US20150086524A1 - Optimised subcutaneous therapeutic agents - Google Patents

Optimised subcutaneous therapeutic agents Download PDF

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US20150086524A1
US20150086524A1 US14/394,437 US201314394437A US2015086524A1 US 20150086524 A1 US20150086524 A1 US 20150086524A1 US 201314394437 A US201314394437 A US 201314394437A US 2015086524 A1 US2015086524 A1 US 2015086524A1
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factor
dosage form
agent
modified
therapeutic agent
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William Henry
Richard Wolf-Garraway
John Charles Mayo
Michael James Earl
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Cantab Biopharmaceuticals Patents Ltd
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Cantab Biopharmaceuticals Patents Ltd
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Priority claimed from GBGB1206628.8A external-priority patent/GB201206628D0/en
Priority claimed from GBGB1213712.1A external-priority patent/GB201213712D0/en
Priority claimed from GBGB1214985.2A external-priority patent/GB201214985D0/en
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Assigned to CANTAB BIOPHARMACEUTICALS PATENTS LIMITED reassignment CANTAB BIOPHARMACEUTICALS PATENTS LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: EARL, Michael James, HENRY, WILLIAM, WOLF-GARRAWAY, Richard, MAYO, JOHN CHARLES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A61K47/48215—
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents

Definitions

  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the agent is 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.
  • 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.
  • 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 ⁇ -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.
  • 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 L , V H , Fab, F(ab′) 2 , Fab′, Fab3, scFv, di-scFv, sdAb, Fc and combinations thereof.
  • 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.
  • 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.
  • blood coagulation factors 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.
  • 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.
  • the blood coagulation factor is suitably Factor VII, Factor VIII or Factor IX.
  • 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 ).
  • FVII blood coagulation Factor VII
  • the protein is organised into four discrete domains: an N-terminal ⁇ -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.
  • TF cofactor tissue factor
  • 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.
  • 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.
  • NovoSeven® Human recombinant FVIIa has been developed and commercialised by Novo Nordisk as NovoSeven® (eptacog alfa [activated], ATC code B02BD08).
  • NovoSeven® 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.
  • Factor VIII is an essential blood clotting factor also known as anti-haemophilic factor (AHF).
  • AHF anti-haemophilic factor
  • 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.
  • 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 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).
  • vWF von Willebrand Factor
  • 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.
  • FIXa The binding of FIXa to FVIII in the presence of Ca 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.
  • activated FVIII is proteolytically inactivated in the process (most prominently by activated Protein C and Factor IXa) and quickly clears from the blood stream.
  • 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).
  • Factor IXa Once activated as Factor IXa and in the presence of Ca 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.
  • 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.
  • 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 is the short half-life and potential “super potency” of which has constrained prophylactic therapy to approximately 3 day intervals.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a larger dose can be administered by subcutaneous delivery such that C 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.
  • the present invention may result in less frequent administration.
  • 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.
  • a lower C max may be of benefit to the patient, as is a lower ratio of C max :C average or C max :C min (i.e. a flattened graph of peaks and troughs when compared to the typical “sawtooth” profile of an intravenously administered drug).
  • 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.
  • the standard protocol is to inject VIIa every two hours and at high dosages (45 to 90 ⁇ g/kg body weight). See, Hedner et al., Transfus. Med. Rev. 7: 78-83 (1993)).
  • procoagulants or anticoagulants in the case of factor VII
  • 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.
  • 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.
  • 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 max :C average ratio is lower than the C max :C 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 max :C average ratio to be reduced.
  • 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.
  • 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 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.
  • 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.
  • hydrophilicity is increased by at least the ratio of the molecular dimensions of the modified agent to the molecular dimensions of the unmodified agent.
  • 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.
  • HLB hydrophilic to lipophilic balance
  • 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.
  • 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.
  • the release from a subcutaneous depot can now be modulated by increasing or decreasing the level of modification of the therapeutic agent.
