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WO2025022005A1 - Administration intranasale de polypeptides du facteur ix - Google Patents

Administration intranasale de polypeptides du facteur ix Download PDF

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Publication number
WO2025022005A1
WO2025022005A1 PCT/EP2024/071359 EP2024071359W WO2025022005A1 WO 2025022005 A1 WO2025022005 A1 WO 2025022005A1 EP 2024071359 W EP2024071359 W EP 2024071359W WO 2025022005 A1 WO2025022005 A1 WO 2025022005A1
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fix
polypeptide
factor
fix polypeptide
wild
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Sandra LE QUELLEC
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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21022—Coagulation factor IXa (3.4.21.22)
    • 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
    • A61K9/0043—Nose

Definitions

  • This invention relates to the intranasal administration of Factor IX (FIX) polypeptides for use in treating bleeding disorders.
  • FIX Factor IX
  • FIX Human coagulation Factor IX plays a key role in the formation of blood clots.
  • FIX has been used in the prophylaxis and treatment of bleeding disorders, such as haemophilia B.
  • Current treatments for haemophilia B include the intravenous administration of wild-type FIX including BeneFIX® and RIXUBIS® and of FIX proteins that have an extended half-life in plasma, including IDELVION®, ALPROLIX® and REBYNIN®.
  • Haemophilia B is a rare inherited X-linked recessive bleeding disorder characterised by coagulation factor IX (FIX) deficiency [1], The bleeding phenotype of haemophilia B is generally correlated with FIX clotting activity (FIX:C) in plasma. Severe haemophilia B (FIX:C ⁇ HU/dL) is characterised by spontaneous and prolonged bleeding episodes, mostly in the joints, but which can be life-threatening such as when intracranial haemorrhage occurs [2],
  • the present invention advantageously provides a new administration route for FIX polypeptides that is non-invasive.
  • the present invention is based on the surprising realisation that large FIX polypeptides can be effectively administered intranasally and that this transport occurs mainly via the paracellular pathway. Understanding how FIX polypeptides are transported across the nasal epithelium opens up new possibilities to identify novel and improved strategies for treating haemophilia B.
  • the present invention provides an advantageous new route for administering FIX polypeptides. It was thought that the transport of drugs across the nasal epithelium, especially large molecular weight biologies, is mediated by the neonatal receptor Fc fragment of immunoglobulin (Ig) G (FcRn) [4], As now shown in the present disclosure, the inventors have advantageously identified for the first time that FIX polypeptides can be effectively delivered intranasally and that this transport occurs mainly via the paracellular pathway which is FcRn receptor independent. This is advantageous because it enables all types of FIX polypeptides to be nasally administered, i.e., irrespective of their ability to bind the FcRn.
  • FIX polypeptides can be effectively transported across the nasal epithelium. This is useful because it enables FIX proteins that have a high molecular weight, for example FIX proteins that have an extended half-life in plasma (e.g. FIX-PEG and FIX-albumin), to be effectively administered intranasally. Therefore, this newnon- invasive route of administration is particularly useful for treating and preventing bleeding disorders, such as haemophilia B.
  • This new non-invasive route of administering FIX polypeptides is also particularly useful for administering FIX polypeptides to children, for example patients who are 18 years old or younger, or patients who are 12 years old or younger.
  • the invention therefore provides a Factor IX (FIX) polypeptide for use in a method of treating or preventing a bleeding disorder by administering the FIX polypeptide intranasally.
  • FIX Factor IX
  • the invention also provides a method of treating or preventing a bleeding disorder in a subject comprising administering a therapeutically or prophylactically effective amount of a FIX polypeptide intranasally to the subject.
  • the invention further provides a use of a FIX polypeptide in the manufacture of a medicament for treating or preventing a bleeding disorder in a subject, wherein the FIX polypeptide is to be administered intranasally to the subject.
  • the invention further provides a FIX polypeptide for treating or preventing a bleeding disorder, wherein the FIX polypeptide is to be administered intranasally to the subject.
  • the bleeding disorder is haemophilia B (also known as congenital factor IX deficiency).
  • the FIX polypeptide is administered as a nasal spray. In some embodiments, the FIX polypeptide is administered directly to the nasal epithelium of a subject. In some embodiments, the FIX polypeptide is administered by a nasal inhaler.
  • the FIX polypeptide can be administered at a concentration of 500-20,000 U/ml, preferably at a concentration of 500-5,000 U/ml.
  • the FIX polypeptide can be administered in combination with a surfactant including a surfactant (e.g. polysorbate) and/or with water.
  • a surfactant e.g. polysorbate
  • the FIX polypeptide can be administered in combination with sodium citrate, polysorbate (e.g. polysorbate 80 and/or polysorbate 20), mannitol, sucrose and hydrochloric acid.
  • the FIX polypeptide is administered in combination with an agent that disrupts epithelial barrier function, such as an agent that increases the permeability of tight junction proteins.
  • the Factor IX polypeptide can have an amino acid sequence that is at least 95%, 96%, 97%, 98% or at least 99% identical to SEQ ID NO: 1 , across the full length of SEQ ID NO: 1 ; or the FIX polypeptide can be defined by SEQ ID NO: 1.
  • the FIX polypeptide can be linked to a half-life enhancing portion, in particular wherein the half-life enhancing portion is selected from the group consisting of albumin including variants and derivatives thereof, polypeptides of the albumin family including variants and derivatives thereof, immunoglobulins without antigen binding domain (e.g., the Fc portion), and polyethylene glycol.
  • the FIX polypeptide is linked to albumin.
  • the FIX polypeptide is linked to the half-life enhancing portion via a linker e.g., a cleavable or non-cleavable linker, preferably a cleavable linker.
  • the FIX polypeptide can be a high-activity variant.
  • the FIX polypeptide can comprise a leucine at a position corresponding to position 338 of wild-type Factor IX.
  • the FIX polypeptide comprises the amino acid tyrosine at a position corresponding to position 318 of wild-type Factor IX, the amino acid glutamic acid at a position corresponding to position 338 of wildtype Factor IX and the amino acid arginine at a position corresponding to position 343 of wild-type Factor IX.
  • the FIX polypeptide can have decreased binding to extracellular matrix, as compared to wild-type FIX.
  • the FIX polypeptide can comprise the amino acid alanine at a position corresponding to position 5 of wild-type Factor IX and/or lysine at a position corresponding to position 10 of wild-type Factor IX.
  • the FIX polypeptide can be administered at a concentration that results in at least a 3-30% bioavailability in the blood plasma.
  • the FIX polypeptides can be administered intranasally to children, for example the FIX polypeptides can be administered to a subject who is 18 years old or younger, or a patient who is 12 years old or younger.
  • polypeptide and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids
  • polypeptides of this invention may be based, for example, upon antibodies or other members of the immunoglobulin superfamily, in certain embodiments, a “polypeptide” can occur as a single chain or as two or more associated chains.
  • a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids is the same in comparing the two sequences.
  • the percentage sequence identity is calculated as the percentage of identical amino acids within the aligned sequences.
  • a sequence that “has” (or “having”) x % sequence identity to another sequence means that the sequence is x % identical to that other sequence.
  • the Factor IX polypeptide may be provided as an “isolated” or as a “purified” polypeptide.
  • This term may refer to a polypeptide produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated (e.g., so as to exist in “substantially pure” form).
  • isolated is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, or the addition of stabilizers.
  • FIX protein or “FIX polypeptide” herein refers to the weight of the FIX portion (e.g., as defined in SEQ ID NO: 1, 9, 10 or 19) in the protein/polypeptide, i.e., excluding the weight of any additional portions such as fusion partners (e.g., albumin).
  • FIX polypeptides described herein have FIX clotting activity, e.g., they have the clotting activity of wild-type FIX or clotting activity that is similar to wild-type FIX, or they may even have a higher clotting activity than wild-type FIX; clotting activity can be measured by standard assays known to those skilled in the art.
  • the FIX polypeptides can have clotting activity that is below the level of wild-type FIX, e.g. FIX polypeptides that are linked to a half-enhancing portion.
  • administration or “administering” or “administered” are used interchangeably herein. Unless specifically stated otherwise the term administration refers to intranasal administration.
  • treatment refers to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder.
  • treatment may include prophylaxis, unless indicated otherwise.
  • treatment also include on-demand treatment.
  • a disorder is treated or prevented if administration of a Factor IX polypeptide as described herein to a subject (e.g. , a human with Factor IX deficiency such as haemophilia B) results in a therapeutic or prophylactic effect.
  • the plasma level of Factor IX activity in the subject is increased following treatment, at least temporarily, when measured with at least one Factor IX assay.
  • the Factor IX activity can be determined using an in vitro aPTT -based one stage clotting assay (ref 5 and 6), a chromogenic FIX assay (ref 7 or 64) or a tail clip model (ref 8).
  • the increase may be clinically relevant, e.g., a reduction in the frequency or intensity of bleeding events.
  • a “therapeutically effective amount” it is meant that the administration of that amount of Factor IX polypeptide to a subject, either in a single dose or as part of a series, is effective for treatment.
  • a “prophylactically effective amount” it is meant that the administration of that amount of Factor IX polypeptide to a subject, either in a single dose or as part of a series, is effective for prevention.
  • Such methods have efficacy in the treating or preventing disorders where a pro-coagulant activity is needed (e.g., to prevent, reduce or inhibit bleeding) and include, without limitation, haemophilia, particularly haemophilia B.
  • references to methods of treating or preventing a bleeding disorder in a subject include methods of treating or preventing a bleeding disorder in a subject in need thereof.
  • reduced binding refers to Factor IX polypeptides that have reduced FIX binding to extracellular matrix compared to wild-type FIX, and includes FIX variants that exhibit no binding to extracellular matrix.
  • FIX binding to extracellular matrix can be determined by various known biological assays, for example the competitor binding assay as described in (ref 9).
  • FIX polypeptides with reduced binding retain FIX clotting activity, e.g., they have the clotting activity of wild-type FIX , or clotting activity that is similar to wild-type FIX, or they may even have a higher clotting activity than wild-type FIX. Clotting activity may be assessed by assays known in the art.
  • Any reference to a method for treatment comprising administering FIX polypeptide to a subject also covers the FIX polypeptide for use in said method for treatment, as well as the use of the FIX polypeptide in said method for treatment, and the use of the FIX polypeptide in the manufacture of a medicament for treating a disease.
