GB2247239A - Hirudin conjugates with polyoxyethylene - Google Patents
Hirudin conjugates with polyoxyethylene Download PDFInfo
- Publication number
- GB2247239A GB2247239A GB9018464A GB9018464A GB2247239A GB 2247239 A GB2247239 A GB 2247239A GB 9018464 A GB9018464 A GB 9018464A GB 9018464 A GB9018464 A GB 9018464A GB 2247239 A GB2247239 A GB 2247239A
- Authority
- GB
- United Kingdom
- Prior art keywords
- hirudin
- desulphatohirudin
- gly
- gln
- asn
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- WQPDUTSPKFMPDP-OUMQNGNKSA-N hirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(OS(O)(=O)=O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]2CSSC[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N2)=O)CSSC1)C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)CSSC1)C(C)C)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 WQPDUTSPKFMPDP-OUMQNGNKSA-N 0.000 title claims abstract description 49
- 108010007267 Hirudins Proteins 0.000 title claims abstract description 45
- 102000007625 Hirudins Human genes 0.000 title claims abstract description 45
- 229940006607 hirudin Drugs 0.000 title claims abstract description 45
- 229920003171 Poly (ethylene oxide) Polymers 0.000 title claims abstract description 18
- -1 polyoxyethylene Polymers 0.000 title claims abstract description 10
- 239000003146 anticoagulant agent Substances 0.000 claims abstract description 5
- 229940127219 anticoagulant drug Drugs 0.000 claims abstract description 5
- 210000004369 blood Anatomy 0.000 claims abstract description 3
- 239000008280 blood Substances 0.000 claims abstract description 3
- XYWBJDRHGNULKG-OUMQNGNKSA-N desirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]2CSSC[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N2)=O)CSSC1)C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)CSSC1)C(C)C)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 XYWBJDRHGNULKG-OUMQNGNKSA-N 0.000 claims description 18
- 108010073652 desirudin Proteins 0.000 claims description 15
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- 101710154944 Hirudin variant-1 Proteins 0.000 description 8
- FIBJDTSHOUXTKV-BRHMIFOHSA-N lepirudin Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H]1CSSC[C@@H]2NC(=O)[C@@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CSSC[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)CNC2=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](Cc2ccc(O)cc2)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)[C@@H](C)O)[C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N1)C(C)C)C(C)C)[C@@H](C)O)[C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O FIBJDTSHOUXTKV-BRHMIFOHSA-N 0.000 description 8
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
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- 108090000623 proteins and genes Proteins 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 5
- 229920001184 polypeptide Polymers 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- YGYAWVDWMABLBF-UHFFFAOYSA-N Phosgene Chemical compound ClC(Cl)=O YGYAWVDWMABLBF-UHFFFAOYSA-N 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
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- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
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- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
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- 229910021653 sulphate ion Inorganic materials 0.000 description 2
- 229960004072 thrombin Drugs 0.000 description 2
- KTPDNYVWXJTUKG-UHFFFAOYSA-O 1-methyl-2-[2-(2-phenyl-1h-indol-3-yl)ethenyl]quinolin-1-ium Chemical compound C1=CC2=CC=CC=C2[N+](C)=C1\C=C\C(C1=CC=CC=C1N1)=C1C1=CC=CC=C1 KTPDNYVWXJTUKG-UHFFFAOYSA-O 0.000 description 1
- 102000004411 Antithrombin III Human genes 0.000 description 1
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- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
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- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- NJMYZEJORPYOTO-BQBZGAKWSA-N Gln-Pro Chemical compound NC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O NJMYZEJORPYOTO-BQBZGAKWSA-N 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 101710154919 Hirudin variant-2 Proteins 0.000 description 1
- 241000545744 Hirudinea Species 0.000 description 1
- 241000237902 Hirudo medicinalis Species 0.000 description 1
- JYOAXOMPIXKMKK-YUMQZZPRSA-N Leu-Gln Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@H](C([O-])=O)CCC(N)=O JYOAXOMPIXKMKK-YUMQZZPRSA-N 0.000 description 1