  • 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.
  • 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.
  • PEG polyethylene glycol
  • the process of covalently attaching a PEG molecule to another molecule is termed PEGylation.
  • PEGylation The process of covalently attaching a PEG molecule to another molecule.
  • nine PEGylated products have received FDA market approval, with four being blockbuster drugs: PegIntron® (Schering-Plough), Pegasys® (Hoffman-La Roche), Neulasta® (Amgen) and Micera® (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 ⁇ -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.
  • 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).
  • polyethyleneglycol is added in a random, non-specific manner to reactive residues on a peptide backbone.
  • 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.
  • recombinant blood clotting factors such as FVIIa, FVIII and FIX
  • 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.
  • 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.
  • TheraPEGTM Another approach to PEGylation of proteins has been developed by Polytherics and is known as TheraPEGTM 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).
  • modified therapeutic agents such as PEGylated forms of blood clotting factors
  • 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.
  • a lower C max :C 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 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.
  • 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.
  • 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.
  • 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-arth
  • 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.
  • EPO ery
  • FSH follicle stimulating hormone
  • 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.
  • the blood coagulation factor is suitably Factor VII, Factor VIII or Factor IX.
  • 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 ⁇ -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.
  • 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 L , V H , Fab, F(ab′) 2 , Fab′, Fab3, scFv, di-scFv, sdAb, Fc and combinations thereof.
  • 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.
  • 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.
  • the subcutaneous delivery volume of the modified therapeutic agent is no more than 2 ml.
  • the delivery volume may be 5 ⁇ l, 10 ⁇ l, 25 ⁇ l, 50 ⁇ l, 100 ⁇ l, 250 ⁇ l, 500 ⁇ l, 750 ⁇ l, or 1 ml.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • intravenous re-dosing with its immediate C 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 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.
  • 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.
  • 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.
  • 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 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.
  • the C max is from 20% to 80%, or from 30% to 70%, or from 40% to 60%.
  • the blood coagulation factor may be FVII, FVIII, or FIX.
  • 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 max , more frequent dosing may occur without adverse effects to the patient.
  • 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.
  • 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.
  • 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.
  • 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.
  • the blood clotting factor may be FVII, FVIII, or FIX.
  • the dosage form of the invention may be modified by any biocompatible polymer, as defined herein.
  • the modification is PEGylation.
  • the dosage form may have a C max of at least 10% and no more than 90% compared to an equivalent reference dosage form when administered intravenously.
  • the dosage form may have a C max of from 10% to 25% compared to an equivalent reference dosage form when administered intravenously.
  • the dosage form may have a C max of from 40% to 60% compared to an equivalent reference dosage form when administered intravenously.
  • the dosage form may have a C max of from 75% to 80% compared to an equivalent reference dosage form when administered intravenously.
  • the dosage form may have a C max of 75% or of 78.8% compared to an equivalent reference dosage form when administered intravenously.
  • C max is 75 to 80% and the blood factor may be FVII.
  • C max is 10% to 25% and the blood factor may be FVIII.
  • C max is 40% to 60% and the blood factor may be FIX.
  • 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.
  • 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.
  • 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.
  • the dosage form is administered less frequently than would be predicted by the plasma half life of a bolus intravenous injection.
  • 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.
  • 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.
  • WBCT Whole Blood Clotting Time
  • the formulations of the invention have a C max of at least 10%, to no more than 90% compared to an equivalent reference dosage form when administered intravenously.
  • the value may be at least 75%, 78% or 80%, and the blood factor may be FVII.
  • the value may be at least 15%, 18% or 20% and the blood factor may be FVIII.
  • the value may be 40%, 45% or 50% and the blood factor may be FIX.
  • 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.
  • 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.
  • the formulation 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.
  • the formulation may be for administration no more than once every two weeks.
  • 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.
  • 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.
  • 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.
  • 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.
  • a reference formulation formulated for intravenous administration comprising the same modified blood coagulation factor in order to achieve the same therapeutic effect.