  • subject refers to any animal (e.g., a mammal), including, but not limited to, humans, nonhuman primates, canines, felines, rabbits, rodents, fish (e.g., zebrafish) and the like, which is to be the recipient of a particular treatment.
  • the subject is preferably a human.
  • the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
  • pharmaceutically acceptable refers to a substance approved or approvable by a regulatory agency of the Federal government or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.
  • pharmaceutically acceptable excipient, carrier, or adjuvant or “acceptable pharmaceutical carrier” refer to an excipient, carrier, or adjuvant that can be administered to a patient, together with at least one agent of the present disclosure, and which does not destroy the pharmacological activity thereof and is non-toxic when administered in doses sufficient to deliver a therapeutic effect.
  • pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation.
  • substantially pure refers to a preparation comprising at least 75% by weight of Factor IX polypeptide, particularly at least 80% by weight, at least 85% by weight, at least 90% by weight, at least 95% by weight, or at least 96%, 97%, 98%, or 99% by weight, e.g., 90-99% or more by weight of Factor IX polypeptide. Purity may be measured by methods appropriate for the compound of interest (e.g., chromatographic methods, polyacrylamide gel electrophoresis, HPLC analysis, and the like).
  • composition “comprising” encompasses “including” as well as “consisting”, “consisting of’ and/or “consisting essentially of’, e.g., a composition “comprising” X may consist exclusively of X or may include something additional, e.g., X + Y. It is also understood that wherever embodiments are described herein with the language “consisting essentially of’ otherwise analogous embodiments described in terms of “consisting of’ are also provided.
  • the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • the verb “to consist” may be replaced, if necessary, by “to consist essentially of’ meaning that a product as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
  • the invention relates to the intranasal administration of FIX polypeptides for use in therapy.
  • the invention is based on the surprising realisation that FIX polypeptides can be effectively administered intranasally.
  • intranasal administration is an attractive administration route. It has many advantages compared to oral administration, such as a rapid onset of drug action, less drug degradation, and a high rate of absorption. Intranasal administration is also advantageous compared to intravenous administration because it can be self-administered, it is easy to use and has a reduced risk of infection because no needles are required [10], Furthermore, nasal administration is a more convenient administration route, in particular for children, e.g. patients at or under the age of 18 or patients at or under the age of 12. Intranasal administration is also useful for patients that have poor venous access or who find it difficult to self-infuse, such as children.
  • Intranasal administration is therefore particularly useful for patients who are 18 years old or younger, preferably patients who are 12 years old or younger.
  • Intranasal administration (also referred to as nasal administration) is a term that is understood by those skilled in the art (https://www.fda.gov/drugs/data-standards-rnanual-rnonographs/route- admim strati on). It can result in systemic and local pharmacological effect and therefore covers administration to the nose and administration by way of the nose.
  • the FIX polypeptides are for administration to a subject, such as an animal, typically a human subject.
  • the principal structure of the nose is in general comparable between rodents, which are commonly used as laboratory animals, and humans.
  • the nasal cavity is divided into two areas which reach from the nostrils towards the nasopharynx. They can be separated into three regions: the vestibular region, the respiratory region and the olfactory region [11], Therefore, the conclusions reached in the examples, based on experiments conducted in mice, can be extrapolated to the nasal administration of FIX polypeptides to humans.
  • the FIX polypeptide can be administered directly to the nasal epithelium of a subject, for example as a powder, a liquid preparation, a spray or a gel.
  • Devices that can be used for nasal drug delivery include nose drops, nasal sprays, nasal inhaler and nasal douches.
  • Nasal drops are administered by drawing liquid into a glass dropper, inserting the dropper into the nostril, and squeezing the top rubber valve to release the liquid.
  • Nasal sprays provide measured doses of drugs that are dissolved or suspended in the excipient solution or in a mixture of non-pressurized dispensers for drug delivery to the nasal cavity.
  • Nasal sprays have the advantage of being non-invasive, avoiding the first-pass hepatic effect, with a fast onset of action, and good patient compliance [10],
  • the FIX polypeptide is administered as a nasal spray.
  • Intranasal administration normally involves applying the drug to both nostrils, e.g. a nasal spray is normally applied to both nostrils.
  • nasal administration can also occur in only one nostril, i.e. only the left nostril or only the right nostril.
  • Intranasal administration can require multiple applications per day.
  • the FIX polypeptides can be administered intranasally (for example into one or both nostrils) two times, three times or four times daily. If the FIX polypeptide is linked to a half-life enhancing portion, then the administration frequency can be reduced.
  • the FIX polypeptide linked to a half-life enhancing portion can be administered once a day or twice a day, respectively.
  • the FIX polypeptide linked to a half-life enhancing portion can be administered every other day, every third day, or once per week.
  • the volume of solution or suspension delivered per dose may be anywhere from 10 to 1000 uL and preferably between 50 and 300 uL. Delivery systems for these various dosage forms may be dropper bottles, plastic squeeze units, atomizers, nebulizers, metered nasal sprayers, or pharmaceutical aerosols in either unit dose or multiple dose packages. Aerosol systems require a propellant to be inert towards the formulation. Suitable propellants may be selected among such gases as fluorocarbons, hydrocarbons, nitrogen and dinitrogen oxide or mixtures thereof.
  • the maximum volume that can be administered to a human nose is usually about 0.2 ml - 0.4 ml per nostril.
  • the typical volume of liquid that is dispensed in a single spray actuation is from 0.01 to 0.14 ml, for example from 0.05 to 0.14 ml, such as 0.1 ml. It is a practical proposition to administer up to about 0.2 ml into each nostril (i.e. two times 0.1 ml sprays) to provide a therapeutic dose of drug, although the most acceptable dosing regimen would be one spray into one or both nostrils.
  • the FIX polypeptide is preferably administered at a concentration that results in at least a 5% bioavailability in the blood plasma, for example, 5-15% bioavailability in the blood plasma or 3-30% bioavailability in the blood plasma.
  • the bioavailability of the administered FIX polypeptide in the blood plasma is at least 5% e.g. between 5-30% or 10-20%.
  • the FIX polypeptide can be administered at a concentration of 500-20,000 U/ml, preferably narrower at a concentration of 500-5,000 U/ml.
  • a lower amount of the high activity variants discussed herein can be used to effectively treat a bleeding disorder.
  • the FIX polypeptide is a high activity variant with a 5-fold increase in activity
  • 1/5 of the wild-type amount would be required to effectively treat or prevent a bleeding disorder, e.g. if the wild-type and high activity variant polypeptides have the same concentration e.g. 500 -5,000 U/ml, then a 1/5 less volume of the high activity variant is required to achieve the same amount of FIX polypeptide activity..
  • Transcellular transport involves movement of molecules through cells and is mediated by apical and basolateral transmembrane transporters.
  • Paracellular transport refers to the transfer of substances across an epithelium by passing through the intercellular space between the cells via the pore pathway or the leak pathway. Both pore and leak pathways are size-selective, and the pore pathway is charge- selective. The rate of paracellular transport depends to the permeability of tight junctions, which seal the space between adjacent epithelial cells [12],
  • the neonatal receptor Fc fragment of immunoglobulin (Ig) G (FcRn) is important for the nasal delivery of biologies.
  • This receptor acts as transporter, allowing transcytosis across epithelial barriers, i.e. the transport occurs via a transcellular pathway[13].
  • the herein examples demonstrate that nasal administration of FIX polypeptides occurs mainly via the paracellular pathway rather than the FcRn mediated transcytosis. This is advantageous because it enables all types of FIX polypeptides to be nasally administered, irrespective of their ability to bind FcRn, for example wildtype FIX polypeptides and FIX polypeptides that are linked to PEG.
  • FIX polypeptides are transported by the paracellular pathway means that higher doses of FIX polypeptides can be administered intranasally, because transport of the FIX polypeptides across the nasal epithelium will not be limited by FcRn availability.
  • Administering higher doses of FIX polypeptides intranasally should result in a higher concentration of FIX polypeptides in the blood plasma, which leads to a more effective treatment for bleeding disorders.
  • the intranasal administration of FIX polypeptides occurs mainly via a paracellular pathway, which is FcRn independent.
  • the invention therefore provides a Factor IX (FIX) polypeptide for use in a method of treating or preventing a bleeding disorder by administering the FIX polypeptide intranasally, wherein the intranasal administration occurs mainly via the paracellular pathway.
  • the invention also provides a Factor IX (FIX) polypeptide for use in a method of treating or preventing a bleeding disorder by administering the FIX polypeptide intranasally, wherein the intranasal administration occurs via a FcRn independent pathway.
  • the invention provides a Factor IX (FIX) polypeptide for use in a method of treating or preventing a bleeding disorder by administering the FIX polypeptide intranasally, wherein the intranasal administration occurs via the paracellular pathway and wherein the FIX polypeptide is linked, e.g., to an Fc portion or albumin.
  • FIX Factor IX
  • Absorption enhancers are functional excipients included in formulations to improve the absorption of drugs across biological barriers.
  • Various strategies are known in the art to improve drug absorption across the nasal epithelium, including preventing degradation of the drug in the nasal epithelium; enhancing the permeability of the nasal epithelium via transient opening of tight junctions, disrupting the lipid bilayer of the nasal epithelium; and enhancing the residence time of the drug by slowing down mucociliary clearance.
  • the FIX polypeptides can be administered in combination with an absorption enhancer, such as those described in ref [14],
  • FIX polypeptides are intranasally administered mainly via the paracellular pathway also opens up the possibility of co-administering FIX polypeptides with agents that disrupt the nasal epithelium or increase the permeability of the nasal epithelium, for example agents that increase the permeability of tight junction proteins. Co-administering FIX polypeptides with such agents would increase the rate of transport across the nasal epithelium and result in higher levels of FIX in the blood plasma, which would lead to a more effective treatment of bleeding disorders.
  • the FIX polypeptides can be administered in combination with an agent that increases the permeability of tight junction proteins.
  • Agents that are known to modulate tight junction proteins include Clostridium perfringens enterotoxin (CPE) and Zonula occludens toxin (ZOT).
  • CPE Clostridium perfringens enterotoxin
  • ZOT Zonula occludens toxin
  • the C-terminal fragment of CPE is known to modulate the barrier function of claudin.
  • Claudin is one of the key structural and functional components of the tight junction seal.
  • ZOT and Zot derivatives are reversible tight junction proteins openers that enhance the delivery of drugs through the paracellular route [14], Formulating FIX polypeptides for intranasal administration
  • the FIX polypeptides of the invention are formulated so that they can be administered intranasally.
