction.
The binding of FIXa to FVIII in the presence of Ca.sup.2+ on the
platelet surface increases the proteolytic activity of FIXa by
approximately 200,000-fold. This complex then activates FX to FXa.
Factor Xa, with its cofactor Factor Va, then activates more
thrombin. Thrombin in turn cleaves fibrinogen into fibrin which
then polymerizes and crosslinks (using Factor XIII) into a fibrin
blood clot.
[0015] No longer protected by vWF, activated FVIII is
proteolytically inactivated in the process (most prominently by
activated Protein C and Factor IXa) and quickly clears from the
blood stream.
[0016] Factor IX (also known as Christmas factor) is a serine
protease of the coagulation system and deficiency of this protein
causes hemophilia B. Factor IX is produced as an inactive zymogen
precursor which is subsequently processed to remove the signal
peptide, followed by further glycosylation and subsequent cleavage
by Factor XIa or Factor VIIa to produce a two-chain form linked by
a disulfide bridge (Scipio et al J Clin Invest. 1978;
61(6):1528-1538). Once activated as Factor IXa and in the presence
of Ca.sup.2+, membrane phospholipids, and a Factor VIII cofactor,
it hydrolyses an arginine-isoleucine bond in Factor X to form
Factor Xa. Factor IX is inhibited by antithrombin.
[0017] Haemophilia B is an X-linked bleeding disorder caused by a
plethora of mutations in the factor IX gene, resulting in a
deficiency of effective procoagulant protein. Haemophilia B which
is also known as Christmas disease, is the consequence of
non-functional or deficient FIX which prevents normal initiation of
the intrinsic cascade. Serious and potentially life threatening
bleeding events can develop with this condition which can be
corrected by timely administration of an adequate amount of FIX.
Haemostasis can be maintained for as long as the circulating
zymogen is in the therapeutic range.
[0018] Historically, Haemophilia B has been treated by intravenous
delivery of plasma FIX or prothrombin complex concentrates and more
recently by highly purified plasma derived and recombinant FIX. The
advent of recombinant human FIX from Chinese hamster ovary cells
(CHO cells) has transformed the treatment of Christmas disease to
the point where prophylactic therapy is now possible particularly
in small children. The limiting factor in this regard however is
the short half-life and potential "super potency" of which has
constrained prophylactic therapy to approximately 3 day
intervals.
[0019] One of the problems faced by physicians seeking to treat
patients with blood clotting and other disorders is how to achieve
a long-lasting therapeutic dosage of a therapeutic agent, such as a
blood clotting factor composition administered to such patients.
Another problem, particularly around the prophylactic use of such
agents is maintaining a predictable, steady state level of
infusion, distribution and elimination of therapeutic agents in the
body, thus avoiding the sawtooth "bursts" or "peaks" of levels of
both the agent and its effects.
[0020] For example, the regulation of blood coagulation is a
process that presents a number of leading health problems,
including both the failure to form blood clots as well as
thrombosis, the formation of unwanted blood clots. Agents that
prevent unwanted clots are used in many situations and a variety of
agents are available. Unfortunately, most current therapies have
undesirable side effects. Orally administered anticoagulants such
as Warfarin act by inhibiting the action of vitamin K in the liver,
thereby preventing complete carboxylation of glutamic acid residues
in the vitamin K-dependent proteins, resulting in a lowered
concentration of active proteins in the circulatory system and
reduced ability to form clots. Warfarin therapy is complicated by
the competitive nature of the drug with its target. Fluctuations of
dietary vitamin K can result in an over-dose or under-dose of
Warfarin. Fluctuations in coagulation activity are an undesirable
outcome of this therapy.
[0021] Injected substances such as heparin, including low molecular
weight heparin, also are commonly used anticoagulants. Again, these
compounds are subject to overdose and must be carefully
monitored.
[0022] Another phenomenon that limits the usefulness of therapeutic
peptides is the relatively short in vivo half-life exhibited by
some of these peptides. Overall, the problem of short in vivo
half-life means that therapeutic glycopeptides must be administered
frequently and in high dosages, which ultimately translate to
higher risk of local adverse reactions and higher health care costs
than might be necessary if a more efficient method for maintaining
therapeutically effective levels of glycoprotein therapeutics for
longer was available.
[0023] The ability to ensure the delivery of therapeutic agents via
the lymphatic system provides controlled infusion of the agent. The
increased hydrophilicity also assists in concealing the molecule
from damage by degrading enzymes, the immune system etc.
Furthermore, the increased mobility in water renders the
therapeutic agents more bioavailable, leading to lower dosage
requirements. This in turn may result in fewer side effects, more
efficient treatment and less time spent in a physician's care.
[0024] The inventors have surprisingly shown that a more consistent
`steady state` level of therapeutic agent can be achieved
systemically when modified in accordance with the invention and
delivered to the subcutaneous space. This increased consistency in
`steady state` can be attributed to a combination of rate of
introduction into the vascular system via the lymphatic system
(i.e. infusion), balanced against the rates of metabolism and/or
immune system degradation, and rate of elimination via the kidneys
or GI tract.
[0025] The subcutaneous delivery of a modified agent in accordance
with the present invention may, therefore, allow the `sawtooth`
peaks and troughs commonly seen with repeated bolus injection
delivery to be mitigated. However a larger dose can be administered
by subcutaneous delivery such that C.sub.max is the same as
achieved by intravenous injection, in which case a longer duration
of the therapeutic effect of the modified agent will be achieved
due to the slower rate of infusion via the lymphatic system into
the vascular system. Thus, the present invention may result in less
frequent administration. Alternatively, the same administration
frequency could be envisaged with a lower dose when subcutaneous
delivery is employed in accordance with the invention, instead of
intravenous delivery.
[0026] In other words, over a given duration (such as 4 days) the
ratio of C.sub.max:C.sub.average of a subcutaneously administered
dose of a modified agent is lower than when the same dose is
administered intravenously. This is clearly an advantage since the
levels of the modified agent in the bloodstream are more
consistent.
[0027] As one of skill in the art will appreciate, a lower
C.sub.max may be of benefit to the patient, as is a lower ratio of
C.sub.max:C.sub.average or C.sub.max:C.sub.min (i.e. a flattened
graph of peaks and troughs when compared to the typical "sawtooth"
profile of an intravenously administered drug).
[0028] Factor VIIa, for example, illustrates this problem and the
modification shows the inventive solution thereto. Factor VII and
VIIa have circulation half-times of about 2-4 hours in the human.
That is, within 2-4 hours, the concentration of the peptide in the
serum is reduced by half. When Factor VIIa is used as a
procoagulant to treat certain forms of haemophilia, the standard
protocol is to inject VIIa every two hours and at high dosages (45
to 90 .mu.g/kg body weight). See, Hedner et al., Transfus. Med.
Rev. 7: 78-83 (1993)). Thus, use of these proteins as procoagulants
or anticoagulants (in the case of factor VII) requires that the
proteins be administered at frequent intervals and at high
dosages.
[0029] The conjugation of biopharmaceuticals to biocompatible
polymers has previously been used successfully to improve the
physicochemical characteristics of such therapeutic products.
Characteristics of proteins which have been improved through
conjugation include PK, PD and immunogenicity. The attachment of a
chemical moiety to a protein can significantly increase its
circulation half-life (Jevsevar et al., Biotechnol. J., 5: 113-128
(2010)). For molecular species with molecular weights below the
glomerular filtration limit the conjugation of a large molecular
weight moiety prevents renal clearance of the product. Also,
addition of chemical moieties to pharmaceutical products can
prevent receptor mediated removal of the molecule through steric
hindrance.
[0030] The use of modifying molecules, such as biocompatible
polymers to render the therapeutic agents more hydrophilic may also
assist in the reduction or a prevention of an immune response to
the introduced therapeutic agent. The modification provides a
`shield of water` around the agent, which may `hide` any epitopes
to which the immune system may otherwise respond. The presence of
water molecules around the modified therapeutic agent may form a
clathrate structure when in aqueous solution.
[0031] Furthermore, the use of the modification to allow
subcutaneous delivery of the agent enables the gradual introduction
of the therapeutic agent into the body via the lymphatic system,
avoiding the reaction associated with bolus injections or
intravenous infusion of large dosages, such as "red-man syndrome"
associated with the intravenous administration of certain
antibiotics.
[0032] Thus, many advantages can be envisaged by modifying such
therapeutic agents for subcutaneous delivery and thereby subsequent
infusion into the vascular system via the lymphatic system.
[0033] Accordingly, the present invention provides, as a first
aspect a method of administering a therapeutic agent to a patient,
comprising subcutaneously administering the therapeutic agent to
the patient, such that the C.sub.max:C.sub.average ratio is lower
than the C.sub.max:C.sub.average ratio of the agent when delivered
intravenously, and wherein the agent is modified in order to
increase the hydrophilicity and modify the molecular dimensions in
relation to the native state of the therapeutic agent. The
subcutaneous administration is such that the agent is at a more
consistent concentration in the patient's bloodstream during the
treatment period when compared to intravenous administration, which
enables the C.sub.max:C.sub.average ratio to be reduced.
[0034] Also provided is a method of administering a therapeutic
agent to the lymphatic system of a patient, comprising the step of
subcutaneously administering the therapeutic agent, such that it
does not directly enter the circulatory system of the patient at
the site of injection, and wherein the agent is modified in order
to increase the hydrophilicity and modify the molecular dimensions
in relation to the native state of the therapeutic agent, such that
the modified agent is unable to enter the circulation directly from
the site of administration.
[0035] Further provided is a method of preventing entry of a
therapeutic agent directly into the local circulatory system of a
patient upon subcutaneous administration of the therapeutic agent
to a patient, the method comprising the step of subcutaneously
administering the modified agent to the patient and wherein the
agent is modified in order to increase the hydrophilicity and
modify the molecular dimensions in relation to the native state of
the therapeutic agent.
[0036] The subcutaneous administration of the modified agent
enables a higher dose of the agent to be administered to the
patient than by intravenous bolus injection; the patient to be
re-dosed earlier than if the modified agent is administered
intravenously; a lesser or equivalent immunogenic response than the
intravenous administration of the modified agent to be achieved;
provides a therapeutic benefit to the patient for a duration of at
least 12 hours longer than the therapeutic benefit of the modified
agent when administered intravenously; and the agent is deliverable
at a concentration higher than the concentration of the modified
agent that can be safely delivered intravenously.
[0037] The hydrophilicity is increased by at least the ratio of the
molecular dimensions of the modified agent to the molecular
dimensions of the unmodified agent. By hydrophilicity it is meant
the hydrophilic to lipophilic balance (HLB), which may be defined
as the affinity for water which in the context of this invention
implies a lower capacity for surface adhesion and a higher
dispersion in water.
[0038] The methods of the invention provide for modulating the
speed of delivery of a therapeutic agent from a subcutaneous depot
in a subject, comprising modifying the therapeutic agent to alter
the hydrophilicity of the agent, wherein the level of
hydrophilicity is proportional to the level of bioavailability.
[0039] It has been surprisingly found that to achieve the longest
duration of depot release from the subcutaneous space, a lesser
degree of modification is required. Without being bound by theory,
this can be rationalised by the lesser degree of modification
exposing some of the therapeutic agent to the subcutaneous tissue
which confers a slow rate on the diffusion through the lymph. By
contrast the higher degree of modification covers the therapeutic
agent completely leaving the product free to quickly enter the
blood circulation.
[0040] It has also been shown that the bioavailability favours the
therapeutic agents which have been more highly modified, namely di-
or tri-modified species compared to mono-modified species. The
present inventors have therefore confirmed that the higher degrees
of modification and hydration levels promote a higher degree of
mobility and therefore bioavailability.
[0041] Consequently, for any given therapeutic agent the release
from a subcutaneous depot can now be modulated by increasing or
decreasing the level of modification of the therapeutic agent.
[0042] In accordance with the invention, subcutaneous delivery may
be by subcutaneous injection, topical application, transdermal
patch, microdermal abrasion, high pressure dry powder delivery, or
any other method for introducing a therapeutic to the subcutaneous
space.
[0043] A further aspect of the invention provides a modified agent
comprising a therapeutic agent and a modification, wherein the
modification increases the hydrophilicity and modifies the
molecular dimensions of the agent in relation to the native state
of the therapeutic agent for use in a method according to the first
and further aspects. Modification of the agent may increase the
hydrophilicity by at least 50% and the molecular dimensions by at
least 50% of the agent in relation to the native state of the
therapeutic agent.
[0044] An example of a biocompatible polymer which has been used in
several marketed biopharmaceutical products is polyethylene glycol
(herein referred to as PEG). The process of covalently attaching a
PEG molecule to another molecule is termed PEGylation. To date,
nine PEGylated products have received FDA market approval, with
four being blockbuster drugs: PegIntron.RTM. (Schering-Plough),
Pegasys.RTM. (Hoffman-La Roche), Neulasta.RTM. (Amgen) and
Micera.RTM. (Hoffman-La Roche). A number of different chemistries
have been used to conjugate protein therapeutics to activated PEG
molecules. Random PEGylation has been used successfully to
covalently link PEG moieties to proteins through amino groups on
proteins. The attachment sites have most frequently, but not
exclusively, been the .epsilon.-amino group on the side chains of
lysine residues. Such random reactions can produce very complex
mixtures of conjugates varying in the number and site of PEG moiety
attachment. Even following purification of random conjugation
reactions, positional isomers can be present which demonstrate very
different physicochemical and pharmaceutical characteristics. A
number of site-specific PEGylation techniques have been developed
and are now being exploited to produce better defined
biopharmaceuticals. Approaches taken to render site-specific
PEGylation include N-terminal, cysteine, glycan, disulphide and
poly-histidine targeted PEGylation.
[0045] The use of PEG to derivatize peptide therapeutics has been
demonstrated to reduce the immunogenicity of the peptides. For
example, U.S. Pat. No. 4,179,337 discloses non-immunogenic
polypeptides such as enzymes and peptide hormones coupled to
polyethylene glycol (PEG) or polypropylene glycol. In addition to
reduced immunogenicity, the clearance time in circulation is
prolonged due to the increased size of the PEG-conjugate of the
polypeptides in question.
[0046] The principal mode of attachment of PEG, and its
derivatives, to peptides is a non-specific bonding through a
peptide amino acid residue (see U.S. Pat. No. 4,088,538, U.S. Pat.
No. 4,496,689, U.S. Pat. No. 4,414,147, U.S. Pat. No. 4,055,635,
and WO 87/00056). Another mode of attaching PEG to peptides is
through the non-specific oxidation of glycosyl residues on a
glycopeptide (see WO 94/05332).
[0047] In these non-specific methods, polyethyleneglycol is added
in a random, non-specific manner to reactive residues on a peptide
backbone. Of course, random addition of PEG molecules has its
drawbacks, including a lack of homogeneity of the final product,
and the possibility for reduction in the biological or enzymatic
activity of the peptide. Therefore, for the production of
therapeutic peptides, a derivitization strategy that results in the
formation of a specifically labelled, readily characterizable,
essentially homogeneous product is superior.
[0048] The state of the art in PEGylation of therapeutic agents,
such as recombinant blood clotting factors, such as FVIIa, FVIII
and FIX can be summarised as follows. WO 98/32466 suggests that
FVII may be PEGylated, but does not contain any further information
on the subject. US 2008/0200651 suggests that FVII polypeptides
with wild-type, or increased, activity which have a PEG molecule
conjugated via an artificially introduced cysteine residue
demonstrate increased in vivo half-life. US 2008/0221032 describes
the production of a FVIIa-polysialic acid conjugate which resulted
in the molecule demonstrating a significantly increased in vivo
half-life. US 2009/0176967 teaches that enzymes can be used to
introduce specific functional groups at the C-terminus of the FVII
polypeptide to which biocompatible polymers such as PEG can be
coupled. US 2009/0227504 describes preparations of FVIIa (or
FVIIa-like molecules) where one, or more, asparagine--and/or
serine-linked oligosaccharide chains are covalently modified with
at least one polymeric group which demonstrate improved serum
half-life. US 2010/0028939 describes how natural glycoproteins can
be modified using the enzyme galactose oxidase to produce reactive
aldehyde functionalities on the glycan termini. The reactive
aldehydes can then be used to conjugate polymeric moieties to the
protein producing a product with improved pharmacological
characteristics. US 2010/0056428 suggests that improved
pharmacokinetic characteristics can be achieved in FVIIa by the
derivitization of the glycoprotein by an oxime of a polymeric
moiety such as PEG at a glycosyl group. Corresponding reports have
been published in relation to FVIII and FIX, see US 2008/0255026
and U.S. Pat. No. 7,683,158 respectively.
[0049] Another approach to PEGylation of proteins has been
developed by Polytherics and is known as TheraPEG.TM. in which a
PEG polymer is attached to the protein of interest via a reduced
disulphide bond of a pair of cysteine residues in the protein (WO
2005/007197). The technique has been used to prepare a PEGylated
version of Factor IX free of contamination from Factor FIXa (WO
2009/130602), PEGylated Factor VII (WO 2011/135308) and PEGylated
Factor VIII (WO 2011/135307).