- BJFJQOMZCSHBMY-YUMQZZPRSA-N Met-Val Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(O)=O BJFJQOMZCSHBMY-YUMQZZPRSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
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- 101000712605 Theromyzon tessulatum Theromin Proteins 0.000 description 1
- WCRFXRIWBFRZBR-GGVZMXCHSA-N Thr-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 WCRFXRIWBFRZBR-GGVZMXCHSA-N 0.000 description 1
- 229940122388 Thrombin inhibitor Drugs 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- NKUGCYDFQKFVOJ-JYJNAYRXSA-N Tyr-Leu-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NKUGCYDFQKFVOJ-JYJNAYRXSA-N 0.000 description 1
- YKZVPMUGEJXEOR-JYJNAYRXSA-N Val-Val-Tyr Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N YKZVPMUGEJXEOR-JYJNAYRXSA-N 0.000 description 1
- 125000003545 alkoxy group Chemical class 0.000 description 1
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- FZFAMSAMCHXGEF-UHFFFAOYSA-N chloro formate Chemical compound ClOC=O FZFAMSAMCHXGEF-UHFFFAOYSA-N 0.000 description 1
- AOGYCOYQMAVAFD-UHFFFAOYSA-N chlorocarbonic acid Chemical class OC(Cl)=O AOGYCOYQMAVAFD-UHFFFAOYSA-N 0.000 description 1
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- YWEUIGNSBFLMFL-UHFFFAOYSA-N diphosphonate Chemical compound O=P(=O)OP(=O)=O YWEUIGNSBFLMFL-UHFFFAOYSA-N 0.000 description 1
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- 150000004676 glycans Chemical class 0.000 description 1
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- STKYPAFSDFAEPH-LURJTMIESA-N glycylvaline Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CN STKYPAFSDFAEPH-LURJTMIESA-N 0.000 description 1
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- DLYUQMMRRRQYAE-UHFFFAOYSA-N phosphorus pentoxide Inorganic materials O1P(O2)(=O)OP3(=O)OP1(=O)OP2(=O)O3 DLYUQMMRRRQYAE-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/55—Protease inhibitors
- A61K38/57—Protease inhibitors from animals; from humans
- A61K38/58—Protease inhibitors from animals; from humans from leeches, e.g. hirudin, eglin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
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Abstract
A conjugate of hirudin and polyoxyethylene (which has a molecular weight of from 20,000 to 50,000 Da) may be used as a blood anticoagulant.
Description
Hirudin Derivatives
The present invention relates to derivatives of hinidin, and especially to conjugates of hirudin.
Hirudin which has been known for a long time and which occurs naturally in leeches (Hirudo medicinalis) (Walsman, P. and Markwardt, F. (1981) Pharmazie 36, 653) is the strongest thrombin inhibitor of all naturally occurring and synthetic anticoagulants known with a complex dissociation constant of 2 x 10-14 M, thus preventing the formation of fibrin from its precursor fibrinogen. Other enzymes of the blood coagulation cascade are not inhibited by hirudin. In contrast to heparin which is the preferred anticoagulant in conventional anticoagulation therapy, hirudin exerts its inhibiting action directly on thrombin and, unlike the former, does not act through antithrombin III. No effect on heart rate, respiration, blood pressure, thrombocyte count, fibrinogen and haemoglobin could be observed after intravenous administration of hirudin to dogs, even in high doses.In tests on rats, pigs and dogs, hirudin has proved effective in experimental thrombosis (induced either by stasis, vascular damage or by the injection of thrombin), in endotoxin shock, and also in DIC (disseminated intravascular coagulation).
Hirudin is not a single polypeptide species but a class of equally acting polypeptides consisting of at least four representatives designated hirudin variant 1 (HV1), hirudin variant 2 (HV2; EP Application 0 158 564), hirudin variant PA (PCT Application WO 88/03493), and "des-(Val)2-hirudin" (EP Application 0 158 986). The variants differ from each other by a number of amino acids, for example at the N-terminal sequence which is
Val-Val-Tyr for HV 1, lle-Thr-Tyr for HV2 and PA and Thr-Tyr for "des-(Val)2-hirudin".
Based on NMR studies, HV1 is composed of an N-terminal core domain with a protruding
"finger" (residues 31-36), and an acidic terminal loop (Clore et al., EMBO Journal 6, 529,
1987). All above-mentioned hirudin variants have an accumulation of hydrophobic amino acids at the N-terminus and an accumulation of polar amino acids at the C-terminus, a tyrosine residue (Tyr 63) present as sulphate monoester, three disuiphide bridges and the
anticoagulant activity in common.
