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WO1999013063A1 - Fragments du facteur vii et leurs analogues, utilisation dans le traitement des troubles dus aux caillots sanguins - Google Patents

Fragments du facteur vii et leurs analogues, utilisation dans le traitement des troubles dus aux caillots sanguins Download PDF

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Publication number
WO1999013063A1
WO1999013063A1 PCT/GB1998/002701 GB9802701W WO9913063A1 WO 1999013063 A1 WO1999013063 A1 WO 1999013063A1 GB 9802701 W GB9802701 W GB 9802701W WO 9913063 A1 WO9913063 A1 WO 9913063A1
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Prior art keywords
gly
tyr
glu
gln
cys
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PCT/GB1998/002701
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English (en)
Inventor
Kjell Steinar Sakariassen
Lars ØRNING
Peter Martin Fischer
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Nycomed Imaging As
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Priority claimed from GBGB9719162.1A external-priority patent/GB9719162D0/en
Application filed by Nycomed Imaging As filed Critical Nycomed Imaging As
Publication of WO1999013063A1 publication Critical patent/WO1999013063A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6437—Coagulation factor VIIa (3.4.21.21)
    • 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)
    • C12Y304/21021—Coagulation factor VIIa (3.4.21.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is concerned with reagents and compositions thereof which reduce blood clot formation.
  • Blood clotting relies upon a series or cascade of activating reactions to produce the ultimate fibrin clot.
  • the cascade leading to fibrin formation may be triggered initially in two different ways - by contact with abnormal surfaces (the "intrinsic pathway") or by traumatization of blood vessels which causes secretion of the lipoprotein known as "tissue factor” or TF (the “extrinsic pathway”).
  • the present invention is primarily concerned with the extrinsic blood clotting pathway.
  • the zymogen form is converted to the fully enzymatically active form FVIIa by factor X and other coagulation proteases through hydrolysis of a single peptide bond Arg 152 - lie 153 .
  • the light chain of 152 amino acid residues contains at its amino terminal part the y- carboxy-glutamic acid (Gla) domain, followed by two epidermal growth factor-like domains (EGF-l and EGF-2) .
  • the heavy chain consists of 254 residues and contains the trypsin- like catalytic domain.
  • FVIIa The function of FVIIa is activation of factor X (FX) by complexation with tissue factor (TF) in the presence of Ca 2+ on a phospholipid membrane surface. Activation of FX to FX a leads to formation of blood clots by the extrinsic pathway. Additionally, the complex FVII a /TF can activate factor IX to factor IX a and lead to clotting through the intrinsic pathway.
  • TF is an integral membrane protein which appears on many cell types. However, cells which constitutively express TF, for example the muscle cells of vessels intima, are not normally exposed to blood (see Edgington et al., Thromb. Haemostas. 66 (1) : 67-69 (1991)). Thus initiation of the extrinsic blood clotting pathway appears to require either the disruption of blood vessel walls (see Almus et al . , Blood 26: 354-360 (1990)) and/or activation of endothelial cells or monocytes to express TF (see Edwards et al . , Blood 5_4: 359-370 (1979) and Bevilaqua et al .
  • TF may also be exposed following injury to blood vessels during thrombolytic therapy, surgery for grafting, mechanical restoration of vessel patency or other similar techniques.
  • TF expression in endothelial cells or in monocytes may be induced during sepsis due to production of tumour necrosis factor- ⁇ or interleukin-1 (see Edwards et al . , supra and Gregory et al., J. Clin. Invest. 76.: 2440-2445 (1985)).
  • One proposed way of providing potential therapeutic agents capable of preventing the primary event in blood clot formation through the extrinsic pathway would thus be to identify peptides derived from the primary structure of FVII which are capable of inhibiting the complex formation between FVII, TF and FX, which is necessary for FX activation.
  • Peptides corresponding to sequence portions between the Gla and EGF-1 domains as well as from the catalytic domain were disclosed in WO91/07432 (Board of Regents, The University of Texas System) as being useful in the treatment of blood clotting disorders. Although inhibition of the FVIIa/TF complex is discussed, those peptides which cause an effect do so by inhibition of the Gla function. Such peptides are therefore unspecific in their action since other physiological proteins have Gla domains. Hence the function of proteins with Gla domains would be disturbed by administration of the disclosed peptides.
  • a particular peptide from the EGF-2 domain was disclosed in WO90/03390 (Corvas, Inc.) as having potential uses in preventing the formation of the fully formed FVIIa/TF complex.
  • the sequence -SDHTGTKRSCR- which is located at amino acids 103-113 of FVII or analogues thereof is said to inhibit the cascade reaction initited by FVIIa/TF complex.
  • Other regions, namely from amino acids 50 to 101 and 114 to 127 in the EGF-2 domain were shown to be inactive in the inhibition of FX activation by FVIIa/TF.
  • WO95/00541 Novartis a number of small peptides from the EGF-1 and EGF-2 domains are disclosed which may be useful in treating blood clotting disorders. Particularly, peptides corresponding to the amino acids 91 to 104 and 114-127 of the FVII sequence are said to be active.