  • the blood coagulation factor is Factor IX.
  • 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.
  • 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.
  • 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® or any other form of Tween®) 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.
  • 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.
  • 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”.
  • Modified blood coagulation 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.
  • the blood coagulation factor is suitably Factor VII, Factor VIII or Factor IX.
  • 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.
  • Factor VIIa Factor VIIa
  • Factor VII Factor VII
  • FVIII is used as an abbreviation for Factor VIII
  • FIX is used as an abbreviation for Factor IX, and so on for blood factors described herein.
  • 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).
  • 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.
  • 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”).
  • modifying agents such as polyethylene glycol polymers (“PEGylation”).
  • PEGylation polyethylene glycol polymers
  • Tween® is currently extensively used in the formulation of blood products.
  • Tween® 80 is a PEGylated fatty acid which carries a molecular weight equivalent of PEG of approximately 0.8 kilo Daltons per Tween® molecule.
  • 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® 80).
  • polysorbate e.g. Tween® 80
  • 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® is necessary.
  • PEG-FVIII 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.
  • 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.
  • 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.
  • a product to have a greater water carrying characteristic 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.
  • the polyethylene glycol (PEG) when the modification is PEG, may have a linear or branched structure and may be attached to the therapeutic agent via any convenient route.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ -amino group, while the other PEG chain is bound to the ⁇ -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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the blood factor conjugates contain multiple linear PEG polymers, where each linear PEG has a molecular weight of about 5-30 kDa.
  • 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.
  • 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.
  • each branched PEG has a molecular weight that is less than about 10 kDa.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • R1 represents a substituent which can be a direct bond, an alkylene group (preferably a C 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.
  • 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 2 , —CO 2 R, —COH, —CH 2 OH, —COR, —OR, —OCOR, —OCO 2 R, —SR, —SOR, —SO 2 R, —NHCOR, —NRCOR, —NHCO 2 R, —NR′CO 2 R, —NO, —NHOH, —NR′OH, —C ⁇ N—NHCOR, —C ⁇ N—NR′COR, —N + R 3 , —N + H 3 , —N + HR 2 , —N + H 2 R, halogen, for example fluorine or chlorine, —C ⁇ CR, —C ⁇ CR 2 and 13 C ⁇ CHR, in which each R or R′ independently represents a hydrogen atom or an alkyl (preferably C 1-6 ) or
  • 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.
  • NHCO amide
  • 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.
  • 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 2 ) 2 CHC(O)—.
  • 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:
  • the structure of the conjugate protein, where R3 is as defined below, may be as follows:
  • R3 represents a substituent which can be a direct bond, an alkylene group (preferably a C 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.
  • 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 2 , —CO 2 R, —COH, —CH 2 OH, —COR, —OR, —OCOR, —OCO 2 R, —SR, —SOR, —SO 2 R, —NHCOR, —NRCOR, —NHCO 2 R, —NR′CO 2 R, —NO, —NHOH, —NR′OH, —C ⁇ N—NHCOR, —C ⁇ N—NR′COR, —N + R 3 , —N + H 3 , —N + HR 2 , —N + H 2 R, halogen, for example fluorine or chlorine, —C ⁇ CR, —C ⁇ CR 2 and 13 C ⁇ CHR, in which each R or R′ independently represents a hydrogen atom or an alkyl (preferably C 1-6 ) or
  • 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.
  • 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.
  • the dosage form may be prepared for subcutaneous administration by formulation in a suitable aqueous vehicle.
  • 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® 80) and optionally a stabiliser (for example benzamidine or a benzamidine derivative, see U.S. Pat. No. 7,612,066 for example).
  • Nonionic surfactants/emulsifiers which can be used according to the present invention include polysorbates such as polyoxyethylene sorbitan monooleate (polysorbate 80, Tween® 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 diste
  • 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® 80 (suitably 0.005% to 0.008% Tween® 80) and optionally 0.5 mM to 5 mM benzamidine (suitably 1 mM to 2 mM benzamidine).