  • Many types of formulations have been developed to apply drugs intranasally. These include nanoparticles (e.g. polymer-based nanoparticles), solid formulations (e.g. gels) and lipid-based formulations.
  • nanoparticles e.g. polymer-based nanoparticles
  • solid formulations e.g. gels
  • lipid-based formulations include emulsions, solid lipid nanoparticles and liposomes [15], Therefore, the FIX polypeptides can be formulated as a nanoparticle, a solid formulation, or a lipid-based formulations.
  • the FIX polypeptides can be formulated with agents that are conventionally used in nasal administration, such as surfactants, preservatives, pH-adjusting agents, viscosity modifiers, hydrating agents, solvents, solubilisers, decongestants such as a-sympathomimetic drugs, essential oils such as peppermint oil, or cooling agents such as menthol.
  • agents that are conventionally used in nasal administration such as surfactants, preservatives, pH-adjusting agents, viscosity modifiers, hydrating agents, solvents, solubilisers, decongestants such as a-sympathomimetic drugs, essential oils such as peppermint oil, or cooling agents such as menthol.
  • the FIX polypeptides are administered in an aqueous solution.
  • Aqueous solutions are preferred as the FIX polypeptides are compatible with water as a carrier or a diluent.
  • FIX polypeptides are stable in aqueous formulations.
  • Aqueous solutions are especially preferred because they are compatible with the spray mechanism and the materials of most kinds of commercially available nasal spray devices. Aqueous solutions are also preferred because of the low risk of side effects.
  • the aqueous solution can be isotonic. Isotonic solutions are preferred as they will not, when applied to the nasal epithelia, result in osmotic pressure, negative or positive, on the epithelia.
  • polysorbate is known to improve intranasal drug delivery [16], Therefore, in preferred embodiments, the FIX polypeptide is administered with polysorbate (e.g. polysorbate 80 or polysorbate 20).
  • the examples demonstrate that intravenous formulations can be suitable for intranasal administration.
  • the FIX polypeptides used in the examples were formulated with either (a) sucrose, glycine, L-histidine and polysorbate 80; (b) sucrose, histidine, mannitol, polysorbate 20, sodium chloride and hydrochloric acid; or (c) sodium citrate, polysorbate 80, mannitol, sucrose and hydrochloric acid.
  • the standard treatment for haemophilia B is repetitive intravenous injections of recombinant FIX. Therefore, it is advantageous that, as discovered by the present inventors, in certain situations no reformulation is necessary to administer the FIX polypeptides via a different administration route (i.e.
  • the FIX polypeptides can be administered in combination with polysorbate (e.g. polysorbate 80 and/or 20), sucrose, glycine, histidine, mannitol, sodium chloride, sodium citrate and/or hydrochloric acid, for example 0.004-0.016% polysorbate 80, 0.5-3.5% sucrose, 200-850 mM glycine, 5-35 mM histidine, 0.05-5% mannitol, 0.05-0.25% sodium chloride and/or 0.05-0.5% sodium citrate.
  • Hydrochloric acid can used to adjust the pH of the formulation.
  • the FIX polypeptide is administered in combination with sucrose, glycine, histidine and polysorbate.
  • the FIX polypeptide is administered in combination with sucrose, histidine, mannitol, polysorbate, sodium chloride and hydrochloric acid. In some embodiments, the FIX polypeptide is administered in combination with sodium citrate, polysorbate, mannitol, sucrose and hydrochloric acid.
  • Various formulation strategies are known in the art to increase the potency of intranasal formulations, these include using solubilizers, employing nanocarriers and formulating the drugs as salts or prodrugs as described in ref [17],
  • the FIX polypeptides can be formulated for intranasal delivery as a pharmaceutical composition.
  • the pharmaceutical composition may be formulated with a pharmaceutically acceptable carrier.
  • the invention therefore also provides a pharmaceutical composition comprising a Factor IX (FIX) polypeptide for use in a method of treating or preventing a bleeding disorder by administering the pharmaceutical composition intranasally.
  • the pharmaceutical composition may contain the formulations described above (e.g., polysorbate 80, glycine, histidine, polysorbate 20 and mannitol).
  • the invention also provides a method of treating or preventing a bleeding disorder in a subject comprising administering a therapeutically or prophylactically effective amount of a pharmaceutical composition comprising a FIX polypeptide intranasally to the subject.
  • the invention further provides a use of a pharmaceutical composition comprising a FIX polypeptide in the manufacture of a medicament for treating or preventing a bleeding disorder in a subject, wherein the pharmaceutical composition is administered intranasally.
  • the pharmaceutical composition is for administration to a subject, such as an animal, typically a human subject.
  • the pharmaceutical composition is pharmaceutically acceptable and typically includes a suitable carrier.
  • a suitable carrier typically includes a suitable carrier.
  • the composition is preferably sterile, pyrogen- and/or preservative-free.
  • the pharmaceutical composition can have a pH of 4-7.2, for example the pharmaceutical composition can have a pH 6 to pH 7 or pH 5.5 to pH 6.5.
  • the Factor IX polypeptide may be provided in buffered liquid form, e.g., in a citrate buffer, optionally containing a stabiliser and/or a bulking agent.
  • An exemplary pharmaceutical composition for use in the invention comprises a Factor IX polypeptide, tri-sodium citrate dihydrate, polysorbate 80, mannitol, sucrose, hydrochloric acid, and sterile water.
  • the Factor IX polypeptide in the composition is lyophilized but is reconstituted with liquid diluent prior to administration.
  • the reconstituted composition can be administered at a concentration of 50, 100, 125, 200, 250, 300, 400, 500, 600, 700 or 750 lU/ml.
  • Suitable excipients in a composition comprising lyophilized Factor IX polypeptide include tri-sodium citrate dihydrate, polysorbate 80, mannitol, sucrose, and/or hydrochloric acid.
  • compositions may be prophylactic (to prevent bleeding) or therapeutic (to treat bleeding).
  • the pharmaceutical composition can comprise sodium chloride.
  • Sodium chloride may be used as a tonicity agent. Adjusting the osmolality of pharmaceutical compositions for nasal administration is beneficial to preserve the integrity of the nasal epithelia and is well-known in the art.
  • Sodium chloride is preferred as the ions of this salt are present ubiquitously in the human body and no adverse effects are associated with intranasal administration of sodium chloride solutions.
  • sodium chloride solution is safe for ingestion and has an acceptable taste and odour. This is important for compliance, as after nasal administration, especially nasal spray administration, the applied dose of the composition may end up in the pharyngeal region of the subject and may get into contact with the taste buds before being swallowed.
  • Sodium chloride is furthermore preferred because FIX polypeptides are stable in sodium chloride formulations.
  • the Factor IX polypeptides described herein are for treating or preventing a bleeding disorder.
  • the bleeding disorder may be any disorder which requires pro-coagulant (e.g., to prevent, reduce or inhibit bleeding).
  • An exemplary bleeding disorder is haemophilia, particularly haemophilia B.
  • the invention therefore provides a FIX polypeptide for use in a method of treating or preventing a bleeding disorder by administering the FIX polypeptide intranasally.
  • the invention also provides a method of treating or preventing a bleeding disorder in a subject by administering a therapeutically or prophylactically effective amount of a FIX polypeptide intranasally to the subject.
  • the invention further provides a use of a FIX polypeptide in the manufacture of a medicament for treating or preventing a bleeding disorder in a subject, wherein the FIX polypeptide is administered intranasally to the subject.
  • Bleeding disorders include haemophilia (haemophilia A, haemophilia B, haemophilia A and B patients with inhibitory antibodies; in particular haemophilia B), deficiencies in at least one coagulation factor (e.g., Factors VII, VIII IX, X, XI, V, XII, II, and/or von Willebrand factor; in particular Factor IX), combined FV/FVIII deficiency, vitamin K epoxide reductase CI deficiency, gamma-carboxylase deficiency; bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy (hypocoagulability), disseminated intravascular coagulation (DIC); over-anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotics (i.e., FXa inhibitors); and plate
  • the method or use described herein is for treatment or prevention of bleeding in a subject with haemophilia B, which is also known in the art as congenital factor IX deficiency.
  • the treating or preventing may include on-demand control of bleeding episodes, perioperative management of bleeding, and/or routine prophylaxis to prevent or reduce the frequency of bleeding episodes.
  • treatment may include on-demand control of bleeding episodes or perioperative management of bleeding.
  • Prevention may include prevention of bleeding episodes or reducing the frequency of bleeding episodes.
  • FIX polypeptides Physiological levels of FIX polypeptides have been shown to be a limiting factor for the coagulation capacity of emicizumab.
  • the addition of FIX polypeptides has been shown to enhance emicizumab clotting activity [19], Therefore, in some embodiments the FIX polypeptides can be used to treat breakthrough bleeds in patients with haemophilia A who are receiving emicizumab.
  • the subject is typically a human.
  • the subject may be an adult (e.g. 18 years old or older) or a child (e.g. 18 years old or younger, or 12 years or younger).
  • the subject may have a basal (without prophylaxis or treatment) plasma Factor IX activity of 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, between 1- 5%, or 1% or less, compared to the plasma Factor IX activity of a healthy subject (i.e. a subject without a bleeding disorder).
  • a healthy subject i.e. a subject without a bleeding disorder
  • the subject is a paediatric subject (a child), e.g., 18 years or younger.
  • Intranasal administration of FIX polypeptides can be useful for patients who are not eligible for AAV gene therapy, which includes patients who have developed inhibitors against FIX and patients who are under 18 years old.
  • Intravenous administration of FIX polypeptides can lead to the development of inhibitors against FIX in approximately 3-5% of patients with haemophilia B.
  • intranasal administration is known to be tolerogenic (see refs [20] and [21]) and is also thought to reduce the development of inhibitors compared to intravenous administration [22], Therefore, intranasal administration of FIX polypeptides is particularly suitable for patients who are not eligible for AAV gene therapy.
  • the subject is not eligible to receive FIX gene therapy, such as patients who have developed inhibitors against FIX or patients who are under 18 years old.
  • the FIX polypeptides can be administered to patients who are eligible to receive FIX gene therapy, or have previously received FIX gene therapy.
  • the FIX polypeptides can be administered to patients who have been treated with gene therapy as an on- demand treatment for breakthrough bleeds.