  • the FVII:EGF-2 like domain and in particular the primary binding loop is essential to the tripod interaction.
  • the EGF-2 domain interacts with TF via the C-terminal part of the loop, residues 88-93 and with the catalytic domain via the N-terminal part of the loop, residues 98-103.
  • the EGF-2 domain and in particular the primary binding loop is sandwiched between the cofactor and the catalytic domain (See figure 2) .
  • the interdomain interaction is basically a ligand/receptor interaction.
  • the EGF-2 loop structure is the ligand and the internal receptor in the catalytic domain is defined by residues Leu 263 , Pro 264 , Glu 265 , Phe 268 , Ser 269 , Tyr 357 and Arg 353 .
  • the main interaction is an aromatic stacking involving the side chains of Tyr 101 in the EGF-2 domain and Phe 268 , and Tyr 357 in the catalytic domain (See figure 3) .
  • Gin 100 makes hydrophobic contact with His 115 and Tyr 118 in the EGF-2 domain and Thr 267 in the catalytic domain.
  • the side chain of Arg 100 is incorrectly positioned and disturbs the important interaction between EGF-2 and the catalytic domain.
  • the FVII/EGF-2 domain appears critical in the formation of a functional FVII/TF complex.
  • any compound, peptide or non-peptide, natural or synthetic which is capable of interacting with the internal receptor in the catalytic domain of FVII defined by the residues Leu 263 , Pro 264 , Glu 265 , Phe 268 , Ser 269 , Tyr 357 and Arg 353 or the ligand defined by residues Cys 98 -Cys 102 of the EGF-2 domain could be effective in inhibiting the formation of a functional FVIIa/TF complex.
  • the invention provides a compound which is capable of interacting with the internal receptor in the catalytic domain of FVII defined by the residues Leu 263 , Pro 264 , Glu 265 , Phe 268 , Ser 269 , Tyr 357 and Arg 353 or the ligand defined by residues Cys 98 -Cys 102 of the EGF-2 domain of FVII to prevent the formation of a functional FVIIa/TF complex, with the exclusion of the peptide from amino acids 82-128 of FVII and functional equivalents thereof in which amino acids in the sequence are modified or absent, and disulphide- cyclo- [H-Cys-Glu-Gln-Tyr-Cys-OH] .
  • the invention provides a pharmaceutical composition containing one or more compounds capable of interacting with the internal receptor in the catalytic domain of FVII defined by the residues Leu 263 , Pro 264 , Glu 265 , Phe 268 , Ser 269 , Tyr 357 and Arg 353 or the ligand defined by residues Cys 98 -Cys 102 of the EGF-2 domain of FVII to prevent the formation of a functional FVIIa/TF complex.
  • the invention provides the use of a compound capable of interacting with the internal receptor in the catalytic domain of FVII defined by the residues Leu 263 , Pro 264 , Glu 265 , Phe 268 , Ser 269 , Tyr 357 and Arg 353 or the ligand defined by residues Cys 98 -Cys 102 of the EGF-2 domain of FVII for use in the prevention of the formation of a functional FVIIa/TF complex.
  • the invention provides the use of a compound capable of interacting with the internal receptor in the catalytic domain of FVII defined by the residues Leu 263 , Pro 264 , Glu 265 , Phe 268 , Ser 269 , Tyr 357 and Arg 353 or the ligand defined by residues Cys 98 -Cys 102 of the EGF-2 domain of FVII for use in the manufacture of a medicament for the prevention of the formation of a functional FVIIa/TF complex.
  • the invention provides a method of treatment of the human or animal body to combat or prevent blood clotting disorders said method comprising administering to said body one or more compounds capable of interacting with the internal receptor in the catalytic domain of FVII defined by the residues Leu 263 , Pro 264 , Glu 265 , Phe 268 , Ser 269 , Tyr 357 and Arg 353 or the ligand defined by residues Cys 98 -Cys 102 of the EGF-2 domain of FVII to prevent the formation of a functional FVIIa/TF complex.
  • the compounds capable of interacting with the internal receptor in the catalytic domain or its ligand can have a wide variety of structures and will be readily determined by the person skilled in the art .
  • the compounds must be capable of interacting with the internal receptor or its ligand in order to prevent the formation of a functional FVII/TF complex.
  • Non-peptide compounds suitable for use according to the invention are those with complementary conformations to the target receptor sites or its ligand and can be readily prepared by conventional combinatorial chemical approaches .
  • Preferred compounds according to the invention are linear peptides of the general structure 1, disulphide- cyclic peptides of the general structure 2, lactam- cyclic peptides of the general structures 3a ("head-to- tail” amide bond), 3b ("side chain-to-tail” amide bond) and 3c ("head-to-side chain” amide bond), as well as cyclic peptides of the general structure 4.
