WO2008107362A2 - Nouveaux inhibiteurs du facteur de coagulation sanguine - Google Patents

Nouveaux inhibiteurs du facteur de coagulation sanguine Download PDF

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WO2008107362A2
WO2008107362A2 PCT/EP2008/052409 EP2008052409W WO2008107362A2 WO 2008107362 A2 WO2008107362 A2 WO 2008107362A2 EP 2008052409 W EP2008052409 W EP 2008052409W WO 2008107362 A2 WO2008107362 A2 WO 2008107362A2
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phe
phg
homoarg
phenylmethanesulfonyl
tyr
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PCT/EP2008/052409
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WO2008107362A3 (fr
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Florencio Zaragoza DÖRWALD
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Novo Nordisk Health Care Ag
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • 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

Definitions

  • the present invention relates to new inhibitors of the proteases involved in the blood coagulation cascade and to their use as stabilizers of blood coagulation factors such as serine proteases and vitamin K-dependent polypeptides, as additives or formulation-aids for blood coagulation factors.
  • the invention relates to stabilizing Factor Vila or other Factor VII polypeptides against chemical and/or physical degradation, particularly in aqueous liquid compositions thereof.
  • the serine protease Factor VII is one of the plasma glycoproteins involved in the blood coagulation process. FVII is mainly present in plasma as single-chain zymogen, and is cleaved by another protease (FXa), to give its two-chain, activated form, FVIIa.
  • FXa protease
  • the tissue factor/factor Vila (TF/FVIIa) complex is the main trigger of thrombotic events. This complex is part of the extrinsic pathway of blood coagulation and mediates the activation of factors IX and X, ultimately leading to the generation of thrombin.
  • Factor Vila has proven to be a valuable therapeutic agent for the treatment of haemophilia and bleeding. It is desirable to have administration forms of Factor Vila suitable for both storage and for delivery. Ideally, the drug product is stored and administered as a liquid. Alternatively, the drug product is lyophilized, i.e. freeze-dried, and then reconstituted by adding a suitable diluent prior to patient use. It is desirable that the drug product has sufficient stability to enable long-term storage, e.g. for more than six months.
  • Protein stability can be affected, inter alia, by such factors as ionic strength, pH, temperature, repeated cycles of freeze/thaw, and exposure to shear forces. Active protein may be lost as a result of physical instabilities, e.g. via denaturation and/or aggregation (both soluble and insoluble aggregate formation), as well as chemical instabilities, including, for example, instability towards hydrolysis, deamidation and/or oxidation, to name just a few.
  • physical instabilities e.g. via denaturation and/or aggregation (both soluble and insoluble aggregate formation)
  • chemical instabilities including, for example, instability towards hydrolysis, deamidation and/or oxidation, to name just a few.
  • Factor Vila undergoes degradation by several pathways, especially aggregation
  • compositions of Factor Vila should be stable for more than 6 months over a wide range of protein concentrations. This allows for flexibility in methods of administration. Generally, more highly concentrated forms allow for the administration of lower volumes, which is highly desirable from the patients' point of view. Liquid compositions can have many advantages over freeze-dried products with regard to ease of administration and use.
  • liquid stability When developing a liquid composition, many factors are taken into consideration. Short- term, i.e. less than six months, liquid stability generally depends on avoiding gross structural changes, such as denaturation and aggregation. These processes are described in the literature for a number of proteins, and many examples of stabilizing agents exist. It is well-known that an agent effective in stabilizing one protein actually acts to destabilize another. Once the protein has been stabilized against gross structural changes, developing a liquid composition for long-term stability (e.g., greater than six months) depends on further stabilizing the protein from types of degradation specific to that protein. More specific types of degradation may include, for example, disulfide bond scrambling, oxidation of certain residues, deamidation, cyclization. Although it is not always possible to pinpoint the individual degradation species, assays are developed to monitor subtle changes so as to monitor the ability of specific excipients to uniquely stabilize the protein of interest.
  • FVII polypeptide composition Today, the only commercially available, recombinantly produced FVII polypeptide composition is a freeze-dried Factor FVIIa product which is reconstituted before use; it contains a relatively low Factor Vila concentration, e.g., about 0.6 mg/mL.
  • a vial (1.2 mg) of NovoSeven® NovoSeven® (Novo Nordisk A/S, Denmark) contains 1.2 mg recombinant human Factor Vila (rhFVIIA), 5.84 mg NaCI, 2.94 mg CaCI2.2H2O, 2.64 mg glycylglycine (GIyGIy), 0.14 mg polysorbate 80, and 60.0 mg mannitol; it is reconstituted to pH 5.5 by addition of 2.0 ml_ water for injection (WFI). When reconstituted, the protein solution is stable for use for 24 hours at room temperature. Thus, no liquid, ready-for-use or concentrated Factor VII products are currently commercially available.
  • the present invention provides compounds of formula (I)
  • X 1 represents lower alkyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, said groups optionally substituted with halogen, hydroxyl, lower alkyl, lower alkoxy, cyano, -CONH 2 , nitro, dimethylamino, acetyl, cyclopropanoyl, carboxyalkyl, or carboxyl;
  • X 2 represents D-Phe, D-IIe, D-Leu, D-Tyr, D-AIa, D-VaI, Aib, D-Phg, D-Trp, D-Abu, D- AIIe, D-Cha, D-Hph, D-NIe, D-Nva, GIu, Phe, He, Leu, Tyr, Ala, VaI, Pro, or Trp;
  • X 3 represents GIn, Phe, Thr, Ser, Tyr, Asn, Asp, (2-fluoro)Phe, (3-fluoro)Phe, (4- fluoro)Phe, GIy, Pro, He, or Ala;
  • X 5 represents Phg, D-Phg, Phe, VaI, He, Leu, Tyr, N-Me-Tyr, Lys, Ala, GIy, Aib, Trp, Abu, AIIe, Cha, Hph, NIe, or Nva;
  • X 6 represents Ala, GIy, GIu, VaI, GIn, He, Thr, Tyr, or is absent;
  • X 7 represents Ala, GIy, Thr, or is absent
  • X 8 represents hydrogen, cycloalkylalkyl, or lower alkyl; including any and all stereoisomer ⁇ form or forms thereof; and any mixture of two or more such compounds of formula I in any ratio; and physiologically tolerable salts and prodrugs thereof.
  • compositions of general formula (I) are inhibitors of the proteases involved in the blood coagulation cascade, and may therefore be used as stabilizing additives for formulations of FVII polypeptides, notably aqueous solutions of FVIIa.
  • Blood coagulation Factor VII or analogues thereof (“Factor VII polypeptides"), when formulated as liquid, aqueous pharmaceutical compositions together with at least one stabilising agent with formula (I), exhibit improved stability and thereby allow for prolonged storage before and/or during actual use.
  • the present invention further provides:
  • compositions comprising a FVII polypeptide, such as wild-type FVIIa, rhFVIIa, or analogs or derivatives thereof, and a compound of formula (I);
  • methods for inhibiting a FVII polypeptide such as wild-type FVIIa, rhFVIIa, or analogs or derivatives thereof;
  • the present invention provides novel compounds with formula (I) (see above) that inhibit degradation of serine proteases, such as e.g. Factor VII polypeptides in liquid (particularly aqueous liquid) or solid administration forms.
  • the invention provides aqueous liquid pharmaceutical compositions of serine proteases, in particular Factor VII polypeptides, which provide acceptable control of chemical and/or physical degradation processes such as those outlined above.
  • Factor VII or analogues thereof (“Factor VII polypeptides")
  • aqueous pharmaceutical compositions together with at least one compound with formula (I) exhibits improved stability and thereby allow for prolonged (e.g. 6 months or more) storage before actual use.
  • Compounds of formula (I) are capable of reversibly inhibiting Factor Vila and may be employed as stabilizers in formulations or compositions, notable aqueous formulations or compositions, comprising a Factor VII polypeptide (see below).
  • compounds of the invention frequently exhibit favourable solubility in water or other aqueous media.
  • tz he term 'proteinogenic amino acid 1 refers to L-alanine, L-arginine, L-asparagine, L- aspartic acid, L-cysteine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L- tryptophan, L-tyrosine, or L-valine.
  • unnatural amino acid refers to any compound comprising at least one primary or secondary amino group and at least one carboxyl group, without being L-alanine, L- arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L- serine, L-threonine, L-tryptophan, L-tyrosine, or L-valine.
  • alkyl is intended to indicate a straight or branched saturated monovalent hydrocarbon radical.
  • alkylene indicates the corresponding diradical.