Recently, cDNAs and synthetic genes coding for hirudin variants have been cloned and expressed in microbial hosts. Although the expression products lack the sulphate monoester group at Tyr 63 - and were, therefore, designated "desulphato hirudins1 - they turned out to exhibit approximately the same biological activity as the natural sulphated hirudins. Desulphatohirudin variant HV1 has been expressed in Escherichia coli (European Patent Applications No. 158 564 and 168 342) and in Saccharomvces cerevisiae (European Patent Applications No. 168 342,200 655, 225 633 and 252 854).
Similarly, desulphatohirudin HV2 has been expressed in Escherichia coli (European
Patent Application No.200655, PCT-Application No. 86/01224) and des-(Val)2-desulphatohirudin has been expressed in Escherichia coli (European Patent
Application No. 158 986).
In the present application, the term hirudin, when not otherwise stated, is intended to embrace (1) all naturally occurring or synthetic hirudin variants and hirudin derivatives, such as hirudin fragments which retain the anticoagulant activity, and (2) all desulphatohirudin variants and desuiphatohirudin derivatives, such as C-terminally shortened sulphatohirudins, which are described in the literature or are obtainable by methods of recombinant DNA technology.
Examples of such hirudins are: (1) hirudin or a hirudin variant of type HV1 with the formula
10 H-4-Zl-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly- 20 -Gln-A sn-lLeu-Cys-Leu-Cys -Glu-Gly-Ser-Asn- 30 -Val-Cys-Gly-Gln-Gly-Asn-Z2-Cys-2e-Leu- (I)
40 -Gly-Ser-Asp-Gly-Glu-5-Asn-Gln-Cys-Val- 50 -Thr-Gly-Glu-Gly-Thr-Pro-Z4--Z6-S er- 60 -Z7-Z8-Asp-GIy-Asp-Phe-Glu-Glu-ne-Pro- -Glu-Glu-Tyr(R)-Leu-Gln-OH, wherein - (R)is the phenolic hydroxygroup of Tyr (desulphatohirudin) or a -0-S03H group, and - the whole molecule can be shortened by the C-terminal amino acid Gln, the C-terminal dipeptide Leu-Gln, the C-terminal tripeptide Tyr-Leu-Gln or the C-terminal tetrapeptide
Glu-Tyr-Leu-Gln, and wherein Zo is a direct bond or represents Val, Ile or Gly or the dipeptidyl radicals
Gly-Val or Met-Val, Z1 is Val, ne or Thr, Z2 is Lys, Gln, Asn, Leu, Arg or Val, Z3 represents Lys, Arg, Asn, Val, Leu, Gln, Z4 represents Lys, Arg, Asn, Val or Leu, 5 represents Pro or Gly, Z6 and Z8 independently from each other represent Gln, Asn or Met, and Z7 represents His, Gln or Asn, (2) desulphatohirudin variants of type HV1 with the formula
10
H-Val-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly
20 -Gln-Asn-Leu-Cys-Leu-Cys-Glu-Gly-Ser-Asn- 30 -Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu- (11) 40 -Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val
50 -Thr-Gly-Glu- Gly-Thr-Pro-Lys -Pro-Gln-S er- 60 His-Asn-Y1-Gly-Asp-Phe-Y2-Y3-lle-Pro- -Y4-Y5-Y6-Leu-Y7-OH, wherein Y1 represents Asp or the radical of a neutral genetically encoded amino acid, Y2 and Y3 independently from each other represent Giu, Gln, Asn or the radical of a lipophilic genetically encoded amino acid, Y4 and Y5 independently from each other represent Glu, Gln or the radical of a neutral genetically encoded amino acid, Y6 represents Tyr or the radical of an acidic genetically encoded amino acid and Y7 represents Gln or the dipeptidyl radical Gln-Pro,