  • these structures Saa, Vaa, Waa, Xaa, Yaa and Zaa denote specified (see below) amino acid residues (e . g. -NH-CH 2 - CO- for a Gly residue) , such residues being of any stereochemistry and optionally containing C ⁇ - and/or N ⁇ - methyl substituents additional to the amino acid side chain:
  • one of the three residues Waa, Xaa and Yaa is an amino acid residue from group I below.
  • the remaining two residues are from groups II and III, respectively, or they are both from group III.
  • Group I denotes aromatic amino acid residues, including Tyr, Aph and Phe. Also included are side-chain constrained analogues of Tyr such as Tic (OH) and Hat. Aromatic amino acids containing a phenol function may be O-alkylated.
  • Group II denotes amino acid residues containing side-chain functional groups which are ionized under physiological conditions, including Glu, Asp, Dap, Dab, Cit, Orn, Lys and Arg. Also included are side-chain constrained analogues of Asp and Glu, such as 1- aminocyclo-pentane-1, 3-dicarboxyl and 1- aminocyclohexane-1, 3-dicarboxyl .
  • Group III denotes neutral and hydrophobic amino acid residues, including Gin, Asn, Ser, Thr, Leu and Nle.
  • peptides H-Ala-Glu-Gln-Tyr-Val-OH and Ac-Ala-Glu- Gln-Tyr-Val-NH 2 are the peptides H-Ala-Glu-Gln-Tyr-Val-OH and Ac-Ala-Glu- Gln-Tyr-Val-NH 2 .
  • the two residues Saa in 2 are amino acid residues containing thiol functions in the side chain, these thiol functions being linked intramolecularly through a disulphide bond.
  • the two residues Saa may independently correspond to Cys, homocysteine or penicillamine .
  • the residues Vaa and Zaa in 1 and 3 may independently correspond to hydrophobic amino acid residues, including Gly, Ala, ⁇ Ala, Abu, Val, norvaline, Pro, aminoalkanoyl (4-8 C atoms) and o- , m- or p- (aminomethyl) benzoyl . Furthermore, one or both of Vaa and Zaa may correspond to Asp, Glu, Dap, Dab, Orn or Lys. In the case of general structures 3b and 3c the terminal residues participating in a "side chain-to- tail" or "head-to-side chain” amide bond (Vaa and Zaa, respectively) correspond exclusively to amino acids residues containing side-chain amine (3b) or carboxyl (3c) groups.
  • the substituent R 1 in 1-3 may correspond to the following: H- , acetyl, R 6 -Gly-, R 6 -Gly-Gly-, R 6 -Asn-Gly- Gly-, R 6 -Dab-Gly-Gly-, Glp-Gly-Gly- , succinyl-Gly-Gly- or amidosuccinyl-Gly-Gly- , where R 6 may be H- or acetyl.
  • the substituent R 2 in 1-3 may correspond to -OH, -NH 2 , -Ser-R 7 , -Ser-Asp-R 7 , -Ala-Asp-R 7 , -Ser-Abu-R 7 , -Ser-Lys-R 7 , -Ser-Dab-R 7 or -Ser-Ile-R 7 , where R 7 may be - OH or -NH 2 .
  • n in 3a may be 1 or 2.
  • R 3 may be -H or methyl.
  • R 4 may be - H, methyl, -NH 2 , -NH-Ac or -NH-R 1
  • R 5 may be -H, methyl -COOH, -COOMe, -CONH 2 or -CO-R 2 .
  • the moiety A in 4 may correspond to a saturated or unsaturated carbon chain of length Ci-Cg, optionally interrupted by a phenyl group or a heteroatom (N, O or S) . Both the chain and the optional phenyl group may bear (C 1 -C 4 ) -alkyl, amino, hydroxyl , carboxyl or carboxamide substituents.
  • the peptides of the present invention are up to several tenfold more potent inhibitors than the previously disclosed peptide H-Asn-Gly-Gly-Cys (Acm) -Glu-Gln-Tyr- Cys (Acm) -Ser-Asp-OH, despite the fact that they are structurally simpler by virtue of having lower molecular mass and, in some cases, by virtue of possessing fewer chiral atoms. Furthermore, those cyclic peptides of the present invention not containing disulphide bonds can be expected to possess enhanced physiological stability due to the fact that they cannot be processed by amino- and carboxypeptidases .
  • Salts of the peptides of the invention include physiologically acceptable salts such as acid addition salts, for example the hydrochlorides .
  • the pharmaceutical compositions comprising the peptides of the invention and/or salts thereof may be administered together with any physiologically acceptable excipient known to those skilled in the art . Examples of excipients include water and oil .
  • compositions according to the invention may be presented, for example in a form suitable for oral, nasal, parenteral or rectal administration.
  • the term "pharmaceutical administration” includes veterinary applications of the invention.
  • the compounds according to the invention may be presented in the conventional pharmacological forms of administration, such as tablets, coated tablets, nasal sprays, solutions, emulsions, powders, capsules or sustained release forms.