  • 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.
  • muteins are prepared by known synthesis and/or by site-directed mutagenesis techniques, or any other known technique suitable therefore.
  • 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.
  • 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)).
  • stringent conditions include washing conditions 12-20° C. below the calculated Tm of the hybrid under study in, e.g., 2 ⁇ SSC and 0.5% SDS for 5 minutes, 2 ⁇ SSC and 0.1% SDS for 15 minutes; 0.1.times.SSC and 0.5% SDS at 37° C. for 30-60 minutes and then, a 0.1 ⁇ SSC and 0.5% SDS at 68° C. for 30-60 minutes.
  • 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.
  • TMAC tetramethyl ammonium chloride
  • 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.
  • 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.
  • the mutein has substantial blood factor activity, it can be considered to have substantially similar activity to blood factor.
  • 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.
  • 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.
  • 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.
  • a “percent identity” may be determined.
  • 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.
  • 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.
  • 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.
  • 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.
  • amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains).
  • amino acids having aliphatic side chains amino acids having aliphatic side chains.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • “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.
  • 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.
  • acyl moieties e.g. alkanoyl or carboxylic aroyl groups
  • 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.
  • an “active fragment of blood factor” 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.
  • 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.
  • 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.
  • mineral acids such as, for example, hydrochloric acid or sulfuric acid
  • organic acids such as, for example, acetic acid or oxalic acid.
  • any such salts must retain the biological activity of blood factors as described herein.
  • AUC area under the curve
  • concentration of a drug in systemic circulation in the 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.
  • concentration or “renal clearance” is defined as the volume of plasma that contains the amount of drug excreted per minute.
  • half-life 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 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.
  • 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.
  • molecular dimensions means the weight, size and/or shape of an agent.
  • “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 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.
  • 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.
  • 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 max /subcutaneous C max .
  • 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.
  • subcutaneous space means the connective tissue under the skin. It excludes blood vessels, the blood stream and internal organs.
  • “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.
  • 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.
  • 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.
  • 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.
  • 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.
  • excipients may include, but are not limited to, saline, buffered saline (such as phosphate buffered saline), dextrose, liposomes, water, glycerol, ethanol and combinations thereof.
  • 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.
  • the active agent administered to an individual as an injectable composition may be, for example, a sterile aqueous dispersion, preferably isotonic.
  • 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 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.
  • 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.
  • 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 max of at least 10% and no more than 90% of that achieved by intravenous administration of the modified blood factor.
  • the C max is from 20% to 80%, or from 30% to 70%, or from 40% to 60%.
  • C max is 75 to 80% and the blood factor may be FVII.
  • C max is 10% to 25% and the blood factor may be FVIII.
  • C max is 40% to 60% and the blood factor may be FIX.
  • Blood clotting diseases or disorders may be characterised by a loss of function of a blood clotting factor, or the generation of auto-antibodies.
  • blood clotting diseases include haemophilia A and haemophilia B.
  • 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.
  • 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.
  • 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.
  • 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).
  • WBCT Whole Blood Clotting Time
  • APTT Activated Partial Thromboplastin Time
  • 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.
  • 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.
  • APTT Activated Partial Thromboplastin Time
  • 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.
  • 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.
  • 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 max of at least 10% and no more than 90% compared to an equivalent reference dosage form when administered intravenously.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 max of at least 10% and no more than 90% for use in the treatment of a blood clotting disorder.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • BU Bethesda unit
  • BU blood coagulation inhibitor activity
  • 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° C.” (Schumacher, Harold Robert (2000). Handbook of Hematologic Pathology . Informa Health Care, p. 583).
  • 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.
  • 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.
  • 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.
  • FIG. 1 shows the blood coagulation cascade.
  • HMWK High Molecular Weight Kininogen
  • PK Prekallikrein
  • PL Phospholipid.