  • a "lower alkyl” is an alkyl having from 1 to 6 carbon atoms, also denoted as Ci- 6 -alkyl.
  • Ci- 6 -alkyl groups include for instance methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 4-methylpentyl, n-hexyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl (neopentyl) and 1,2,2-trimethylpropyl.
  • cycloalkyl is intended to indicate a cyclic saturated monovalent hydrocarbon radical.
  • a "lower cycloalkyl” is an cycloalkyl having from three to six carbon atoms, also denoted as Ci- 6 -cycloalkyl.
  • Ci- 6 -cycloalkyl groups include for instance cyclopropyl, cyclobutyl, cyclopentyl, 2-methyl-cyclopentyl, and cyclohexyl.
  • alkoxy is intended to indicate a radical wherein an alkyl group in either a linear or branched or cyclic configuration is linked through an ether oxygen and having its free valence bond from the ether oxygen (alkyl-O).
  • a "lower alkoxy” is an alkoxy radical wherein the alkyl group has from 1 to 6 carbon atoms, also denoted as Ci- 6 - alkoxy.
  • Examples of linear alkoxy groups are methoxy, ethoxy, propoxy, butoxy, pentoxy and hexoxy.
  • Examples of branched alkoxy include isopropoxy, sec-butoxy, tert-butoxy, isopentoxy and isohexoxy.
  • Examples of cyclic alkoxy include cyclopropyloxy, cyclobutyloxy, cyclopentyloxy and cyclohexyloxy.
  • a “lower alkoxycarbonyl” is an alkoxycarbonyl radical wherein the alkyl group has from 1 to 6 carbon atoms, also denoted as Ci- 6 -alkoxycarbonyl.
  • methyloxycarbonylmethyl methoxycarbonyethyl , carbethoxy, propoxycarbonylmethyl, propoxycarbonylethyl, isopropoxycarbonylmethyl, isopropoxycarbonylethyl, n-butoxycarbonylmethyl, n- butoxycarbonylethyl, sec-butoxycarbonylmethyl, sec-butoxycarbonylethyl, tert- butoxycarbonylmethyl, tert-butoxycarbonylethyl, 3-methylbutoxycarbonylmethyl, 3- methylbutoxycarbonylethyl, n-hexoxycarbonylmethyl, n-hexoxycarbonylethyl and the like.
  • a “lower alkoxycarbonylalkyl” is an alkoxycarbonylalkyl radical wherein the two alkyl groups independently have from 1 to 6 carbon atoms, also denoted as Ci- 6 - alkoxycarbonyl-Ci- 6 -alkyl.
  • cycloalkylalkyl is intended to represent a branched or straight alkyl group substituted at a carbon with a cycloalkyl group.
  • examples of such groups include, but are not limited to, cyclopropylethyl, cyclobutylmethyl, 2-(cyclohexyl)ethyl, cyclohexylmethyl, 3-(cyclopentyl)-l-propyl, and the like.
  • arylalkyl is intended to refer to a straight or branched saturated carbon chain containing from 1 to 6 carbons substituted with an aromatic carbohydride; such as benzyl, phenethyl, 3-phenylpropyl, 1-naphtylmethyl, 2-(l-naphtyl)ethyl and the like.
  • arylalkyl is intended to refer to a straight or branched saturated carbon chain containing from 1 to 6 carbons substituted with an aromatic carbohydride; such as benzyl, phenethyl, 3-phenylpropyl, 1-naphtylmethyl, 2-(l-naphtyl)ethyl and the like.
  • heteroarylalkyl is intended to refer to a straight or branched saturated carbon chain containing from 1 to 6 carbons substituted with a heteroaryl group; such as (2-furyl)methyl, (3-furyl)methyl, (2-thienyl)methyl, (3-thienyl)methyl, (2-pyridyl)methyl, l-methyl-l-(2-pyrimidyl)ethyl and the like.
  • aryl as used herein is intended to indicate a mono- or polycyclic carbocyclic aromatic ring radical with for instance 6 to 10 member atoms, or an aromatic ring system radical with for instance from 10 to 22 member atoms.
  • Aryl is also intended to include the partially hydrogenated derivatives of the carbocyclic systems, wherein at least one ring is aromatic. Examples of such partially hydrogenated derivatives include 1,2,3,4-tetrahydronaphthyl, fluorenyl and 1,4-dihydronaphthyl.
  • heteroaryl as used herein is intended to indicate a mono- or polycyclic heterocyclic aromatic ring radical with for instance 5 to 13 member atoms, or a heterocyclic aromatic ring system radical with for instance from 13 to 21 member atoms, containing one or more heteroatoms selected from nitrogen, oxygen, and sulfur, wherein N-oxides and sulfur monoxides and sulfur dioxides are permissible heteroaromatic substitutions, such as for instance furyl, thienyl, thiophenyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl isoxazolyl, oxadiazoly, thiadiazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4
  • Heteroaryl is also intended to include the partially hydrogenated derivatives of the heterocyclic systems, provided at least one ring comprising a hetero atom is aromatic.
  • Examples of such partially hydrogenated derivatives include 2,3-dihydrobenzofuranyl, pyrrolinyl, pyrazolinyl, indolinyl, oxazolidinyl, oxazolinyl and oxazepinyl.
  • aryl and “heteroaryl” include, but are not limited to phenyl, biphenylyl, indenyl, fluorenyl, phenanthrenyl, azulenyl, naphthyl (1-naphthyl, 2-naphthyl), anthracenyl (1-anthracenyl, 2-anthracenyl, 3-anthracenyl), thienyl (2-thienyl, 3-thienyl), furyl (2-furyl, 3-furyl), indolyl, oxadiazolyl, isoxazolyl, thiadiazolyl, oxatriazolyl, thiatriazolyl, quinazolin, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (1-pyrrolyl, 2-pyrrolyl, 3-pyrrol,
  • prodrug includes biohydrolyzable amides and biohydrolyzable esters, acetals, aminals, carbamates, thioaminals, thioacetals, and hydrazones, and also encompasses a) compounds in which the biohydrolyzable functionality in such a prodrug is encompassed in the compound according to the present invention, and b) compounds which may be oxidized or reduced biologically at a given functional group to yield drug substances according to the present invention.
  • the invention also comprises prodrugs of compounds of formula (I)-
  • Stereogenic atoms present in the compounds of formula (I) can independently of each other have R configuration or S configuration.
  • the compounds of formula (I) can be pure enantiomers or mixtures of enantiomers, e.g. racemates, or pure diastereomers or mixtures of diastereomers.
  • the invention also comprises all tautomeric forms of compounds of formula (I).
  • salts which are not harmful to the patient.
  • Such salts include pharmaceutically tolerable acid addition salts, pharmaceutically tolerable metal salts, ammonium and alkylated ammonium salts.
  • Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like.
  • suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p- toluenesulfonic acids and the like.
  • compositions of formula (I) include the pharmaceutically tolerable salts listed in J. Pharm. Sci. 66, 2 (1977), which is incorporated herein by reference.
  • metal salts include lithium, sodium, potassium, magnesium salts and the like.
  • ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts and the like.
  • the present invention also comprises pharmaceutically tolerable salts of compounds of formula (I).
  • solvate is a complex of defined stoichiometry formed by a solute and a solvent.
  • Solvents may be, by way of example, water, ethanol, or acetic acid.
  • the invention relates to compounds of formula (I), wherein X 1 represents lower alkyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein said groups are optionally substituted with halogen, lower alkyl, lower alkoxy, cyano, dimethylamino, acetyl, cyclopropanoyl, carboxyalkyl, or carboxyl.
  • X 1 represents lower alkyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, or arylalkyl, wherein said groups are optionally substituted with halogen, lower alkyl, lower alkoxy, dimethylamino, or carboxyl.
  • X 1 represents benzyl (phenylmethyl or C 6 H 5 -CH 2 -), 3-carboxybenzyl, phenyl, 4- chlorophenyl, 4-methylphenyl, 4-methoxyphenyl, 3-carboxyphenyl, dansyl, 6-quinolinyl, cyclohexyl, cyclohexylmethyl, methyl, ethyl, propyl, butyl, 3-carboxypropyl, or 5- carboxypenyl.
  • the invention relates to compounds of formula (I), wherein X 2 represents a D-amino acid (amino acid in the D-form).
  • the invention relates to compounds of formula (I), wherein X 2 represents D-Phe, D-IIe, D- Leu, D-Tyr, D-AIa, D-VaI, Aib, D-Phg, D-Trp, D-Abu, D-AIIe, D-Cha, D-Hph, D-NIe, D-Nva, or GIu.