  • Conventional pharmaceutical excipients as the usual methods of production may be employed for the preparation of these forms .
  • Tablets may be produced, for example, by mixing the active ingredient or ingredients with known excipients, such as for example with diluents, such as calcium carbonate, calcium phosphate or lactose, disintegrants such as corn starch or alginic acid, binders such as starch or gelatine, lubricants such as magnesium stearate or talcum, and/or agents for obtaining sustained release, such as carboxypolymethylene, carboxymethylcellulose, cellulose acetate phthalate, or polyvinylacetate .
  • diluents such as calcium carbonate, calcium phosphate or lactose
  • disintegrants such as corn starch or alginic acid
  • binders such as starch or gelatine
  • lubricants such as magnesium stearate or talcum
  • agents for obtaining sustained release such as carboxypolymethylene, carboxymethylcellulose, cellulose acetate phthalate, or polyvinylacetate .
  • the tablets may if desired consist of several layers.
  • Coated tablets may be produced by coating cores, obtained in a similar manner to the tablets, with agents commonly used for tablet coatings, for example, polyvinyl pyrrolidone or shellac, gum arabic, talcum, titanium dioxide or sugar.
  • the core may consist of several layers in order to obtain sustained release, in which case the excipients mentioned above for tablets may be used.
  • Organ specific carrier systems may also be used.
  • Injection solutions may, for example, be produced in the conventional manner, such as by the addition of preservation agents, such as p-hydroxybenzoates, or stabilizers, such as EDTA. The solutions are then filled into injection vials or ampoules.
  • Nasal sprays may be formulated similarly in aqueous solution and packed into spray containers either with an aerosol propellant or provided with means for manual compression.
  • Capsules containing one or several active ingredients may be produced, for example, by mixing the active ingredients with inert carriers, such as lactose or sorbitol, and filling the mixture into gelatine capsules .
  • Suitable suppositories may, for example, be produced by mixing the active ingredient or active ingredient combinations with the conventional carriers envisaged for this purpose, such as natural fats or polyethylene glycol or derivatives thereof .
  • Dosage units containing the compounds of this invention preferably contain 0.1-1.0 mg, for example 1-5 mg of the peptide of formula 1-4 or salt thereof.
  • Blood clotting disorders in which the compounds of the invention may be used include thrombosis (particularly vascular thrombosis or deep vein thrombosis) , acute myocardial infarction, restenosis, angina, reclosure, cerebrovasular disease, peripheral arterial occlusive disease, hypercoagulability and pulmonary embolism.
  • the peptides according to the invention can also be used to prevent occurrence of blood clotting problems caused by, for example, grafting surgery, vessel wall patency restoration, etc. Blood clotting disorders may be triggered by sepsis due to production of tissue necrosis factor- ⁇ or interleukin-1.
  • the present invention provides a process for the preparation of peptides of the general structures 1-4 as defined above.
  • the peptides of the present invention may be synthesised in any convenient way.
  • the reactive groups present e . g. amino, thiol, carboxyl, etc.
  • the final step in the synthesis will normally be deprotection of a protected derivative of the peptides of the invention.
  • disulphide-cyclic peptides of general structure 2 the final step in the synthesis will normally be cyclisation following deprotection of a protected derivative of the peptides of the invention.
  • This derivative will have a free amino group and can be reacted with the free or activated carboxyl group of a protected amino acid derivative corresponding to the penultimate residue of the desired sequence.
  • the resulting dipeptide intermediate may be purified, for example by chromatography, and then selectively deprotected at the terminal amino group to permit addition of a further amino acid residue. This procedure is continued until the required amino acid sequence is assembled.
  • Carboxylic acid activating substituents which may, for example, be employed include symmetrical anhydrides or mixed anhydrides, or activated esters such as for example the p-nitrophenyl ester, 2, 4, 5-trichlorophenyl ester, N-hydroxysucccinimidyl ester or N- hydroxybenzotriazolyl ester.
  • Amino components may be acylated directly by amino acid derivatives possessing a free carboxyl group which the aid of coupling reagents such as 2- (lH-benzotriazole-1-yl) -1, 1, 3, 3- tetramethyluroniu hexafluorophosphate and other reactive species derived from 1-hydroxybenzotriazole, as well as carbodiimides, 2-ethoxy-l-ethoxycarbonyl-l, 2 , - dihydroquinoline, etc.
  • catalysts which possess racemisation-suppressing properties are added to the acylation mixtures, e . g. 1-hydroxybenzotriazole.
  • Chloromethylated polystyrene cross- linked with 1 % divinylbenzene
  • a solid support Chloromethylated polystyrene (cross- linked with 1 % divinylbenzene) is one useful type of support; in this case the synthesis will start at the carboxyl terminus, for example by coupling an N- protected derivative of the terminal amino acid residue to the support.
  • Preferred supports are p-alkoxybenzyl alcohol resins (Wang, J. Am . Chem . Soc , 1973, 95:1328- 33) and trityl resins (Barlos et al . , Tetrahedron Lett . , 1989, 30:3943-6) for the synthesis of peptides containing free carboxyl termini.