(3) a hirudin variant of type HV2 with the formula
10
H-Ile-Thr-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly
20 -Gln-Asn-Leu-Cys-Leu-Cys-Glu-Gly-Ser-Asn- 30 -Val-Cys-Gly-Lys-Gly-Asn-Lys-CysJie-Leu- (III) 40 -Gly-Ser-Asn-Gly-Lys-Gly-Asn-Gln-Cys-Val- 50 -Thr-Gly-Glu-Gly-Thr-Pro-Asn-Pr > Glu-Ser- 60 -His-Asn-Asn-Gly-Asp-Phe-Glu-Glu-lle-Pro- -Glu-Glu-Tyr (R)-Leu-Gln-OH, wherein - (R)is the phenolic hydroxygroup of Tyr (desulphatohirudin) or a -0-S03H group, and - Ile 1 can be replaced by Val and Thr 2 by Val (HV2 modified) or - Asn 47 can be replaced by Lys or Arg or His or - Tyr 63 can be replaced by Glu or Asp, (4) a hirudin variant of type PA (HV3) with the formula
10 H-Ile-Thr-Tyr-Thr-A sp- Cys -Thr-Glu- S er-Gly- 20 -Gln-Asn-Leu-Cys-Leu-Cys-Glu-Gly-Ser-Asn
30 -Val-Cys-Gly-Lys-Gly-Asn-Lys-Cys-Ile-Leu- (IV)
40 -Gly-Ser-Gln-Gly-Lys-Asp-Asn-Gln-Cys-Val- 50 -Thr-Gly-Glu-Gly-Thr-Pro-Lys -Pro-Gln-Ser- 60 -His-Asn-Gln-Gly-Asp-Phe-Glu-Pro-Ile-Pro- -Glu-Asp-Ala-Tyr (R)-Asp-Glu-OH, wherein - (R)is the phenolic hydroxygroup of Tyr (desulphatohirudin) or a -0-S03H group, and - the polypeptide chain can be shortened at the C-terminius by 18, 10, 9, 6,4 or 2 amino acids, or - the polypeptide chain can be shortened at the N-terminus by 1 or 2 amino acids.
Examples of hirudins of the formula (I) are desulphatohirudin HV1 in which (R) is the phenolic hydroxygroup of Tyr, Z0 represents Val, Z1 is Val, Z2, Z3 and Z4 are each Lys, Z5 is Pro, Z6 is Gln, Z7 is His and Z5 represents Asn, or des-(Val)2-desulphatohirudin, in which 4 represents a direct bond, Z1 is Thr and Z2-z8 and (R) are as defined for HV1.
Further examples are variants of HV1 like [Asn27]-desulphatohirudin, [Asn36]-desulphatohirudin, [Val36] -desuiphatohirudin, [Gly48]-desulphatohirudin, [Met49]desulphatohirudin, [Me2] -desuiphatohirudin, [Asn51]-desulphatohirudin, [Gin27, Arg47]-desulphatohirudin, [Gly27, Gin36, Arg47]-desulphatohirudin, [Arg36, Arg47]-desulphatohirudin, [Arg27, Arg47]-desulphatohirudin, Glycyl-[Gln27,Gln36, Arg47]-desulphatohirudin, Methionyl-[Gln27Arg47]-desulphatohirudin, [Ilel, Ile2]-desulphatohirudin and [Glyl]-desulphatohirudin.
Neutral genetically encoded amino acids are the following L-amino acids: Ala, Ser, Thr,
Val, Leu, Ile, Asn, Gln, Met, Phe, Try and Pro, furthermore the amino acid Gly.
Lipophilic genetically encoded amino acids are the following L-amino acids: Ala, Val,
Leu, Ile, Phe and Gly.
Acidic genetically encoded amino acids are Asp and Glu.
Examples of desulphatohirudin variants of the formula (II) are [Gly61.62] -desulphatohirudin, [Leu61,62]-desulphatohirudin, [Asn61,62]-desulphatohirudin, [1-eu57,58,6 61,62]-desulphatohirudin, [Asn57,58,61,65 -desulphatohirudin, [Ala53]-desulphatohirudin, [Asp63] -desuiphatohirudin, [Glu63]-desulphatohirudin, [Pro66]-desulphatohirudin, rGln57,58,61,623 -desulphatohirudin.
Examples of hirudin variants of type HV2 of the formula (I) are desulphatohirudin HV2 or desulphatohirudin HV2 (Lys47).
An example of a hirudin variant of type PA of the formula aV) is desulphatohirudin PA.