  • 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).
  • FIG. 3 shows Whole Blood Clotting Times (WBCT) following subcutaneous (SQ) administration of PEGhrFIX to subject Dog 1 and hrFIX to subject Dog 2.
  • FIG. 4 shows APTT (Activated Partial Thromboplastin Time) Values with Time Following SQ Administration.
  • FIG. 5 shows APTT of Retained Plasma Following SQ Administration.
  • FIG. 6 shows APTT Relative Values to Baseline Following SQ Administration.
  • FIG. 7 shows subject Dog 9 WBCT following 25 IU/Kg SQ Administration.
  • FIG. 8 shows PK profiles and parameters of FVIIa following 200 ug/kg rFVIIa.
  • FIG. 9 shows PK profiles and parameters of FVIIa following 800 ug/kg TheraPEG-rFVIIa.
  • FIG. 10 shows PK profiles and parameters of FVIIa following 1600 ug/kg TheraPEG-rFVIIa.
  • FIG. 11 shows concentration of FVIII in Plasma (all dogs).
  • FIG. 12 shows concentration of FVIII in Plasma (SQ administered dogs only).
  • FIG. 13 shows immune data (Bethesda value) for PEGFVIII administered subcutaneously (SQ) compared to intravenous (IV), number of subjects is given by “n”.
  • 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.
  • 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.
  • 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® 80. 1 mM benzamidine was added as a stabiliser.
  • 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.
  • 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.
  • 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.
  • Table 1 is a summary of the titres measured at 22 hours as circulating FIX following intravenous administration.
  • Table 2 shows comparison of measured Circulating FIX Titre at 22 Hours Following subcutaneous (SQ) Administration.
  • 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.
  • test subject Dog 9 was a na ⁇ ve 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.
  • 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.
  • 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
  • Results show a C 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 max value given.
  • 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%.
  • 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.
  • 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.
  • Polysorbate, Tween® 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)).
  • each FVIII molecule has the equivalent of an associated 516 kDa PEG.
  • the PEGFVIII dosage formulation prepared according to the present invention has a single 20 kDa PEG.
  • 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.
  • TheraPEGylation of transgenic FVIIa 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
  • the experimental animals were Lhasa Apso-Basenji cross dogs with congenital severe haemophilia B caused by a 5-bp deletion and a C ⁇ 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.
  • 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.
  • Plasma samples 4 ml were 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° C. An aliquot of plasma was assayed for FVIIa concentration by ELISA.
  • 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
  • PEG-rFVIIa (0.78 to 50 ng/ml) for assay of plasma from dogs that were administered PEG-rFVIIa.
  • 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.
  • 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 ⁇ higher than that of the 800 ug/kg IV dose.
  • the AUC for the 800 ug/kg IV dose of TheraPEG-rFVIIa is 84 ⁇ 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 ⁇ that of 200 ug/kg SQ non-PEGylated rFVIIa.
  • TheraPEG-pdFVIII TheraPEGylated plasma derived FVIII
  • TheraPEG-pdFVIII was prepared as described in WO 2011/135307 with a 20 kDa linear PEG and further purified to yield purified TheraPEG-pdFVIII.
  • 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 na ⁇ ve 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.
  • 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.
  • IV Intravenous
  • SQ Sub cutaneous
  • 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° C. for the FVIII antigen ELISA.
  • ACT Activated Clotting Time
  • APTT Activated Partial Thromboplastin Time
  • the ACT and APTT tests were carried out using a Haemachron Jr coagulation analyzer (International Technidyne Corporation.) according to the manufacturer's instructions.
  • 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.
  • Haemostasis 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.
  • 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.
  • a lower dose of TheraPEG-pdFVIII (HA6) given SQ maintained ACT below 200 seconds for at least 36 h.
  • non-PEGylated FVIII given SQ reduced the ACT, it did not result in a sustained ACT below 200 seconds.
  • 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.