  • p- nitrobenzophenone oxime resin Keriser et al . , Science, 1989, 243:187-92 is preferred for the synthesis of lactam-cyclic peptides.
  • amino protecting groups for amino acids are known (Bodanszky, Principles of peptide synthesis, Berlin: Springer-Verlag, 1984) .
  • amino protecting groups which may be employed include groups such as benzyloxycarbonyl, t-butoxycarbonyl and 9- fluorenylmethoxycarbonyl . It will be appreciated that when the peptide is built up from the carboxyl terminus, an amine-protecting group will be present on the amino acid group of each new residue added and will need to be removed selectively prior to the next coupling step.
  • Particularly useful groups for such temporary amine protection are the Fmoc group which can be removed selectively by treatment with piperidine in an organic solvent and the t-butoxycarbonyl group which may be removed with the aid of acids such as trifluoroacetic acid.
  • Carboxyl protecting groups which may, for example, be employed include readily cleaved ester groups such as benzyl and t-butyl, as well as the linkers on solid supports, for example p-alkoxybenzyl alcohol linked to polystyrene.
  • Thiol protecting groups include p- methoxybenzyl , trityl, acetamidomethyl and 3-nitro-2- pyrdylsulphenyl .
  • the amide groups of e When desired, the amide groups of e .
  • g. asparagine and glutamine side chains can be protected with a trityl group. It will be appreciated that a wide range of other such protecting groups exist, and the use of all such groups in hereinbefore described processes fall within the scope of the present invention. A wide range of procedures exist for removing peptide protecting groups. These must, however, be consistent with the synthetic strategy employed. The side-chain protecting groups must be stable to the conditions used to remove the temporary amino protecting group prior to the next coupling step.
  • Disulphide-cyclic peptides (2) of the present invention may be obtained after assembly of linear S- protected precursors through cyclisation.
  • the cyclisation may be effected using oxidation, e . g. with oxygen at high dilution under basic conditions or under acidic conditions in trifluoroacetic acid with dimethylsulphoxide.
  • oxidation e . g. with oxygen at high dilution under basic conditions or under acidic conditions in trifluoroacetic acid with dimethylsulphoxide.
  • deprotection and oxidation ca be effected simultaneously using e . g. iodine or thallium(III) trifluoroacetate.
  • the two thiol- containing residues of the precursors to the disulphide- cyclic peptides are protected separately, one with an acid-labile group such as trityl and one with an acid- stable disulphide-forming group such as sulphonate or 3- nitro-2-pyridylsulphenyl (Npys) .
  • an acid-labile group such as trityl
  • an acid- stable disulphide-forming group such as sulphonate or 3- nitro-2-pyridylsulphenyl (Npys) .
  • the peptide is assembled using acid-labile protecting groups and, where a solid support is used, an acid-labile ester linkage, conventional acidolysis (King et al . , Int . J. Peptide Protein Res .
  • Cyclic peptides of the present invention other than those containing disulphide bonds (i.e. peptides of general structures 3 and 4) may be synthesised through known procedures for cyclisation of a precursor in which all reactive groups are protected with the exception of the amino and carboxyl groups to be condensed.
  • the fully protected peptides can be obtained by conventional solution synthesis or preferably by solid-phase synthesis on super acid-labile linkers (Mergler et al . , Tetrahedron Lett . , 1988, 29:4005-4008; Barlos et al . , ibid. , 1989, 30:3943-6).
  • the amino and carboxyl functions taking part in the prospective lactam bond are protected in such a way as to be compatible with both temporary amino protection as well as semi-permanent side-chain protection.
  • temporary amino protection is through Fmoc and semi-permanent side-chain protection is through t- butyl-type groups
  • the amino and carboxyl groups participating in the prospective lactam bond may be protected through allyl carbamate ⁇ and esters, respectively, which can be removed selectively with palladium(0) catalysts.
  • the condensation reaction to form the lactam bond can be achieved through the wide range of coupling procedures described above for peptide bond formation.
  • condensations will be carried out in a suitable organic solvent at high peptide dilution in order to prevent polymerisation.
  • a suitable organic solvent at high peptide dilution in order to prevent polymerisation.
  • such cyclisation can be performed with the protected linear peptide precursor still attached to a solid synthesis support.
  • linear precursors to lactam-cyclic peptides are assembled as described (Osapay et al., in: Techniques in Protein Chemistry II, edited by J.J. Villafranca, New York:Academic Press, Inc. 1991, p.
  • This starting material can be selectively Boc-deprotected, followed by conventional solution synthesis to afford Boc-Waa-Xaa-Yaa-NH-CH(COOBn) - (CH 2 ) 4 -CH(NH-Z) -COOPha.