The desulphatohirudin variants of the formulae ('), (11), (m) and JV) can be prepared by conventional recombinant DNA technology well known in the art. Following the isolation and cloning of the hirudin gene mutation of defined codons (like e.g. base exchanges, base deletions or base extensions) within the cloned DNA is achieved in vitro by the method of site-directed mutagenesis using suitable mutagenic primers. The resulting mutant gene is integrated in an appropriate expression vector and transformed in a microbial host like e.g.
Escherichia coli, Bacillus subtilis or Saccharomvces cerevisiae. Transformants carrying the hybrid vector which preferably comprises a signal sequence linked in the proper reading frame to the DNA-sequence encoding the mutant gene are cultivated by employing conventional techniques. The desulphatohirudin variants are isolated from the culture broth and purified by means well known to anybody of ordinary skill in the art.
Protein or polypeptide drugs are generally by necessity administered parentally in vivo.
Intravenously administered peptide drugs are, in general rapidly eliminated from the bloodstream via a process which is a combination of metabolism and of excretion.
Large proteins having sizes above the glomerulan filtration limit (i.e. diameter 745 AO) are eliminated from the body by metabolism. Small proteins/peptides, of a size below the glomerular filtration limit can be either filtered out of the body, metabolised by the body or both. Hirudin is known to be almost fully removed from the blood by the kidney. This removal is relatively rapid (tV2".5h), limiting the duration of biological action of the compound.
Various methods for modifying proteins to increase the time during which they remain in circulation have been proposed. In the case of hirudin, it has been proposed to link it to a carrier such as polysaccharide such as dextran or polyethylene glycol with a molecular weight of 1500-15000 Da - see EP 0 345 616.
In order to limit the rate of filtration of any compound through the kidneys, its effective size needs to be increased close to the size cut-off of the kidney membrane (i.e., 45 A).
Therefore, the size of the polymer to be attached to hirudin needed to be selected accordingly.
We have now found that when hirudin is conjugated with polyoxyethylene (the term to include alkoxy derivatives) having a molecular weight of from 20,000 to 50,000 Da, the circulating half life is much extended.
Accordingly, the present invention provides a conjugate of hirudin and polyoxyethylene which has a molecular weight of from 20,000 to 50,000 Da The conjugate has an extended plasma half-life and can be used as a long-acting anticoagulant for therapeutic purposes.
The polyoxyethylene is conjugated to the hirudin via the available amino groups of the hirudin. Usually between three and four amino groups are substituted with polyoxyethylene, making the molecular weight of the product > 100 k Da.
The conjugate may be prepared by first forming a chloroformate derivative of polyoxyethylene (POE-OCOCl) by reacting POE with phosgene in dry acetonitrile by the method described by Muther et al., Reactive Polymers 6, 99-107 (1987). This is then reacted with hirudin at an alkaline pH e.g. 7.5-10, to give a hirudin -POE conjugate free from cross-linked material.
The therapeutically effective amount of hirudin is normally in the dosage range from about 0.001 to 10 mgkg of body weight, e.g. from 0.01 to 3 mg/kg. Because of the longer life of the product of the invention in the body, the dosage range may be lowered from that used with hirudin itself. Administration is made by intravenous, intramuscular or subcutaneous injection.
The following Examples illustrate the invention. In the Examples the hirudin used is desulphatohirudin HV1 made by the method of Example 1 of EP 0 367 713.
Example
Polyoxyethylene (POE) having a molecular weight of 35 k Da is dissolved in tetrahydrofuran and precipitated by the addition of diethyl ether. This is repeated twice and the precipitated material is collected and dried over phosphorus pentoxide in a vacuum desiccator.
The dry POE (33g) is dissolved in dry acetonitrile (200ml) and reacted with (lOg) phosgene. The resulting bifunctional POE chloroformate is isolated by removal of solvent and excess phosgene, and precipitation of the product from dichloromethane into ether.
The bifunctional POE chioroformate (910mg) is reacted with hirudin (9mg) in aqueous phosphate buffer at a pH of 8.5. The resulting conjugate is isolated and purified by ultra filtration through a 100 kDa membrane using a 50 x cell volume wash.
Size exclusion chromatography shows the conjugate to be polydisperse with a peak molecular weight of about 100 kDa. Elemental analysis (C/N ratio) indicates the substitution of between three and four amino groups (C/N =52.13=28.54 mol % Hirudin and 71.46 mol % POE., by weight 7.3% Hirudin and 92.7% POE).