  • TheraPEG-pdFVIII (HA6) given SQ maintained APTT at less than 60 seconds for 40 h.
  • 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.
  • 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.
  • 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.
  • 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.
  • the PK profile of FVIII following SQ administration of TheraPEG-pdFVIII had a much reduced C max and AUC compared to IV administration and bioavailability was determined to be 32%.
  • C max and AUC 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 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.
  • 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 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.
  • the plots in FIG. 13 demonstrate how much inhibitor activity has been found in blood plasma over time, as stimulated by the treatments.
  • 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.
  • 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.
  • rFVIIa 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.
  • 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 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.
  • Formulations at a dose of 0.5 mg/kg were administered either IV or SQ to Healthy rat subjects.
  • IV administration the appropriate volume of test article was injected into the tail vain.
  • SQ administration the appropriate volume of test article was injected into the scruff of the neck.
  • blood samples were taken at the following time intervals:
  • Plasma samples were 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.
  • 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.
  • IV intravenous
  • SQ subcutaneous
  • 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.
  • 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.
  • 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.
  • 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.

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Publication number Priority date Publication date Assignee Title
CN109498815B (zh) * 2013-12-30 2022-07-22 江苏众红生物工程创药研究院有限公司 重组人激肽释放酶的化学修饰物及其应用
GB201417589D0 (en) * 2014-10-06 2014-11-19 Cantab Biopharmaceuticals Patents Ltd Pharmaceutical Formulations
FR3034669B1 (fr) * 2015-04-07 2020-02-14 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Nouvelle utilisation du facteur von willebrand
US20180169297A1 (en) * 2015-06-12 2018-06-21 Laboratoire Français Du Fractionnement Et Des Biotechnologies Injectable composition of factor vii and fillers
GB201518171D0 (en) 2015-10-14 2015-11-25 Cantab Biopharmaceuticals Patents Ltd Colloidal particles for topical administration with therapeutic agent
GB201518172D0 (en) 2015-10-14 2015-11-25 Cantab Biopharmaceuticals Patents Ltd Colloidal particles for use in medicine
GB201518170D0 (en) 2015-10-14 2015-11-25 Cantab Biopharmaceuticals Patents Ltd Colloidal particles for subcutaneous administration with intravenous administration of therapeutic agent
CN109998994B (zh) * 2018-02-13 2021-12-07 四川大学 包含药物的柔性脂质体及其制备方法
CN114929261A (zh) 2020-02-17 2022-08-19 生物测试股份公司 凝血因子viii的皮下施用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5925739A (en) * 1994-03-31 1999-07-20 Pharmacia & Upjohn Ab Pharmaceutical formulation for subcutaneous intramuscular or intradermal administration of factor VIII
US7199223B2 (en) * 2003-02-26 2007-04-03 Nektar Therapeutics Al, Corporation Polymer-factor VIII moiety conjugates