[0050] It has now been discovered by the present inventors that
subcutaneous administration of modified therapeutic agents such as
PEGylated forms of blood clotting factors can result in improved
half-lives and prolonged activity in plasma compared to equivalent
forms delivered by intravenous administration, particularly when
"dose adjusted". The specific location at which the subcutaneous
injection is given may either increase or decrease the onset time
in which the modified agent appears in the blood system. In any
event, a lower C.sub.max:C.sub.average ratio is achieved; similar
pharmacokinetic profiles are seen usually associated with sustained
release formulations and the like. The disadvantage with
administering unmodified therapeutic agents subcutaneously is that
they are able to enter directly into the cardiovascular system, and
thereby the resultant C.sub.max and duration depends largely on the
vascular condition of the site of subcutaneous injection. A highly
vascularised region will clearly take up more quickly an amount of
agent when administered by a subcutaneous injection into that area
than an injection into a less vascularised area. Such
inconsistencies may be overcome with the use of the modified agents
of the invention for subcutaneous delivery.
[0051] The provision of a modified therapeutic agent in accordance
with the present invention results in a molecule being delivered to
the cardiovascular system via the lymph system and therefore is
independent of the vasculature at the site of injection, leading to
a more predictable, consistent rate of delivery into the
circulation, via the lymphatic system.
[0052] Prior speculation in the art about formulations of
therapeutic agents, including blood clotting factors, does not
appreciate the advantages that could be derived from formulating
such factors for subcutaneous administration. In particular, there
is no hint or suggestion that such formulations when administered
subcutaneously could deliver and maintain normal haemostasis for
prolonged periods of time or that they could deliver a steadier
level of drug bioavailability (lower C.sub.max:C.sub.average
ratio), which is due to the steady infusion effect achieved. The
lymph system provides an aqueous fluid in which the vessel walls
are collagen containing. Any molecule that is too big to go through
blood vessel walls must rely on lymphatic drainage to reach to the
bloodstream. However, if a degree of hydrophobic character exists
in the molecule, it is likely to adhere to tissue both before it
enters the lymph system and to the lymph vessel walls and will,
thus, be immobile in the fluid. By contrast when a hydrophilic
moiety is provided, the modified agents will more readily disperse
in the aqueous phase of the lymph and drain easily into the system
to enter the bloodstream at the thoracic duct.
[0053] The therapeutic agent of any aspect may be small molecule,
macromolecule, polymer and polypeptide, wherein a small molecule
includes hypnotics and sedatives, antiarrhythmics, antioxidants,
anti-asthma agents, hormonal agents including contraceptives,
sympathomimetics, diuretics, lipid regulating agents,
antiandrogenic agents, antiparasitics, anticoagulants, neoplastics,
antineoplastics, hypoglycemics, psychic energizers, tranquilizers,
respiratory drugs, anticonvulsants, muscle relaxants,
anti-Parkinson agents (dopamine antagnonists), cytokines, growth
factors, anti-cancer agents, antithrombotic agents,
antihypertensives, cardiovascular drugs, analgesics,
anti-inflammatories, antianxiety drugs (anxiolytics), appetite
suppressants, anti-migraine agents, muscle contractants,
anti-infectives (antibiotics, antivirals, antifungals, vaccines)
anti-arthritics, anti-malarials, anti-emetics, anepileptics,
bronchodilators, nutritional agents and supplements, growth
supplements, anti-enteritis agents, vaccines, antibodies,
diagnostic agents, and contrasting agents.
[0054] Examples of agents suitable for use in the invention
include, but are not limited to, calcitonin, erythropoietin (EPO),
ceredase, cerezyme, cyclosporin, granulocyte colony stimulating
factor (GCSF), thrombopoietin (TPO), alpha-1 proteinase inhibitor,
elcatonin, granulocyte macrophage colony stimulating factor
(GMCSF), growth hormone, human growth hormone (HGH), growth hormone
releasing hormone (GHRH), heparin, low molecular weight heparin
(LMWH), interferon alpha, interferon beta, interferon gamma,
interleukin-1 receptor, interleukin-2, interleukin-1 receptor
antagonist, interleukin-3, interleukin-4, interleukin-6,
luteinizing hormone releasing hormone (LHRH), factor IX insulin,
pro-insulin, insulin analogues (e.g., mono-acylated insulin as
described in U.S. Pat. No. 5,922,675), amylin, C-peptide,
somatostatin, somatostatin analogs including octreotide,
vasopressin, follicle stimulating hormone (FSH), insulin-like
growth factor (IGF), insulintropin, macrophage colony stimulating
factor (M-CSF), nerve growth factor (NGF), tissue growth factors,
keratinocyte growth factor (KGF), glial growth factor (GGF), tumor
necrosis factor (TNF), endothelial growth factors, parathyroid
hormone (PTH), glucagon-like peptide thymosin alpha 1, Ilb/Illa
inhibitor, alpha-1 antitrypsin, phosphodiesterase (PDE) compounds,
VLA-4 inhibitors, bisphosphonates, respiratory syncytial virus
antibody, cystic fibrosis transmembrane regulator (CFTR) gene,
deoxyreibonuclease (Dnase), antipseudomonal penicillins like
carbenicillin, ticarcillin, azlocillin, mezlocillin, and
piperacillin; cephalosporins like cefpodoxime, cefprozil,
ceftbuten, ceftizoxime, ceftriaxone, cephalothin, cephapirin,
cephalexin, cephradrine, cefoxitin, cefamandole, cefazolin,
cephaloridine, cefaclor, cefadroxil, cephaloglycin, cefuroxime,
ceforanide, cefotaxime, cefatrizine, cephacetrile, cefepime,
cefixime, cefonicid, cefoperazone, cefotetan, cefmetazole,
ceftazidime, loracarbef, and moxalactam, monobactams like
aztreonam; bactericidal/permeability increasing protein (BPI),
anti-CMV antibody, 13-cis retinoic acid, macrolides such as
erythromycin, oleandomycin, troleandomycin, roxithromycin,
clarithromycin, davercin, azithromycin, flurithromycin,
dirithromycin, josamycin, spiramycin, midecamycin, leucomycin,
miocamycin, rokitamycin, andazithromycin, and swinolide A;
fluoroquinolones such as ciprofloxacin, ofloxacin, levofloxacin,
trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin, enoxacin,
grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin,
temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin,
prulifloxacin, irloxacin, pazufloxacin, clinafloxacin, and
sitafloxacin, aminoglycosides such as gentamicin, netilmicin,
paramecin, tobramycin, amikacin, kanamycin, neomycin, and
streptomycin, vancomycin, teicoplanin, rampolanin, mideplanin,
colistin, daptomycin, gramicidin, colistimethate, polymixins such
as polymixin B, capreomycin, bacitracin, penems; penicillins
including penicllinase-sensitive agents like penicillin G,
penicillin V, penicllinase-resistant agents like methicillin,
oxacillin, cloxacillin, dicloxacillin, floxacillin, nafcillin; gram
negative microorganism active agents like ampicillin, amoxicillin,
and hetacillin, cillin, and galampicillin; and carbapenems such as
imipenem, meropenem, pentamidine isethiouate, albuterol sulfate,
lidocaine, metaproterenol sulfate, beclomethasone diprepionate,
triamcinolone acetamide, budesonide acetonide, fluticasone,
ipratropium bromide, flunisolide, cromolyn sodium, ergotamine
tartrate and where applicable, analogues, agonists, antagonists,
inhibitors, and pharmaceutically acceptable salt forms of the above
an antibiotic, a blood factor, a hormone, a growth factor, another
therapeutic peptide or protein, or a monoclonal antibody or a small
molecule. Suitably, the agent to be modified may be selected from
the group consisting of Factor VII, Factor VIII, Factor IX, Factor
X, Factor Xa, Factor XI, Factor VIIa, Factor V, Factor XIII, von
Willebrand's Factor and Protein C. In some embodiments the blood
coagulation factor is suitably Factor VII, Factor VIII or Factor
IX.
[0055] The agent in accordance with the invention may be modified
by any biocompatible polymer, such as polyethylene glycol (PEG),
poly-phosphatidyl choline (PC), polypropylene glycol (PPG),
copolymers of ethylene glycol and propylene glycol, polyethylene
oxide (PEO), polyoxyethylated polyol, polyolefinic alcohol,
polyhydroxyalkylmethacrylate, polysaccharides, poly .alpha.-hydroxy
acid, polyvinyl alcohol, polyphosphosphasphazene, poly
N-acryloylmorpholine, polyalkyene oxide polymers, polymaleic acid,
poly DL-alanine, carboxymethylcellulose, dextran, starch or starch
derivatives, hyaluronic acid, chitin, polymethacrylates, polysialic
acid (PSA), polyhydroxy alkanoates, poly amino acids and
combinations thereof. The biocompatible polymer may have a linear
or branched structure.
[0056] In a further embodiment, the biocompatible polymer is a
protein selected from, but not limited to, the group consisting of
albumin, transferrin, immunoglobulins including monoclonal
antibodies, antibody fragments for example; single-domain
antibodies, V.sub.L, V.sub.H, Fab, F(ab').sub.2, Fab', Fab3, scFv,
di-scFv, sdAb, Fc and combinations thereof.
[0057] In some embodiments the increased hydrophilicity/solubility
of the modified therapeutic agent delivered subcutaneously enables
that agent to be constituted in a higher concentration in a
delivery medium than if delivered intravenously. In the case where
the drug product is administered by injection, this may enable a
smaller injection volume to be used, which is more suitable to
subcutaneous administration. In addition, at higher concentrations,
where an unmodified agent might be expected to auto-catalyze, the
modification prevents the agent from auto-digestion, which in the
unmodified form might have led to undesirable, dangerous
by-products. For example, unmodified blood factor IX will
auto-catalyze at high concentrations to produce factor IXa, which
is dangerously thrombogenic.
[0058] Accordingly, in another aspect of the present invention, the
subcutaneous delivery volume of the modified therapeutic agent is
no more than 2 ml. Suitably, the delivery volume may be 5 .mu.l, 10
.mu.l, 25 .mu.l, 50 .mu.l, 100 .mu.l, 250 .mu.l, 500 .mu.l, 750
.mu.l, or 1 ml. In alternative embodiments the delivery volume of
the agent may be no more than 1.5 ml, 2 ml, 2.5 ml, 3.0 ml or 3.5
ml. It is important to note that the present invention allows for a
higher concentration of an active agent to be delivered in a single
subcutaneous injection more safely than by intravenous injection,
since it is not delivered directly into the bloodstream of the
patient. This is particularly important when dealing with blood
clotting factors, since high concentration of blood clotting
factors administered intravenously can result in undesirable and
dangerous blood clots in the patient. Subcutaneous delivery allows
the steady infusion of the active agent into the blood stream via
the lymphatic system, thus avoiding the effect of dangerous levels
of an active agent being delivered directly into the blood system.
Therefore, since the concentration of delivery of the agent into
the blood stream is regulated by the lymph system of the patient, a
higher concentration may be delivered in a subcutaneous
administration dose, which allows for smaller volumes to be used
than traditionally used with intravenous delivery.
[0059] Within the scope of the present invention is included
therapeutic agents that are able to be modified by hydrophilic
modification to increase hydrophilicity and modify molecular
dimensions in order to prevent direct entry into the vascular
system through the blood vessel walls and that are administrable to
the patient via subcutaneous delivery, in order to reach the
circulatory system via the lymphatic system. Methods of modifying
such agents are also included in the invention.
[0060] The dosage forms of the invention may be for administration
at least once per day, at least twice per day, about once per week,
about twice per week, about once per two weeks, or about once per
month. The ability to modulate the release rate of the modified
therapeutic agent from the subcutaneous depot means that the
administration may be controlled more conveniently.
[0061] For certain therapeutic substances, a dosage regime of once
per day will be sufficient, but for others a more frequent dosage
regime may be more appropriate or desirable, where the amount
delivered in each dosage administered subcutaneously may be reduced
relative to a standard intravenous dosage. So for example a dosage
form of the invention may be administered once per day, twice per
day (or more if required).
[0062] The present invention allows the prevention of the rapid
rise and subsequent fall (i.e. a "sawtooth") in the concentration
of an agent in the blood. The present invention provides a more
consistent, predictable concentration of the agent in the blood of
a patient over a longer period of time than is traditionally seen
with unmodified agents or the same modified product when repeatedly
delivered intravenously.
[0063] A further benefit of the present invention is that it
enables a higher dose of the agent to be administered
subcutaneously than may be safely administered intravenously. This
results in the provision of a longer duration of the therapeutic
benefit than could ordinarily and safely be achieved by higher
dosing or more frequent dosing via intravenous delivery. For
example, in the case of blood factors, because the products are
being delivered via the thoracic duct into the subclavian vein, the
method enables a larger amount of product to be administered at a
single time point as a single dose subcutaneously than could be
administered at a single time point intravenously into a vein.
Delivery of a high dose bolus into a vein may cause an undesirable
thrombotic event.
[0064] A further benefit of the present invention enables the agent
to be re-dosed at intervals to allow blood concentration of the
agent to be maintained at a consistent level, providing a sustained
constant and predictable therapeutic effect without the need to
wait to re-dose until the concentration of the agent in the blood
falls to therapeutically irrelevant levels. In traditional
practice, intravenous re-dosing, with its immediate C.sub.max and
onset of action, is delayed until it has been estimated that the
level of the therapeutic has dropped to a level at which the
addition of the C.sub.max from the new injection will not reach a
potentially thrombogenic level (i.e. reducing the risk of an
adverse event), but which means that the patient has reached an
"unhealthy" range of a level of an agent in his or her bloodstream.
In other words, subsequent doses of an agent are not normally given
to the patient while "healthy levels", or therapeutically effective
levels, of the agent are still present in the bloodstream. However,
the present invention enables re-dosing of the agent to occur while
blood levels of the agent are still in a therapeutic effective
range, thus the invention provides for a more consistent
therapeutic level of protein in the bloodstream, that is more
ideally suited to prophylaxis. Due to the consistent delivery of
the agent into the bloodstream via the thoracic duct, the problem
of increasing the agent in the bloodstream to undesirably high
levels is avoided.
[0065] According to an aspect of the invention, there is provided a
dosage form of a pharmaceutical composition of a modified blood
coagulation factor for subcutaneous administration which when
formulated for subcutaneous administration to a subject provides a
no more than once per month dosage form sufficient to maintain a
whole blood clotting time in said subject of no more than 20
minutes. Also provided is a liquid dosage form of a PEGylated blood
coagulation factor for subcutaneous administration no more than
once per month wherein the dosage form has a C.sub.max of at least
10% and no more than 90% compared to an equivalent reference dosage
form when administered intravenously, for use in the treatment of a
blood clotting disorder. Suitably, the C.sub.max is from 20% to
80%, or from 30% to 70%, or from 40% to 60%. Suitably, the blood
coagulation factor may be FVII, FVIII, or FIX.
[0066] By "no more than" it is meant that the dosage form may be
administered more frequently than the time period specified, but it
is not necessary to do so; the effect of the subcutaneous
administration of such a dosage form means that the effects are
seen for the duration of the time period. However, due to the lower
and consistent C.sub.max, more frequent dosing may occur without
adverse effects to the patient.
[0067] Suitably, the dosage form of a blood clotting factor may be
sufficient to maintain a whole blood clotting time in said subject
of less than 15 minutes, or suitably, less than 12 minutes. In an
embodiment, the dosage form of a blood clotting factor is an at
least once per week dosage form, or at least once per month, at
least once per two weeks, at least once per half week dosage
form.
[0068] A dosage form according to the invention may comprise a
blood clotting factor selected from the group consisting of Factor
VIIa, Factor VII, Factor VIII, Factor IX, Factor X, Factor Xa,
Factor XI, Factor XIII, Factor V, von Willebrand's Factor and
Protein C. Suitably, the blood clotting factor may be FVII, FVIII,
or FIX.
[0069] The dosage form of the invention may be modified by any
biocompatible polymer, as defined herein. Suitably, the
modification is PEGylation.
[0070] The dosage form may have a C.sub.max of at least 10% and no
more than 90% compared to an equivalent reference dosage form when
administered intravenously. In particular embodiments, the dosage
form may have a C.sub.max of from 10% to 25% compared to an
equivalent reference dosage form when administered intravenously.
In particular embodiments, the dosage form may have a C.sub.max of
from 40% to 60% compared to an equivalent reference dosage form
when administered intravenously. In particular embodiments, the
dosage form may have a C.sub.max of from 75% to 80% compared to an
equivalent reference dosage form when administered intravenously.
In particular embodiments, the dosage form may have a C.sub.max of
75% or of 78.8% compared to an equivalent reference dosage form
when administered intravenously. In one embodiment, C.sub.max is 75
to 80% and the blood factor may be FVII. In another embodiment
C.sub.max is 10% to 25% and the blood factor may be FVIII. In yet
another embodiment C.sub.max is 40% to 60% and the blood factor may
be FIX.
[0071] Also provided is a dosage formulation according to the
invention, in which the dosage is of from 1 to 1000 IU/kg, or from
5 to 500 IU/kg, or from 100 to 250 IU/kg or from 25 to 50
Ill/kg.
[0072] The dosage form of the present invention allows for a less
frequent dosing of the dosage form, which is still sufficient to
maintain the whole blood clotting time in a subject of no more than
20 minutes, or no more than 15 minutes, or no more than 10 minutes.
In one embodiment, the dosage form is sufficient to maintain whole
blood clotting time of less than 12 minutes. The dosage form may
provide a no more than once a fortnight, no more than once a week,
no more than twice a week, no more than once every three days, no
more than once every 2 days, no more than once a day or a more or
less frequent dosage form.