Example 2
A comparative duration of action study is carried out following intravenous administration in rats (4 used per experiment). A dosage of 100 mgRg of conjugate is given to provide an initial anticoagulant activity (measured after 2 minutes) equivalent to 0.1 mg/kg of
Hirudin. After 30 minutes the activities of Hirudin and Hirudin-POE are 23% and 91% of the initial value. After 60 min the activities are 15% and 62% respectively. These figures illustrate the significant increase in half life of the conjugate compared to hirudin itself.
Claims (6)
1. A conjugate of hirudin and polyoxyethylene which has a molecular weight of from 20,000 to 50,000 Da.
2. A conjugate as claimed in claim 1 which has between three and four polyoxyethylene chains per molecule conjugated via the available amino groups on hirudin.
3. A conjugate as claimed in claim 1 or 2 having a molecular weight of > 100 kDa.
4. A conjugate as claimed in any preceding claim in which the hirudin is selected from naturally occurring or synthetic variants, derivatives and fragments as well as desulphatohirudin variants and derivatives.
5. A conjugate as claimed in any preceding claim in which the hirudin is desulphatohirudin HV1.
6. The use of a conjugate as claimed in any preceding claim as a long acting blood anticoagulant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9018464A GB2247239A (en) | 1990-08-22 | 1990-08-22 | Hirudin conjugates with polyoxyethylene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9018464A GB2247239A (en) | 1990-08-22 | 1990-08-22 | Hirudin conjugates with polyoxyethylene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| GB9018464D0 GB9018464D0 (en) | 1990-10-03 |
| GB2247239A true GB2247239A (en) | 1992-02-26 |
Family
ID=10681080
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB9018464A Withdrawn GB2247239A (en) | 1990-08-22 | 1990-08-22 | Hirudin conjugates with polyoxyethylene |
Country Status (1)
| Country | Link |
|---|---|
| GB (1) | GB2247239A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0503829A3 (en) * | 1991-03-08 | 1993-06-02 | Ciba-Geigy Ag | Hirudin for the inhibition of cancer metastasis |
| EP0658585A3 (en) * | 1993-12-10 | 1995-10-18 | Behringwerke Ag | Aminophenylalanine derivatives, process for their production, their use and agents containing these as anticoagulants. |
| WO2000061121A3 (en) * | 1999-04-08 | 2001-04-26 | Max Planck Gesellschaft | Use of molecular weight-amplified hirudin as an anticoagulant in extracorporeal renal replacement therapy |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0333356A2 (en) * | 1988-03-04 | 1989-09-20 | Biogen, Inc. | Hirudin peptides |
| EP0345616A2 (en) * | 1988-06-04 | 1989-12-13 | Hoechst Aktiengesellschaft | Hirudin derivatives with delayed action |
-
1990
- 1990-08-22 GB GB9018464A patent/GB2247239A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0333356A2 (en) * | 1988-03-04 | 1989-09-20 | Biogen, Inc. | Hirudin peptides |
| EP0345616A2 (en) * | 1988-06-04 | 1989-12-13 | Hoechst Aktiengesellschaft | Hirudin derivatives with delayed action |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0503829A3 (en) * | 1991-03-08 | 1993-06-02 | Ciba-Geigy Ag | Hirudin for the inhibition of cancer metastasis |
| EP0658585A3 (en) * | 1993-12-10 | 1995-10-18 | Behringwerke Ag | Aminophenylalanine derivatives, process for their production, their use and agents containing these as anticoagulants. |
| US5571844A (en) * | 1993-12-10 | 1996-11-05 | Behringwerke Aktiengesellschaft | Amidinophenylalanine derivatives, a process for their preparation, their use and compositions containing these as anticoagulants |
| JP3448119B2 (en) | 1993-12-10 | 2003-09-16 | ヴエルナー・シユテユーバー | Amidinophenylalanine derivative, method for producing the same, and anticoagulant containing the same |
| WO2000061121A3 (en) * | 1999-04-08 | 2001-04-26 | Max Planck Gesellschaft | Use of molecular weight-amplified hirudin as an anticoagulant in extracorporeal renal replacement therapy |
Also Published As
| Publication number | Publication date |
|---|---|
| GB9018464D0 (en) | 1990-10-03 |
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