  • X 2 represents D-Phe, D-IIe, D-Leu, D-Tyr, D-AIa, D-VaI, Aib, D-Phg, D-Trp, or GIu. In yet another embodiment, X 2 represents D-Phe, D-IIe, D- Leu, D-Tyr, or D-VaI.
  • the invention relates to compounds of formula (I), wherein X 3 represents GIn, Phe, Thr, Ser, Tyr, Asn, Asp, He, GIy, or Ala. In another embodiment, X 3 represents GIn, Phe, Thr, Ser, Tyr, or He.
  • X 4 represents Arg, homoArg, (4-amidino)Phe, (4-guanidino)Phe, (N- amidinopiperidin-4-yl)Ala, or (N-amidinopiperidin-4-yl)homoAla.
  • the invention relates to compounds of formula (I), wherein X 5 represents Phg, D-Phg, Phe, VaI, He, Leu, Tyr, N-Me-Tyr, Lys, Ala, Trp, or GIy. In another embodiment, X 5 represents Phg, D-Phg, Phe, VaI, He, Tyr, N-Me-Tyr, Lys, Ala, or GIy. In yet another embodiment, X 5 represents Phg, Phe, He, VaI, Tyr, or N-Me-Tyr.
  • the invention relates to compounds of formula (I), wherein X 6 represents Ala, GIy, GIu, VaI, He, or is absent. In another embodiment, X 6 represents Ala, GIu, VaI, or is absent. In yet another embodiment, X 6 is absent.
  • the invention relates to compounds of formula (I), wherein X 7 is absent.
  • the invention relates to compounds of formula (I), wherein X 8 represents hydrogen, methyl, ethyl, propyl, or cyclopropylmethyl. In another embodiment, X 8 represents hydrogen.
  • the compound with formula (I) is selected from the list of: N-Phenylmethanesulfonyl-(D-Tyr)-Thr-homoArg-Phg-NH2; N-Phenylmethanesulfonyl-(D-Ile)-Gln-homoArg-Tyr-NH2; N-Phenylmethanesulfonyl-(D-Tyr)-Thr-homoArg-Tyr-NH2; N-Phenylmethanesulfonyl-(D-Phe)-Phe-homoArg-Tyr-l ⁇ IH2;
  • the compounds of formula I are reversible inhibitors of Factor VII polypeptides (in their activated form). Preferably, they are specific inhibitors of Factor VII polypeptides.
  • the term specific when used in reference to the inhibition of Factor Vila activity means that a compound of the formula I can inhibit Factor VII activity without substantially inhibiting the activity of other specified proteases involved in the blood coagulation and/or the fibrinolysis pathway including, for example, Factor Xa, plasmin, thrombin, Factor IXa, Factor XIa, Factor XIIa and tissue-plasminogen activator (tPA) (using the same concentration of the inhibitor).
  • tPA tissue-plasminogen activator
  • the present specification describes assays and methods (see below) for determining the inhibition constant (K 1 ) for a Factor VII polypeptide as well as other specified proteases involved in the blood coagulation and/or the fibrinolysis pathway.
  • the compounds of formula I exhibit at least one of the following :
  • (activated Factor VII polypeptide) of 1/10 or less such as 1/20 or less, 1/50 or less, or 1/100 or less) of K,(Factor Xa) (using the same concentration of the inhibitor); • a K,(activated Factor VII polypeptide) of 1/10 or less (such as 1/20 or less, 1/50 or less, or 1/100 or less) of K,(plasmin) (using the same concentration of the inhibitor);
  • (activated Factor VII polypeptide) of 1/10 or less such as 1/20 or less, 1/50 or less, or 1/100 or less) of K,(Factor XIIa) (using the same concentration of the inhibitor); • a K,(activated Factor VII polypeptide) of 1/10 or less (such as 1/20 or less, 1/50 or less, or 1/100 or less) of K,(tPA) (using the same concentration of the inhibitor).
  • Factor VII polypeptide or "FVII polypeptide” means any protein comprising the amino acid sequence 1-406 of wild-type human Factor Vila (i.e., a polypeptide having the amino acid sequence disclosed in U.S. Patent No. 4,784,950), variants thereof as well as Factor VII-related polypeptides, Factor VII derivatives and Factor VII conjugates. This includes FVII variants, Factor VII-related polypeptides, Factor VII derivatives and Factor VII conjugates exhibiting substantially the same or improved biological activity relative to wild-type human Factor Vila.
  • Factor VII is intended to encompass Factor VII polypeptides in their uncleaved (zymogen) form, as well as those that have been proteolytically processed to yield their respective bioactive forms, which may be designated Factor Vila.
  • Factor VII is cleaved between residues 152 and 153 to yield Factor Vila.
  • variants of Factor VII may exhibit different properties relative to human Factor VII, including stability, phospholipid binding, altered specific activity, and the like.
  • Fractor VII or “Factor Vila” within the above definition also includes natural allelic variations that may exist and occur from one individual to another. Also, degree and location of glycosylation or other post-translation modifications may vary depending on the chosen host cells and the nature of the host cellular environment.
  • wild type human FVIIa is a polypeptide having the amino acid sequence disclosed in U.S. Patent No. 4,784,950.
  • Factor VII-related polypeptides encompasses polypeptides, including variants, in which the Factor Vila biological activity has been substantially modified, such as reduced, relative to the activity of wild-type Factor Vila.
  • These polypeptides include, without limitation, Factor VII or Factor Vila into which specific amino acid sequence alterations have been introduced that modify or disrupt the bioactivity of the polypeptide.
  • Fractor VII derivative is intended to designate a FVII polypeptide exhibiting substantially the same or improved biological activity relative to wild-type Factor VII, in which one or more of the amino acids of the parent peptide have been genetically and/or chemically and/or enzymatically modified, e.g. by alkylation, glycosylation, PEGylation, acylation, ester formation or amide formation or the like. This includes but is not limited to PEGylated human Factor Vila, cysteine-PEGylated human Factor Vila and variants thereof.
  • Non-limiting examples of Factor VII derivatives includes GlycoPegylated FVII derivatives as disclosed in WO 03/31464 and US Patent applications US 20040043446, US 20040063911, US 20040142856, US 20040137557, US 20040132640 and WO 2007/022512 (Neose Technologies, Inc.); FVII conjugates as disclosed in WO 01/04287, US patent application 20030165996, WO 01/58935, WO 03/93465 (Maxygen ApS) and WO 02/02764, US patent application 20030211094 (University of Minnesota).
  • PEGylated human Factor Vila encompasses any Factor Vila to which one or more PEG moieties has been attached.
  • the PEG molecule may be attached to any part of the Factor Vila polypeptide, including any amino acid residue or carbohydrate moiety of the Factor Vila polypeptide.
  • the term "cysteine-PEGylated human Factor Vila” refers to Factor Vila having a PEG molecule conjugated to a sulfhydryl group of a cysteine introduced in human Factor Vila to form a Factor Vila sequence variant.
  • the PEG may be any branched or unbranched poly(ethylenglycol) having any chain length, including, without limitation, branched PEGs having a molecular weight of from about 5KDa to about 80 KDa, such as, e.g., about 1 KDa, 2 KDa, 5 KDa, 10 KDa, 20 KDa, 30 KDa, and 40 KDa).
  • improved biological activity refers to FVII polypeptides with i) substantially the same or increased proteolytic activity compared to recombinant wild type human Factor Vila or ii) to FVII polypeptides with substantially the same or increased TF binding activity compared to recombinant wild type human Factor Vila or iii) to FVII polypeptides with substantially the same or increased half life in blood plasma compared to recombinant wild type human Factor Vila.
  • Non-limiting examples of Factor VII variants having substantially the same or increased proteolytic activity compared to recombinant wild type human Factor Vila include S52A- FVIIa, S60A-FVIIa ( Lino et al., Arch. Biochem. Biophys. 352: 182-192, 1998); FVIIa variants exhibiting increased proteolytic stability as disclosed in U.S. Patent No. 5,580,560; Factor Vila that has been proteolytically cleaved between residues 290 and 291 or between residues 315 and 316 (Mollerup et al., Biotechnol. Bioeng.
  • Non-limiting examples of FVII variants having increased biological activity compared to wild-type FVIIa include FVII variants as disclosed in WO 01/83725, WO 02/22776, WO 02/077218, WO 03/027147, WO 04/029090, WO 05/075635, and European patent application with application number 05108713.8 (Novo Nordisk A/S), WO 02/38162 (Scripps Research Institute); and FVIIa variants with enhanced activity as disclosed in JP 2001061479 (Chemo-Sero-Therapeutic Res Inst.).