  • FIX polypeptides Children suffering from bleeding disorders, such as haemophilia B, are on average exposed to clotting factors, such as FIX polypeptides, around 0.8 years old [23], It is therefore important to have a route of administration for FIX polypeptides that is suitable for patients who are younger than one year, e.g. neonates. Intranasal administration of FIX polypeptides is also suitable for patients that have difficult venous access, which includes neonates and children.
  • intranasal administration of FIX polypeptides is particularly suitable for children, for example intranasal administration of FIX polypeptides is particularly suitable for patients who are 18 years old or younger, patients who are 12 years old or younger, patients who are 6 years old or younger or patients who are 1 year old or younger. In preferred embodiments, the subject is 12 years old or younger. In some embodiments, the FIX polypeptide is administered at a dose of 20 lU/kg to 350 lU/kg.
  • the FIX polypeptide is administered at a dose of 30 lU/kg to 300 lU/kg, 30 lU/kg to 250 lU/kg, 50 lU/kg to 200 lU/kg or 50 lU/kg to 150 lU/kg. In some embodiments, the FIX polypeptide is administered at a dose of about 25 lU/kg, 30 lU/kg, 50 lU/kg, 75 lU/kg, 100 lU/kg, 150 lU/kg, 200 lU/kg, 250 lU/kg, 300 lU/kg or 350 lU/kg. In certain embodiments, the FIX polypeptide is administered at a dose of about 50 lU/kg, 100 lU/kg or 150 lU/kg.
  • the FIX polypeptide is administered in a composition that does not contain antithrombotic substances (e.g., heparin).
  • antithrombotic substances e.g., heparin
  • Factor IX activity in plasma is as a percentage relative to normal human plasma.
  • Another way of expressing Factor IX activity in plasma is in International Units (IU) relative to an International Standard for Factor IX in plasma.
  • IU International Units
  • One IU of Factor IX activity in plasma is equivalent to that quantity of Factor IX in one mL of normal human plasma.
  • One way of checking efficacy of prophylaxis or treatment is by measuring the plasma Factor IX activity in the subject after prophylaxis or treatment, and comparing it to the plasma Factor IX activity in that subject before prophylaxis or treatment.
  • An increase in Factor IX activity after prophylaxis or treatment e.g., from ⁇ 1%, or l%-5%, or 5-40% of normal human plasma to e.g., 15%, 20%, >25%, >30%, >35%, >40%, >50%, or >60% peak levels of normal human plasma, e.g., from ⁇ 5% to >5% such as to 5-40%) indicates a prophylactic or therapeutic effect.
  • Factor IX levels of 3-10% of normal human serum have been targeted in clinical trials for achieving bleeding control while on prophylaxis.
  • a prophylactic or therapeutic effect is also achieved where the Factor IX activity after prophylaxis or treatment is sufficient to prevent, reduce or inhibit bleeding.
  • the Factor IX activity after prophylaxis or treatment may results in troughs of at least 3% or at least 5%, for example 3-40% or 5-40%.
  • Factor IX activity can be measured using any Factor IX activity assay known to the skilled person, for example using an aPTT assay (a decrease in aPTT value indicates increased Factor IX activity) or a chromogenic assay.
  • Factor IX activity is determined using an in vitro aPTT-based one stage clotting assay [ref 5 and 6]
  • Factor IX activity is determined using a chromogenic assay [ref 64]
  • a Factor IX polypeptide for use in the invention may have a higher specific molar activity when administered in vivo to a subject than the corresponding wild-type Factor IX polypeptide.
  • Such high- activity variants are described herein.
  • the % increase in plasma Factor IX activity e.g., measured using an in vitro aPTT-based one stage clotting assay
  • Another way of describing this is that the aPTT time in a serum sample after administering a Factor IX variant polypeptide as described herein is shorter as compared with the same molar amount of the corresponding wild-type Factor IX polypeptide.
  • FIX Factor IX
  • the Factor IX polypeptide can be derived from a Factor IX polypeptide sequence of any mammalian species.
  • the Factor IX polypeptide is derived from a Factor IX polypeptide sequence of human origin. Gene ID: 2158 (https://www.ncbi.nlm.nih.gov/gene/2158), GenBank Accession Nos.
  • NM_000133.4 https://www.ncbi.nlm.nih.gov/nuccore/NM_000133
  • NP_000124.1 https://www.ncbi.nlm.nih.gOv/protein/NP_000124.l
  • UniProt entry P00740 https://www.uniprot.org/uniprotkb/P00740/entry
  • the Factor IX polypeptides according to the invention may be derived from mature (z.e., excluding signal peptide and propeptide) Factor IX, for example of human origin, the amino acid sequence of which is shown in SEQ ID NO: 1.
  • An exemplary polynucleotide coding sequence for the polypeptide of SEQ ID NO: 1 is provided by SEQ ID NO: 2. That polypeptide sequence is ‘isoform 1’ of human Factor IX (also referred to herein as wild-type Factor IX).
  • wild-type Factor IX refers to a Factor IX polypeptide sequence that occurs naturally and has a FIX activity that is typical of natural FIX such as that found in standard human plasma.
  • the sequence has not been artificially modified relative to the sequence of the naturally occurring polypeptide sequence. This means that none of the amino acids in the naturally occurring polypeptide sequence has been substituted with a different amino acid.
  • SEQ ID NO: 1 is an example of a wild-type polypeptide sequence, but functional fragments, truncations, etc. are also encompassed by the term, as exemplified below.
  • the term includes polypeptides with a modified N-terminal or C -terminal end including terminal amino acid deletions or additions, as long as those polypeptides substantially retain the activity of wild-type Factor IX.
  • the term also includes any natural polymorphic variant of Factor IX.
  • a common natural polymorphic variant which occurs with a frequency of 33% is a Factor IX polypeptide presenting an alanine (A) in a position corresponding to position T148 in SEQ ID NO: 1.
  • This T148A polymorphic variant is shown in SEQ ID NO: 20. All references to SEQ ID NO: 1 herein may therefore also refer to SEQ ID NO: 20.
  • the Factor IX polypeptide can have an amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or at least 99% identical to SEQ ID NO: 1, across the full length of SEQ ID NO: 1.
  • the Factor IX polypeptide is defined by SEQ ID NO: 1.
  • the Factor IX polypeptide may also be derived from a wild-type Factor IX that includes the signal and/or the propeptide, as shown in SEQ ID NO: 3.
  • SEQ ID NO: 3 includes both the signal peptide (aa 1-28) and the propeptide (aa 29-46).
  • the polypeptide of SEQ ID NO: 3 is known in the art as the precursor of human Factor IX, or as the prepropeptide Factor IX.
  • Factor IX with propeptide but lacking the signal peptide is also known as a propeptide Factor IX.
  • An exemplary polynucleotide coding sequence encoding the polypeptide of SEQ ID NO: 3 is shown in SEQ ID NO: 4.
  • the Factor IX polypeptide may also be derived from one or more functional fragments of wild-type Factor IX, for example it may be derived from activated Factor IX which contains two fragments of Factor IX (it is missing the intervening ‘activation peptide’ that is present in SEQ ID NO: 1).
  • SEQ ID NOs 17 and 18 show the light chain and heavy chain, respectively, of human activated Factor IX, which are held together by a disulphide bridge.
  • Another example is isoform 2 of human Factor IX, which lacks the 38-aa stretch at positions 47-84 of SEQ ID NO: 1.
  • the Factor IX polypeptide may be derived from a truncation or a fusion of wild-type Factor IX.
  • the term “derived from a polypeptide sequence of wild-type Factor IX” means that the Factor IX polypeptide has some degree of sequence identity with wild-type Factor IX polypeptide when the two sequences are aligned.
  • the Factor IX polypeptide may have at least 70% etc. sequence identity to SEQ ID NO: 1, as described herein.
  • the Factor IX polypeptide is biologically active, i.e., it is capable of activating Factor X (z.e., generating Factor Xa). Fusions and conjugates
  • a Factor IX polypeptide for use in the invention can also be provided as part of a fusion with another moiety, e.g., with an Fc portion, PEG or albumin (for example attached via a cleavable linker).
  • FIX polypeptides fused to an Fc portion (98 kDa) or fused to albumin (125 kDa) can reach the blood compartment following intranasal administration. Based on these data it is likely that other high molecular weight FIX polypeptides, such as a FIX polypeptide fused to PEG (98 kDa), can also be administrated intranasally.
  • the Factor IX polypeptide may be provided in fusion with, or it may be conjugated to, one or more additional portions.
  • the terms “fused with”, “attached to”, “linked to”, “linked via” and “conjugated to” are used interchangeable herein.
  • the FIX polypeptide can be directly attached to another moiety or can be indirectly attached to another moiety via a linker.
  • the one or more additional portions are typically different from Factor IX, i.e., they do not have the biological function of Factor IX as defined herein (they do not have the ability to generate Factor Xa).
  • fragments of Factor IX e.g., linkers comprising a fragment of a Factor IX-derived polypeptide sequence, but which do not on their own have the function of Factor IX, may be such “one or more additional portions”, i.e., they are not part of the Factor IX portion, but they may be part of the molecule that comprises the Factor IX portion.
  • the FIX polypeptide is linked to a half-life enhancing portion.
  • the half- life enhancing portion may comprise one or more polypeptides (half-life enhancing polypeptides, HLEPs).
  • the HLEP is albumin, e.g., recombinant human albumin.
  • the HLEP is a fragment of an antibody (immunoglobulin), such as the Fc fragment, e.g., IgG Fc, such as IgGl Fc.
  • the HLEP may be a C-terminal peptide of human chorionic gonadotropin (CTP).
  • the HLEP may also be an unstructured recombinant polypeptide (e.g., XTEN).
  • the HLEP can be another coagulation factor such as FXIII subunit B [24], Such molecules are also referred to in the art as fusion polypeptides.
  • the FIX polypeptide may be linked to the HLEP via a cleavable linker, in particular a cleavable peptide linker.
  • a cleavable linker is cleavable by the same protease that activates Factor IX or any other blood coagulation factors (e.g. Factors II, V, VII, VIII, X, XI, XU, XIII).
  • Such cleavable linkers therefore provide a high molar specific activity of the fusion polypeptide or a specific activity that is similar to wild-type FIX polypeptide.
  • the FIX polypeptide is directly linked to the HLEP, i.e. there is no linker connecting the FIX polypeptide to the HLEP.
  • the FIX polypeptide is linked to the HLEP via a non-cleavable linker, e.g. the FIX polypeptide is linked to an Fc portion via a non- cleavable linker.