[0073] It is important to note that one benefit of the present
invention is that the dosage form when the agent is a blood
clotting factor, does not need to be administered to the patient
more frequently than these intervals in order to continue to
maintain whole blood clotting time in a healthy range, but it may
be administered more frequently in order to help to provide a
"steady state" similar to that of a controlled release formulation.
A `normal` whole blood clotting time is generally considered by one
skilled in the art to be 10 to 12 minutes, and anything under 15
minutes is considered to be healthy in a non-haemophiliac human.
Once whole blood clotting time is over 20 minutes, it is considered
to be in an unhealthy range. Between 15 and 20 minutes is
considered to indicate that although bleeding is under control, it
is not normal.
[0074] In another embodiment the dosage form is administered less
frequently than would be predicted by the plasma half life of a
bolus intravenous injection. For example, a bolus injection of
modified Factor IX may be required once a week, whereas the same
agent delivered subcutaneously in accordance with the invention,
may only be required once per ten days, or less.
[0075] According to a further aspect of the invention, there is
provided a dosage form of a pharmaceutical composition of 25 to 50
IU/kg of a modified blood coagulation factor for subcutaneous
administration at the same or with less frequency than the blood
coagulation factor administered intravenously.
[0076] Formulations of the present invention are therefore able to
maintain a normal value for haemostasis of up to seven days in
which a normal value is defined as a Whole Blood Clotting Time
(WBCT) of less than 15 minutes, suitably, about 12 minutes or
less.
[0077] The formulations of the invention have a C.sub.max of at
least 10%, to no more than 90% compared to an equivalent reference
dosage form when administered intravenously. In some embodiments of
the invention, the value may be at least 75%, 78% or 80%, and the
blood factor may be FVII. In some embodiments of the invention, the
value may be at least 15%, 18% or 20% and the blood factor may be
FVIII. In some embodiments of the invention the value may be 40%,
45% or 50% and the blood factor may be FIX.
[0078] The formulations of specific embodiments of the invention
wherein the modified agent is a PEGylated blood factor when
formulated for subcutaneous administration no more than once per
month comprise a dosage of from 25 to 50 IU/kg. In some embodiments
the dosage may be 25, 30, 35, 40, 45, or 50 IU/kg. The dosage may
be from 25 IU/Kg to 30 IU/Kg, 35 IU/Kg to 40 IU/Kg, or 40 IU/Kg to
50 IU/Kg.
[0079] In one embodiment, when the dosage form is prepared as a
dose of 150 IU/Kg, the formulation may be suitable for
administration once every two weeks to a subject in need thereof.
Suitably, the formulation may be for administration no more than
once every two weeks.
[0080] According to an embodiment of the invention, a dosage form
of a modified blood coagulation factor when formulated for
subcutaneous administration can result in normal haemostasis being
maintained for at least one half of a week.
[0081] Dosage forms in accordance with the invention, when
administered subcutaneously result in lower amounts of the modified
blood coagulation (clotting) factor being required to achieve the
same therapeutic end-point thus providing safer products for
subjects in need of treatment. In one embodiment half the adjusted
dose of modified blood clotting factor administered intravenously
is sufficient to achieve normal haemostasis for at least one week
in subjects, particularly wherein the blood coagulation factor is
Factor VIIa or Factor VIII. A suitable value for normal haemostasis
is a Whole Blood Clotting Time (WBCT) of about 12 minutes, as
described above.
[0082] Formulations of the invention may suitably comprise less
than half the dose adjusted therapeutically effective amount of a
reference formulation formulated for intravenous administration
comprising the same modified blood coagulation factor in order to
achieve the same therapeutic effect. For example, in an embodiment
wherein the blood coagulation factor is Factor IX.
[0083] The invention therefore also provides for a dosage form of a
modified blood coagulation factor for subcutaneous administration
in which the dosage form comprises 50% of the dose adjusted amount
required for intravenous administration in order to achieve the
same duration of effective action.
[0084] A formulation suitable for subcutaneous administration may
suitably be prepared as an aqueous or substantially aqueous
formulation. The formulation may comprise such additional salts,
preservatives and stabilisers and/or excipients or adjuvants as
required. The dosage forms of the invention may be provided as
anhydrous powders ready for extemporaneous formulation in a
suitable aqueous medium.
[0085] It may be generally preferred to formulate such dosage forms
as a buffered aqueous formulation. Suitable buffer solutions may
include, but are not limited to amino acids (for example
histidine), salts of inorganic acids and alkali metals or alkaline
earth metals, (for example sodium salts, magnesium salts, potassium
salts, lithium salts or calcium salts--exemplified as sodium
chloride, sodium phosphate). Other components such as detergents or
emulsifiers (for example, Tween 80.RTM. or any other form of
Tween.RTM.) may be present and stabilisers (for example benzamidine
or a benzamidine derivative). Excipients such as sugars, (for
example sucrose) may also be present. Suitable values for pH are
physiological pH, e.g. pH 6.8 to 7.4. Liquid dosage forms may be
prepared ready for use in such administration vehicles.
[0086] A "modified blood coagulation factor" is a blood coagulation
factor (blood clotting factor) which has been linked to one or more
modifying agents as described above. In some embodiments, the
modification is PEG. The PEG molecule may be linked directly or
indirectly to the blood coagulation factor. The PEGylated blood
coagulation factor can also be defined as a "blood coagulation
factor conjugated to a PEG molecule" or a "blood coagulation
factor-PEG conjugate".
[0087] Modified blood coagulation factors (blood clotting factors)
suitably comprise at least one of Factor VII, Factor VIII, Factor
IX, Factor X, Factor Xa, Factor XI, Factor VIIa, Factor V, Factor
XIII, von Willebrand's Factor and Protein C. In some embodiments
the blood coagulation factor is suitably Factor VII, Factor VIII or
Factor IX.
[0088] As used herein, the term "blood factor conjugate" refers to
a blood clotting factor protein that has been modified to include a
modification, such as a PEG moiety, other conjugated moiety as
defined above.
[0089] The terms Factor VIIa (FVIIa) and Factor VII (FVII) are also
used interchangeably unless the context specifies otherwise. FVIII
is used as an abbreviation for Factor VIII and FIX is used as an
abbreviation for Factor IX, and so on for blood factors described
herein.
[0090] The blood coagulation (clotting) factor may be from any
suitable source and may be a recombinant protein produced by
recombinant DNA technology using molecular biological techniques or
synthesised chemically or produced transgenically in the milk of a
mammal, or the factor may be isolated from natural sources (e.g.
purified from blood plasma). Suitably the factor is a mammalian
blood clotting factor, such as a human blood clotting factor.
References to a blood clotting factor include a blood coagulation
factor.
[0091] As indicated herein the present invention relates to
formulations of blood clotting factors which have been modified by
conjugation with one or more modifying agents, such as polyethylene
glycol polymers ("PEGylation"). The modification of the blood
clotting factor may be by any convenient means.
[0092] Tween.RTM. is currently extensively used in the formulation
of blood products. Tween.RTM. 80 is a PEGylated fatty acid which
carries a molecular weight equivalent of PEG of approximately 0.8
kilo Daltons per Tween.RTM. molecule.
[0093] As discussed above, blood factors are all characterised
inter alia by the property of surface adhesion. This is a necessary
feature of the coagulation cascade which requires that enzymes and
cofactors adhere to other participants in the cascade, to the
surface of platelets and to tissue at the site of injury. Indeed it
is particularly important that a blood clot remains at the site of
injury and does not drift to cause a dangerous thrombosis. This
property presents a challenge in the formulation of drug products,
since blood factors such as VIIa VIII and IX will adhere
excessively to any glass and plastic surfaces. In practical terms
this is mitigated by the extensive use of polysorbate (e.g.
Tween.RTM. 80).
[0094] In one embodiment of the present invention, FVIII has a 20
kDa straight chain polyethylene glycol moiety conjugated to it. The
conjugation of PEG mitigates the surface adhesion property of this
factor to the extent that no further use of Tween.RTM. is
necessary.
[0095] When activated in the process of coagulation, PEG-FVIII
still adheres to the surface of platelets and is a small component
in the overall clotting process. In this regard, blood clots will
form in the normal manner on platelets at the site of injury.
[0096] By having a mono-PEGylated factor and thereby obviating the
requirement for additional Tween.RTM. in the formulation, a
decrease in the amount of polyethylene glycol can be achieved. A
calculation using Kogenate.RTM. FS (Bayer FVIII) was performed to
identify the total amount of PEG per mol of FVIII used in the
formulation and make a comparison to a single conjugated 20 kDa
moiety which does not require any further Tween.RTM. in its
formulation. Thus, on a dose-for-dose basis, an embodiment of the
present invention provides a 25.8 fold reduction in polyethylene
glycol, which, when the reduced frequency of dosing is also taken
into account, may result in an overall reduction in the
administration of PEG of approximately 80-fold.
[0097] The present inventors have found that increasing the
water-carrying capability of the target therapeutic (for example
via di-PEGylating a product versus mono-PEGylating it), the passage
of the product into the bloodstream, following subcutaneous
administration, can be accelerated. Conversely, decreasing the
water-carrying capability (for example mono-PEGylating the products
versus di-PEGylating it), the passage of the product into the
bloodstream, following subcutaneous administration, can be slowed,
giving a depot effect. Without wishing to be bound by theory, it
would appear that the same product with a lesser water-carrying
ability (e.g. via mono-PEGylation or with a smaller PEG molecule)
resists being dispersed through the subcutaneous space for longer
than the same product modified to have a greater water-carrying
capability (e.g. via multi-PEGylation or the attachment of a larger
PEG molecule), thus providing the enhanced depot effect.
[0098] Without wishing to be bound by theory, designing a product
to have a greater water carrying characteristic (for example by
increasing its PEG coverage via di- or multi-PEGylation, increasing
the size of the PEG or using branched vs. straight PEG molecules)
would seem to render it more water dispersible within the
subcutaneous space, leading to a faster rate of entry via the
lymphatic vessels into the plasma; the reduced hydrophilicity of
products designed to have a lesser water-carrying characteristic
(for example via mono-PEGylation or via the use of smaller PEG
molecules), would seem to leave more of the hydrophobic therapeutic
agent exposed reducing its dispersibility and slowing its entry
into the plasma via the aqueous lymphatic system.
[0099] This ability to modify the dispersion characteristics of a
molecule for subcutaneous administration, by selectively adjusting
the balance between hydrophilicity and hydrophobicity, provides an
exquisite degree of control over the controlled release of a
product from the subcutaneous space to the plasma via the lymph,
which may be adjusted according to the characteristics of the
therapeutic agent, the needs and physiology of the patient or a
combination of these or other influencing factors.
[0100] In some embodiments, when the modification is PEG, the
polyethylene glycol (PEG) may have a linear or branched structure
and may be attached to the therapeutic agent via any convenient
route. Where the therapeutic agent is a protein, e.g. a blood
clotting factor or other therapeutic protein as described herein,
conjugation of PEG may be via a serine or threonine residue in the
native protein, via a hydroxyl residue on a sugar residue attached
to the native protein, or via one or more cysteine residues. The
PEG moiety may be attached via such residues which occur in the
native or the recombinant forms of the protein. Proteins made by
recombinant expression allow for site specific engineering to
insert desired amino acid residues into a protein sequence and/or
to control patterns of glycosylation with specific glycosylase
enzymes. Other routes for PEGylation include amide or N-terminal
amino group PEGylation, or carboxyl group PEGylation.
[0101] The PEG moiety may also be conjugated to the blood clotting
factor, i.e. Factor VII, Factor VIII, Factor IX, Factor X, Factor
Xa, Factor XI, Factor VIIa, Factor V, Factor XIII, von Willebrand's
Factor or Protein C, via one or more reduced cysteine disulphide
bonds. A free cysteine residue is the result of reducing a cystine
disulphide bond in the protein. For example, the conjugation may be
by means of a linker group bridging the sulphur residues of two
cysteine residues that formed a disulphide bond in Factor VII,
Factor VIII, Factor IX, Factor X, Factor Xa, Factor XI, Factor
VIIa, Factor V, Factor XIII, von Willebrand's Factor or Protein C.
The disulphide bond may therefore be a native disulphide bond or a
recombinantly introduced disulphide bond.
[0102] In one embodiment of the invention, the hydrophilic moiety,
such as the polyethylene glycol chain is attached via a bivalent
linker moiety across two cysteine residues that normally form a
disulphide bridge in the native form of the blood clotting
factor.
[0103] The PEG molecule may be of any suitable molecular weight,
for example from 1 kDa to 100 kDa, 10 to 500 kDa, suitably 5 to 30
kDa or 20 to 30 kDa. Some suitable molecular weights include 5, 10,
20, or 30 kDa. Suitably, the PEG molecule may be from 5 kDa to 40
kDa.
[0104] There are several different types of polyethylene glycol
polymers that will form conjugates with Factor VII, Factor VIII,
Factor IX, Factor X, Factor Xa, Factor XI, Factor VIIa, Factor V,
Factor XIII, von Willebrand's Factor or Protein C. There are linear
PEG polymers that contain a single polyethylene glycol chain, and
there are branched or multi-arm PEG polymers. Branched polyethylene
glycol contains 2 or more separate linear PEG chains bound together
through a unifying group. For example, two PEG polymers may be
bound together by a lysine residue. One linear PEG chain is bound
to the .alpha.-amino group, while the other PEG chain is bound to
the .epsilon.-amino group. The remaining carboxyl group of the
lysine core is left available for covalent attachment to a protein.
Both linear and branched polyethylene glycol polymers are
commercially available in a range of molecular weights.
[0105] In one embodiment of the invention, the Factor VII, Factor
VIII, Factor IX, Factor X, Factor Xa, Factor XI, Factor VIIa,
Factor V, Factor XIII, von Willebrand's Factor or Protein
C-conjugate contains one or more linear polyethylene glycol
polymers bound to Factor VII, Factor VIII, Factor IX, Factor X,
Factor Xa, Factor XI, Factor VIIa, Factor V, Factor XIII, von
Willebrand's Factor and Protein C. In some embodiments the blood
coagulation factor is Factor III, Factor VIII or Factor IX, in
which each PEG has a molecular weight between about 2 kDa to about
100 kDa. In another aspect of the invention, a Factor VII, Factor
VIII, Factor IX, Factor X, Factor Xa, Factor XI, Factor VIIa,
Factor V, Factor XIII, von Willebrand's Factor or Protein
C-conjugate contains one or more linear polyethylene glycol
polymers bound to Factor VII, Factor VIII, Factor IX, Factor X,
Factor Xa, Factor XI, Factor VIIa, Factor V, Factor XIII, von
Willebrand's Factor or Protein C, wherein each linear PEG has a
molecular weight between about 1 kDa to about 40 kDa. In certain
embodiments, each linear PEG has a molecular weight between about
10 kDa to about 30 kDa. In certain embodiments, each linear PEG has
a molecular weight that is about 20 kDa. In certain embodiments,
each linear PEG has a molecular weight that is about 10 kDa. In
certain embodiments, each linear PEG has a molecular weight that is
less than 10 kDa. In particular embodiments, where the blood factor
conjugate contains more than one linear PEG polymers bound to a
blood coagulation factor, for example two, three, or up to eight
linear PEG polymers bound to Factor VII, Factor VIII, Factor IX,
Factor X, Factor Xa, Factor XI, Factor VIIa, Factor V, Factor XIII,
von Willebrand's Factor or Protein C. In some embodiments, the
blood factor conjugates contain multiple linear PEG polymers, where
each linear PEG has a molecular weight of about 5-30 kDa.
[0106] A blood factor conjugate of this invention may contain one
or more branched PEG polymers bound to Factor VII, Factor VIII,
Factor IX, Factor X, Factor Xa, Factor XI, Factor VIIa, Factor V,
Factor XIII, von Willebrand's Factor or Protein C, wherein each
branched PEG has a molecular weight between about 2 kDa to about
100 kDa. In another aspect of the invention, a blood factor
conjugate contains one or more branched polyethylene glycol
polymers bound to Factor VII, Factor VIII, Factor IX, Factor X,
Factor Xa, Factor XI, Factor VIIa, Factor V, Factor XIII, von
Willebrand's Factor or Protein C, wherein each branched PEG has a
molecular weight between about 1 kDa to about 100 kDa. In certain
embodiments, each branched PEG has a molecular weight between about
5 kDa to about 40 kDa. In certain embodiments, each branched PEG
has a molecular weight that is about 10 kDa, 20 kDa, or about 30
kDa. In certain embodiments, each branched PEG has a molecular
weight that is less than about 10 kDa. In particular embodiments,
where the blood factor conjugate contains more than one branched
PEG polymers bound to Factor VII, Factor VIII, Factor IX, Factor X,
Factor Xa, Factor XI, Factor VIIa, Factor V, Factor XIII, von
Willebrand's Factor or Protein C, for example two, three, or up to
eight branched PEG polymers bound to Factor VII, Factor VIII,
Factor IX, Factor X, Factor Xa, Factor XI, Factor VIIa, Factor V,
Factor XIII, von Willebrand's Factor or Protein C. In a some
embodiments, the Factor VII, Factor VIII, Factor IX, Factor X,
Factor Xa, Factor XI, Factor VIIa, Factor V, Factor XIII, von
Willebrand's Factor or Protein C-PEG conjugates contains up to
eight branched PEG polymers, where each branched PEG has a
molecular weight of about 5-40 kDa, suitably 10 to 30 kDa.