  • Boc-Waa-Xaa-Yaa-NH-CH(COOBn) - (CH 2 ) 4 -CH(NH-Z) -COOPha After selective removal of the Boc group with trifluoroacetic acid and the phenacyl ester group with zinc in acetic acid, the resulting free amino and carboxyl groups are condensed to afford the cyclic protected peptide, from which the Z and Bn groups, together with any benzyl-type side chain protecting groups in Waa, Xaa and Yaa, are removed through hydrogenolysis .
  • the synthesis was carried out on a 0.1 mmol scale with Fmoc-Asp (OBu t ) - [p-alkoxybenzyl alcohol resin] (0.6 mmol/g) using an Applied Biosystems model 433A peptide synthesiser and standard Fmoc-chemistry program cycles.
  • the final H-Asn(Trt) -Gly-Gly-Abu-GlufOBu* 1 ) -Gln(Trt) - Tyr (Bu 1 ") -Abu-Ser (Bu fc ) -Asp (0Bu fc ) -peptidyl resin was washed with CH 2 C1 2 and Et 2 0 and was dried.
  • the entire mixture was stirred for 1.5 h at room temperature.
  • the resulting Fmoc-Abu resin was collected by filtration, was washed with DMF and CH 2 C1 2 and was dried.
  • the resin was then treated with benzoyl chloride and Pr ⁇ NEt (10 eq each) in DMF for 2 h in order to cap unreacted hydroxyl groups before- being washed and dried again.
  • This material was redissolved in 0.1 % aq CF 3 COOH (250 mL) , filtered and pumped (at 10 mL/min) onto a prep. RP-HPLC column (Vydac 218 ' TP1022, 2.5 x 25 cm). The column was then eluted at 10 mL/min using a gradient from 0 to 12 % MeCN in 0.1 % aq CF 3 COOH over 60 min, followed by isocratic elution. The eluant was monitored (260 nm) and appropriate peak fractions were collected, pooled and lyophilised to afford pure title compound (44.7 mg) . Anal.
  • the chain assembly was performed using Fmoc- Cys (Trt) -Tentagel S Trityl resin (1.39 g, 0.25 mmol; from Rapp Polymere GmbH, Tubingen, Germany) . Fmoc- deprotection was carried out with 20 % piperidine in DMF during 20 min.
  • Fmoc-Cys (Trt) - [p-alkoxybenzyl alcohol resin] (0.18 g, 0.55 mmol/g) was deprotected with 50 % piperidine in DMF for 15 min and was then washed with DMF.
  • Fmoc-D/L- Hat(Me)-OH (synthesised according to Rastogi et al . , Indian J. Chem. , 1971, 9:1175-82, Cardinaux & Pless, in: Peptides 1984 , edited by U. Ragnarssson, Sweden: Almgyist & Wiksell International, 1984, p.
  • RP-HPLC indicated complete oxidation after 36 h, after which time the solution was dried by vacuum centrifugation. The residue was redissolved (5 mL of 10 % MeCN, 0.1 % CF 3 C00H in H 2 0) and chromatographed by prep. RP-HPLC (conditions as above except 7.5 to 15.5 % MeCN gradient over 100 min) . Appropriate peak fractions were collected, pooled and lyophilised to afford the pure cyclic peptide diastereomers (9.27 mg, isomer 1; 2.70 mg, isomer 2). Anal.
  • This peptide was synthesised using chain assembly (using Fmoc-Orn (Boc) -OH and Fmoc-Tyr (Bu fc ) -OH in the appropriate acylation cycles) , cleavage, deprotection, cyclisation (of the fully deprotected linear precursor at high dilution in basic aqueous solution) and purification methods analogous to those described in Example 6.
  • the following analytical details for the title compound were recorded: Anal.
  • RP-HPLC: t R 15.6 min, 99% purity at 215 nm (Vydac 218TP54, 1 mL/min, 0 to 12% MeCN in 0.1% aq CF 3 COOH over 20 min) .
  • Peptide chain assembly The capped Boc-Abu-resin was then deprotected by treatment with 25 % CF 3 COOH / CH 2 C1 2 during 30 min. After draining, the resin was washed with CH 2 C1 2 and was then reacted with Boc- Tyr(2BrZ)-0H (5 eq) , HOBt (5 eq) , PyBOP (5 eq) and Pr ⁇ NEt (20 eq) in DMF for 2 h. Further peptide chain extension was then achieved similarly by applying alternating deprotection and coupling steps. The amino acid derivatives Boc-Gln-OH, Boc-Glu (OBn) -OH and Boc- Abu-OH were used successively. The Boc-Abu-Glu (OBn) -Gln- Tyr(2BrZ) -Abu-resin was finally obtained after washing with CH 2 C1 2 , Et 2 0 and drying.
  • Peptide chain assembly The capped Boc-Abu-resin was then deprotected by treatment with 25 % CF 3 COOH/ CH 2 C1 2 during 30 min. After draining, the resin was washed with CH 2 C1 2 and was then reacted with Boc- Tyr(2BrZ)-OH (5 eq) , HOBt (5 eq) , PyBOP (5 eq) and Pr ⁇ NEt (20 eq) in DMF for 2 h. Further peptide chain extension was then achieved similarly by applying alternating deprotection and coupling steps. The amino acid derivatives Boc-Gln-OH, Boc-Glu (OBn) -OH and Boc- Gly-OH were used successively. The Boc-Gly-Glu (OBn) -Gln- Tyr (2BrZ) -Gly-resin was finally obtained after washing with CH 2 C1 2 , Et 2 0 and drying.