  • the FIX polypeptide may also be PEGylated, i.e., one or more polyethylene glycol moieties are conjugated to the FIX polypeptide, using methods known in the art.
  • a FIX polypeptide for use in the invention may be linked to one half-life enhancing portion, or more than one half-life enhancing portions.
  • the wording “a half-life enhancing portion” therefore covers one or more half-life enhancing portions.
  • the half-life enhancing portions may be of the same type.
  • the half-life enhancing portions may be of different types.
  • the FIX polypeptide may be linked to XTEN (e.g., XTEN72) and additionally to an Fc domain (e.g., human IgGl Fc).
  • the half-life enhancing portion is capable of extending the half-life of the FIX polypeptide in vivo (in plasma) by at least about 25% as compared to the non-fused FIX polypeptide.
  • the half-life enhancing portion is capable of extending the half-life of the FIX polypeptide in vivo (in plasma) by at least about 50%, and more preferably by more than 100%.
  • the in vivo half-life is generally determined as the terminal half-life or the P-half-life.
  • albumin refers collectively to an albumin polypeptide or amino acid sequence, or an albumin fragment, variant or analogue having one or more functional activities (biological activities) of albumin.
  • albumin may refer to human albumin (HA) or a fragment thereof, especially the mature form of human albumin as shown in SEQ ID NO: 5 herein.
  • the albumin may also be derived from other species, in particular other vertebrates.
  • the albumin portion of the fusion polypeptide may comprise the full length of the HA sequence as described in SEQ ID NO: 5, or it may include one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity of the Factor IX polypeptide.
  • Such fragments may be of 10 or more amino acids in length or may include about 15, 20, 25, 30, 50, or more contiguous amino acids from the HA sequence or may include part or all of the specific domains of HA.
  • suitable albumin portions including variants are described in reference 26.
  • Structurally related family members of the albumin family may also be used as HLEPs.
  • alpha-fetopolypeptide AFP, reference 25
  • AFP alpha-fetopolypeptide
  • AFM vitamin D binding polypeptide
  • DBP vitamin D binding polypeptide
  • Fragments of these polypeptides may also be used.
  • the albumin is typically provided as a genetic fusion with the Factor IX portion. This means that a single cDNA molecule encodes the Factor IX portion and the albumin portion, optionally with an intervening sequence encoding a linker, such as a cleavable linker.
  • the invention therefore provides a Factor IX (FIX) polypeptide for use in a method of treating or preventing a bleeding disorder by administering the FIX polypeptide intranasally, wherein the FIX polypeptide is linked to albumin.
  • FIX Factor IX
  • the invention also provides a method of treating or preventing a bleeding disorder in a subject comprising administering a therapeutically or prophylactically effective amount of a FIX polypeptide intranasally to the subject, wherein the FIX polypeptide is linked to albumin.
  • the invention further provides a use of a FIX polypeptide in the manufacture of a medicament for treating or preventing a bleeding disorder in a subject, wherein the FIX polypeptide is to be administered intranasally to the subject and wherein the FIX polypeptide is linked to albumin.
  • the invention further provides a FIX polypeptide for treating or preventing a bleeding disorder, wherein the FIX polypeptide is to be administered intranasally to the subject and wherein the FIX polypeptide is linked to albumin.
  • An immunoglobulin (Ig) or a fragment thereof may also be used as a HLEP.
  • An example of a suitable immunoglobulin is IgG, or an IgG-fragment, such as an Fc region.
  • the Fc region may be an Fc domain (e.g., two polypeptide chains each of which comprises the hinge region (or part of the hinge region), the CH2 region and the CH3 region).
  • a Factor IX polypeptide may be fused to an Fc domain, directly or via a linker.
  • the linker may be cleavable.
  • the Factor IX polypeptide may be a heterodimer comprising two polypeptide chains, wherein the first chain comprises a Factor IX portion linked to the hinge region (or part of the hinge region), the CH2 region and the CH3 region of an immunoglobulin (e.g., IgGl), and the second chain comprises the hinge region (or part of the hinge region), the CH2 region and the CH3 region of an immunoglobulin (e.g., IgGl).
  • the first chain comprises a Factor IX portion linked to the hinge region (or part of the hinge region), the CH2 region and the CH3 region of an immunoglobulin (e.g., IgGl)
  • the second chain comprises the hinge region (or part of the hinge region), the CH2 region and the CH3 region of an immunoglobulin (e.g., IgGl).
  • the Factor IX polypeptide is a homodimer comprising two polypeptide chains, wherein each chain comprises a Factor IX portion linked to the hinge region (or part of the hinge region), the CH2 region and the CH3 region of an immunoglobulin (e.g., IgGl).
  • each chain comprises a Factor IX portion linked to the hinge region (or part of the hinge region), the CH2 region and the CH3 region of an immunoglobulin (e.g., IgGl).
  • the Factor IX polypeptide is a monomer comprising a Factor IX portion linked to the hinge region (or part of the hinge region), the CH2 region and the CH3 region of an immunoglobulin (e.g., IgGl).
  • an immunoglobulin e.g., IgGl
  • suitable Factor IX IgG Fc fusion molecule configurations are found, e.g., in reference 29.
  • Fc polypeptide (derived from the human IgGl Fc domain) is shown in SEQ ID NO: 6.
  • Another exemplary Fc polypeptide (derived from the human IgGl Fc domain) is shown in SEQ ID NO: 7.
  • the Factor IX portion may be linked directly or via a linker to the Fc portion.
  • the linker may be cleavable or non-cleavable.
  • the linker is cleavable.
  • An exemplary cleavable linker is shown in SEQ ID NO: 8.
  • An exemplary Fc portion is the Fc portion of Eftrenonacog alfa (Alprolix®). See also references 30, 31 or 32.
  • the invention therefore provides a Factor IX (FIX) polypeptide for use in a method of treating or preventing a bleeding disorder by administering the FIX polypeptide intranasally, wherein the FIX polypeptide is linked to an Fc portion.
  • FIX Factor IX
  • the invention also provides a method of treating or preventing a bleeding disorder in a subject comprising administering a therapeutically or prophylactically effective amount of a FIX polypeptide intranasally to the subject, wherein the FIX polypeptide is linked to an Fc portion.
  • the invention further provides a use of a FIX polypeptide in the manufacture of a medicament for treating or preventing a bleeding disorder in a subject, wherein the FIX polypeptide is to be administered intranasally to the subject and wherein the FIX polypeptide is linked to an Fc portion.
  • the invention further provides a FIX polypeptide for treating or preventing a bleeding disorder, wherein the FIX polypeptide is to be administered intranasally to the subject and wherein the FIX polypeptide is linked to an Fc portion.
  • CTP human chorionic gonadotropin
  • CTP human chorionic gonadotropin
  • hCG human chorionic gonadotropin
  • One or more units of CTP can be fused to a Factor IX portion.
  • the one or more units of CTP can be fused to the N-terminus and/or to the C-terminus of Factor IX, preferably to the C-terminus.
  • the Factor IX polypeptide is a CTP-modified Factor IX comprising a Factor IX polypeptide as described herein linked with three to five CTPs, optionally wherein the CTPs are attached to the C-terminus of the Factor IX polypeptide.
  • three tandem units of CTP are attached the Factor IX polypeptide, optionally at the C-terminus of the Factor IX polypeptide.
  • At least one of the CTP may be attached to the Factor IX portion via a linker.
  • the linker may be a peptide bond.
  • the linker may be cleavable.
  • the CTP sequence comprises SEQ ID NO: 11. In another exemplary embodiment, the CTP sequence comprises SEQ ID NO: 12. In another exemplary embodiment, the CTP sequence comprises SEQ ID NO: 13.
  • Another exemplary half-life enhancing portion is an unstructured recombinant polypeptide.
  • An example of such an unstructured recombinant polypeptide is XTEN, see, e.g., reference 36.
  • the Factor IX polypeptide is therefore a Factor IX polypeptide fused with at least one XTEN.
  • XTEN may be fused to the Factor IX portion by insertion into the Factor IX polypeptide sequence while maintaining the biological activity of Factor IX.
  • the XTEN may be inserted between two neighbouring amino acids in the activation peptide of Factor IX at a position that does not prevent cleavage of the activation peptide during coagulation when XTEN is inserted.
  • XTEN may fused to the C-terminus and/or N-terminus of the Factor IX, preferably the C-terminus.
  • XTEN may be fused to the C-terminus and/or N-terminus (preferably C-terminus) of the Factor IX via a linker, e.g., a cleavable linker.
  • the linker may be cleavable by thrombin.
  • a preferred XTEN is XTEN72.
  • An exemplary XTEN72 sequence is shown in SEQ ID NO: 14.
  • An alternative XTEN sequence is shown in SEQ ID NO: 15.
  • Other suitable sequences and methods are disclosed in, e.g., references 37, 38 or 39.
  • the Factor IX polypeptide comprises XTEN72 linked to the activation peptide of Factor IX and wherein the Factor IX portion is furthermore linked to a human IgGl Fc domain at the C-terminus of the Factor IX portion.
  • PEG polyethylene glycol
  • GlycoPEGylation is within the scope of the term “PEGylation” as used herein.
  • a ca. 40 kDa PEG portion may be covalently attached to the Factor IX polypeptide, for example via a specific N-linked glycan within the activation peptide.
  • glycoPEG moiety is the gly coPEG moiety of nonacog beta pegol (Refixia®) (see also reference 40), in which an average of one non-reducing end of a glycan at N157 or N167 of Factor IX (numbering according to SEQ ID NO: 1) is attached to neuraminic acid conjugated to two PEG polymers (total average molecular weight of the polymers is ca. 42 kDa) via the amino group.
  • Refixia® nonacog beta pegol
  • the molecular weight of nonacog beta pegol is approximately 98 kDa.
  • the examples demonstrate that other FIX polypeptides that have a similar molecular wight can be transported across the nasal epithelium mainly via the paracellular pathway. It is therefore plausible that FIX polypeptides that are linked to PEG, such as nonacog beta pegol, will also be transported via the same pathway. The inventors have therefore identified a new non-invasive pathway for administering FIX polypeptides that are linked to PEG - i.e. intranasal administration.
  • the invention further provides a FIX polypeptide for treating or preventing a bleeding disorder, wherein the FIX polypeptide is to be administered intranasally to the subject and wherein the FIX polypeptide is linked to PEG.
  • the invention therefore provides a Factor IX (FIX) polypeptide for use in a method of treating or preventing a bleeding disorder by administering the FIX polypeptide intranasally, wherein the FIX polypeptide is linked to PEG.