[0107] The blood factor-PEG conjugates may be purified by
chromatographic methods known in the art, including, but not
limited to ion exchange chromatography and size exclusion
chromatography, affinity chromatography, precipitation and
membrane-based separations.
[0108] Suitably, the PEG moiety of the Factor VII, Factor VIII,
Factor IX, Factor X, Factor Xa, Factor XI, Factor VIIa, Factor V,
Factor XIII, von Willebrand's Factor or Protein C-conjugate may be
bound to two cysteine residues, which form a disulphide bond in the
blood coagulation factor. Therefore, the PEG containing linker
bridges the disulphide bond. Examples of such conjugation
procedures are described in WO 2005/007197, WO 2009/047500 and WO
2010/010324.
[0109] As discussed above, other routes of PEGylation may include
standard glycoPEGylation procedures as described in Stennicke et al
(Thromb. Haemost. 2008, 100(5), 920-8), or N-terminal amide
PEGylation as described in U.S. Pat. No. 5,644,029.
[0110] In one embodiment of the invention, a PEG moiety can be
conjugated to Factor VII, Factor VIII, Factor IX, Factor X, Factor
Xa, Factor XI, Factor VIIa, Factor V, Factor XIII, von Willebrand's
Factor or Protein C according to the scheme set out in FIG. 2. In
FIG. 2, a group R1 is shown between the PEG moiety and the linker
group spanning the sulphur atoms of the disulphide bond on the
blood factor molecule.
[0111] R1 represents a substituent which can be a direct bond, an
alkylene group (preferably a C.sub.1-10 alkylene group), or an
optionally-substituted aryl or heteroaryl group; wherein the aryl
groups include phenyl, benzoyl and naphthyl groups; wherein
suitable heteroaryl groups include pyridine, pyrrole, furan, pyran,
imidazole, pyrazole, oxazole, pyridazine, pyrimidine and purine;
wherein linkage to the polymer may be by way of a hydrolytically
labile bond, or by a non-labile bond.
[0112] Particular substituents which may be present on the
optionally substituted aryl or heteroaryl group include for example
one or more of the same or different substituents selected from
--CN, --NO.sub.2, --CO.sub.2R, --COH, --CH.sub.2OH, --COR, --OR,
--OCOR, --OCO.sub.2R, --SR, --SOR, --SO.sub.2R, --NHCOR, --NRCOR,
--NHCO.sub.2R, --NR'CO.sub.2R, --NO, --NHOH, --NR'OH,
--C.dbd.N--NHCOR, --C.dbd.N--NR'COR, --N.sup.+R.sub.3,
--N.sup.+H.sub.3, --N.sup.+HR.sub.2, --N.sup.+H.sub.2R, halogen,
for example fluorine or chlorine, --C.ident.CR, --C.dbd.CR.sub.2
and .sup.13C.dbd.CHR, in which each R or R' independently
represents a hydrogen atom or an alkyl (preferably C.sub.1-6) or an
aryl (preferably phenyl) group. The presence of electron
withdrawing substituents is especially preferred. In one
embodiment, the optionally-substituted aryl or heteroaryl group in
R1 includes aryl or heteroaryl groups substituted by an amide
(NHCO) group which connects the R1 unit to the PEG moiety.
[0113] The linker group between the two sulphur atoms of the
original disulphide bond between the cysteine residues of Factor
VII, Factor VIII, Factor IX, Factor X, Factor Xa, Factor XI, Factor
VIIa, Factor V, Factor XIII, von Willebrand's Factor or Protein C
may therefore comprise a 3-carbon bridge. In one embodiment, the
linker group between the two sulphur atoms of the original
disulphide bond between the cysteine residues of Factor VII, Factor
VIII, Factor IX, Factor X, Factor Xa, Factor XI, Factor VIIa,
Factor V, Factor XIII, von Willebrand's Factor or Protein C is
(CH.sub.2).sub.2CHC(O)--.
[0114] In one embodiment of the invention, the PEG moiety may be
conjugated as described above wherein the composition comprising
Factor VII, Factor VIII, Factor IX, Factor X, Factor Xa, Factor XI,
Factor VIIa, Factor V, Factor XIII, von Willebrand's Factor or
Protein C conjugated to a PEG moiety has the structure:
##STR00001##
[0115] Where R1 is as defined above, and "Factor" represents a
blood clotting factor.
[0116] In embodiments where the optionally-substituted aryl or
heteroaryl group in R1 as defined above includes aryl or heteroaryl
groups substituted by an amide (NHCO) group, the structure of the
conjugate protein, where R3 is as defined below, may be as
follows:
##STR00002##
[0117] R3 represents a substituent which can be a direct bond, an
alkylene group (preferably a C.sub.1-10 alkylene group), or an
optionally-substituted aryl or heteroaryl group; wherein the aryl
groups include phenyl, benzoyl and naphthyl groups; wherein
suitable heteroaryl groups include pyridine, pyrrole, furan, pyran,
imidazole, pyrazole, oxazole, pyridazine, pyrimidine and purine;
wherein linkage to the polymer may be by way of a hydrolytically
labile bond, or by a non-labile bond, and "Factor" represents a
blood clotting factor.
[0118] Particular substituents which may be present on the
optionally substituted aryl or heteroaryl group include for example
one or more of the same or different substituents selected from
--CN, --NO.sub.2, --CO.sub.2R, --COH, --CH.sub.2OH, --COR, --OR,
--OCOR, --OCO.sub.2R, --SR, --SOR, --SO.sub.2R, --NHCOR, --NRCOR,
--NHCO.sub.2R, --NR'CO.sub.2R, --NO, --NHOH, --NR'OH,
--C.dbd.N--NHCOR, --C.dbd.N--NR'COR, --N.sup.+R.sub.3,
--N.sup.+H.sub.3, --N.sup.+HR.sub.2, --N.sup.+H.sub.2R, halogen,
for example fluorine or chlorine, --C.ident.CR, --C.dbd.CR.sub.2
and .sup.13C.dbd.CHR, in which each R or R' independently
represents a hydrogen atom or an alkyl (preferably C.sub.1-6) or an
aryl (preferably phenyl) group. The presence of electron
withdrawing substituents is especially preferred.
[0119] In some embodiments, dosage forms of the present invention
may be composed of PEGylated forms of blood clotting factors as
defined herein in which the polyethyleneglycol molecule is a
straight-chain, (suitably mono-disperse) form. The PEG may be
conjugated to the blood clotting factor via a three carbon bridge
moiety. For example, the PEG may be 1 to 100 kDa; in some
embodiments, 5 to 30 kDa; in some embodiments 10 kDa and in other
embodiments 20 kDa.
[0120] The dosage form may be prepared for subcutaneous
administration by formulation in a suitable aqueous vehicle. In
most embodiments, the suitable aqueous solution is buffered to
physiological pH (for example to pH 6.8) with a composition
comprising one or more amino acids and/or salts (for example
histidine and NaCl) and in the presence of a non-ionic surfactant
(for example Tween.RTM. 80) and optionally a stabiliser (for
example benzamidine or a benzamidine derivative, see U.S. Pat. No.
7,612,066 for example).
[0121] Nonionic surfactants/emulsifiers which can be used according
to the present invention include polysorbates such as
polyoxyethylene sorbitan monooleate (polysorbate 80, Tween.RTM.
80), polysorbate 65, polysorbate 65, polysorbate 61, polysorbate
60, polysorbate 40, polysorbate 21, polysorbate 20, polysorbate 81,
polysorbate 85, and polysorbate 120, and polyoxyethylene stearates
such as polyoxyl 8 stearate (PEG 400 monostearate), polyoxyl 2
stearate, polyoxyl 4 stearate, polyoxyl 6 stearate, polyoxyl 12
stearate, polyoxyl 20 stearate, polyoxyl 30 stearate, polyoxyl 40
stearate, polyoxyl 50 stearate, polyoxyl 100 stearate, polyoxyl 150
stearate, and polyoxyl 4 distearate, polyoxyl 8 distearate,
polyoxyl 12 distearate, polyoxyl 32 distearate, polyoxyl 150
distearate.
[0122] Suitable concentration ranges for the components in the
composition may be for example 5 mM to 25 mM histidine (suitably 10
mM to 15 mM histidine), 10 mM to 50 mM NaCl (suitably 30 mM to 40
mM NaCl) and 0.001 to 0.01% Tween.RTM. 80 (suitably 0.005% to
0.008% Tween.RTM. 80) and optionally 0.5 mM to 5 mM benzamidine
(suitably 1 mM to 2 mM benzamidine).
[0123] As used herein the term "muteins" refers to analogs of an
Factor VII, Factor VIII, Factor IX, Factor X, Factor Xa, Factor XI,
Factor VIIa, Factor V, Factor XIII, von Willebrand's Factor or
Protein C, in which one or more of the amino acid residues of the
naturally occurring components of Factor VII, Factor VIII, Factor
IX, Factor X, Factor Xa, Factor XI, Factor VIIa, Factor V, Factor
XIII, von Willebrand's Factor or Protein C are replaced by
different amino acid residues, or are deleted, or one or more amino
acid residues are added to the original sequence of a blood factor,
without changing considerably the activity of the resulting
products as compared with the original blood factor. These muteins
are prepared by known synthesis and/or by site-directed mutagenesis
techniques, or any other known technique suitable therefore.
[0124] Muteins in accordance with the present invention include
proteins encoded by a nucleic acid, such as DNA or RNA, which
hybridizes to DNA or RNA, which encodes an Factor VII, Factor VIII,
Factor IX, Factor X, Factor Xa, Factor XI, Factor VIIa, Factor V,
Factor XIII, von Willebrand's Factor or Protein C in accordance
with the present invention, under stringent conditions. The term
"stringent conditions" refers to hybridization and subsequent
washing conditions, which those of ordinary skill in the art
conventionally refer to as "stringent" (Ausubel et al., Current
Protocols in Molecular Biology, Interscience, N.Y., sections 63 and
6.4 (1987, 1992); Sambrook et al. (Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y (1989)).
[0125] Without limitation, examples of stringent conditions include
washing conditions 12-20.degree. C. below the calculated Tm of the
hybrid under study in, e.g., 2.times.SSC and 0.5% SDS for 5
minutes, 2.times.SSC and 0.1% SDS for 15 minutes; 0.1.times.SSC and
0.5% SDS at 37.degree. C. for 30-60 minutes and then, a
0.1.times.SSC and 0.5% SDS at 68.degree. C. for 30-60 minutes.
Those of ordinary skill in this art understand that stringency
conditions also depend on the length of the DNA sequences,
oligonucleotide probes (such as 10-40 bases) or mixed
oligonucleotide probes. If mixed probes are used, it is preferable
to use tetramethyl ammonium chloride (TMAC) instead of SSC.
[0126] Any such mutein preferably has a sequence of amino acids
sufficiently duplicative of that of an Factor VII, Factor VIII,
Factor IX, Factor X, Factor Xa, Factor XI, Factor VIIa, Factor V,
Factor XIII, von Willebrand's Factor or Protein C such as to have
substantially similar, or even better, activity to Factor VII,
Factor VIII, Factor IX, Factor X, Factor Xa, Factor XI, Factor
VIIa, Factor V, Factor XIII, von Willebrand's Factor or Protein
C.
[0127] One characteristic activity of Factor VII, Factor VIII,
Factor IX, Factor X, Factor Xa, Factor XI, Factor VIIa, Factor V,
Factor XIII, von Willebrand's Factor or Protein C is its capability
of participate in the blood coagulation cascade and assays to
detect Factor VII, Factor VIII, Factor IX, Factor X, Factor Xa,
Factor XI, Factor VIIa, Factor V, Factor XIII, von Willebrand's
Factor or Protein C. As long as the mutein has substantial blood
factor activity, it can be considered to have substantially similar
activity to blood factor. Thus, it can be determined whether any
given mutein has at least substantially the same activity as Factor
VII, Factor VIII, Factor IX, Factor X, Factor Xa, Factor XI, Factor
VIIa, Factor V, Factor XIII, von Willebrand's Factor or Protein C
by means of routine experimentation comprising subjecting such a
mutein to assays as described herein.
[0128] In a preferred embodiment, any such mutein has at least 40%
identity or homology with the amino acid sequence of Factor VII,
Factor VIII, Factor IX, Factor X, Factor Xa, Factor XI, Factor
VIIa, Factor V, Factor XIII, von Willebrand's Factor or Protein C.
More preferably, it has at least 50%, at least 60%, at least 70%,
at least 80% or, most preferably, at least 90%, 95%, 98% or 99%
identity or homology thereto.
[0129] Identity reflects a relationship between two or more
polypeptide sequences or two or more polynucleotide sequences,
determined by comparing the sequences. In general, identity refers
to an exact nucleotide to nucleotide or amino acid to amino acid
correspondence of the two polynucleotides or two polypeptide
sequences, respectively, over the length of the sequences being
compared.
[0130] For sequences where there is not an exact correspondence, a
"percent identity" may be determined. In general, the two sequences
to be compared are aligned to give a maximum correlation between
the sequences. This may include inserting "gaps" in either one or
both sequences, to enhance the degree of alignment. A percent
identity may be determined over the whole length of each of the
sequences being compared (so-called global alignment), that is
particularly suitable for sequences of the same or very similar
length, or over shorter, defined lengths (so-called local
alignment), that is more suitable for sequences of unequal
length.
[0131] Methods for comparing the identity and homology of two or
more sequences are well known in the art. Thus for instance,
programs available in the Wisconsin Sequence Analysis Package,
version 9.1 (Devereux, et al., Nucleic acids Research, 12: 387
(1984)), for example the programs BESTFIT and GAP, may be used to
determine the percentage identity between two polynucleotides and
the percentage identity and the percentage homology between two
polypeptide sequences. BESTFIT uses the "local homology" algorithm
of Smith and Waterman (Advances in Applied Mathematics, 2; 482-489
(1981)) and finds the best single region of similarity between two
sequences. Other programs for determining identity and/or
similarity between sequences are also known in the art, for
instance the BLAST family of programs (Atschul et al., J. Molec.
Biol., 215: 403 (1990), accessible through the home page of the
NCBI, at www.ncbi.nlm.nih.gov) and FASTA (Pearson W R, Methods in
Enzymology, 183: 63-98 (1990)).
[0132] Muteins of Factor VII, Factor VIII, Factor IX, Factor X,
Factor Xa, Factor XI, Factor VIIa, Factor V, Factor XIII, von
Willebrand's Factor or Protein C, which can be used in accordance
with the present invention include a finite set of substantially
corresponding sequences as substitution peptides which can be
routinely obtained by one of ordinary skill in the art, without
undue experimentation, based on the teachings and guidance
presented herein.
[0133] Preferred changes for muteins in accordance with the present
invention are what are known as "conservative" substitutions.
Conservative amino acid substitutions of Factor VII, Factor VIII,
Factor IX, Factor X, Factor Xa, Factor XI, Factor VIIa, Factor V,
Factor XIII, von Willebrand's Factor or Protein C, may include
synonymous amino acids within a group which have sufficiently
similar physicochemical properties that substitution between
members of the group will preserve the biological function of the
molecule. It is clear that insertions and deletions of amino acids
may also be made in the above-defined sequences without altering
their function, particularly if the insertions or deletions only
involve a few amino acids, e.g., under thirty, and preferably under
ten, and do not remove or displace amino acids which are critical
to a functional conformation, e.g., cysteine residues. Proteins and
muteins produced by such deletions and/or insertions come within
the scope of the present invention.
[0134] Thus the amino acids glycine, alanine, valine, leucine and
isoleucine can often be substituted for one another (amino acids
having aliphatic side chains). Of these possible substitutions it
is preferred that glycine and alanine are used to substitute for
one another (since they have relatively short side chains) and that
valine, leucine and isoleucine are used to substitute for one
another (since they have larger aliphatic side chains which are
hydrophobic). Other amino acids which can often be substituted for
one another include: phenylalanine, tyrosine and tryptophan (amino
acids having aromatic side chains); lysine, arginine and histidine
(amino acids having basic side chains); aspartate and glutamate
(amino acids having acidic side chains); asparagine and glutamine
(amino acids having amide side chains); and cysteine and methionine
(amino acids having sulphur containing side chains). Substitutions
of this nature are often referred to as "conservative" or
"semi-conservative" amino acid substitutions.
[0135] Amino acid changes relative to the sequence for the fusion
protein of the invention can be made using any suitable technique
e.g. by using site-directed mutagenesis.
[0136] It should be appreciated that amino acid substitutions or
insertions within the scope of the present invention can be made
using naturally occurring or non-naturally occurring amino acids.
Whether or not natural or synthetic amino acids are used, it is
preferred that only L-amino acids are present.
[0137] In addition fusion proteins comprising Factor VII, Factor
VIII, Factor IX, Factor X, Factor Xa, Factor XI, Factor VIIa,
Factor V, Factor XIII, von Willebrand's Factor or Protein C, fused
with another peptide or protein fragment may be also be used
provided that the fusion protein retains the activity of Factor
VII, Factor VIII, Factor IX, Factor X, Factor Xa, Factor XI, Factor
VIIa, Factor V, Factor XIII, von Willebrand's Factor or Protein C.