Family Cites Families (56)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1479268A (en) 1973-07-05 1977-07-13 Beecham Group Ltd Pharmaceutical compositions
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
CH596313A5 (fr) 1975-05-30 1978-03-15 Battelle Memorial Institute
US4414147A (en) 1981-04-17 1983-11-08 Massachusetts Institute Of Technology Methods of decreasing the hydrophobicity of fibroblast and other interferons
US4496689A (en) 1983-12-27 1985-01-29 Miles Laboratories, Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
JP2524586B2 (ja) 1985-06-26 1996-08-14 シタス コーポレイション ポリマ−接合を利用する医薬組成物用蛋白質の可溶化
WO1990015628A1 (fr) * 1989-06-14 1990-12-27 Cetus Corporation Conjugue polymere antibiotique
WO1994005332A2 (fr) 1992-09-01 1994-03-17 Berlex Laboratories, Inc. Glycolation de macromolecules glycosylees
DE69332928T2 (de) 1992-09-28 2004-05-06 Research Corp. Technologies, Inc., Tucson Peptidekupplungsreagentien
US5621039A (en) * 1993-06-08 1997-04-15 Hallahan; Terrence W. Factor IX- polymeric conjugates
JPH0892294A (ja) * 1994-09-29 1996-04-09 Teijin Ltd ヒト活性化プロテインｃ誘導体
US5693609A (en) 1994-11-17 1997-12-02 Eli Lilly And Company Acylated insulin analogs
JPH08325270A (ja) * 1995-05-31 1996-12-10 Bristol Myers Squibb Co β−ラクタマーゼに対するポリマープロドラッグおよびその使用
SE9503380D0 (sv) * 1995-09-29 1995-09-29 Pharmacia Ab Protein derivatives
EP0921817B1 (fr) 1997-01-29 2001-03-28 PolyMASC Pharmaceuticals plc Procede de p.e.g.ylation
WO2001082943A2 (fr) 2000-05-03 2001-11-08 Novo Nordisk A/S Administration sous-cutanee de facteur coagulant vii
AU2605099A (en) * 1998-02-25 1999-09-15 Micrologix Biotech, Inc. Indolicidin and cationic peptides conjugated with polymers
ES2325877T3 (es) * 2000-02-11 2009-09-23 Bayer Healthcare Llc Moleculas de tipo factor vii o viia.
US20030211094A1 (en) * 2001-06-26 2003-11-13 Nelsestuen Gary L. High molecular weight derivatives of vitamin k-dependent polypeptides
KR101186140B1 (ko) * 2002-06-21 2012-09-27 노보 노르디스크 헬스 케어 악티엔게젤샤프트 페길화된 인자 ｖｉｉ 글리코형태
DE60336555D1 (de) * 2002-06-21 2011-05-12 Novo Nordisk Healthcare Ag Pegylierte glykoformen von faktor vii
WO2004089280A2 (fr) * 2003-04-08 2004-10-21 Yeda Research And Development Co. Ltd. Medicaments pegyles reversibles
US20130137157A1 (en) * 2003-04-09 2013-05-30 Novo Nordisk A/S Glycopegylated factor vii and factor viia
GB0316294D0 (en) 2003-07-11 2003-08-13 Polytherics Ltd Conjugated biological molecules and their preparation
US20060198819A1 (en) 2003-08-08 2006-09-07 Novo Nordisk Healthcare A/G Use of galactose oxidase for selective chemical conjugation of protractor molecules to proteins of therapeutic interest
JPWO2005084679A1 (ja) 2004-03-04 2007-11-29 エーザイ・アール・アンド・ディー・マネジメント株式会社 ベンズアミジン誘導体含有組成物及びベンズアミジン誘導体の安定化方法
MXPA06015234A (es) * 2004-06-30 2007-11-22 Nektar Therapeutics Al Corp Conjugados de fraccion polimero-factor ix.
JP2008507990A (ja) 2004-08-02 2008-03-21 ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト Ｆｖｉｉの抱合体
JP5216580B2 (ja) 2005-05-25 2013-06-19 ノヴォ ノルディスク アー／エス グリコペグ化第ｉｘ因子
EP1893632B1 (fr) 2005-06-17 2015-08-12 Novo Nordisk Health Care AG Reduction et derivation séléctives de proteines facteur vii comprenant au moins une cysteine non native
JP2009515508A (ja) * 2005-08-19 2009-04-16 ネオス テクノロジーズ インコーポレイテッド グリコｐｅｇ化因子ｖｉｉおよび因子ｖｉｉａ
AU2007245190B2 (en) * 2006-03-31 2011-07-21 Takeda Pharmaceutical Company Limited Pegylated factor VIII
US20100056428A1 (en) 2006-09-01 2010-03-04 Novo Nordisk Health Care Ag Modified proteins
ES2521490T3 (es) 2006-12-15 2014-11-12 Baxter International Inc. Conjugado de factor VIIa - ácido (poli)siálico con una vida media in vivo prolongada.