  • FIX Factor IX
  • the invention also provides a method of treating or preventing a bleeding disorder in a subject comprising administering a therapeutically or prophylactically effective amount of a FIX polypeptide intranasally to the subject, wherein the FIX polypeptide is linked to PEG.
  • the invention further provides a use of a FIX polypeptide in the manufacture of a medicament for treating or preventing a bleeding disorder in a subject, wherein the FIX polypeptide is to be administered intranasally to the subject and wherein the FIX polypeptide is linked to PEG.
  • the invention further provides a FIX polypeptide for treating or preventing a bleeding disorder, wherein the FIX polypeptide is to be administered intranasally to the subject and wherein the FIX polypeptide is linked to PEG.
  • a Factor IX polypeptide may be connected to a half-life enhancing portion via a cleavable linker or non-cleavable linker, in particular a proteolytically cleavable linker.
  • the linker is generally positioned between the Factor IX polypeptide and the half-life enhancing portion.
  • the linker may liberate the Factor IX upon cleavage of the linker by a protease of the coagulation cascade, e.g., a protease that is also capable of converting Factor IX to its activated form, e.g., FXIa or Vlla/tissue factor (TF).
  • Cleavable linkers are particularly useful when the HLEP is albumin.
  • a cleavable linker links the Factor IX polypeptide to a half-life enhancing portion, thereby providing a Factor IX polypeptide with a longer half-life relative to a non-fusion polypeptide.
  • a protease of the coagulation cascade activates the Factor IX polypeptide which has increased specific activity relative to, e.g., the corresponding wild-type Factor IX.
  • the linker is cleaved and the activated Factor IX polypeptide is liberated from the half-life enhancing portion, thereby reducing the risk of a prothrombotic effect due to any prolonged increased Factor IX activity.
  • the linker may be a fragment of Factor IX, preferably a fragment that is involved in Factor IX activation.
  • the linker may comprise such a fragment of a Factor IX sequence, extended by an N-terminal residue, such as a proline residue.
  • An exemplary cleavable linker is shown in SEQ ID NO: 8. Other cleavable linkers are described in reference 26.
  • a Factor IX polypeptide linked to a half-life enhancing portion via an intervening cleavable linker may have at least 25% higher molar specific activity compared to the corresponding molecule with a non- cleavable linker (e.g., GGGGGGV, SEQ ID NO: 16), when measured in at least one coagulation- related assay, examples of which are known to the skilled person in the art, e.g., an aPTT one-stage assay.
  • a Factor IX polypeptide linked to a half-life enhancing portion via an intervening cleavable linker has at least 50%, more preferably at least 100% increased molar specific activity compared to the corresponding molecule without cleavable linker.
  • the FIX polypeptides for use in the invention can comprise mutations compared to wild-type Factor IX that can increase coagulation activity (e.g., increase specific activity) relative to wild-type Factor IX.
  • a polypeptide is also referred to herein as a ‘high-activity’ FIX polypeptide, a high-activity variant, or a high-activity FIX variant polypeptide.
  • Other terms are used in the art synonymously, e.g. , ‘hyperactive’ FIX variants. These variants have the biological function of a Factor IX, i.e.
  • the variant is able to generate Factor Xa, optionally after the high-activity Factor IX variant polypeptide has been converted to its active form (Factor IXa) by excision of the activation peptide.
  • the variant is able to generate Factor Xa with a higher activity than wild-type FIX.
  • Activation cleavage of Factor IX can be achieved in vitro, e.g., by Factor Xia or Factor VIIa/TF.
  • Suitable in vitro assays to measure Factor IX activity are known to the person skilled in the art (e.g., one-stage clotting assay such as an aPTT assay, chromogenic assay, etc.).
  • FIX polypeptides are derived from a polypeptide sequence of wild-type Factor IX (SEQ ID NO:1) and differ at one or more amino acid positions from the corresponding positions in the wildtype Factor IX, i.e., the variant has one or more amino acid substitutions relative to the corresponding positions in the wild-type Factor IX.
  • the numbering refers to the amino acid positions in wild-type Factor IX as defined in SEQ ID NO: 1.
  • An exemplary high-activity Factor IX polypeptide comprises leucine (L) at a position corresponding to position 338 of wild-type Factor IX, which typically has an arginine (R) at that position (“R338L”).
  • L leucine
  • R arginine
  • One such exemplary polypeptide is the ‘Padua’ mutant, described in ref 44. See SEQ ID NO: 10. The specific activity of the ‘Padua’ mutant is typically at least around 5-8 fold higher compared to wildtype Factor IX.
  • the Factor IX (FIX) polypeptides for use in the invention comprise the amino acid leucine at a position corresponding to position 338 of wild-type Factor IX.
  • exemplary high-activity Factor IX variants are E410H, E410K, R338V, and R338L + E410K, and those described in ref. 45, e.g. , comprising the amino acid H at a position corresponding to position 410 of wild-type Factor IX, and comprising an amino acid other than R at a position corresponding to position 338 of wild-type Factor IX, for example comprising an amino acid selected from the group consisting of V, T and W at a position corresponding to position 338 of wild-type Factor IX, e.g., R338V + E410H, R338T + E410H, R338W + E410H, and R338L + E410H.
  • the FIX polypeptides for use in the invention comprise an amino acid selected from valine, threonine and tryptophan at aposition corresponding to position 338 of wildtype Factor IX and the amino acid histidine at a position corresponding to position 410 of wild-type Factor IX.
  • the FIX polypeptides for use in the invention comprise the amino acid valine at a position corresponding to position 338 of wild-type Factor IX and the amino acid histidine at a position corresponding to position 410 of wild-type Factor IX.
  • a further high-activity Factor IX variant for use in the invention is the Dalcinonacog alfa variant (also known as CB 2679d), see SEQ ID NO: 19.
  • Dalcinonacog alfa has three amino acid substitutions in two loops within the FIX protein. Based on mature FIX sequence numbering, (1) R318Y located in the ‘150-loop’, stabilizes activated FIX (FIXa), directly interacts with the substrate factor X (FX) and provides resistance to antithrombin; (2) R338E, and (3) T343R, both located in the ‘170-loop’, significantly enhance affinity to the cofactor, activated factor VIII (FVIIIa) and increase the catalytic activity FIXa.
  • R318Y/R338E/T343R refer to R150Y/R170E/T175R in classic chymotrypsin numbering [ref 46] and R364Y/R384E/T389R in the Human Genome Variation Society (HGVS) nomenclature, which includes the 46 amino acid propeptide [ref 47], Therefore, in some embodiments, the FIX polypeptides for use in the invention comprise the amino acid tyrosine at a position corresponding to position 318 of wild-type Factor IX, the amino acid glutamic acid at a position corresponding to position 338 of wild-type Factor IX and the amino acid arginine at a position corresponding to position 343 of wild-type Factor IX.
  • a further high-activity Factor IX variant for use in the invention is the R338Q variant (known as the Shanghai variant - ref [48]), which has 5 fold higher activity compared to wild-type FIX. Therefore, in some embodiments, the FIX polypeptides for use in the invention comprise the amino acid glutamine at a position corresponding to position 338 of wild-type Factor IX.
  • the FIX polypeptides for use in the invention comprise the amino acid alanine at a position corresponding to position 338 of wild-type Factor IX.
  • the FIX polypeptides for use in the invention comprise the amino acid alanine at position 86 of wild-type Factor IX, the amino acid alanine at a position corresponding to position 227 of wild-type Factor IX and the amino acid alanine at a position corresponding to position 338 of wild-type Factor IX.
  • High-activity Factor IX polypeptides include those listed in Table 1 below (ref 49) and described in ref 50.
  • Table 1 refers to the positions in the mature FIX protein without propeptide sequence (SEQ ID NO: 1). The activity was determined with a one-stage clotting assay.
  • the skilled person is able to identify and verify these and other high-activity Factor IX polypeptides, by determining the specific (molar) activity of a Factor IX polypeptide using methods known in the art, and comparing that activity with wild-type Factor IX.
  • the FIX polypeptides for use in the invention can comprise mutations compared to wild-type Factor IX that decrease binding to extracellular matrix.
  • Such polypeptides include full length FIX proteins or fragments of FIX proteins that are biologically active, i.e., the polypeptide is capable of activating Factor X (i.e., generating Factor Xa).
  • These “decreased binding” FIX polypeptides are derived from a polypeptide sequence of wild-type Factor IX (SEQ ID NO: 1) and differ at one or more amino acid positions from the corresponding positions in the wild-type Factor IX.
  • FIX polypeptides that have decreased binding to the extracellular matrix include a FIX polypeptide comprising an amino acid with any hydrophobic or uncharged side chain at a position corresponding to position 5 of wild-type Factor IX, or a FIX polypeptide comprising an amino acid with a positively charged side chain at a position corresponding to position 10 of wild-type Factor IX, as long as they retain FIX clotting activity, e.g., they have the clotting activity of wild-type FIX, or they may even have a higher clotting activity than wild-type FIX; clotting activity can be measured by standard assays known to those skilled in the art.
  • Amino acids that comprise a hydrophobic side chain include Alanine, Valine, Isoleucine, Leucine, Methionine, Phenylalanine, Tyrosine and Tryptophan.
  • Amino acids that comprise an uncharged side chain include Serine, Threonine, Asparagine and Glutamine.
  • Amino acids that comprise a positively charged side chain include Lysine, Arginine and Histidine.
  • the FIX polypeptide comprises the amino acid alanine at a position corresponding to position 5 of wild-type Factor IX.
  • the FIX polypeptide can also comprise two or more mutations (e.g. , at positions 5 and 10) that decrease the binding of the polypeptide to extracellular matrix.
  • the FIX polypeptide comprises the amino acid alanine at a position corresponding to position 5 of wild-type Factor IX and the amino acid lysine at a position corresponding to position 10 of wild-type Factor IX.
  • a Factor IX polypeptide for use in the invention can be made using standard techniques well known to the skilled person in the art.
  • the cDNA sequence of a wild-type Factor IX e.g., SEQ ID NO: 2
  • SEQ ID NO: 2 may be modified using standard mutagenesis techniques (e.g., site-directed mutagenesis) so that it encodes the desired Factor IX polypeptide.
  • An N-terminal leader peptide for the purposes of recombinant protein production can be used, based on the natural Factor IX leader peptide (as shown in SEQ ID NO: 3) or alternatives known to the skilled person in the art.