The term "fusion protein" in this text means, in general terms, one
or more proteins joined together by chemical means, including
hydrogen bonds or salt bridges, or by peptide bonds through protein
synthesis or both.
[0138] "Functional derivatives" as used herein cover derivatives of
Factor VII, Factor VIII, Factor IX, Factor X, Factor Xa, Factor XI,
Factor VIIa, Factor V, Factor XIII, von Willebrand's Factor or
Protein C, and their muteins, which may be prepared from the
functional groups which occur as side chains on the residues or are
additions to the N- or C-terminal groups, by means known in the
art, and are included in the invention as long as they remain
pharmaceutically acceptable, i.e. they do not destroy the activity
of the protein which is substantially similar to the activity of
blood factors, and do not confer toxic properties on compositions
containing it.
[0139] These derivatives may, for example, include aliphatic esters
of the carboxyl groups, amides of the carboxyl groups by reaction
with ammonia or with primary or secondary amines, N-acyl
derivatives of free amino groups of the amino acid residues formed
with acyl moieties (e.g. alkanoyl or carboxylic aroyl groups) or
O-acyl derivatives of free hydroxyl groups (for example that of
seryl or threonyl residues) formed with acyl moieties, including
for example glycosylation of available hydroxyl residues.
[0140] An "active fragment of blood factor" according to the
present invention may be a fragment of Factor VII, Factor VIII,
Factor IX, Factor X, Factor Xa, Factor XI, Factor VIIa, Factor V,
Factor XIII, von Willebrand's Factor or Protein C or a mutein as
defined herein. The term fragment refers to any subset of the
molecule, that is, a shorter peptide that retains the desired
biological activity. Fragments may readily be prepared by removing
amino acids from either end of the blood factor molecule and
testing the resultant fragment for its properties as described
herein. Proteases for removing one amino acid at a time from either
the N-terminal or the C-terminal of a polypeptide are known, and so
determining fragments, which retain the desired biological
activity, involves only routine experimentation.
[0141] As active fractions of an Factor VII, Factor VIII, Factor
IX, Factor X, Factor Xa, Factor XI, Factor VIIa, Factor V, Factor
XIII, von Willebrand's Factor and Protein C, muteins and active
fragments thereof, the present invention further covers any
fragment or precursors of the polypeptide chain of the protein
molecule alone or together with associated molecules or residues
linked thereto, e.g., sugar or phosphate residues, or aggregates of
the protein molecule or the sugar residues by themselves, provided
said fraction has substantially similar activity to Factor VII,
Factor VIII, Factor IX, Factor X, Factor Xa, Factor XI, Factor
VIIa, Factor V, Factor XIII, von Willebrand's Factor or Protein
C.
[0142] The term "salts" herein refers to both salts of carboxyl
groups and to acid addition salts of amino groups of the Factor
VII, Factor VIII, Factor IX, Factor X, Factor Xa, Factor XI, Factor
VIIa, Factor V, Factor XIII, von Willebrand's Factor or Protein C
molecule or analogs thereof. Salts of a carboxyl group may be
formed by means known in the art and include inorganic salts, for
example, sodium, calcium, ammonium, ferric or zinc salts, and the
like, and salts with organic bases as those formed, for example,
with amines, such as triethanolamine, arginine or lysine,
piperidine, procaine and the like. Acid addition salts include, for
example, salts with mineral acids, such as, for example,
hydrochloric acid or sulfuric acid, and salts with organic acids,
such as, for example, acetic acid or oxalic acid. Of course, any
such salts must retain the biological activity of blood factors as
described herein.
[0143] The "area under the curve" or "AUC", as used herein in the
context of administering a therapeutic agent to a patient, is
defined as total area under the curve that describes the
concentration of a drug in systemic circulation in the patient as a
function of time from zero to infinity. As used herein the term
"clearance" or "renal clearance" is defined as the volume of plasma
that contains the amount of drug excreted per minute.
[0144] As used herein the term "half-life" or "t1/2", in the
context of administering a peptide drug to a patient, is defined as
the time required for plasma concentration of a drug in a patient
to be reduced by one half. There may be more than one half-life
associated with the peptide drug depending on multiple clearance
mechanisms, redistribution, and other mechanisms well known in the
art. Usually, alpha and beta half-lives are defined such that the
alpha phase is associated with redistribution, and the beta phase
is associated with clearance. However, with protein drugs that are,
for the most part, confined to the bloodstream, there can be at
least two clearance half-lives. The precise impact of PEGylation on
alpha phase and beta phase half-lives will vary depending upon the
size and other parameters, as is well known in the art. Further
explanation of "half-life" is found in Pharmaceutical Biotechnology
(1997, DFA Crommelin and RD Sindelar, eds., Harwood Publishers,
Amsterdam, pp 101-120).
[0145] As used herein the term "residence time", in the context of
administering a peptide drug to a patient, is defined as the
average time that drug stays in the body of the patient after
dosing.
[0146] As used herein the term "immunogenicity", in the context of
administering a peptide drug to a patient, is defined as the
propensity of that peptide drug to illicit an immune response in
the patient after dosing, or after repeat dosing.
[0147] As used herein the term "molecular dimensions" means the
weight, size and/or shape of an agent. Thus, "increasing the
molecular dimensions by modification" means that the molecular
dimensions are increased such that the agent is too large in
physical size to pass through the blood vessel walls into the blood
stream. The molecular dimensions, however do not necessarily mean
an increase in molecular weight, if, for example, an agent is
truncated prior to modification. Molecular dimensions may include
molecular/weight, size and/or conformation provide that the
modified agent retains activity and cannot pass directly into the
blood vessels without being delivered thereto by the lymphatic
system.
[0148] As used herein, the term "subcutaneous delivery" or
"subcutaneous administration" means delivery by any suitable means
such that the therapeutic agent is delivered through the skin
directly to the subcutaneous space.
[0149] As used herein, "dose adjusted" in the context of
subcutaneous doses of the modified agent means the intravenous dose
for the modified agent multiplied by the fraction intravenous
C.sub.max/subcutaneous C.sub.max. As explained herein, the methods
of the present invention allow for less frequent dosing and/or
higher doses to be given to a patient when compared to the
unmodified or modified agent administered intravenously. "Dose
unadjusted" in the context of subcutaneous doses means the same
dose of intravenous of the modified agent is delivered as would be
delivered intravenously.
[0150] As used herein, the term "subcutaneous space" means the
connective tissue under the skin. It excludes blood vessels, the
blood stream and internal organs.
[0151] By "native state" it is meant the state in which an agent
exists prior to modification and in the state in which it is
generally intravenously administered to a patient in a
pharmaceutically acceptable form.
[0152] The subcutaneous dosage forms of the invention may further
comprise a pharmaceutically acceptable diluent, adjuvant or
carrier. Subcutaneous dosage forms adapted for subcutaneous
administration can include aqueous and/or non-aqueous sterile
injection solution(s) which may contain anti-oxidants, buffers,
bacteriostats and solutes which render the formulation
substantially isotonic with the blood of the intended recipient;
and aqueous and non-aqueous sterile suspensions which may include
suspending agents and thickening agents. Excipients which may be
used for injectable solutions include water, alcohols, polyols,
glycerine and vegetable oils, for example. The compositions may be
presented in unit-dose or multi-dose containers, for example sealed
ampoules and vials, and may be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carried, for example water for injections, immediately prior
to use. Extemporaneous injection solutions and suspensions may be
prepared from sterile powders, granules and tablets.
[0153] In general, the subcutaneous dosage forms may contain
preserving agents, solubilising agents, stabilising agents, wetting
agents, emulsifiers, colourants, salts (active substances of the
present invention may themselves be provided in the form of a
pharmaceutically acceptable salt), buffers, or antioxidants. They
may also contain therapeutically active agents in addition to the
substance of the present invention. The subcutaneous dosage forms
of the invention may be employed in combination with
pharmaceutically acceptable diluents, adjuvants, or carriers. Such
excipients may include, but are not limited to, saline, buffered
saline (such as phosphate buffered saline), dextrose, liposomes,
water, glycerol, ethanol and combinations thereof.
[0154] Subcutaneous administration of the subcutaneous dosage forms
described herein may be undertaken in any effective, convenient
manner effective for treating a patient's disease. The dosage form
may be a liquid form or a solid form. Liquid forms may be ready for
use or prepared as concentrates which are then diluted prior to
subcutaneous administration. Solid forms may suitably be
reconstituted in an appropriate administration vehicle for
subcutaneous administration. In therapy or as a prophylactic, the
active agent administered to an individual as an injectable
composition may be, for example, a sterile aqueous dispersion,
preferably isotonic.
[0155] According to a further aspect of the invention, there is
provided a liquid dosage form of a modified blood coagulation
factor for subcutaneous administration no more than once a month
wherein the dosage form has a C.sub.max of at least 10% and no more
than 90% of that achieved by intravenous administration of the
modified blood factor for use in the treatment of a blood clotting
disorder.
[0156] This aspect of the invention also includes methods of
treatment of a blood clotting disease or trauma in a subject
comprising administering subcutaneously a dosage form of a modified
blood clotting factor as defined herein to a subject in need
thereof.
[0157] The invention therefore also provides the use of a modified
blood clotting factor in the manufacture of a medicament comprising
a dosage form as defined herein for the treatment of a blood
clotting disorder in a subject wherein said medicament is for
subcutaneous administration and has a C.sub.max of at least 10% and
no more than 90% of that achieved by intravenous administration of
the modified blood factor. Suitably, the C.sub.max is from 20% to
80%, or from 30% to 70%, or from 40% to 60%. In one embodiment,
C.sub.max is 75 to 80% and the blood factor may be FVII. In another
embodiment C.sub.max is 10% to 25% and the blood factor may be
FVIII. In yet another embodiment C.sub.max is 40% to 60% and the
blood factor may be FIX.
[0158] Blood clotting diseases or disorders may be characterised by
a loss of function of a blood clotting factor, or the generation of
auto-antibodies. Examples of blood clotting diseases include
haemophilia A and haemophilia B.
[0159] Factor VIIa can be used in the treatment of bleeding
episodes in haemophilia A or B, or in treatment of patients who
have developed inhibitory antibodies against FVIII or IX,
respectively. Factor VIII can be used in the treatment of bleeding
episodes in patients with haemophilia A and Factor IX can be used
in the treatment of patients with haemophilia B.
[0160] As used herein, the term "treatment" includes any regime
that can benefit a human or a non-human mammal. The treatment of
"non-human mammals" extends to the treatment of domestic mammals,
including horses and companion animals (e.g. cats and dogs) and
farm/agricultural animals including members of the ovine, caprine,
porcine, bovine and equine families. The treatment may be in
respect of any existing condition or disorder, or may be
prophylactic (preventive treatment). The treatment may be of an
inherited or an acquired disease. The treatment may be of an acute
or chronic condition.
[0161] The subcutaneous dosage forms of the invention may be
employed alone or in conjunction with other compounds, such as
therapeutic compounds or molecules, e.g. anti-inflammatory drugs,
analgesics or antibiotics, or other pharmaceutically active agents
which may promote or enhance the activity of Factor VII, Factor
VIII, Factor IX, Factor X, Factor Xa, Factor XI, Factor VIIa,
Factor V, Factor XIII, von Willebrand's Factor or Protein C, for
example another blood coagulation factor. Such administration with
other compounds may be simultaneous, separate or sequential. The
components may be prepared in the form of a kit which may comprise
instructions as appropriate.
[0162] Levels of activity in the blood coagulation cascade may be
measured by any suitable assay, for example the Whole Blood
Clotting Time (WBCT) test or the Activated Partial Thromboplastin
Time (APTT).
[0163] The Whole Blood Clotting Time (WBCT) test measures the time
taken for whole blood to form a clot in an external environment,
usually a glass tube or dish.
[0164] The Activated Partial Thromboplastin Time (APTT) test
measures a parameter of part of the blood clotting pathway. It is
abnormally elevated in Haemophilia and by intravenous heparin
therapy. The APTT requires a few millilitres of blood from a vein.
The APTT time is a measure of one part of the clotting system known
as the "intrinsic pathway". The APTT value is the time in seconds
for a specific clotting process to occur in the laboratory test.
This result is always compared to a "control" sample of normal
blood. If the test sample takes longer than the control sample, it
indicates decreased clotting function in the intrinsic pathway.
General medical therapy usually aims for a range of APTT of the
order of 45 to 70 seconds, but the value may also be expressed as a
ratio of test to normal, for example 1.5 times normal. A high APTT
in the absence of heparin treatment can be due to Haemophilia,
which may require further testing.
[0165] The invention also provides a kit of parts comprising a
subcutaneous dosage form of invention, and an administration
vehicle including injectable solutions for subcutaneous
administration, said kit suitably comprising instructions for use
thereof.
[0166] In one embodiment of the invention, there is provided a
dosage form of a pharmaceutical composition of a modified blood
coagulation factor (suitably, Factor VII, Factor VIII, Factor IX,
Factor X, Factor Xa, Factor XI, Factor VIIa, Factor V, Factor XIII,
von Willebrand's Factor or Protein C) for subcutaneous
administration which when formulated for subcutaneous
administration to a subject provides an no more than once per month
dosage form sufficient to maintain a whole blood clotting time in
said subject of less than 15 minutes. The dosage formulation may
suitably have a C.sub.max of at least 10% and no more than 90%
compared to an equivalent reference dosage form when administered
intravenously.
[0167] The invention therefore provides a dosage form of a
pharmaceutical composition of a modified blood coagulation factor
selected from the group consisting of Factor VII, Factor VIII,
Factor IX, Factor X, Factor Xa, Factor XI, Factor VIIa, Factor V,
Factor XIII, von Willebrand's Factor or Protein C for subcutaneous
administration which when formulated for subcutaneous
administration to a subject provides an no more than once per month
dosage form sufficient to maintain a whole blood clotting time in
said subject of less than 12 minutes.
[0168] In one embodiment the invention therefore provides a dosage
form of a pharmaceutical composition of 25 to 50 IU/kg of a
PEGylated blood coagulation factor selected from the group
consisting of Factor VIIa, Factor VIII and Factor IX for
subcutaneous administration no more than once per week.
[0169] A liquid dosage form of the invention may comprises a
modified blood coagulation factor as defined herein for
subcutaneous administration no more than once per month wherein the
dosage form has a C.sub.max of at least 10% and no more than 90%
for use in the treatment of a blood clotting disorder.
[0170] Such compositions may find particular utility in methods of
treatment of a blood clotting disease or trauma in a subject
comprising administering subcutaneously a dosage form of a blood
clotting factor according to the invention to a subject in need
thereof.
[0171] The dosage forms of the invention when administered
subcutaneously have a bioavailability and efficacy comparable to
the levels the respective modified analogue blood clotting factor
when administered intravenously by both circulating titre and
clotting activity.
[0172] In another embodiment of the invention, there is provided a
dosage form for subcutaneous administration comprising a blood
clotting factor as defined herein modified to a straight-chain,
mono-disperse polyethyleneglycol molecule via a three carbon bridge
moiety to a single disulphide bond in the protein.
[0173] A liquid dosage form of the invention may be prepared by
formulating the PEG-conjugated blood clotting factor in an aqueous
solution, buffered to physiological and in the presence of a
non-ionic surfactant and optionally a stabiliser.
[0174] Preferred features for the second and subsequent aspects of
the invention are as for the first aspect mutatis mutandis.
[0175] The product impact of a modified agent in accordance with
the invention has been shown to be superior to the same modified
agent delivered intravenously. Product impact can be defined as
being the improvement in, for example, the WBCT. This defined as
initial WBCT divided by the WBCT at a particular time point. Using
this method, modified blood clotting agents delivered
subcutaneously consistently showed a higher product impact than the
same product delivered intravenously at the same time point.
[0176] According to the present invention, there is a lower immune
response arising from subcutaneous administration of therapeutic
agents which have been modified, for example by the addition of a
biocompatible polymer. This effect is diametrically opposite to
what would be expected prior to the present invention by someone of
ordinary skill in the art of administration of pharmaceutical
formulations. For example, in the field of blood factor
formulations, it is generally accepted that administration of an
unmodified blood factor subcutaneously would be expected to
stimulate an immunogenic response (creation of FVIII inhibitors) or
to trigger an immune response by the existing population of FVIII
inhibitors.
[0177] Relative immune response to blood clotting factors can be
measured in Bethesda units. A Bethesda unit (BU) is a measure of
blood coagulation inhibitor activity. According to Practical
Haemostasis, "1 Bethesda Unit (Bu) is defined as the amount of
inhibitor in a plasma sample which will neutralise 50% of 1 unit of
Factor VIII:C in normal plasma after 2 hr incubation at 37.degree.
C." (Schumacher, Harold Robert (2000). Handbook of Hematologic
Pathology. Informa Health Care, p. 583).
[0178] In the present invention, a very surprising outcome has been
found. In order to lower the incidence of immune (inhibitor)
responses it is proposed to adopt subcutaneous administration where
the level of immune response is directly related to the level of
systemic exposure. By providing a subcutaneous delivery, the
C.sub.max can be radically lowered and in so doing there is a
lowering of immune response.
[0179] As an example, the present invention describes the
surprising depot effect encountered with blood factors when
conjugated to polymers such as PEG. Moreover, the results show that
it is possible to engineer the rate at which blood factors are made
available from the subcutaneous space by manipulating the level of
hydration imposed on the protein from the size (or amount) of
PEG.