  • Amino acid analysis Glx 2.01 (2), Tyr 1.01 (1), Gly 1.98 (2) for monomer; Glx 4.00 (4), 2.02 (2) Tyr, Gly 3.98 (4) for dimer.
  • Amino acid analysis Glx 2.00 (2), Tyr 0.99 (1), Gly 1.01 (1), 3-Amb 1.00 (1) for monomer; Glx 4.00 (4), Tyr 1.96 (2), Gly 2.01 (2), 3-Amb 2.04 (2) for dimer.

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Abstract

Cette invention a trait à un composé capable d'interagir avec le récepteur interne du domaine catalytique du facteur VII défini par les restes Leu?263, Pro264, Glu265, Phe268, Ser269, Tyr357 et Arg353¿ ou avec le ligand défini par les restes Cys?98-Cys102¿ du domaine EGF-2 du facteur FVII et ce, afin d'empêcher la formation d'un complexe fonctionnel FVIIa/TF, à l'exclusion du peptide des acides aminés 82 à 128 du facteur FVII et de ses équivalents fonctionnels chez qui des acides aminés de la séquence sont modifiés ou bien sont absents. Elle a, notamment, trait au composé bisulfure-cyclo-[H-Cys-Glu-Gln-Tyr-Cys-OH].
PCT/GB1998/002701 1997-09-09 1998-09-08 Fragments du facteur vii et leurs analogues, utilisation dans le traitement des troubles dus aux caillots sanguins WO1999013063A1 (fr)

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US6197997P 1997-10-16 1997-10-16

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Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002077218A1 (fr) * 2001-03-22 2002-10-03 Novo Nordisk Health Care Ag Derives du facteur vii de coagulation
US7235638B2 (en) 2001-03-22 2007-06-26 Novo Nordisk Healthcare A/G Coagulation factor VII derivatives
JP2009521505A (ja) * 2005-12-23 2009-06-04 ブリストル−マイヤーズ スクイブ カンパニー 抗凝血剤として有用な大環状第ｖｉｉａ因子阻害剤
US8716240B2 (en) 2001-10-10 2014-05-06 Novo Nordisk A/S Erythropoietin: remodeling and glycoconjugation of erythropoietin
US8716239B2 (en) 2001-10-10 2014-05-06 Novo Nordisk A/S Granulocyte colony stimulating factor: remodeling and glycoconjugation G-CSF
US8841439B2 (en) 2005-11-03 2014-09-23 Novo Nordisk A/S Nucleotide sugar purification using membranes
US8853161B2 (en) 2003-04-09 2014-10-07 Novo Nordisk A/S Glycopegylation methods and proteins/peptides produced by the methods
US8911967B2 (en) 2005-08-19 2014-12-16 Novo Nordisk A/S One pot desialylation and glycopegylation of therapeutic peptides
US8916360B2 (en) 2003-11-24 2014-12-23 Novo Nordisk A/S Glycopegylated erythropoietin
US8969532B2 (en) 2006-10-03 2015-03-03 Novo Nordisk A/S Methods for the purification of polypeptide conjugates comprising polyalkylene oxide using hydrophobic interaction chromatography
US9005625B2 (en) 2003-07-25 2015-04-14 Novo Nordisk A/S Antibody toxin conjugates
US9029331B2 (en) 2005-01-10 2015-05-12 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
US9050304B2 (en) 2007-04-03 2015-06-09 Ratiopharm Gmbh Methods of treatment using glycopegylated G-CSF
US9150848B2 (en) 2008-02-27 2015-10-06 Novo Nordisk A/S Conjugated factor VIII molecules
US9187546B2 (en) 2005-04-08 2015-11-17 Novo Nordisk A/S Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
US9187532B2 (en) 2006-07-21 2015-11-17 Novo Nordisk A/S Glycosylation of peptides via O-linked glycosylation sequences
US9200049B2 (en) 2004-10-29 2015-12-01 Novo Nordisk A/S Remodeling and glycopegylation of fibroblast growth factor (FGF)
US9493499B2 (en) 2007-06-12 2016-11-15 Novo Nordisk A/S Process for the production of purified cytidinemonophosphate-sialic acid-polyalkylene oxide (CMP-SA-PEG) as modified nucleotide sugars via anion exchange chromatography
US10022256B2 (en) 2012-11-30 2018-07-17 Novartis Ag Methods for making conjugates from disulfide-containing proteins
US10172948B2 (en) 2013-11-26 2019-01-08 Novartis Ag Methods for oxime conjugation to ketone-modified polypeptides

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WO1990003390A1 (fr) * 1988-09-23 1990-04-05 Corvas, Inc. Inhibiteurs peptidyles de l'initiation de la coagulation
WO1995000541A1 (fr) * 1993-06-18 1995-01-05 Hafslund Nycomed A/S Peptides derives du facteur vii