CN101678119B (zh) * 2006-12-27 2014-02-12 尼克塔治疗公司 具有可释放的键合的血管性血友病因子-和因子ⅷ-聚合物轭合物
EP2099475B1 (fr) 2007-01-03 2016-08-24 Novo Nordisk Health Care AG Administration sous-cutanée de polypeptides liés au facteur viia de la coagulation
WO2008119815A1 (fr) * 2007-04-02 2008-10-09 Novo Nordisk A/S Administration sous-cutanée du facteur ix de la coagulation
WO2008127702A2 (fr) 2007-04-13 2008-10-23 Catalyst Biosciences, Inc. Polypeptides du facteur vii modifiés et leurs utilisations
EP2014299A1 (fr) 2007-07-11 2009-01-14 Novo Nordisk A/S Administration sous-cutanée du facteur VII de coagulation
US20100239517A1 (en) 2007-10-09 2010-09-23 Stephen Brocchini Novel conjugated proteins and peptides
JP5733744B2 (ja) 2007-10-16 2015-06-10 ファーマサイクリックス，インク． 凝血因子ＶＩＩａモジュレーターの製造、組成物及び使用
EP2257311B1 (fr) 2008-02-27 2014-04-16 Novo Nordisk A/S Molécules de facteur viii conjuguées
TWI538916B (zh) 2008-04-11 2016-06-21 介控生化科技公司 經修飾的因子vii多肽和其用途
JP2011517950A (ja) * 2008-04-16 2011-06-23 バイエル・ヘルスケア・エルエルシー 第ｉｘ因子の部位特異的修飾
CN102046205A (zh) * 2008-04-24 2011-05-04 凯尔特药物Peg有限公司 具有延长的半衰期的因子ix缀合物
JP5642065B2 (ja) * 2008-05-23 2014-12-17 ノボ ノルディスク ヘルス ケア アーゲー ペグ化されたｇｌａ−ドメイン含有タンパク質を含む低粘度組成物
US8969626B2 (en) 2008-07-21 2015-03-03 Polytherics Limited Reagents and method for conjugating biological molecules
DK2349341T3 (da) * 2008-10-15 2014-01-06 Baxter Int Pegylering af rekombinante blodkoagulationsfaktorer i nærvær af bundne antistoffer
ES2692172T3 (es) * 2008-10-17 2018-11-30 Baxalta GmbH Factores sanguíneos modificados que comprenden un bajo grado de polímero soluble en agua
WO2010083536A1 (fr) * 2009-01-19 2010-07-22 Bayer Healthcare Llc Conjugué protéique ayant une fraction bio-protectrice pouvant être clivée par une endopeptidase
WO2011017055A2 (fr) * 2009-07-27 2011-02-10 Baxter International Inc. Conjugués de protéine de coagulation sanguine
WO2011101242A1 (fr) * 2010-02-16 2011-08-25 Novo Nordisk A/S Molécules de facteur viii avec liaison réduite au vwf
GB201007357D0 (en) 2010-04-30 2010-06-16 Leverton Licence Holdings Ltd Conjugated factor VIII
GB201007356D0 (en) * 2010-04-30 2010-06-16 Leverton Licence Holdings Ltd Conjugated factor VIIa
DK2598172T3 (da) * 2010-07-30 2019-07-01 Baxalta GmbH Nukleofile katalysatorer til oximforbindelse
WO2012088123A1 (fr) * 2010-12-22 2012-06-28 Nektar Therapeutics Conjugués polymère-fraction de facteur vii

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5925739A (en) * 1994-03-31 1999-07-20 Pharmacia & Upjohn Ab Pharmaceutical formulation for subcutaneous intramuscular or intradermal administration of factor VIII
US7199223B2 (en) * 2003-02-26 2007-04-03 Nektar Therapeutics Al, Corporation Polymer-factor VIII moiety conjugates

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Peng, A., et al. 2012 The AAPS Journal 14(1): 35-42. *

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