  • variants of factor VII include, without limitation, PlOQ-FVII, K32E-FVII, P10Q/K32E-FVII, L305V-FVII, L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII, V158D/E296V/M298Q/L305V/
  • L305V/K337A/E296V-FVII L305V/K337A/V158D-FVII, L305V/V158D/M298Q-FVII, L305V/V158D/E296V-FVII, L305V/V158T/M298Q-FVII, L305V/V158T/E296V-FVII, L305V/E296V/M298Q-FVII, L305V/V158D/E296V/M298Q-FVII, L305V/V158T/E296V/M298Q-FVII, L305V/V158T/E296V/M298Q-FVII, L305V/V158T/K337A/M298Q-FVII, L305V/V158T/E296V/K337A-FVII, L305V/V158D/K337A/M298Q-FVII, L305
  • K316Q/L305V/K337A/M298Q-FVII K316Q/L305V/K337A/E296V-FVII, K316Q/L305V/V158D/M298Q-FVII, K316Q/L305V/V158D/E296V-FVII, K316Q/L305V/V158T/M298Q-FVII, K316Q/L305V/V158T/E296V-FVII, K316Q/L305V/E296V/M298Q-FVII, K316Q/L305V/V158D/E296V/M298Q-FVII, K316Q/L305V/V158D/E296V/M298Q-FVII, K316Q/L305V/V158T/E296V/M298Q-FVII, K316Q/L305V/V158T/E296V/M298Q-FVII, K
  • substitution variants in a factor VII polypeptide include, without limitation substitutions in positions PlO, K32, L305, M306, D309, L305, L305, F374, V158, M298, V158, E296, K337, M298, M298, S336, S314, K316, K316, F374, S52, S60, R152, S344, T106, K143, N145, V253, R290, A292, G291, R315, V317, and substitutions, additions or deletions in the amino acid sequence from T233 to N240 or from R304 to C329; or from 1153 to R223, or combinations thereof, in particular variants such as PlOQ, K32E, L305V, M306D, D309S, L305I, L305T, F374P, V158T, M298Q, V158D, E296V, K337A, M298Q, M298K, S336G, S314E, K316H, K316Q
  • Factor VII (as Factor Vila) in blood clotting derives from its ability to (i) bind to tissue factor (TF) and (ii) catalyze the proteolytic cleavage of Factor IX or Factor X to produce activated Factor IX or X (Factor IXa or Xa, respectively).
  • Human Factor Vila biological activity may be quantified by an assay measuring the ability of a preparation to promote blood clotting using Factor VII-deficient plasma and thromboplastin, as described, e.g., in U.S. Patent No. 5,997,864.
  • biological activity is expressed as the reduction in clotting time relative to a control sample and is converted to "Factor VII units" by comparison with a pooled human serum standard containing 1 unit/ml Factor VII activity.
  • Factor VII polypeptides may also be assayed for specific activities ("clot activity") by using a one-stage coagulation assay.
  • the sample to be tested is diluted in 50 mM PIPES- buffer (pH 7.5), 0.1% BSA and 40 ⁇ l is incubated with 40 ⁇ l of Factor VII deficient plasma and 80 ⁇ l of human recombinant tissue factor containing 10 mM Ca2+ and synthetic phospholipids.
  • Coagulation times are measured and compared to a standard curve using a reference standard in a parallel line assay.
  • Factor Vila biological activity may be quantified by (i) measuring the ability of Factor Vila to produce Factor Xa in a system comprising TF embedded in a lipid membrane and Factor X. (Persson et al., J. Biol. Chem. 272: 19919-19924, 1997); (ii) measuring Factor X hydrolysis in an aqueous system; (iii) measuring its physical binding to TF using an instrument based on surface plasmon resonance (Persson, FEBS Letts. 413 :359-363, 1997) and (iv) measuring hydrolysis of a synthetic substrate.
  • Factor VII variants having, in the activated (FVIIa) form, substantially the same or improved biological activity relative to wild-type Factor Vila encompass those that exhibit at least about 10%, preferably at least about 25%, more preferably at least about 50%, even more preferably at least about 75% and most preferably at least about 90% of the specific activity of Factor Vila that has been produced in the same cell type, when tested in one or more of a clotting assay, proteolysis assay or TF binding assay as described above.
  • the Factor VII polypeptide is human Factor Vila (hFVIIa), preferably recombinantly made human Factor Vila (rhVIIa).
  • the Factor VII polypeptide is a Factor VII sequence variant.
  • the Factor VII polypeptide has a glycosylation different from wild-type human Factor VII.
  • the Factor VII polypeptide is a Factor VII derivative, in particular a PEGylated Factor VII polypeptide, including a glycopegylated Factor VII polypeptide.
  • hFVIIa human Factor Vila
  • rhVIIa recombinantly made human Factor Vila
  • the Factor VII polypeptide is a Factor VII sequence variant.
  • the Factor VII polypeptide has a glycosylation different from wild-type human Factor VII.
  • the Factor VII polypeptide is a Factor VII derivative, in particular a PEGylated Factor VII polypeptide, including a glycopegylated Fact
  • the ratio between the activity of the Factor VII polypeptide and the activity of native human Factor Vila is at least about 1.25, preferably at least about 2.0, or 4.0, most preferred at least about 8.0, when tested in the "In Vitro Proteolysis Assay" (Assay 2) as described in the present specification.
  • the Factor VII polypeptides are Factor VII-related polypeptides, in particular variants, wherein the ratio between the activity of said Factor VII polypeptide and the activity of native human Factor Vila (wild-type FVIIa) is at least about 1.25 when tested in the "In Vitro Hydrolysis Assay” (see Assay 1 below); in other embodiments, the ratio is at least about 2.0; in further embodiments, the ratio is at least about 4.0.
  • the product is an oil, it can be extracted with DCM.
  • the peptides were synthesized on Fmoc protected Rink amide resin (Novabiochem), a H- N(X 8 )-CH 2 -(C 6 H 2 (OR) 3 )-resin, or an equivalent amine-functionalized resin suitable for the preparation of N-alkyl or N-aryl amides (see, e.g. Boojamra et al., J. Org. Chem. 1997, 62, 1240-1256; Estep et al., J. Org. Chem.
  • the peptide can be cleaved from the resin by means of conventional methods, such as, e.g., by stirring at room temperature with a mixture of trifluoroacetic acid, water and triisopropylsilane (95:2.5:2.5).
  • an aqueous, liquid Factor VII polypeptide formulation or composition of the invention (aqueous pharmaceutical composition of the invention) will - irrespective of whether the aqueous formulation is present in aqueous liquid form from the start, or is produced by dissolution/reconstitution of a substantially solid formulation (e.g. a lyophilized preparation) by addition of water or another aqueous carrier or vehicle - in general, suitably be administered parenterally, i.e., intravenously, subcutaneously, or intramuscularly, or by continuous or pulsatile infusion.
  • a substantially solid formulation e.g. a lyophilized preparation
  • Factor VII polypeptide compositions of the invention for parenteral administration will, in addition to a compound of formula I or a physiologically tolerable salt thereof in an appropriate concentration, normally comprise the Factor VII polypeptide in combination with, preferably dissolved in, a pharmaceutically acceptable aqueous carrier.
  • aqueous carriers such as water, buffered water, 0.4% saline, 0.3% glycine and the like.
  • Factor VII polypeptides in the context of the invention may also be formulated into liposome preparations for delivery or targeting to the sites of injury. Liposome preparations are generally described in, e.g., US 4,837,028, US 4,501,728 and US 4,975,282.
  • compositions may be sterilised by conventional, well-known sterilisation techniques.
  • the resulting aqueous solutions may be packaged for use as such, or they may be filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with sterile water or a sterile aqueous solution (carrier, vehicle) prior to administration.
  • the compositions may further contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions and/or to enhance the chemical and/or physical stability of the the composition. These include: pH-ad ⁇ ustina and/or buffering agents, e.g.
  • the buffer concentration range is chosen to maintain the preferred pH of the solution.
  • the buffering agent may also be a mixture of two or more buffering agents, e.g.
  • the buffer is a mixture of citrate and at least one of the buffers acetate (ammonium, sodium or calcium), histidine (L-histidine), malate, phosphate (sodium or potassium), tartaric acid, succinic acid, MES, HEPES, imidazole, TRIS, lactate and glutamate.
  • the total concentration of buffer agent(s) is typically in the range of from about 1 mM to about 100 mM, such as from about 1 mM to about 50 mM, often from about 1 mM to about 25 mM, e.g. from about 2 mM to about 20 mM.