[0180] From the results shown in the present application it can be
seen that for therapeutic agents modified by one polymer chain that
such agents have a slower rate of entry into the plasma than the
corresponding di-conjugated forms where two polymer chains are
added.
[0181] In other words, the mono-conjugated products would appear to
have more of the protein exposed by comparison to the di-conjugated
products. This condition would mean that the higher-order
conjugated forms would be more water dispersible and therefore a
fast rate of entry via the lymphatic vessels into the plasma.
[0182] Surprisingly therefore, to achieve the longest duration of
depot release, a lesser degree of modification is required. Without
being bound by theory, this can be rationalised by the lesser
degree of modification exposing some of the therapeutic agent to
the subcutaneous tissue which confers a slow rate on the diffusion
through the lymph. By contrast the higher degree of modification
covers the therapeutic agent completely leaving the product free to
quickly enter the blood circulation.
[0183] Overall, there is a very surprising total effect whereby the
combination of modification followed by subcutaneous delivery,
renders an observed 35-fold increase in apparent half-life
following subcutaneous (SQ) administration).
[0184] Finally, it can be seen overall that the bioavailability
favours the higher order conjugated forms, confirming that the
higher the level of modification and hydration levels promote a
higher degree of mobility and therefore bioavailability.
[0185] All features of each aspect apply to all other aspects of
the invention, mutatis mutandis.
[0186] Reference is also made herein to the following drawings in
which:
[0187] FIG. 1 shows the blood coagulation cascade. Abbreviations:
HMWK--High Molecular Weight Kininogen; PK--Prekallikrein;
PL--Phospholipid.
[0188] FIG. 2 shows the steps involved in disulphide-specific
biopolymer conjugation chemistry with the use of a PEGylation
reagent as an example of a conjugation reagent (from Shaunak et al.
in Nat. Chem. Biol. 2006; 2(6):312-313).
[0189] FIG. 3 shows Whole Blood Clotting Times (WBCT) following
subcutaneous (SQ) administration of PEGhrFIX to subject Dog 1 and
hrFIX to subject Dog 2.
[0190] FIG. 4 shows APTT (Activated Partial Thromboplastin Time)
Values with Time Following SQ Administration.
[0191] FIG. 5 shows APTT of Retained Plasma Following SQ
Administration.
[0192] FIG. 6 shows APTT Relative Values to Baseline Following SQ
Administration.
[0193] FIG. 7 shows subject Dog 9 WBCT following 25 IU/Kg SQ
Administration.
[0194] FIG. 8 shows PK profiles and parameters of FVIIa following
200 ug/kg rFVIIa.
[0195] FIG. 9 shows PK profiles and parameters of FVIIa following
800 ug/kg TheraPEG-rFVIIa.
[0196] FIG. 10 shows PK profiles and parameters of FVIIa following
1600 ug/kg TheraPEG-rFVIIa.
[0197] FIG. 11 shows concentration of FVIII in Plasma (all
dogs).
[0198] FIG. 12 shows concentration of FVIII in Plasma (SQ
administered dogs only).
[0199] FIG. 13 shows immune data (Bethesda value) for PEGFVIII
administered subcutaneously (SQ) compared to intravenous (IV),
number of subjects is given by "n".
[0200] The invention will now be further described by way of
reference to the following Examples which are included for the
purposes of illustration only and should not be construed as being
limiting. References to subcutaneous administration of dosage
formulations of the invention are given as SQ (s.c.) and
intravenous administration as IV (i.v.).
EXAMPLE 1
Preparation of Dosage Forms and Administration Subcutaneously
[0201] The study includes an assessment of the bioavailability and
efficacy of hrFIX following subcutaneous administration. Naked
(unPEGylated) hrFIX was compared to its PEGylated analogue by both
circulating titre and clotting activity.
[0202] 10 kDa PEGylated hrFIX was prepared following standard
technology whereby 10 kDa, straight-chain, mono-disperse
polyethyleneglycol was conjugated via a three carbon bridge to a
single disulphide bond.
[0203] The test article was prepared for administration by forming
a suitable aqueous solution, buffered to pH 6.8 with 10 mM
histidine, 40 mM NaCl and 0.005% Tween.RTM. 80. 1 mM benzamidine
was added as a stabiliser.
[0204] On the basis that dilution studies of hrFIX showed
comparable clotting times with PEGrFIX at 25% dilution, the
allocated potency for this study was 4.times. protein equivalents.
The control article was supplied as a lyophilised powder and
prepared for administration following the enclosed instructions for
reconstitution. The delivery vehicle is identical to that described
above for PEG.
[0205] As an adjunct to this study it was decided to explore the
possibility of subcutaneous (SQ) administration of rFIX. The
prospect of the PEGylated form of rFIX being suitable for SQ
administration emerged from the above observation that PEG provided
a shielding effect of the protein. Historically the SQ route was
considered unavailable for FIX since there was the concern that
this would exacerbate the incidence of antibody production and
would not translate into meaningful quantities in the blood.
[0206] In this part of the study PEGhrFIX 50 IU/Kg was administered
subcutaneously to an additional test animal (Dog 1) and compared to
2 SQ administrations of naked hrFIX) to 2 other test subjects,
namely Dog 6 and Dog 2 respectively.
[0207] In this particular representation, each animal had a
slightly different baseline so for ease of comparison, the
pre-administration APTT level was normalised to 1. Dog 1 and Dog 2
were the test subjects in the previous PEGrFIX trial in January
2010 from which the recorded plasma titres following intravenous
administration were available for comparison.
[0208] Blood samples were taken over a regular time course to
follow the decay of titre and the effect on blood coagulation.
Table 1 is a summary of the titres measured at 22 hours as
circulating FIX following intravenous administration.
TABLE-US-00001 TABLE 1 Subject Article Dose (IU/Kg) Titre Dog 1
PEGhrFIX 50 9.9 Dog 2 Benefix .RTM. 50 5.6 Dog 3 hrFIX 50 5.1 Dog 4
PEGhrFIX 50 9.7 Dog 5 PEGhrFIX 100 11.1 Dog 6 PEGhrFIX 100 10.2 Dog
7 PEGhrFIX 150 17.6 Dog 8 PEGhrFIX 150 75.5
[0209] Table 2 shows comparison of measured Circulating FIX Titre
at 22 Hours Following subcutaneous (SQ) Administration.
TABLE-US-00002 TABLE 2 Subject Article Dose (IU/Kg) Titre Dog 1
PEGhrFIX 50 7.8 Dog 6 hrFIX 50 1.7 Dog 2 Benefix .RTM. 25 ND
[0210] It can be seen that 25 IU/Kg of Benefix.RTM. by the SQ route
was undetectable in circulation and 50 IU/Kg of PEGhrFIX was barely
detectable in plasma. In stark contrast the SQ administration of
PEGhrFIX was at a level (7.8) approaching that of the IV
administered product (9.9).
[0211] The effect of these titres on the correction of clotting
times was then investigated. In the first instance the whole blood
clotting times were recorded. The WBCT following subcutaneous
administration is displayed in Table 3 and in FIG. 3. In addition,
APTT on Hemochron.RTM. Junior was also recorded and the values are
shown in Table 4 below (Whole blood citrated) and in FIG. 4 after
subcutaneous administration. The APTT values on retained plasma
samples are displayed in Table 5 and in FIG. 5 also following
subcutaneous administration.
TABLE-US-00003 TABLE 3 Whole Blood Clotting Time (minutes)**
Following Subcutaneous Administration Hours Dog 1 (PEGhrFIX) Dog 2
(hrFIX) 6 4 45* 22 3.5 45 48 8 45 72 9.5 45 120 4 45 144 4 45 168
45 45 192 45 45 216 45 45 *Note - 45 minutes was the time at which
monitoring was ceased, due to no clot having been formed, according
to standard procedures. **both dogs were naive dogs, meaning they
had not previously been exposed to FIX.
TABLE-US-00004 TABLE 4 Citrated APTT Following Subcutaneous
Administration Dog 1 (PEGhrFIX Dog 2 (hrFIX) Pre 279.7 225.1 6 99.4
375.5 22 75.6 328.7 48 88.6 72 90.1 261.7 120 124.4 274.5 144 138.9
301 168 300 372.5 192 254.2 216 300 298.3
TABLE-US-00005 TABLE 5 APTT Following Subcutaneous Administration
Dog1 (PEGhrFIX Dog 2 (hrFIX) Pre 67.2 66.3 6 44.6 71.9 22 40.2 61.1
48 45.3 64.7 72 43.9 60.7 120 49.6 59.6 144 -- 58.5 168 55.4
64.9
[0212] FIG. 6--This collection of data clearly shows that naked
rFIX is (practically) not bioavailable from subcutaneous injection.
This is entirely expected from published literature and general
knowledge of the art. It is all the more surprising then such high
circulating titres of rFIX can be detected following subcutaneous
injection of PEGhrFIX. Indeed it can be seen in table 10 that ca
80% of the subcutaneously injected PEGrFIX is available for
participation in haemostatic control.
[0213] The contrast is starkest in the measured clotting times,
both WBCT and APTT for rFIX are barely corrected, whereas PEGhrFIX
from subcutaneous injection corrects clotting times immediately.
The duration of haemostasis by these measurements is prolonged to
approximately 1 week from a single 50 IU/Kg subcutaneous
injection.
EXAMPLE 2
Dog 9 Subcutaneous Administration (SQ) of hrFIX
[0214] Given the success of the above SQ studies it was decided to
conduct a further single SQ administration of PEGhrFIX and
similarly follow haemostatic control over an extended period. The
test subject chosen was a naive subject Dog 9 to explore the
influence of neutralising antibodies on the SQ route of
administration.
[0215] Studies of human blood factors in dogs are confounded by the
response of the canine immune system to a human protein. Human rFIX
is a xenoprotein therefore in canine studies and neutralising
antibodies should be expected at some point following
administration of the test article. Indeed when test subjects are
reintroduced to human blood factors the production of antibodies is
more pronounced and speedier. The subjects Dog 1 and Dog 7
following subcutaneous administration have a shortened haemostasis
period as a consequence.
[0216] The test subject Dog 9 was a naive animal and was given a
small subcutaneous dose and therefore revealed the true sustained
protection that PEGylated blood factors of this invention can
provide. Since Dog 9 had no previous exposure to human blood
factors the true underlying (and highly surprising) result was
observed.
[0217] FIG. 7 shows results for WBCT following 25 IU/Kg
subcutaneous (SQ) administration of PEGylated IB1001 to Dog 9 of a
dose of 1 ml (volume 1 ml)) and also in Table 6 below.
TABLE-US-00006 TABLE 6 Time WBCT FIX Titre APTT (hours) (minutes)
(% Normal) (seconds) Pre 45 67.2 6 1.75 0.68 53.8 24 6 2.46 50.9 48
9.5 2.42 49.8 72 3 1.69 55.6 94 9 1.24 57.7 118 5.5 1.13 53.2 142 8
0.66 56.3 168 9 0.28 61.4 189 18 ND 64.3 216 22.5 ND 47.7 240 25 ND
59.7 336 61.4
EXAMPLE 3
Comparative Example
[0218] Comparison of intravenous and subcutaneous administration of
FIX and PEG-FIX.
TABLE-US-00007 TABLE 7* Dose IV SQ SQ/IV Animal IU/kg Type
C.sub.max ng/ml C.sub.max ng/ml % Cmax Beagle 200 BFIX 4517.5 550.7
12% Haemophilia 200 BFIX 7916 658.3 8.2% B (HB) dog *from McCarthy
et al Thromb. Haemost. 87(5) 824-830, (2002).
TABLE-US-00008 TABLE 8 Dose IV SQ SQ/IV Animal IU/kg Type % Normal
% Normal % Cmax Dog 1 50 PEGFIX 9.9 7.8 78.8% Dog 7 50 PEGFIX 7.8
Dog 6 50 hrFIX 1.7 Dog 2 25 BFIX ND BFIX = Benefix .RTM. PEGFIX =
PEG-hrFIX
[0219] Results show a C.sub.max of the subcutaneous dose of 78.8%
of the intravenous dose. The percentage values for IV and SQ
compared to normal appear to be low but are actually experimental
artefacts. It is assumed that the FIX in each case is being spun
down with the cells as the samples are prepared. It can be seen
that the value of 9.9% for an intravenous dose is actually a
representation of a good result. Consequently, the comparison with
7.8% for a subcutaneous dose is favourable as indicated by the
calculated C.sub.max value given.
CONCLUSIONS
[0220] Administration of hrFIX by subcutaneous injection of both 25
and 50 IU/Kg resulted in a barely detectable circulating titre and
did not correct haemophilia in the canine subjects.
[0221] In stark contrast to the above, subcutaneous dosing of 50
IU/Kg of PEGhrFIX gave rise to approximately 80% bioavailability
and corrected clotting times to be within the normal range for
duration of 1 week.
EXAMPLE 4
Factor VIIa with 20 kDa PEG
[0222] This example reports a study on PEGFVIIa Bioavailability of
Blood Factor from Subcutaneous Injection. Two haemophilic dogs (HB)
were treated with equipotent quantities of PEGFVIIa at time 0; one
intravenously (IV), one subcutaneously (SQ). Blood samples were
taken and the plasma recovered to be measured for FVIIa protein.
The table of results display a bioavailability from subcutaneous
administration of 89.5%.
TABLE-US-00009 TABLE 9 Time PEGylated blood factor VIIa plasma
titres (hours) IV SQ 0 9.5 9.5 4 167.5 73.9 (max) 12 122.2 62.4 24
45.7 57.5 48 23.1 39.6 72 9.3 22.5 Average 62.88 44.23 Max/ 2.66
1.67 Average
[0223] The presence of PEG confers aqueous solubility which
facilitates mobility in lymph vessels. The data shows a steady
controlled infusion of FVIIa rather than the bolus peak and trough
associated with the IV injection.
[0224] The area under the curve indicates 89.5% bioavailability for
PEGylated FVIIa and a more steady state of the level of FVIIa when
delivered subcutaneously.
EXAMPLE 5
PEGFVIII Drug Products
[0225] To make the comparison, reference is made to Kogenate.RTM.
FS (a commercially available recombinant FVIII). The PEGylated
excipient, Tween.RTM. 80 is used in large quantity.
[0226] Polysorbate, Tween.RTM. 80, has a molecular weight of 1310
g/mol, 880 g of which is derived from PEGylation (total monomer
units of 20 which each carry 44 g/mol, (CH2-CH2-O)).
[0227] The calculation is thus:
[0228] Molar PEG Length Equivalent:
[0229] Reference: Product Monograph Example taken 250 IU Vial
TABLE-US-00010 FVIII Molecular weight 3.00E+05 g/mol IU/g 4.00+06
IU/g IU/Vial 2.50E+02 IU/Vial Vial volumes 2.50E+00 ml/vial
Polysorbate concentration 6.40E-05 g/ml Molecular weight
Polysorbate 1.31E+03 g/mol Molecular weight PEG per mol Polysorbate
880 g/mol
TABLE-US-00011 TABLE 10 FVIII Kogenate .RTM. Polysorbate 4.00E+06
IU/g 1.31E+03 g/mol 3.00E+05 g/mol 6.40E-05 g/ml 1.2E+12 IU/Mol
2.50E+00 ml/vial 1.60E-04 g/vial 2.50E+0.2 IU/Vial 2.08E-10
Mol/vial 1.22E-07 mol/vial Ratio of Tween .RTM./FVIII 5.86E+02 PEG
equivalent Mol Wt. 5.16E+05
[0230] Therefore, in Kogenate.RTM. FS, each FVIII molecule has the
equivalent of an associated 516 kDa PEG. By comparison, the
PEGFVIII dosage formulation prepared according to the present
invention has a single 20 kDa PEG.
[0231] Conclusions: On a dose-for-dose basis there is a 25.8 fold
reduction in polyethylene glycol; given the PEG-FVIII dosage
formulation of the present invention may be administered once per
week versus a prophylactic use of Kogenate.RTM. on a three times a
week basis, there is a potential overall reduction of ca 80-fold
reduction in the administration of PEG; and the amount of PEG
administered by the FVIII dosage formulation of the present
invention over a dosing period is 1.25% of that administered by
Kogenate.RTM..
EXAMPLE 6
Subcutaneous administration FVIIa
[0232] The objectives of this study were to investigate the
pharmacokinetics of TheraPEGylated and non-TheraPEGylated
recombinant human FVIIa (TheraPEGrFVIIa and FVIIa respectively)
following intravenous and subcutaneous administration in
haemophilic B dogs.
[0233] TheraPEGylation of transgenic FVIIa (rFVIIa) was carried out
according to WO 2011/135308. TheraPEGrFVIIa was supplied to the
test site as a lyophile in multiple batches which, on
reconstitution with high purity water, resulted in 1 mg/ml
TheraPEGrFVIIa in a physiologically acceptable buffer which
maintained activity of FVIIa
[0234] The experimental animals were Lhasa Apso-Basenji cross dogs
with congenital severe haemophilia B caused by a 5-bp deletion and
a C.fwdarw.T transition in the F9 gene that results in an early
stop codon and unstable FIX transcript. Prior to dosing, all dogs
were tested to verify normal health status, including complete
blood chemistry, serum chemistry profile fibrinogen, fibrinogen
derived peptides (FDPs), thrombin time and urinary analysis. Drugs
given intravenously (IV) were given as a bolus injection into the
cephalic vein. Subcutaneous (SQ) doses were given between the
scapula as a single dose.