  • the cDNA sequence may be inserted into a suitable expression plasmid to express the recombinant Factor IX polypeptide (rFIX).
  • rFIX recombinant Factor IX polypeptide
  • This is typically performed using mammalian cells (e.g., HEK for transient expression or a CHO cell line for stable expression), although other types of cells that can produce glycosylated and correctly folded proteins can also be used.
  • the recombinant Factor IX polypeptide may subsequently be purified, for example using anion exchange chromatography.
  • the Factor IX polypeptide may be combined with other agents and/or with a pharmaceutically acceptable carrier.
  • FIG. 1 Pharmacokinetic profile of FIX polypeptides in wild-type (WT) mice after intravenous (IV) administration.
  • FIG. 2 Pharmacokinetic profile of FIX polypeptides in WT and transgenic mice after intranasal (IND) administration.
  • the transgenic mice tested had a C57BL/6J background and (1) expressed humanised FcRn (hFcRn) (2) were a FcRn knock-out (FcRn KO) or (3) a FIX knock out (FIX KO) .
  • Plasma FIX antigen levels (FIX:Ag) in ng/ml of rFIX and rFIX-Fc were measured 15 min after intranasal administration.
  • the circles and triangles correspond to individual mice.
  • N 7- 10.
  • One-way ANOVA statistical analysis was applied and completed by a Dunnett’s multiple comparison test. Data are presented as mean ⁇ SEM.
  • FIG. 3 Histological analysis of the mucosal uptake of rFIX and rFIX-Fc after intranasal administration.
  • A Section of the nasal cavity in wild-type mice with respiratory epithelium (RE), olfactory epithelium (OE), and turbinate (T) visible;
  • B Section of the olfactory epithelium: absence of labelling on the PBS control sections (a and d); results with a humanised FcRn mouse (‘hFcRN’)showed a strong labelling on the outer side of the epithelium within the mucus covering the ciliated cell layer, as well as between the sustentatory cells and neurons up to the lamina limba, and in the vasculature, showing evidence of passive passage with no clear differences between rFIX and rFIX-Fc (a vs d).
  • hFcRN humanised FcRn mouse
  • FIG. 4 Western blot analysis of rFIX and rFIX-Fc after intravenous (IV) and intranasal administration (IN) to WT mice. Both rFIX and rFIX-Fc proteins produced a single band at their expected molecular weights of 55kDa and 98kDa, respectively. PBS represents the control.
  • FIG. 5 FIX:Ag plasma levels in ng/ml in FIX KO mice at 15min after intranasal administration of rFIX and rFIX-Fc.
  • FIG. 6 FIX clotting activity (FIX:C) in lU/dL measured in FIX KO mice at 15min after intranasal administration with rFIX and rFIX-Fc.
  • FIG. 7 Pharmacokinetic profile of FIX polypeptides in wild-type mice after intravenous (IV) administration of rFIX, rFIX-Fc, and FIX linked to albumin (rFIX-Alb).
  • One-way ANOVA statistical analysis was applied and completed by a Dunnett’s multiple comparison test. Data are presented as mean ⁇ SEM.
  • concentration of rFIX [rFIX]) is the same as FIX:Ag levels.
  • Example 1 Evaluating the effectiveness of intranasal delivery of rFIX and rFIX fused to an Fc portion (rFIX-Fc)
  • the aim of this study was to evaluate whether the high-molecular weight proteins rFIX and rFIX fused to Fc portion (rFIX-Fc) can be effectively administered intranasally.
  • mice Male and female wild-type C57BL/6J mice (WT), and transgenic mice with C57BL/6J background that were either (1) FcRn knock-out (FcRn KO), (2) FcRn KO and expressed humanised FcRn (hFcRn), or (3) FIX KO were purchased from the Charles River Laboratory (Les Oncins, Saint Germain Nuelles, France). All mice were either 8-12 weeks or 76 weeks.
  • rFIX Nonacog alfa, BENEFIX®, Pfizer, New- York, USA
  • rFIX-Fc Eftrenonacog alfa, ALPROLIX®, SOBI, Sweden
  • the same formulation was used for both intravenous and intranasal administration.
  • the FIX in BENEFIX® was formulated with sucrose, glycine, L-histidine, polysorbate 80 (see BENEFIX SmPC https://www. ema. etiropa. en/ documents/product-information/benefix-epar-product- information en.
  • mice were weighed (verification of sufficient weight 20-3 Og), and then anaesthetised by intraperitoneal injection (mixture of ketamine and xylazine) to obtain sedation and an analgesic effect.
  • a maximum volume of lOpL was administered as a drop into each nostril (20pL of FIX polypeptide (rFIX and rFIX-Fc) corresponding to 10IU for a 20-3 Og mice). Sham treatment consisted of a saline solution.
  • Three IU of rFIX and rFIX-Fc were also administered via intravenous injection in the retro-orbital vein of mice that served as controls.
  • Plasma FIX antigen levels were quantified using the Asserachrom® IX:Ag (Stago, New Jersey, USA) enzyme-linked immunosorbent assay kit, as described previously [62,63], FIX: C was measured using a validated chromogenic assay [64] (Biophen Hyphen Biomed, Neuville-sur-Oise, France).
  • Murine plasma samples were loaded onto a Mini -Protean TGX 4-15% Gel (BioradTM) for electrophoresis under reducing conditions (Tris 25mmol/L, glycine 192mmol/L, SDS 0.1%, pH 8.3 running buffer) and then transferred onto a nitrocellulose membrane (Trans-Blot Turbo Mini 0.2pm NC Transfer pack, Biorad) according to the manufacturer's instructions.
  • the nitrocellulose membrane was then blocked with 6% skimmed milk-PBS and exposed to horseradish peroxidase (HRP)-conjugated affinity -purified goat anti -human FIX (Img/mL) IgG (1:2000 dilution; Affinity Biologicals, Ancaster, Canada).
  • HRP horseradish peroxidase
  • Sections were successively incubated at RT with acetone, 3% hydrogen peroxide (Sigma), PBS containing 3% bovine serum albumin and 5% normal goat serum, and for Ih with HRP-conjugated anti-FIX antibody (1:100, goat polyclonal, Cedarlane, Burlington, Canada). HRP was revealed using 3-amino-9-ethylcarbazole (AEC) substrate (Vector Laboratories, Burlingame, CA, USA). Sections were counterstained with Hematoxylin QS (Vector Laboratories), mounted with Glycergel (Agilent Technologies, Santa Clara, CA, USA). Images of nasal sections were captured using an inverted microscope (40x oil lens and 10pm scale bar, Nikon Ti-E microscope).
  • mice A total of 95 mice (85 were 8-12weeks old and 10 were older) of 4 different strains (WT, hFcRn, FcRn KO, and FIX KO) were used to study the intranasal administration of two high-molecular weight proteins, rFIX (55 kDa) and rFIX-Fc (98 kDa).
  • FcRn has been described as playing a key role in the mucosal uptake of biologies [66], However, the absence of significant differences between rFIX and rFIX-Fc regarding FIX:Ag levels 15min post- intranasal administration in WT mice suggests that the uptake of FIX is independent of FcRn.
  • the nasal mucosal uptake of rFIX and rFIX-Fc was thus tested in two FcRn transgenic mouse models, one that lacks the expression of murine FcRn (FcRn KO) and one that expresses human FcRn (hFcRn) instead.
  • FIX KO mice Ag plasma levels ( Figure 5) and FIX clotting activity ( Figure 6) were measured at 15min after intranasal administration. No significant difference was observed between the two FIX polypeptides tested (rFIX and rFIX-Fc).
  • Example 2 Evaluation the effectiveness of intranasal delivery of rFIX, rFIX fused to an Fc portion (rFIX-Fc) and rFIX fused to albumin (rFIX-Alb)
  • mice Male and female wild-type C57BL/6J mice (WT) were purchased from the Charles River Laboratory (Les Oncins, Saint Germain Nuelles, France). All mice were 7-19 week olds. Recombinant rFIX (Nonacog alfa, BENEFIX, Pfizer, New- York, USA), rFIX-Fc (Eftrenonacog alfa, ALPROLIX, SOBI, Swiss, Sweden) and rFIX-albumin (albutrepenonacog alfa, IDELVION, CSL Behring, Pennsylvania, United States) were used.
  • rFIX Nonacog alfa, BENEFIX, Pfizer, New- York, USA
  • rFIX-Fc Eftrenonacog alfa, ALPROLIX, SOBI, Swiss, Sweden
  • rFIX-albumin albutrepenonacog alfa, IDELVION, CSL Behring, Pennsylvania, United States
  • mice were treated with rFIX, rFIX-Fc and rFIX-albumin. Three IU of each of rFIX, rFIX-Fc and rFIX-albumin were administered via intravenous injection in the retro-orbital vein of mice.
  • FIX detection and measurement was conducted as set out in example 1.
  • the FIX in IDELVION was formulated with sodium citrate, polysorbate 80, mannitol, sucrose and hydrochloric acid (see Idelvion SmPC https://www.ema.europa.eu/en/documents/product-information/idelvion-epar-product- information_en.pdf).
  • Figure 8A demonstrates that rFIX, rFIX-Fc and rFIX-albumin proteins can be successfully administered intranasally, which have an approximate molecular weight of 55 kDa, 98 kDa and 125 kDa, respectively. These data demonstrate that FIX polypeptides with a molecular weight of at least 125kDa can be successfully administered intranasally.
  • FIG. 8B demonstrates that mice that were anaesthetised prior to intranasal administration had higher blood plasma concentrations of FIX polypeptides compared to “Vigile” mice that were not anaesthetised. This observation was true for all of the FIX polypeptides tested.
  • the table below shows the bioavailability of different FIX polypeptides at 15 minutes after intranasal administration. Without being bound by any particular theory, this difference in bioavailability is likely to be due to the ease and improved compliance of nasally administering a drug to mice that are anaesthetised.
  • mice When the mice are active, they are more likely to sneeze or swallow which reduces the dose of the FIX polypeptide in the nose. Therefore, the differences in bioavailability observed between the various anaesthesia methods in mice are unlikely to be relevant to human administration, which does not involve any form of anaesthesia. Overall, these data demonstrate that FIX polypeptides with a molecular weight of at least 125kDa can be effectively administered intranasally.
  • a Factor IX (FIX) polypeptide for use in a method of treating or preventing a bleeding disorder by administering the FIX polypeptide intranasally.