WO1995017421A1 (fr) * 1993-12-22 1995-06-29 Temple University - Of The Commonwealth System Of Higher Education ANALOGUES PEPTIDIQUES DU SITE DE FIXATION PLAQUETTAIRE DU FACTEUR IXa
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WO1996018654A1 (fr) * 1994-12-15 1996-06-20 Nycomed Imaging As Fragment 82-128 du facteur vii et son utilisation pour le traitement des troubles de la coagulation sanguine

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WO1990003390A1 (fr) * 1988-09-23 1990-04-05 Corvas, Inc. Inhibiteurs peptidyles de l'initiation de la coagulation
WO1995000541A1 (fr) * 1993-06-18 1995-01-05 Hafslund Nycomed A/S Peptides derives du facteur vii
WO1995017421A1 (fr) * 1993-12-22 1995-06-29 Temple University - Of The Commonwealth System Of Higher Education ANALOGUES PEPTIDIQUES DU SITE DE FIXATION PLAQUETTAIRE DU FACTEUR IXa
WO1996018653A1 (fr) * 1994-12-15 1996-06-20 Nycomed Imaging As Disulfure-cyclo-[h-cys-glu-gln-tyr-cyr-oh] et son utilisation pour le traitement des troubles de coagulation sanguine
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Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7235638B2 (en) 2001-03-22 2007-06-26 Novo Nordisk Healthcare A/G Coagulation factor VII derivatives
WO2002077218A1 (fr) * 2001-03-22 2002-10-03 Novo Nordisk Health Care Ag Derives du facteur vii de coagulation
US8716240B2 (en) 2001-10-10 2014-05-06 Novo Nordisk A/S Erythropoietin: remodeling and glycoconjugation of erythropoietin
US8716239B2 (en) 2001-10-10 2014-05-06 Novo Nordisk A/S Granulocyte colony stimulating factor: remodeling and glycoconjugation G-CSF
US8853161B2 (en) 2003-04-09 2014-10-07 Novo Nordisk A/S Glycopegylation methods and proteins/peptides produced by the methods
US9005625B2 (en) 2003-07-25 2015-04-14 Novo Nordisk A/S Antibody toxin conjugates
US8916360B2 (en) 2003-11-24 2014-12-23 Novo Nordisk A/S Glycopegylated erythropoietin
US10874714B2 (en) 2004-10-29 2020-12-29 89Bio Ltd. Method of treating fibroblast growth factor 21 (FGF-21) deficiency
US9200049B2 (en) 2004-10-29 2015-12-01 Novo Nordisk A/S Remodeling and glycopegylation of fibroblast growth factor (FGF)
US9029331B2 (en) 2005-01-10 2015-05-12 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
US9187546B2 (en) 2005-04-08 2015-11-17 Novo Nordisk A/S Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
US8911967B2 (en) 2005-08-19 2014-12-16 Novo Nordisk A/S One pot desialylation and glycopegylation of therapeutic peptides
US8841439B2 (en) 2005-11-03 2014-09-23 Novo Nordisk A/S Nucleotide sugar purification using membranes
JP2009521505A (ja) * 2005-12-23 2009-06-04 ブリストル−マイヤーズ スクイブ カンパニー 抗凝血剤として有用な大環状第ｖｉｉａ因子阻害剤
US9187532B2 (en) 2006-07-21 2015-11-17 Novo Nordisk A/S Glycosylation of peptides via O-linked glycosylation sequences
US8969532B2 (en) 2006-10-03 2015-03-03 Novo Nordisk A/S Methods for the purification of polypeptide conjugates comprising polyalkylene oxide using hydrophobic interaction chromatography
US9050304B2 (en) 2007-04-03 2015-06-09 Ratiopharm Gmbh Methods of treatment using glycopegylated G-CSF
US9493499B2 (en) 2007-06-12 2016-11-15 Novo Nordisk A/S Process for the production of purified cytidinemonophosphate-sialic acid-polyalkylene oxide (CMP-SA-PEG) as modified nucleotide sugars via anion exchange chromatography
US9150848B2 (en) 2008-02-27 2015-10-06 Novo Nordisk A/S Conjugated factor VIII molecules
US10022256B2 (en) 2012-11-30 2018-07-17 Novartis Ag Methods for making conjugates from disulfide-containing proteins
US10667935B2 (en) 2012-11-30 2020-06-02 Novartis Ag Methods for making conjugates from disulfide-containing proteins
US11311400B2 (en) 2012-11-30 2022-04-26 Novartis Ag Methods for making conjugates from disulfide-containing proteins
US10172948B2 (en) 2013-11-26 2019-01-08 Novartis Ag Methods for oxime conjugation to ketone-modified polypeptides

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