  • the compositions - whether initially in liquid, freeze-dried or reconstituted form - may optionally contain a calcium salt.
  • the calcium salt may be present in a low concentration, such as, e.g., from about 0.1 mM to about 5 mM; it may be present in a medium concentration, such as, e.g., from about 5 mM to about 15 mM; or it may be present in a higher concentration, such as, e.g., from about 15 mM to about 1000 mM.
  • the calcium salt is selected from : calcium chloride, calcium acetate, calcium gluconate and calcium laevulate, and mixtures of two or more thereof.
  • the concentration of calcium ions in the composition may be below 0.1 mM.
  • tonicitv-adiusting agents tonicity-modifying substances which contribute to the osmolality of the the formulation
  • amino acids e.g. amino acids, small peptides (having, e.g., from 2 to 5 amino acid residues), neutral salts, mono- or disaccharides, polysaccharides, sugar alcohols, or mixtures of at least two of such substances.
  • specific examples include, but are not limited to, sodium chloride, potassium chloride, sodium citrate, sucrose, glucose and mannitol.
  • concentration of tonicity-adjusting agent is adjusted to near isotonicity, depending on the other ingredients present in the formulation.
  • tonicity-adjusting agents are incorporated in a concentration of from about 1 to about 500 mM, such as from about 1 to about 300 mM, often from about 10 to about 200 mM, e.g. from about 20 to about 150 mM, depending on the other ingredients present.
  • Neutral salts such as, e.g., sodium chloride or potassium chloride may be used.
  • neutral salt indicates a salt that is substantially neither acidic nor basic, i.e. has little or no effect on formulation pH when dissolved;
  • surfactants typically a non-ionic surfactant, suitably of the polysorbate or TweenTM type (e.g. PolysorbateTM 20 or 80, or TweenTM 80), or of the poloxamer or PluronicTM type (e.g. PoloxamerTM 188 or 407).
  • the amount of surfactant incorporated may typically range from about 0.005 to about 1% weight/weight (w/w), with amounts of from about 0.005 to about 0.1% w/w, such as from about 0.005 to 0.02% w/w, typically being preferred. In some situations, relatively high concentrations, e.g. up to about 0.5% w/w, are desirable to maintain protein stability. However, the levels of surfactant used in actual practice are customarily limited by clinical practice;
  • antioxidants e.g. ascorbic acid, cysteine, homocysteine, cystine, cysstathionine, methionine, glutathione, or peptides containing cysteine or methionine; methionine, in particular L-methionine, is typically a very suitable antioxidant.
  • An antioxidant is typically incorporated in a concentration of from about 0.1 to about 2 mg/ml;
  • preservatives include phenol, benzyl alcohol, ortho-cresol, meta-cresol, para-cresol, methylparaben, propylparaben, benzalconium chloride, and benzethonium chloride.
  • a preservative is typically incorporated in a concentration of from about 0.1 to about 2 mg/ml, depending on the pH range and type of preservative.
  • the concentration of Factor VII polypeptide in the compositions can vary widely, typically from about 0.01% w/w to about 2% w/w (i.e. from about 0.1 mg/ml to about 20 mg/ml), such as from about 0.05% w/w to about 1.5% w/w (i.e. from about 0.5 mg/l to about 15 mg/ml), e.g. from about 0.05% w/w to about 1% w/w (i.e. from about 0.5 mg/ml to about 10 mg/ml), and will be selected primarily on the basis of fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. In the case of Factor Vila, concentration is frequently expressed as mg/ml or as International units/ml (IU/ml). 1 mg of FVIIa usually corresponds to 43000-56000 IU or more.
  • concentration of a compound (or compounds) of formula I of the invention in a liquid, aqueous pharmaceutical composition of the invention will typically be at least 1 ⁇ M.
  • concentration typically depends on the selected compound (or compounds), more specifically on the binding affinity of the selected compound(s) to the Factor VII polypeptide.
  • the compound of formula I may be present in a concentration of at least 5 ⁇ M, at least 10 ⁇ M, at least 20 ⁇ M, at least 50 ⁇ M, at least 100 ⁇ M, at least 150 ⁇ M, at least 250 ⁇ M, at least 500 ⁇ M, at least 1 mM, at least 2 mM, at least 4 mM, at least 5 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 15 mM, or at least 20 mM, such as, e.g., in the range of 1-10000 ⁇ M , 10-10000 ⁇ M, 20-10000 ⁇ M, 50-10000 ⁇ M, 10-5000 ⁇ M, 10-2000 ⁇ M, 20-5000 ⁇ M, 20-2000 ⁇ M, 50-5000 ⁇ M, 0.1-100 mM, 0.1-75 mM, 0.1-50 mM, 0.1-10 mM, 0.2-75 mM, 0.2-50 ⁇ M,
  • the molar ratio between the compound of formula I and FVII polypeptide may be: above 0.1, above 0.5, above 1, above 2, above 5, above 10, above 25, above 100, above 250, above 1000, above 2500, or above 5000, such as, e.g., in the range of 0.1-10000, 0.1-5000, 0.1-2500, 0.1-1000, 0.1-250, 0.1-100, 0.1-25, 0.1-10, 0.5-10000, 0.5-5000, 0.5-2500, 0.5-1000, 0.5-250, 0.5-100, 0.5-25, 0.5-10, 1-10000, 1-5000, 1-2500, 1-1000, 1-250, 1-100; 1-25; 1-10, 10-10000, 10-5000, 10-250, 1000- 10000, or 1000-5000.
  • parenterally administrable compositions will be known or apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Sciences, 18 th edition, Mack Publishing Company, Easton, PA (1990).
  • Factor VII polypeptides will typically be administered within about 24 hours prior to performing the intervention, and for as much as 7 days or more thereafter.
  • the dose of Factor VII polypeptide (e.g. rhFVIIa) will normally range from about 0.05 mg/day to 500 mg/day, preferably from about 1 mg/day to about 200 mg/day, and more preferably from about 10 mg/day to about 175 mg/day for a 70 kg subject as loading and maintenance doses, depending on the weight of the subject and the severity of the condition.
  • compositions containing Factor VII polypeptides may be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a subject already suffering from a condition, as described above, in an amount sufficient to cure, alleviate or partially arrest the condition and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective amount”.
  • amounts effective for this purpose will depend on the severity of the condition or injury, as well as on the body weight and general physical condition of the subject.
  • compositions of FVII polypeptides are generally employed in connection with life-threatening or potentially life- threatening medical conditions or states, and in such circumstances - in view of the general advantages associated with minimizing quantities of extraneous substances, and taking into account the general lack of immunogenicity of human Factor VII polypeptides - it is possible and may be felt desirable by the treating physician to administer a substantial excess of the Factor VII polypeptide in question.
  • compositions containing a Factor VII polypeptide are administered to a subject susceptible to, or otherwise at risk of, a disease state or injury in order to enhance the subject's own coagulative capability.
  • the dosage employed for such purposes (which may be termed a "prophylactically effective dose”) will once again depend on the subject's body weight and general state of health, but will once again generally range from about 0.05 mg/day to about 500 mg/day, more commonly from about 1.0 mg/day to about 200 mg/day for a 70-kilogram subject.
  • dosage levels have generally been in the range of about 90-120 ⁇ g/kg body weight per dose.
  • doses e.g. doses in excess of 150 ⁇ g/kg body weight, and in some cases doses of about 250-300 ⁇ g/kg.
  • Single or multiple administration of the composition in question may be carried out using dose levels and dosing regimens selected by the treating physician.
  • a Factor VII polypeptide may be administered by continuous infusion, e.g. using a portable pump system.
  • a Factor VII polypeptide e.g. topical application
  • a Factor VII polypeptide may be carried out, e.g., by spraying, by perfusion, by use of a double balloon catheter or a stent, by incorporation into vascular grafts or stents, in the form of hydrogels to coat balloon catheters, or by other well established methods.
  • the pharmaceutical composition in question should provide a quantity of Factor VII polypeptide which is adequate to effectively treat the subject.
  • the liquid pharmaceutical preparation should typically be stable for at least six months, and preferably up to 36 months, when stored at temperatures ranging from 2°C to 8°C. It should be understood that the liquid pharmaceutical preparation preferably is stable even at higher temperatures, such as ambient temperature, e.g. 20°C to 30°C, although this often requires a higher content of the inhibitor.
  • stable is intended to denote that after storage for 6 months at 2°C to 8°C the initial liquid pharmaceutical preparation retains at least 50% of its initial biological activity.