  • FIX polypeptide for use according to embodiment 1, wherein the bleeding disorder is haemophilia B.
  • FIX polypeptide for use according to embodiment 1 or embodiment 2, wherein the FIX polypeptide is administered directly to the nasal epithelium of a subject.
  • FIX polypeptide for use according to any one of embodiments 1-3, wherein the FIX polypeptide is administered as a nasal spray.
  • FIX polypeptide for use according to any one of embodiments 1-4, wherein the FIX polypeptide is administered at a concentration of 500-20,000 U/ml, preferably at a concentration of 500-5,000 U/ml.
  • FIX polypeptide for use according to any one of embodiments 1-5, wherein the FIX polypeptide is administered in combination with a surfactant, preferably polysorbate 80 and/or water.
  • a surfactant preferably polysorbate 80 and/or water.
  • FIX polypeptide for use according to any one of embodiments 1-6, wherein the FIX polypeptide is administered in combination with an agent that disrupts epithelial barrier function.
  • FIX polypeptide for use according to any one of embodiments 1-7, wherein the FIX polypeptide is defined by SEQ ID NO: 1 or has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, across the full length of SEQ ID NO: 1.
  • FIX polypeptide for use according to any one of embodiments 1-8, wherein the FIX polypeptide is linked to a half-life enhancing portion, in particular wherein the half-life enhancing portion is selected from the group consisting of albumin including variants and derivatives thereof, polypeptides of the albumin family including variants and derivatives thereof, immunoglobulins without antigen binding domain (e.g., the Fc portion), and polyethylene glycol.
  • FIX polypeptide for use according to embodiment 9, wherein the FIX polypeptide is linked to the half-life enhancing portion via a cleavable or non-cleavable linker.
  • FIX polypeptide for use according to any one of embodiments 1-10, wherein the FIX polypeptide is a high-activity variant.
  • FIX polypeptide comprises a leucine at a position corresponding to position 338 of wild-type Factor IX.
  • FIX polypeptide for use according to any one of embodiments 1-13, wherein the FIX polypeptide comprises the amino acid alanine at a position corresponding to position 5 of wildtype Factor IX and/or lysine at a position corresponding to position 10 of wild-type Factor IX.
  • FIX polypeptide for use according to any preceding embodiment, wherein the FIX polypeptide is administered at a concentration that results in at least 3-30% bioavailability in the blood plasma.
  • FIX polypeptide for use according to any preceding embodiment, wherein the FIX polypeptide is administered to a patient who is 18 years old or younger, preferably a patient who is 12 years old or younger.
  • YNSGKLEEFVQGNLERECMEEKCS FEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDIN

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Abstract

La présente invention concerne l'administration intranasale de facteurs de coagulation sanguine.
PCT/EP2024/071359 2023-07-26 2024-07-26 Administration intranasale de polypeptides du facteur ix Pending WO2025022005A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004091487A2 (fr) * 2003-04-09 2004-10-28 Wyeth Traitement de l'hemophilie par inhalation de facteurs de coagulation
WO2006026998A1 (fr) * 2004-09-10 2006-03-16 Pharmaorigin Aps Procede pour traiter les saignements locaux tracheaux, bronchiques ou alveolaires ou l'hemoptysie
WO2010029178A1 (fr) * 2008-09-15 2010-03-18 Paolo Simioni Mutant polypeptidique du facteur ix, ses utilisations et méthode pour sa production
WO2012061654A1 (fr) * 2010-11-03 2012-05-10 Catalyst Biosciences, Inc. Polypeptides de facteur ix modifiés et leurs applications
US20160122739A1 (en) * 2014-10-31 2016-05-05 Wisconsin Alumni Research Foundation Factor ix variants and methods of use therefor
WO2018102743A1 (fr) * 2016-12-02 2018-06-07 Bioverativ Therapeutics Inc. Méthodes de traitement de l'arthropathie hémophilique à l'aide de facteurs de coagulation chimériques

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004091487A2 (fr) * 2003-04-09 2004-10-28 Wyeth Traitement de l'hemophilie par inhalation de facteurs de coagulation
WO2006026998A1 (fr) * 2004-09-10 2006-03-16 Pharmaorigin Aps Procede pour traiter les saignements locaux tracheaux, bronchiques ou alveolaires ou l'hemoptysie
WO2010029178A1 (fr) * 2008-09-15 2010-03-18 Paolo Simioni Mutant polypeptidique du facteur ix, ses utilisations et méthode pour sa production
WO2012061654A1 (fr) * 2010-11-03 2012-05-10 Catalyst Biosciences, Inc. Polypeptides de facteur ix modifiés et leurs applications
US20160122739A1 (en) * 2014-10-31 2016-05-05 Wisconsin Alumni Research Foundation Factor ix variants and methods of use therefor
WO2018102743A1 (fr) * 2016-12-02 2018-06-07 Bioverativ Therapeutics Inc. Méthodes de traitement de l'arthropathie hémophilique à l'aide de facteurs de coagulation chimériques

Non-Patent Citations (54)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. NP_000124.1
ARYA ET AL., J THROMB HAEMOST, vol. 20, 2013, pages 296 - 306
ASTERMARK ET AL., HAEMOPHILIA, vol. 27, no. 5, 2021, pages 802 - 813
BAKER ET AL., CLIN EXP ALLERGY,, vol. 51, no. 10, 2021, pages 1361 - 1373
BEATTIEDUGAICZYK, GENE, vol. 20, 1982, pages 415 - 422
BENEDICT ET AL., NEUROPSYCHOPHARMACOL, vol. 32, 2007, pages 239 - 43
BOLTON-MAGGS ET AL., LANCET, vol. 361, 2003, pages 1801 - 9
BRAMBILLA ET AL., DRUG DEV RES, vol. 77, 2016, pages 479 - 88
BRUNETTI-PIERRI ET AL., HUM GENE THER., vol. 5, 2009, pages 479 - 85
CALO ET AL., PRECISION MEDICINE, vol. 2, 2015, pages 989
CHEUNG ET AL., PNAS, vol. 93, 1996, pages 11068 - 11073
COLLINS ET AL., BLOOD, vol. 124, 2014, pages 3880 - 3886
COOKEDAVID, J. CLIN. INVEST., vol. 76, 1985, pages 2420 - 2424
DARGAUD ET AL., HAEMATOLOGICA, vol. 103, no. 4, 2018
DEFREES ET AL., GLYCOBIOLOGY, vol. 16, no. 9, 2006, pages 833 - 843
DOLAN ET AL., BLOOD REV., vol. 32, no. 1, 2018, pages 52 - 60
EN DUNNEN ET AL., HUM MUTAT., vol. 37, no. 6, 2016, pages 564 - 569
FARES ET AL., PNAS, vol. 15, no. 10, 1992, pages 4304 - 4308
FIEUX ET AL., INT J MOL SCI, vol. 22, 2021, pages 6475
FIEUX ET AL., INT J MOL SCI,, vol. 22, 2021, pages 6475
GAO ET AL., DRUG DELIV TRANSLRES, vol. 9, no. 1, 2019, pages 311 - 318
GENNARO, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY., 2000
GHADIRI ET AL., PHARMACEUTICS, vol. 11, no. 3, 2019, pages 113
GUI ET AL., J THROMB HAEMOST, vol. 7, 2009, pages 1843 - 1851
HORIWITZ ET AL., NATURE REVIEWS GASTROENTEROLOGY & HEPATOLOGY, vol. 20, 2023, pages 417 - 432
KAO ET AL., THROMB. HAEMOST., vol. 110, 2013, pages 244 - 256
KELLER ET AL., DRUG DELIVERY AND TRANSLATIONAL RESEARCH, vol. 12, 2022, pages 735 - 757
LICHENSTEIN ET AL., J. BIOL. CHEM., vol. 269, 1994, pages 18149 - 18154
LIN ET AL., J. THROMB. HAEMOST., vol. 8, 2010, pages 1773 - 1783
MANCUSO ET AL., LANCET, vol. 397, 2021, pages 630 - 400
MARLAR ET AL., EUR J HAEMATOL., vol. 104, no. 1, 2020, pages 3 - 14
MILANOV ET AL., BLOOD, vol. 119, 2012, pages 602 - 611
MISENHEIMER ET AL., BIOCHEMISTRY, vol. 46, 2007, pages 7886 - 7895
NATHWANI ET AL., BLOOD, vol. 100, 2002, pages 1662 - 9
NATHWANI ET AL., BLOOD, vol. 97, 2001, pages 1258 - 65
NEURATHWALSH, PNAS, vol. 73, no. 11, 1976, pages 3825 - 3832
PEROT ET AL., THROMB. RES., vol. 135, 2015, pages 1017 - 1024
PETERS ET AL., BLOOD, vol. 115, 2010, pages 2057 - 2064
PIRES ET AL., PHARMACEUTICS, vol. 14, no. 5, 2022, pages 1073
POWELL ET AL., N. ENGL. J. MED., vol. 369, 2013, pages 2313 - 2323
QUADE-LYSSY ET AL., J. THROMB. HAEMOST., vol. 12, 2014, pages 932 - 942
QUELLEC ET AL., THROMB HAEMOST., vol. 118, no. 12, 2018, pages 2053 - 2063
S.-B. HONG ET AL., AM. SOC. HEMATOLOGY, 2016
SAMELSON-JONESARRUDA, MOLECULAR THERAPY, vol. 12, 2019, pages 184 - 201
SCHELLENBERGER ET AL., NATURE BIOTECHNOLOGY, vol. 27, 2009, pages 1186 - 1190
SCHMITT ET AL., THROMB HAEMOST, vol. 121, 2021, pages 351 - 60
SICHLER ET AL., J. BIOL. CHEM., vol. 278, 2003, pages 4121 - 4126
SIMIONI ET AL., N ENGL J MED., vol. 361, no. 17, 2009, pages 1671 - 1675
SONG ET AL., BIOMATERIALS, 2018, pages 18078 - 90
SRIVASTAVA ET AL., HAEMOPHILIA, vol. 19, 2013, pages 1 - 47
TSITOURA ET AL., J IMMUNOL, vol. 163, no. 5, 1999, pages 2592 - 2600
WINNER ET AL., PEDIATRICS, vol. 106, 2000, pages 989 - 97
WOENSEL ET AL., CANCERS, vol. 5, no. 3, 2013, pages 1020 - 1048
WU ET AL., HAEMATOLOGICA, vol. 106, no. 1, 2021, pages 264 - 268

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