[0235] Individual batches of TheraPEGrFVIIa were reconstituted and
then combined in order to produce a single dose solution used to
dose the animals as described in Table 11.
TABLE-US-00012 TABLE 11 Dog Dog Dose Dose Subject and Weight Dose
Level Amount Code (Gender) (kg) Drug route (ug/kg) (mg) Dog 9 5.4
TheraPEG- SQ 800 4.32 HB1 rFVIIa (Male) Dog 3 11.4 rFVIIa SQ 200
2.28 HB2 (Male) Dog 5 5.6 TheraPEG- IV 800 4.48 HB3 rFVIIa (female)
Dog 7 10.0 rFVIIa IV 200 2.0 HB4 (female) Dog 10 5.5 TheraPEG- IV
1600 8.8 HB5 rFVIIa (female) Dog 11 4.8 TheraPEG- SQ 1600 7.68 HB6
rFVIIa (male)
[0236] A 5 ml blood sample was protocolled to be taken from each
dog at the following times points: Pre-drug administration and at
10, 30 minutes, 1, 2, 4, 8, 12, 18, 24, 36, 48, 72, 96, 120, 144,
168, 192, 216 and 240 hours post-dose.
[0237] 4 ml of the blood sample was transferred into a tube
containing 0.109M tri-sodium citrate anticoagulant (9:1 v/v) on
ice. Plasma was prepared by centrifugation of the remaining
citrated blood and the resulting plasma samples were stored in
aliquots at -80.degree. C. An aliquot of plasma was assayed for
FVIIa concentration by ELISA.
[0238] The Stago Asserachrom VII:Ag ELISA assay is an enzyme linked
immunoassay procedure for the quantitative determination of Factor
VII/VIIa concentration in plasma samples. The assay is a sandwich
ELISA which comprises of microtitre wells pre-coated with a rabbit
anti-human FVII antibody. Because the antibody has a different
affinity for FVIIa than for PEG-FVIIa, a standard curve was
prepared by dilution of a protein appropriate to the FVIIa that is
present in the test plasma, i.e. rFVIIa (0.78 to 50 ng/ml) for
assay of plasma from dogs that were administered rFVIIa, or
PEG-rFVIIa (0.78 to 50 ng/ml) for assay of plasma from dogs that
were administered PEG-rFVIIa.
[0239] Plasma samples were diluted to an appropriate concentration
to fall within the standard curve. Diluted plasma samples and
standards were loaded and incubated at room temperature before
washing and subsequent development with a rabbit anti-human FVII
HRP conjugate and OPD (a colorimetric HRP substrate). The plate was
read at 492 nm and the concentration of the test samples (ng/ml) is
read from the standard curve.
TABLE-US-00013 TABLE 12 Dose Tmax Cmax AUC(0-t) AUC(0-.infin.) Rate
Half-life Bio. Route (h) (ng/mL) (ng h/mL) (ng h/mL) (/h) (h) (%)
IV 0.16 1643 2467 2534 0.2994 2.3 100 SQ 7.5 31.3 276 -- -- --
11
TABLE-US-00014 TABLE 13 Dose Tmax Cmax AUC(0-t) AUC(0-.infin.) Rate
Half-life Bio. Route (h) (ng/mL) (ng h/mL) (ng h/mL) (/h) (h) (%)
IV 0.5 19372 128305 129646 0.0256 27.0 100 SQ 12.0 1378 84960 87139
0.0262 26.5 67
TABLE-US-00015 TABLE 14 Dose Tmax Cmax AUC(0-t) AUC(0-.infin.) Rate
Half-life Bio. Route (h) (ng/mL) (ng h/mL) (ng h/mL) (/h) (h) (%)
IV 0.5 26609 236116 240449 0.050 13.8 100 SQ 24 2030 107728 108454
0.038 18.3 45.6
[0240] Pharmacokinetics
[0241] The IV and SQ profiles and PK parameters for 200 ug/kg
FVIIa, 800 ug/kg TheraPEG-rFVIIa and 1600 ug/kg TheraPEG-rFVIIa are
shown in FIGS. 8, 9 and 10 (Table 12, Table 13 and Table 14). The
half-life of TheraPEG-rFVIIa was found to be between 14 and 27
hours, which is a clear extension over the 2.3 hour half-life of
non-PEGylated rFVIIa. The AUC of the 1600 ug/kg IV dose of
TheraPEG-rFVIIa was 1.8.times. higher than that of the 800 ug/kg IV
dose. However the 1600 ug/kg SQ dose was only 1.2.times. higher
than that of the 800 ug/kg dose. This is reflected in the
bioavailability calculations of 67% and 45% for the 800 ug/kg and
1600 ug/kg doses respectively, which represented a significant
increase over the 11% SQ bioavailability observed for non-PEGylated
rFVIIa.
[0242] The AUC for the 800 ug/kg IV dose of TheraPEG-rFVIIa is
84.times. that of the AUC following 200 ug/kg IV non-PEGylated
rFVIIa and the AUC for the 800 ug/kg SQ dose of TheraPEG-rFVIIa is
300.times. that of 200 ug/kg SQ non-PEGylated rFVIIa.
EXAMPLE 7
Subcutaneous Administration FVIII
[0243] The objectives of this study were to investigate the
pharmacokinetics and pharmacodynamics of TheraPEGylated plasma
derived FVIII (TheraPEG-pdFVIII) when administered intravenously
and subcutaneously to haemophilic A dogs. TheraPEG-pdFVIII was
prepared as described in WO 2011/135307 with a 20 kDa linear PEG
and further purified to yield purified TheraPEG-pdFVIII.
[0244] The experimental animals were greyhound cross dogs which had
congenital severe haemophilia A and had previously been
administered canine plasma for the treatment of spontaneous bleeds,
but were naive to treatment with human FVIII. Prior to dosing, all
animals were tested to verify normal health status, including
complete blood chemistry, serum chemistry profile fibrinogen,
fibrinogen derived peptides, thrombin time and urinary
analysis.
[0245] Table 15 shows the weight of each dog and the FVIII doses
that were administered. Each dog received a single dose of either
TheraPEG-pdFVIII at a higher (approx. 0.14 mg/kg) or a lower (0.07
mg/kg) dose or non-PEGylated pdFVIII at 0.03 mg/kg. Intravenous
(IV) administration was given as a bolus dose via the cephalic
vein. Sub cutaneous (SQ) administration was given as a single dose
between the scapulae.
TABLE-US-00016 TABLE 15 Dog subject Dose Dose total Dose (gender)
Weight FVIII Conc. volume amount Dose Test article route and code
(kg) (mg/ml) (ml) FVIII (mg) (mg FVIII/kg) TheraPEG- SQ Dog 12 (F)
21.8 0.211 14 2.954 0.135 pdFVIII HA1 TheraPEG- SQ Dog 13 (M) 26.6
0.235 16 3.76 0.141 pdFVIII HA2 TheraPEG- IV Dog 14 (F) 20.6 0.211
14 2.954 0.143 pdFVIII HA3 TheraPEG- IV Dog 15 (M) 31 0.235 17.1
4.019 0.130 pdFVIII HA4 TheraPEG- SQ Dog 16 (M) 28 0.273 7.0 1.911
0.068 pdFVIII HA6 (low dose) Non-PEG'd SQ Dog 17 (F) 27.4 0.090*
9.0 0.810 0.030 pdFVIII HA5
[0246] A blood sample was protocolled to be taken from each dog at
the following times points. Pre-drug administration and at 10, 30
minutes, 1, 2, 4, 8, 12, 18, 24, 36, 48, 72, 96, 120, 144, 168,
192, 216 and 240 hours post-dose. Whole blood (non-citrated) was
used for the whole blood clotting assay and the activated clotting
time assay. The remaining blood sample was transferred into tubes
containing 0.109M tri-sodium citrate anticoagulant (9:1 v/v) on
ice. The activated partial thromboplastin time assay was conducted
on citrated blood. Plasma was prepared by centrifugation of the
citrated blood and the resulting plasma samples were stored in
aliquots at -80.degree. C. for the FVIII antigen ELISA.
[0247] Whole Blood Clotting Time Assay (WBCT)
[0248] Blood samples were divided between 2 vacutubes, (2.times.0.5
ml), and observed carefully with periodic and judicious levelling
of the tube until a clot was determined by interruption of flow in
the fully horizontal position. The quality of the clot was then
observed by holding the tube in the fully inverted position. The
WBCT was recorded as the mean of the total time from sample
extraction until visual observation of blood clot for both samples
and the quality of the clot in the inverted position was also
noted.
[0249] Activated Clotting Time (ACT) and Activated Partial
Thromboplastin Time (APTT)
[0250] The ACT and APTT tests were carried out using a Haemachron
Jr coagulation analyzer (International Technidyne Corps.) according
to the manufacturer's instructions.
[0251] The concentration of FVIII antigen in plasma samples was
determined by ELISA using the Visulize FVIII antigen kit from
Affinity Biologicals (Ancaster, Ontario, Canada) according to the
manufacturer's instructions.
[0252] Results
[0253] Whole Blood Clotting Time (WBCT)
[0254] Haemostasis (WBCT<12 minutes) was maintained in all dogs
that had received the higher dose of TheraPEG-pdFVIII (HA1-4) for
between 80-100 hours. There appeared to be no difference in the
WBCT profile between IV and SQ administration. A lower dose of
TheraPEG-pdFVIII (HA6) given SQ maintained haemostasis for between
56-75 hours. In contrast, although non-PEGylated FVIII administered
SQ reduced the WBCT, it did not result in a sustained WBCT below 12
minutes.
[0255] Activated Clotting Time (ACT)
[0256] ACT was reduced into the normal range of less than 200
seconds in all dogs that had received the higher dose of
TheraPEG-pdFVIII (HA1-4) for approximately 80 hours post-dose.
There was no difference in the ACT profile between IV and SQ
administration.
[0257] A lower dose of TheraPEG-pdFVIII (HA6) given SQ maintained
ACT below 200 seconds for at least 36 h. In contrast, although
non-PEGylated FVIII given SQ reduced the ACT, it did not result in
a sustained ACT below 200 seconds.
[0258] Activated Partial Thromboplastin Time (APTT)
[0259] APTT was reduced to less than 60 seconds in all dogs that
had received the higher dose of TheraPEG-pdFVIII (HA1-4) for
approximately 60 hours post-dose. There was no difference in the
APTT profile between IV and SQ administration.
[0260] A lower dose of TheraPEG-pdFVIII (HA6) given SQ maintained
APTT at less than 60 seconds for 40 h. In contrast, although
non-PEGylated FVIII given SQ reduced the APTT, the shortest APTT
time was 80 seconds. The reason why the APTT for this individual
remained below base-line value for the duration of the study
post-dose is obscure, but may be due to dog-to-dog variation.
[0261] FVIII Plasma Concentrations and Pharmacokinetics
[0262] The FVIII plasma concentration against time in all dogs is
shown in FIG. 11. The data for SQ dosed dogs alone is shown in FIG.
12. Raw data are listed in Tables 23-28. Key PK parameters are
shown in Table 16.
[0263] The half-life of TheraPEG-FVIII administered SQ was 18.3 h
and 16.6 h for HA1 and 2 respectively. When administered IV,
half-lives were slightly shorter at 15.2 h and 13.9 h for HA3 and 4
respectively. Bioavailability was calculated at 32% following SQ
administration. The concentrations of FVIII following SQ
administration of non-PEGylated FVIII (HA5) were mainly below the
level of quantification and therefore no PK parameters could be
calculated.
TABLE-US-00017 TABLE 16 Dose Dog T.sub.max C.sub.max AUC.sub.0-t
AUC.sub.0-.infin. .lamda..sub.z t.sub.1/2 (mg/kg) Ref. (h) (%
Normal) (% Normal h) (% Normal h) (/h) (h) 0.135 HA1 (SQ) 8.00
31.20 871.4 1066.8 0.0379 18.3 0.141 HA2 (SQ) 8.00 32.80 1085.3
1171.7 0.0417 16.6 0.143 HA3 (IV) 0.16 176.40 2929.8 3698.0 0.0456
15.2 0.130 HA4 (IV) 0.16 179.20 3302.8 3522.8 0.0500 13.9 0.068 HA6
(SQ) 4.00 9.01 345.2 510.1* 0.0143* 48.4* *Approximate value due to
variability of data.
CONCLUSIONS
[0264] Sub-cutaneous delivery of the higher dose of TheraPEG-FVIII
resulted in haemostatic control for 80-100 hours following a single
dose as measured by WBCT, APTT and ACT. The profile of SQ in these
assays was indistinguishable from the profile of an equivalent dose
of TheraPEG-FVIII given IV This clearly demonstrated the
feasibility of delivering TheraPEG-FVIII SQ.
[0265] The half-life of TheraPEG-pdFVIII ranged from 13.9 to 18.3
h. This demonstrates a clear extension in half-life compared to
marketed recombinant FVIII which is reported to be 7-11 h in
haemophilia A dogs (Karpf et al., Haemophilia 17, 5 (2011)). Hence,
the TheraPEG-FVIII was not only bioavailable SQ but also
demonstrated an extended half-life.
[0266] The PK profile of FVIII following SQ administration of
TheraPEG-pdFVIII had a much reduced C.sub.max and AUC compared to
IV administration and bioavailability was determined to be 32%.
However, at this dose level, due to the "slow release" nature of
the PK curve, exposures were maintained above the 5% normal level
following SQ administration for a similar amount of time as after
the IV dose which is likely to explain the equivalent functional
responses. The decrease in C.sub.max and AUC, coupled to the
increase in duration of action for SQ delivered TheraPEG-FVIII
highlighted potential, additional safety features of this product
and dosing options.
[0267] Sub-cutaneous administration of non-PEGylated FVIII resulted
in no detectable FVIII in plasma and although clotting times were
reduced, there was no sustainable maintenance of haemostasis. This
demonstrated that non-PEGylated FVIII had a very low SQ
bioavailability, but that very small amounts of FVIII can affect
haemostasis. In contrast to non-PEGylated FVIII, a low dose of
TheraPEG-pdFVIII resulted in plasma levels of up to 9% normal and
haemostasis was maintained for 56-75 hours. Therefore, the addition
of TheraPEG to pdFVIII resulted in a greater bioavailability and
functional response when administered SQ. In conclusion, this study
clearly demonstrated that TheraPEGylation of FVIII resulted in a
superior product that can be administered subcutaneously with an
extended duration of action.
TABLE-US-00018 TABLE 17 HA1 (SQ PEG-pdFVIII) Dog 12 WBCT APTT ACT
ELISA Time (h) (min) (min) (min) (% normal) 0.00 40.00 186.95
378.00 0 0.16 31.00 178.4 400 0 0.50 32.00 168.1 319 0 1.00 8.50
114.1 229 0 2.00 8.75 81.3 241 0 3.50 7.00 57.3 177 7.8 8.00 7.50
40.4 167 31.2 12.00 6.50 51.4 183 29.4 20.00 5.25 33.4 184 23.4
26.50 5.50 37.3 194 21.4 33.50 5.80 40.4 196 15.4 48.50 6.40 81.3
205 7.4 58.00 8.13 58.5 183 3.8 72.00 9.00 71.5 188 0.1 94.00 13.00
119.2 275 0 117.75 29.00 146.4 400 0 145.50 33.00 174.3 386 170.00
31.75 137 360 398.00 32.65 140.8 365 696.00 30.00 133.4 347
TABLE-US-00019 TABLE 18 HA2 (SQ PEG-pdFVIII) Dog 13 WBCT APTT ACT
ELISA Time (h) (min) (min) (min) (% normal) 0.00 36.50 117.50
367.00 0 0.16 12.50 79.8 265 0 0.50 7.50 52.5 197 0 1.00 7.50 39.3
181 4.4 2.16 7.00 40.4 179 12.8 4.00 7.75 33.4 172 24.6 8.00 6.00
30.6 168 32.8 11.00 6.75 35.3 177 26.6 21.00 5.00 36.3 185 29.8
24.75 7.50 32.4 173 26.4 32.50 7.75 51.4 186 18.8 46.00 7.25 47.9
183 9.2 52.25 8.00 50.2 185 1.4 72.75 8.00 56.1 213 3.6 97.00 19.75
122.7 314 0 120.50 24.00 174.3 400 143.50 26.00 176.3 371 165.00
33.50 306.4 400
TABLE-US-00020 TABLE 19 HA3 (IV PEG-pdFVIII) Dog 14 WBCT APTT ACT
ELISA Time (h) (min) (min) (min) (% normal) 0.00 24.00 239.45 0
0.16 4.00 38.3 174 176.4 0.50 4.00 38.3 158 169.4 1.00 4.50 32.4
174 172.2 2.00 5.50 37.3 180 154.8 3.50 6.50 31.5 179 140.6 8.00
4.50 44.6 160 116.4 12.00 4.90 29.6 166 98.6 20.00 5.00 28.7 176
70.2 26.50 6.00 42.5 171 52.2 33.50 6.75 37.3 163 35 48.50 7.63
43.5 179 13.6 58.00 7.13 44.6 184 4 72.00 6.00 209 0 94.00 9.00 234
0 117.75 19.00 112.4 320 0 145.50 26.25 176.3 347 170.00 29.25
114.1 333 398.00 40.00 142.6 362 696.00 30.00 53.7 294
TABLE-US-00021 TABLE 20 HA4 (IV PEG-pdFVIII) Dog 15 WBCT APTT ACT
ELISA Time (h) (min) (min) (min) (% normal) 0.00 26.25 84.2 396 0
0.16 40.00 77 227 179.2 0.50 22.50 62.3 168 170.8 1.00 8.25 33.4
151 166.2 2.16 6.00 24.4 158 151.8 4.00 5.25 30.6 165 129.8 8.00
7.50 29.6 150 115.6 11.00 8.50 31.5 157 100.2 21.00 6.50 35.3 185
68.8 24.75 7.00 34.3 162 55 32.50 7.00 38.3 177 35.8 46.00 6.25
43.5 174 18.4 52.25 6.75 45.7 167 11 72.75 7.00 53.7 203 0 97.00
22.00 79.8 334 0 120.50 20.50 102.5 356 143.50 23.50 178.4 306
165.00 34.50 114.1 371
TABLE-US-00022 TABLE 21 HA5 (SQ pdFVIII) Dog 17 WBCT APTT ACT ELISA
Time (h) (min) (min) (min) (% normal) 0.00 31 104.2 0 0.166 21 137
351 0 0.5 31.5 100.9 311 0 1 19.25 105.8 268 0 2 16 98.5 268 0 4 18
84.2 261 0 8 13.25 105.8 238 0.186 12 13 79.8 200 0 18 12.5 93.1
230 0 24 10.25 82.7 226 0 36 15.5 84.2 247 0 48 18.5 122.7 237 0 56
19.25 148.3 278 0 72 23 364 0.489 96 25.5 122.7 347 0 120 109.1 328
0 192 37.00 87.1 334 0 432 40 128 375 0
TABLE-US-00023 TABLE 22 HA6 (SQ PEG-pdFVIII Low Dose) Dog 16 WBCT
APTT ACT ELISA Time (h) (min) (min) (min) (% normal) 0.00 31.75
81.3 321 0 0.166 39 104.2 0 0.5 33.5 115.8 298 0 1 12.5 94.2 224 0
2 8.25 82.7 190 0.131 4 9 62.3 168 9.006 8 9 44.5 188 8.621 12 7.5
40.4 156 3.053 18 7 49.1 179 5.148 24 7.75 57.3 168 6.167 36 11.5
62.3 180 6.553 48 8.75 68.8 217 2.419 56 15 74.2 178 5.01 72 10.5
88.6 215 2.364 96 27 110.7 281 0 120 90.1 0 192 31.25 100.9 323 0
432 35.5 93.1 333 0
EXAMPLE 8
Immune Response to Subcutaneous Administration in Dogs
[0268] In the present invention, it has been observed that there is
a lower immune response arising from subcutaneous administration.