  • the liquid pharmaceutical preparation retains at least 70%, such as at least 80%, or at least 85%, or at least 90%, or at least 95%, of its initial activity after storage for 6 months at 2 to 8°C.
  • the term “stable” is more particularly intended to denote that (i) after storage for 6 months at 2°C to 8°C the initial liquid pharmaceutical preparation retains at least 50% of its initial biological activity as measured by a one- stage clot assay (Assay 4), or (ii) after storage for 6 months at 2°C to 8°C, the content of heavy chain degradation products (Assay 5) is at the most 40% (w/w) assuming that the initial liquid pharmaceutical preparation comprises no heavy chain degradation products (i.e. only the Factor VII polypeptide is entered into the calculation of the percentage).
  • the initial liquid pharmaceutical preparation retains at least 70%, such as at least 80%, or at least 85%, or at least 90%, or at least 95%, of its initial activity after storage for 6 months at 2 to 8°C.
  • the content of heavy chain degradation products in the initial liquid pharmaceutical preparation is at the most 30% (w/w), at the most 25% (w/w), at the most 20% (w/w), at the most 15% (w/w), at the most 10% (w/w), at the most 5% (w/w), or at the most 3% (w/w).
  • a “stabilized composition” refers to a composition with increased physical stability, increased chemical stability or increased physical and chemical stability.
  • a composition must be stable during use and storage (in compliance with recommended use and storage conditions) until the expiration date is reached.
  • the pharmaceutical composition comprising the FVII polypeptide and a compound of formula (I) is stable for more than 6 months of storage (at 2-8°C) and for more than 1 week of usage (at ambient temperature).
  • the pharmaceutical composition comprising the FVII polypeptide and a compound of formula (I) is stable for more than 24 months of storage (at 2-8°C) and for more than 4 weeks of usage (at ambient temperature).
  • the pharmaceutical composition comprising the FVII polypeptide and a compound of formula (I) is stable for more than 36 months of storage (at 2-8°C) and for more than 6 weeks of usage (at ambient temperature).
  • the liquid, aqueous pharmaceutical compositions defined herein can be used in the field of medicine.
  • the present invention in particular provides the liquid, aqueous pharmaceutical compositions defined herein for use as a medicament, more particular for use as a medicament for treating condition or disorder against which said Factor VII polypeptide is effective.
  • the present invention also provides the use of the liquid, aqueous pharmaceutical composition as defined herein for the preparation of a medicament for treating a condition or disorder against which said Factor VII polypeptide is effective, as well as a method for treating a condition or disorder against which said Factor VII polypeptide is effective, the method comprising administering to a subject in need thereof an effective amount of the liquid, aqueous pharmaceutical composition as defined herein.
  • the preparations of the present invention may be used to treat any condition or disorder against which said Factor VII polypeptide is effective, such as, without limitation, bleeding disorders, including those caused by clotting factor deficiencies (e.g., congenital haemophilia A with and without inhibitors, acquired haemophilia A, congenital haemophilia B with and without inhibitors, acquired haemophilia B, coagulation Factor XI deficiency, coagulation Factor VII deficiency), von Willebrand's disease, platelet disorders or deficiencies (e.g., low platelet count), or thrombocytopenia.
  • clotting factor deficiencies e.g., congenital haemophilia A with and without inhibitors, acquired haemophilia A, congenital haemophilia B with and without inhibitors, acquired haemophilia B, coagulation Factor XI deficiency, coagulation Factor VII deficiency
  • von Willebrand's disease
  • bleeding disorder reflects any defect, congenital, acquired or induced, of cellular or molecular origin that is manifested in bleedings.
  • Conditions or disorders against which said Factor VII polypeptide is effective also include bleedings in subjects who experience extensive tissue damage, e.g. in association with surgery or trauma including, without limitation, bleedings associated with spinal or cardiac surgery, orthopedic surgery (e.g. hips, elbows, knees), or laparoscopic surgery, penetrating or blunt trauma, head trauma including traumatic brain injury, intracerebral haemorrhage, bleedings associated with induced defective haemostasis, such as bleedings associated with anticoagulant therapy or antifibrinolytic therapy, and uncontrolled and excessive bleeding from any cause.
  • the normal haemostatic mechanism may be overwhelmed by the demand of immediate haemostasis and bleedings may develop in spite of an otherwise normal haemostatic mechanism.
  • haemostasis e.g. the brain, inner ear region, eyes, liver, lung, tumour tissue, and gastrointestinal tract as well as when bleeding is diffuse (haemorrhagic gastritis and profuse uterine bleeding).
  • haemostasis e.g. the brain, inner ear region, eyes, liver, lung, tumour tissue, and gastrointestinal tract as well as when bleeding is diffuse (haemorrhagic gastritis and profuse uterine bleeding).
  • surgical techniques sutures, clips, etc.
  • the term "effective amount" is the effective dose to be determined by a qualified practitioner, who may titrate dosages to achieve the desired response. Factors for consideration of dose will include potency, bioavailability, desired pharmacokinetic/pharmacodynamic profiles, condition of treatment, patient-related factors (e.g. weight, health, age, etc.), presence of co-administered medications (e.g., anticoagulants), time of administration, or other factors known to a medical practitioner.
  • treatment is defined as the management and care of a subject, e.g. a mammal, in particular a human, for the purpose of combating the disease, condition, or disorder and includes the administration of a Factor VII polypeptide to prevent the onset of the symptoms or complications, or alleviating the symptoms or complications, or eliminating the disease, condition, or disorder.
  • Pharmaceutical compositions according to the present invention containing a Factor VII polypeptide may be administered parenterally to subjects in need of such a treatment.
  • Parenteral administration may be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a pen-like syringe.
  • parenteral administration can be performed by means of an infusion pump.
  • the pharmaceutical composition is adapted to subcutaneous, intramuscular or intravenous injection according to methods known in the art.
  • the pharmaceutical composition comprising a Factor VII polypeptide and one or more compounds (I) according to the present invention may further contain, or be used in conjunction with, one or more coagulation factors, such as, without limitation, Factor XIII, Factor VIII, Factor IX, another Factor VII polypeptide, or FEIBATM.
  • a pharmaceutical composition e.g. a liquid, aqueous pharmaceutical composition
  • a pharmaceutical composition comprising : one or more compounds, or physiologically tolerable salts thereof, according to the invention; and a Factor VII polypeptide (e.g. wild-type human FVIIa, such as rhFVIIa);
  • a method of preparing a composition comprising a Factor VII polypeptide comprising a Factor VII polypeptide (e.g. wild-type human FVIIa, such as rhFVIIa), comprising : adding a compound, or a physiologically tolerable salt thereof, according to the invention to a sample containing the Factor VII polypeptide; or adding the Factor VII polypeptide to a sample containing a compound, or a physiologically tolerable salt thereof, according to the invention; in a method of this type, the compound or salt thereof and/or the Factor VII polypeptide may be present in a liquid, aqueous medium.
  • a Factor VII polypeptide e.g. wild-type human FVIIa, such as rhFVIIa
  • the compound or salt thereof and/or the Factor VII polypeptide may be present in a liquid, aqueous medium.
  • a method of inhibiting a Factor VII polypeptide comprising : adding a compound, or a physiologically tolerable salt thereof, according to the invention to a sample containing the Factor VII polypeptide; or adding the Factor VII polypeptide to a sample containing a compound, or a physiologically tolerable salt thereof, according to the invention; in a method of this type, the compound or salt thereof and/or the Factor VII polypeptide may be present in a liquid, aqueous medium.
  • a Factor VII polypeptide e.g. wild-type FVIIa, such as rhFVIIa
  • HOBt N-hydroxybenzotriazole, 1 -hydroxy benzotriazole homoArg : homo-arginine, N-epsilon-amidinolysine, H 2 N-CH((CH 2 ) 4 -NH-
  • NMP N -methyl pyrrol idone
  • TIS triisopropylsilane
  • Trt trityl
  • Enzyme Inhibition general The ability of compounds of formula (I) to inhibit FVIIa or other enzymes/factors, such as, e.g., Factor Xa, thrombin, plasmin, or trypsin, is assessed by determining the concentration of the compound of formula (I) that inhibits the activity of the enzyme in question by 50%, i.e. the IC 50 value, which is related to the inhibition constant Ki. This IC 50 value is determined with the aid of a suitable chromogenic substrate, and calculated by linear regression after plotting the relative rates of hydrolysis (compared to the uninhibited control) versus the log of the concentration of compound of formula (I).
  • IC 50 value is determined with the aid of a suitable chromogenic substrate, and calculated by linear regression after plotting the relative rates of hydrolysis (compared to the uninhibited control) versus the log of the concentration of compound of formula (I).
  • Ki IC 5O / ⁇ 1+ (substrate concentration/Km) ⁇
  • Km is the Michaelis-Menten constant [Chen and Prusoff, Biochem. Pharmacol. 22 (1973), 3099-3108; I. H. Segal, Enzyme Kinetics, 1975, John Wiley & Sons, New York, 100-125; both of which references are incorporated herein in their entirety by reference].
  • the inhibitory activity [expressed as inhibition constant Ki(FVIIa)] of compounds of formula I towards Factor Vila/tissue factor activity may be determined using a chromogenic assay essentially as described previously [J. A. Ostrem et al., Biochemistry 37 (1998) 1053-1059, which reference is incorporated herein in its entirety by reference). Kinetic assays are conducted at 25° C. in half-area microtiter plates (Costar Corp., Cambridge, Mass.) using a kinetic plate reader (Molecular Devices Spectramax 250).
  • rhFVIIa and TF final concentrations 5 nM and 10 nM, respectively
  • 40 ⁇ l of inhibitor dilutions in 10% DMSO/TBS-PEG buffer 50 mM Tris, 15 mM NaCI, 5 mM CaCI 2 , 0.05% PEG 8000, pH 8.15.
  • the assay is initiated by the addition of 35 ⁇ l of the chromogenic substrate S-2288 (D-Ile-Pro-Arg-p-nitroanilide, Pharmacia Hepar Inc., 500 ⁇ M final concentration).
  • the compounds of the present invention inhibit FVIIa/TF with IC 50 -values ranging from 3 mM to ⁇ 1 ⁇ M.
  • a TBS-PEG buffer of composition 50 mM Tris-CI, pH 7.8, 200 mM NaCI, 0.05% (w/v)
  • PEG-8000, 0.02% (w/v) NaN 3 ) is used for this assay.
  • the IC 50 is determined by combining :
  • the assay is performed by pre-incubating the compound of formula I plus enzyme for 10 min.
  • the assay is then initiated by adding substrate to obtain a final volume of 100 ⁇ l.
  • the initial velocity of chromogenic substrate hydrolysis is measured by the change in absorbance at 405 nm using a Bio-tek Instruments kinetic plate reader (Ceres UV900HDi) at 25° C. during the linear portion of the time course (usually 1.5 min after addition of substrate).
  • the enzyme concentration is 0.5 nM
  • substrate concentration is 140 ⁇ M.
  • (l)c Thrombin assay TBS-PEG buffer is likewise used in this assay.
  • the IC 50 is determined as above for the Factor Xa assay, except that the substrate employed is S-2366 (L-PyroGlu-L-Pro-L-Arg- p-nitroanilide; Kabi) and the enzyme is human thrombin (Enzyme Research Laboratories, Inc.; South Bend, Ind.).
  • the enzyme concentration is 175 ⁇ M.
  • TBS-PEG buffer is likewise used in this assay.
  • the IC 50 is determined as described above for the Factor Xa assay, except that the substrate employed is S-2251 (D-Val-L-Leu-L- Lys-p-nitroanilide; Kabi) and the enzyme is human plasmin (Kabi).
  • the enzyme concentration is 5 nM and the substrate concentration is 300 ⁇ M.
  • TBS-PEG buffer containing 10 mM CaCI 2 is used for this assay.
  • the IC 50 is determined as described above for the Factor Xa assay, except that the substrate employed is BAPNA (benzoyl-L-Arg-p-nitroanilide; Sigma Chemical Co. ; St. Louis, Mo.) and the enzyme is bovine pancreatic trypsin (Type XIII, TPCK treated; Sigma).
  • the enzyme concentration is 50 nM and the substrate concentration is 300 ⁇ M.
  • Enzymes and substrates are from American Diagnostica; FIXa (cat no 449b), FIXa substrate (cat no 299F), tPA (cat no 170) and tPA substrate (cat no 444LF). Hydrolysis of substrates 299F and 444LF is followed in a Spectramax Fluorimeter at 360 nm excitation and 440 nm emission. Hydrolysis of substrate 251 and S-2288 was followed in a Spectramax Spectrophotometer at 405 nm.
  • All assays are performed in a buffer consisting of 50 mM Hepes pH 7.4, 100 mM NaCI, 5 mM CaCI2, 0.01% Tween ⁇ O. Inhibitors are used at 10, 20, 50, 100, 200 ⁇ M concentration.
  • the FIXa assay is performed using 100 ⁇ M substrate, the tPA assay is performed using 10 ⁇ M substrate.
  • FXIa and FXIIa are from American Diagnostica; FXIa (cat no 4011a), FXIIa (cat no 412HA) and trypsin (cat no 20465) are from Life Technology.
  • the chromogenic substrates (Chromogenix) in use are 2288 for FXIa and 2765 for FXIIa.
  • 50, 100, 200, 500, 1000 ⁇ M are employed for each inhibitor concentration: 25, 50, 100,
  • VO is the rate of hydrolysis without inhibitors present
  • VI is the rate of hydrolysis at the in the presence of inhibitor
  • I is the inhibitor concentration.
  • Km(app)/V is determined from double reciprocal plots of 1/v versus 1/s for each inhibitor concentration. This is followed by plots of Km(app)/V against the inhibitor concentration. Ki is determined as the intercept of the straight line at the i-axis.
  • FVIIa The biological activity of a FVII polypeptide, such as FVIIa, may be measured using a one-stage coagulation assay.
  • the sample to be tested is diluted in 50 mM PlPES-buffer (pH 7.5), 0.1% BSA, and 40 ⁇ l of this solution is incubated with 40 ⁇ l of FVII-deficient plasma and 80 ⁇ l of human recombinant TF containing 10 mM Ca 2+ and synthetic phospholipids. Coagulation times are measured and compared to a standard curve using a reference standard in a parallel line assay.
  • Factor VII polypeptides useful in accordance with the present invention may be selected by suitable assays that can be performed as simple preliminary in vitro tests.
  • suitable assays that can be performed as simple preliminary in vitro tests.
  • the present specification discloses a simple test (entitled “In Vitro Hydrolysis Assay") for the activity of Factor VII polypeptides.
  • the activity of the Factor VII polypeptides may be measured using a one-stage clot assay essentially as described in WO 92/15686 or US 5,997,864. Briefly, the sample to be tested is diluted in 50 mM Tris (pH 7.5), 0.1% BSA and 100 ⁇ l_ is incubated with 100 ⁇ l_ of Factor VII deficient plasma and 200 ⁇ l_ of thromboplastin C containing 10 mM Ca2+. Clotting times are measured and compared to a standard curve using a reference standard or a pool of citrated normal human plasma in serial dilution.
  • Factor Vila Native (wild-type) Factor Vila and Factor VII polypeptide (both hereinafter referred to as "Factor Vila") may be assayed for specific activities. They may also be assayed in parallel to directly compare their specific activities.
  • the assay is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark).
  • the absorbance at 405 nm is measured continuously in a SpectraMaxTM 340 plate reader (Molecular Devices, USA).
  • the absorbance developed during a 20-minute incubation, after subtraction of the absorbance in a blank well containing no enzyme, is used for calculating the ratio between the activities of Factor VII polypeptide and wild-type Factor Vila:
  • Ratio (A405 nm Factor VII polypeptide)/(A405 nm Factor Vila wild-type). Based thereon, Factor VII polypeptides with an activity lower than, comparable to, or higher than native Factor Vila may be identified, such as, for example, Factor VII polypeptides where the ratio between the activity of the Factor VII polypeptide and the activity of native Factor VII (wild-type FVII) is about 1.0 versus above 1.0.
  • the activity of the Factor VII polypeptides may also be measured using a physiological substrate such as Factor X ("In Vitro Proteolysis Assay"), suitably at a concentration of 100-1000 nM, where the Factor Xa generated is measured after the addition of a suitable chromogenic substrate (eg. S-2765).
  • a physiological substrate such as Factor X ("In Vitro Proteolysis Assay")
  • the activity assay may be run at physiological temperature.
  • Factor Vila Native (wild-type) Factor Vila and Factor VII polypeptide (both hereinafter referred to as "Factor Vila") are assayed in parallel to directly compare their specific activities.
  • the assay is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark).
  • Factor X cleavage is then stopped by the addition of 50 ⁇ l_ 50 mM HEPES, pH 7.4, containing 0.1 M NaCI, 20 mM EDTA and 1 mg/mL bovine serum albumin.
  • the amount of Factor Xa generated is measured by the addition of the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide (S-2765, Chromogenix, Sweden), final concentration 0.5 mM.