This effect is diametrically opposite to what would be anticipated
prior to the present invention by someone of ordinary skill in the
art of blood factor administration. It is generally accepted that
by administering subcutaneously the existing very high level of
immune response (FVIII inhibitor frequency) would be
exacerbated.
[0269] In the present invention, a very surprising outcome has been
found. In order to lower the incidence of immune (inhibitor)
responses it is proposed to adopt subcutaneous administration where
the level of immune response is directly related to the level of
systemic exposure. By providing a subcutaneous delivery, the
C.sub.max can be radically lowered and in so doing there is a
lowering of immune response.
[0270] In the examples of the invention, the PEGylated product is
exposed to the most testing of immune environments, namely the dog
system. It can be seen that the Bethesda values (units of inhibitor
quantities) are highest and earliest when given intravenously. By
contrast the subcutaneous deliveries have a very much lower
systemic exposure as evidenced by the C.sub.max and a lower and
later Bethesda response. Indeed the lowest value of all is the
naked FVIII given SQ which has almost no systemic exposure and is
never seen to give an inhibitor value. See FIG. 13/Table 23 for a
representation of the data obtained.
TABLE-US-00024 TABLE 23 Summary N (no. of Bethesda Units Product
& route subjects) Cmax PRE Day 7 Day 14 Day 30 PEGFVIII IV 2
177.8 0 0 20 17.5 PEGFVIII SQ 2 32 0 0 10 17 PEGFVIII SQ (LD) 1 9.0
0 0 0 6 FVIII SQ 1 0.5 0 0 0 0
[0271] The plots in FIG. 13 demonstrate how much inhibitor activity
has been found in blood plasma over time, as stimulated by the
treatments. In the case of the direct IV treatment, there is a more
rapid occurrence of a higher level of inhibitors, compared to SQ
treatment which leaches into the system more slowly and is less
provocative to the immune system.
EXAMPLE 9
Comparative Studies on Subcutaneous Administration in Rats
[0272] This example describes the surprising depot effect
encountered with blood factors when conjugated to polymers such as
PEG. Moreover, the results show that it is possible to engineer the
rate at which blood factors are made available from the
subcutaneous space by manipulating the level of hydration imposed
on the protein from the size (or amount) of PEG.
[0273] The relative pharmacokinetics of Factor VIIa PEGylated via 3
different forms of PEGylation was studied in rat subjects to
compare their performance in terms of delivery from the
subcutaneous space.
[0274] Native, recombinant Factor VIIa was administered to rat
subjects, as well as 3 different PEGylated forms of FVIIa, either
subcutaneously (SQ) or by intravenous (IV) administration: [0275]
a) TheraPEGylated FVIIa: FVIIa was mono-PEGylated to a 20 kDa PEG
molecule using the "TheraPEG" technology of Polytherics Ltd (as
described elsewhere and in WO 2011/135308); [0276] b)
GlycoPEGylated FVIIa: FVIIa was conjugated to PEG via standard
glycoPEGylation technology giving a test product that was dominated
by di-conjugated 20 kDa PEG with also some significant amounts of
higher PEG products: [0277] c) HATU-catalysed PEGylated FVIIa:
FVIIa was monoPEGylated to a 20 kDa PEG (using a conjugation method
derived from one described in U.S. Pat. No. 5,644,029).
[0278] TheraPEG-FVIIa
[0279] 20 kDa PEG was dispersed to 10 mg/mL in 5 mM Na phosphate
pH8.0, 15 mM NaCl, 2 mM EDTA. It was then incubated at 20.degree.
C. for 3 hours. A vial (5.3 mg) of FVIIa was reconstituted to 0.8
mg/mL in 20 mM sodium citrate pH6.0, 0.1M NaCl, 10 mM EDTA. It was
incubated at 20.degree. C. for 10 minutes. TCEP, 1.5 Molar
Equivalents (ME) of 24 mM and 0.025 ME of 0.4 mM SeCM were then
added and incubated at 20.degree. C. for 1 hour. 2 ME of activated
PEG was then added to the reduced FVIIa. The mixture was incubated
at 20.degree. C. for 1 hour, and then at 5.degree. C. for 17 hours.
Size Exclusion Chromatography using a Superdex 200 column was then
carried out in formulation buffer in order to purify the PEGylated
FVIIa.
[0280] For analysis of the product, reconstituted rFVIIa, activated
PEG, reaction mixture, and selected Superdex fractions (25, 30, 35,
39, 45, 51, 80) were run on non-reduced SDS-PAGE gels. Fractions
containing PEGylated FVIIa were pooled and concentrated to
approximately 3 mL prior to lyophilisation. The concentrated SEC
pool was tested by reduced and non-reduced SDS PAGE, clotting
activity and reversed phase HPLC assays both pre- and
post-lyophilisation.
[0281] GlycoPEGylated FVIIa
[0282] A vial (5.3 mg) of rFVIIa was reconstituted in 2.5 mL MOPS
buffered saline. The reconstituted rFVIIa was then buffer exchange
on a PD10 desalting column into MOPS buffered saline and diluted to
1 mg/mL. The buffer exchanged rFVIIa was placed on ice and 100 mM
sodium periodate was added to a final concentration of 2.5 mM. The
mixture was incubated in the dark for a maximum of 30 minutes.
Glycerol (50%) was the added to a final concentration of 3%. The
mixture was then buffer exchanged into 0.1M sodium acetate buffer
using a Zeba spin column. A 50 mg/mL stock solution of Amino oxy
PEG was made and 10 ME of this PEG was added to the desalted FVIIa.
The reaction mixture was incubated at Room Temperature for 1-2
hours before further incubation at 4.degree. C. overnight. The
GlycoPEGylated FVIIa was then purified by SEC chromatography as
described above.
[0283] For analysis of the product, selected SEC fractions (23, 27,
32, 35, 40, and 80) were run on non-reduced SDS-PAGE. Fractions
containing GlycoPEGylated FVIIa were pooled and concentrated to
approximately 3 mL prior to lyophilisation. The concentrated SEC
pool was tested by reduced and non-reduced SDS PAGE, clotting
activity and reversed phase HPLC assays both pre- and
post-lyophilisation.
[0284] HATU PEG-FVIIa
[0285] A vial (5.3 mg) of rFVIIa was reconstituted in 2.5 mL borate
buffer, buffer exchange on a PD10 column into borate buffer and
dilute to 0.5 mg/mL. A stock solution of Methoxy-PEG was made up in
acetonitrile to 16 mg/mL. The buffer exchanged rFVIIa was activated
with 1.0 ME of HATU and 2.5 ME of DIEA for 10 minutes at room
temperature. Following activation 8 ME of Methoxy-PEG was added to
the activated rFVIIa over 2-5 minutes. The reaction mixture was
then incubated at room temperature for 80-100 minutes. The HATU
PEGylated FVIIa was then purified by SEC chromatography as
described above.
[0286] For analysis of the product, selected SEC fractions were run
on non-reduced SDS-PAGE. Fractions containing HATU PEGylated FVIIa
were pooled and concentrated to approximately 3 mL prior to
lyophilisation. The concentrated SEC pool was tested by reduced and
non-reduced SDS PAGE, clotting activity and reversed phase HPLC
assays both pre- and post-lyophilisation.
[0287] Method
[0288] Formulations at a dose of 0.5 mg/kg were administered either
IV or SQ to Healthy rat subjects. For IV administration the
appropriate volume of test article was injected into the tail vain.
For SQ administration the appropriate volume of test article was
injected into the scruff of the neck. Following administration of
the control test articles (native rFVIIa) blood samples were taken
at the following time intervals:
TABLE-US-00025 TABLE 24 Time (h) 0.033 0.25 0.5 1 1.5 2 3 4 6 8 12
18 24 36 48 IV control SQ control
[0289] Following administration of the test articles blood samples
were taken at the following time intervals:
TABLE-US-00026 TABLE 25 Time (h) 0.033 0.25 0.5 1 2 4 6 8 12 18 24
48 72 96 120 IV article SQ article
[0290] At each time point plasma was prepared from the blood sample
and the FVIIa concentration determined using the Stago Asserachrom
VII:Ag ELISA assay. This assay is an enzyme linked immunoassay
procedure for the quantitative determination of Factor VII/VIIa
concentration in plasma samples. The assay is a sandwich ELISA
which comprises of microtitre wells pre-coated with a rabbit
anti-human FVII antibody. Because the antibody has a different
affinity for FVIIa than for PEG-FVIIa, a standard curve was
prepared by dilution of a protein appropriate to the FVIIa that is
present in the test plasma, i.e. rFVIIa (0.78 to 50 ng/ml) for
assay of plasma from rats that were administered rFVIIa, or
PEG-rFVIIa (0.78 to 50 ng/ml) for assay of plasma from dogs that
were administered PEG-rFVIIa.
[0291] Plasma samples were diluted to an appropriate concentration
to fall within the standard curve. Diluted plasma samples and
standards were loaded and incubated at room temperature before
washing and subsequent development with a rabbit anti-human FVII
HRP conjugate and OPD (a colorimetric HRP substrate). The plate was
read at 492 nm and the concentration of the test samples (ng/ml) is
read from the standard curve. Results of the study are as shown in
Table 26(a) and (b) where there are two routes of administration:
intravenous (IV) and subcutaneous (SQ) for each of the PEGylated
FVIIa molecules and a control arm which was the native FVIIa.
[0292] As shown in Table 26(a) and (b), there are 2 routes of
administration, intravenous (IV) and subcutaneous (SQ) for each of
the PEGylated FVIIa molecules and a control arm which was the
native FVIIa.
[0293] This example describes the surprising depot effect
encountered with blood factors when conjugated to polymers such as
PEG. Moreover, the results show that it is possible to engineer the
rate at which blood factors are made available from the
subcutaneous space by manipulating the level of hydration imposed
on the protein from the size (or amount) of PEG.
[0294] From the results shown in Table 26(a) and (b), it can be
seen that: [0295] All the PEGylated proteins have extended plasma
half-lives by comparison to the naked protein
[0296] The mono-PEG products, namely TheraPEG and HATU PEGylated
proteins have a slower rate of entry to the plasma than the di-PEG
conjugate (GlycoPEG) and therefore a more pronounced depot effect.
This can be deduced by comparing the differences in the IV and SQ
half-lives in each product. [0297] For TheraPEG-FVIIa the IV t1/2
was 8.68 hours which compares to 23.2 hours for the same product
given by SQ. This represents a 2.7-fold increase implying a very
large depot effect for this mono-PEGylated product. [0298]
Similarly, for the mono-PEGylated HATU PEG-FVIIa the SQ t1/2 has an
enhanced depot effect represented by a 1.7-fold increase over the
IV t1/2 (24.3/14.07) [0299] In contrast, for the heavily PEGylated
product, GlycoPEG-FVIIa, the half-lives for both products are
closer to parity (22.3/19.34=1.15-fold) implying that the SQ
administration of this product has little depot effect compared to
IV administration.
[0300] In other words, the mono-PEGylated products when
administered SQ would appear to have resisted being dispersed
through the sub-cutaneous space for longer than the di-PEGylated
product, thus providing the enhanced depot effect. The reduced
amount of PEG on the mono-PEGylated products would leave more of
the protein exposed; the greater PEG coverage on the GlycoPEG
product would render it more water dispersible within the
subcutaneous space, leading to a faster rate of entry via the
lymphatic vessels into the plasma.
[0301] Surprisingly therefore, to achieve the longest duration of
depot release, a lesser degree of PEGylation is required. Without
being bound by theory, this can be rationalised by the lesser
PEGylation exposing some of the protein to the subcutaneous tissue
which confers a slow rate on the diffusion through the lymph. By
contrast the higher degree of PEGylation covers the protein
completely leaving the product free to quickly enter the blood
circulation.
[0302] This supports the teaching that the modification of target
molecules, in this case via PEGylation, may be tuned to exquisitely
modify the release characteristics and thereby the concentration of
the product in the blood over time and its bioavailability.
[0303] Overall, there is a very surprising total effect whereby the
combination of PEGylation followed by subcutaneous delivery,
renders an observed 35-fold increase in apparent half-life (0.66
hours for naked FVIIa to 23.2 hours following subcutaneous (SQ)
administration).
[0304] Finally, it can be seen overall that the bioavailability
favours the higher PEGylated species, namely GlycoPEG, confirming
that the higher PEG and hydration levels promote a higher degree of
mobility and therefore bioavailability.
TABLE-US-00027 TABLE 26 (a) Test Dose Cmax AUC0-t AUC0-.infin.
Article route Rat Tmax (h) (ng/ml) (ng h/ml) (ng h/ml) t1/2 (h)
FVIIa IV 1 0.03 2251.8 600 624 0.55 2 0.03 2538.3 711 728 0.75 3
0.03 1892.3 533 561 0.66 Mean 0.03 2227.47 615 638 0.65
TheraPEG-FVIIa IV 4 0.03 10024.5 33598 34021 8.97 5 0.03 8181 22051
22435 9.42 6 0.03 10799.7 23251 23449 7.66 Mean 0.03 9668.40 26300
26635 8.68 GlycoPEG-FVIIa IV 7 0.03 6647.2 55987 57138 21.24 8 0.03
5674.9 46702 47609 21.21 9 0.03 6227.3 47188 47663 15.57 Mean 0.03
6183.13 49959 50803 19.34 HATU catalysed IV 10 0.03 8090.5 31172
31775 13.49 PEG-FVIIa 11 0.03 7586.5 30448 31003 13.07 12 0.03
7557.1 35317 35697 15.66 Mean 0.03 7744.70 32312 32825 14.07
TABLE-US-00028 TABLE 26 (b) Bioavailability Dose Tmax Cmax AUC0-t
AUC0-.infin. (% AUC0-t Test Article route Rat (h) (ng/ml) (ng h/ml)
(ng h/ml) t1/2 (h) SQ vs IV ) TheraPEG-FVIIa SQ 16 18.0 215.5 8015
9557 23.96 30.5 17 12.0 117.6 3695 4839 21.15 14.0 18 18.0 129.3
4227 5805 24.52 16.1 Mean 16.0 154.13 5312 6734 23.21 20.2
GlycoPEG-FVIIa SQ 19 18.0 297.8 16868 17665 22.27 33.8 20 24.0
234.3 12471 13456 23.55 25.0 21 18.0 407.6 22243 22871 21.16 44.5
Mean 20.0 313.23 17194 17997 22.33 34.4 HATU catalysed SQ 22 18.0
224.8 10234 10996 20.85 31.7 PEG-FVIIa 23 18.0 138.7 5544 6750
28.03 17.2 24 18.0 249.1 11277 12147 24.03 34.9 Mean 18.0 204.20
9018 9964 24.30 27.9
* * * * *
References