  • the absorbance at 405 nm is measured continuously in a SpectraMaxTM 340 plate reader (Molecular Devices, USA). The absorbance developed during 10 minutes, after subtraction of the absorbance in a blank well containing no FVIIa, is used for calculating the ratio between the proteolytic activities of Factor VII polypeptide and wild-type Factor Vila:
  • Ratio (A405 nm Factor VII polypeptide)/(A405 nm Factor Vila wild-type). Based thereon, a Factor VII polypeptide with an activity lower than, comparable to, or higher than native Factor Vila may be identified, such as, for example, Factor VII polypeptides where the ratio between the activity of the Factor VII polypeptide and the activity of native Factor VII (wild-type FVII) is about 1.0 versus above 1.0.
  • Assay 3 The ability of a Factor VII polypeptides to generate thrombin can be measured in an assay (Assay 3) comprising all relevant coagulation Factors and inhibitors at physiological concentrations (minus Factor VIII when mimicking hemophilia A conditions) and activated platelets (as described on p. 543 in Monroe et al. (1997) Brit. J. Haematol. 99, 542-547 which is hereby incorporated as reference).
  • Clot Assay 4 One-stage Coagulation Assay (Clot Assay) (Assay 4) Factor VII polypeptides may also be assayed for specific activities ("clot activity") by using a one-stage coagulation assay (Assay 4).
  • the sample to be tested is diluted in 50 mM PlPES-buffer (pH 7.2), 1% BSA and 40 ⁇ l is incubated with 40 ⁇ l of Factor VII deficient plasma and 80 ⁇ l of human recombinant tissue factor containing 10 mM Ca 2+ and synthetic phospholipids.
  • Coagulation times (clotting times) are measured and compared to a standard curve using a reference standard in a parallel line assay.
  • the content of aggregates is determined by non-denaturing size exclusion HPLC: Non-denaturing size exclusion chromatography was run on a Waters Protein Pak 300 SW column, 7,5x300 mm using 0.2 M ammoniumsulfat, 5% 2-propanol pH 7,0 as mobile phase. Flow rate :0.5 ml/min. Detection : 215 nm. Load : 25 ⁇ g FVIIa.
  • HPLC-systems from Merck-Hitachi HibarTM RT 250-4, LichrosorbTM RP 18, 5.0 ⁇ m, 4.0 x 250 mm, gradient elution, 20% to 80% acetonitrile in water within 30 min, 1.0 ml/min, detection at 254 nm
  • Waters SymmetryTM, C18, 3.5 ⁇ m, 3.0 x 150 mm, gradient elution, 5% to 90% acetonitrile in water within 15 min, 1.0 ml/min, detection at 214 nm
  • the peptide was cleaved from the resin by stirring for 180 min at room temperature with a mixture of trifluoroacetic acid, water and triisopropylsilane (95:2.5:2.5). The cleavage mixture was filtered and the filtrate was concentrated to an oil by a stream of nitrogen. The crude peptide was precipitated from this oil with diethyl ether (45 ml) and washed three times with diethyl ether (45 ml each).
  • the crude peptide was purified by semipreparative HPLC on a 20 mm x 250 mm column packed with C-18 silica. Depending on the peptide one or two of the following purification systems were used.
  • TFA After drying, the crude peptide was dissolved in 5 ml 50% acetic acid/H 2 O and diluted to 20 ml with H 2 O, injected, and eluted with a gradient of 40-60 % CH 3 CN in 0.1% TFA 10 ml/min during 50 min at 40 0 C. The peptide-containing fractions were collected. The purified peptide was lyophilized after dilution of the eluate with water. Ammonium sulphate: The column was equilibrated with 40% CH 3 CN in 0.05M (NH 4 ) 2 SO 4 , which was adjusted to pH 2.5 with concentrated H 2 SO 4 .
  • the crude peptide was dissolved in 5 ml 50% acetic acid/H 2 O and diluted to 20 ml with H 2 O, injected, and eluted with a gradient of 40%-60% CH 3 CN in 0.05M (NH 4 ) 2 SO 4 , pH 2.5, at 10 ml/min during 50 min at 40 0 C.
  • the peptide-containing fractions were collected and diluted with H 2 O and passed through a Sep-Pak ® C18 cartridge (Waters part. # : 51910) which had been equilibrated with 0.1% TFA. It was then eluted with 70% CH 3 CN containing 0.1% TFA and the purified peptide was isolated by lyophilisation after dilution of the eluate with water.
  • the final product obtained was characterised by analytical RP-HPLC (retention time) and by LCMS.
  • the RP-HPLC analysis was performed using UV detection at 214 nm and a Vydac
  • Bl Equilibration of the column with 0.1% TFA / H 2 O and elution by a gradient of 0% CH 3 CN / 0.1% TFA/H 2 O to 60% CH 3 CN / 0.1% TFA / H 2 O during 50 min.
  • B6 Equilibration of the column with 0.1% TFA / H 2 O and elution by a gradient of 0% CH 3 CN / 0.1% TFA / H 2 O to 90% CH 3 CN / 0.1% TFA / H 2 O during 50 min.
  • LCMS was performed on a setup consisting of Hewlett Packard series 1100 G1312A Bin Pump, Hewlett Packard series 1100 Column compartment, Hewlett Packard series 1100 G1315A DAD diode array detector, Hewlett Packard series 1100 MSD and Sedere 75 Evaporative Light Scattering detector controlled by HP Chemstation software.
  • the HPLC pump is connected to two eluent reservoirs containing : A: 1OmM NH 4 OH in water B: 1OmM NH 4 OH in 90% acetonitrile
  • R represents hydrogen, lower alkyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl
  • R represents hydrogen, lower alkyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl
  • Example 25 N-Phenylmethanesulfonyl-(D-Ile)-Gln-(N-amidinopiperidin-4-yl)Ala-Phg- NH2

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Abstract

L'invention concerne de nouveaux composés de formule (I) utiles comme inhibiteurs du facteur de coagulation sanguine. Les composés (I) peuvent être utilisés pour le traitement des états thrombotiques ou en tant que stabilisants de formulations liquides des facteurs de coagulation sanguine, en particulier des formulations liquides de FVIIa, des variantes du facteur VII ou des dérivés du facteur VII.
PCT/EP2008/052409 2007-03-07 2008-02-28 Nouveaux inhibiteurs du facteur de coagulation sanguine WO2008107362A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009092758A1 (fr) * 2008-01-23 2009-07-30 Novo Nordisk Health Care Ag Nouveaux inhibiteurs du facteur de la coagulation du sang

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0987274A1 (fr) * 1998-09-15 2000-03-22 Hoechst Marion Roussel Deutschland GmbH Inhibiteurs du facteur VIIa
WO2000058346A1 (fr) * 1999-03-30 2000-10-05 Sanofi-Synthelabo Derives de n-sulfonyl-dipeptides, leur preparation et leur application en therapeutique
WO2002026774A2 (fr) * 2000-09-27 2002-04-04 The Procter & Gamble Company Ligands de recepteurs de la melanocortine
WO2004041302A1 (fr) * 2002-11-06 2004-05-21 Novo Nordisk A/S Composition pharmaceutique comprenant un antagoniste de facteur tissulaire et un regulateur de glycemie
WO2005016365A2 (fr) * 2003-08-14 2005-02-24 Novo Nordisk Health Care Ag Composition pharmaceutique liquide aqueuse de polypeptides du facteur vii

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0987274A1 (fr) * 1998-09-15 2000-03-22 Hoechst Marion Roussel Deutschland GmbH Inhibiteurs du facteur VIIa
WO2000058346A1 (fr) * 1999-03-30 2000-10-05 Sanofi-Synthelabo Derives de n-sulfonyl-dipeptides, leur preparation et leur application en therapeutique
WO2002026774A2 (fr) * 2000-09-27 2002-04-04 The Procter & Gamble Company Ligands de recepteurs de la melanocortine
WO2004041302A1 (fr) * 2002-11-06 2004-05-21 Novo Nordisk A/S Composition pharmaceutique comprenant un antagoniste de facteur tissulaire et un regulateur de glycemie
WO2005016365A2 (fr) * 2003-08-14 2005-02-24 Novo Nordisk Health Care Ag Composition pharmaceutique liquide aqueuse de polypeptides du facteur vii

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009092758A1 (fr) * 2008-01-23 2009-07-30 Novo Nordisk Health Care Ag Nouveaux inhibiteurs du facteur de la coagulation du sang
US8486892B2 (en) 2008-01-23 2013-07-16 Novo Nordisk Health Care Ag Blood coagulation factor inhibitors

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