+

WO1995007295A1 - Analogues d'hirudine - Google Patents

Analogues d'hirudine Download PDF

Info

Publication number
WO1995007295A1
WO1995007295A1 PCT/US1994/010048 US9410048W WO9507295A1 WO 1995007295 A1 WO1995007295 A1 WO 1995007295A1 US 9410048 W US9410048 W US 9410048W WO 9507295 A1 WO9507295 A1 WO 9507295A1
Authority
WO
WIPO (PCT)
Prior art keywords
hirudin
tyr
molecule
thrombin
spacer
Prior art date
Application number
PCT/US1994/010048
Other languages
English (en)
Inventor
Cecilia S. L. Ku
Richard Johnson
Julian Breillatt
Original Assignee
Baxter International Inc.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Baxter International Inc. filed Critical Baxter International Inc.
Priority to AU77215/94A priority Critical patent/AU7721594A/en
Publication of WO1995007295A1 publication Critical patent/WO1995007295A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to analogs of hirudin and in particular relates to analogs of hirudin which have antithrombogenic activity and which can be bound to polymers.
  • Hirudin forms a very tight complex with thrombin, wherein over 40% of the hirudin structure intimately contacts the thrombin molecule and covers both the fibrinogen recognition site of thrombin and the fibrinogen cleaving (active) site of thrombin. Twenty-seven of the sixty-five residues of hirudin have contacts less than 4.0 A with thrombin. This close fit prevents both the binding and cleavage of fibrinogen by thrombin.
  • hirudin Three regions of the hirudin molecule are now believed to be essential to the thrombin-hirudin high affinity interaction based on X-ray crystallography (Rydel, et al., Science 249 (1990) pp. 277 to 280) and structure-activity studies (Krstenansky, et al., J. Med. Chem. 30 (1987) pp. 1688 to 1691).
  • the three NH 2 -terminal amino acid residues at positions 1, 2, and 3 of hirudin form a parallel beta strand with Ser 214 to Glu 217 of thrombin and participate in several dozen non-polar interactions with side chains of amino acids in and around the active site of thrombin.
  • the NH 2 -terminal domain of hirudm from approximately Thr to Pro is a compactly folded structure composed of four loops stabilized by three disulfide bridges and antiparallel beta structures. The main function of this domain is to position and facilitate the interaction of hirudin's NH 2 -terminal tripeptide at the thrombin active site.
  • the COOH-terminal tail of hirudin (Glu 49 -Pro 60 ) binds in the anion-binding exosite of thrombin and terminates in a hydrophobic helical turn defined by the sequence Glu 61 -Leu 64 .
  • the exosite constitutes the fibrinogen binding recognition site of thrombin and is dominated by numerous polar and non-polar interactions. The presence of hirudin in the exosite prevents fibrinogen from being recognized.
  • hirudin Most research on the hirudin molecule has focussed on (i) determining the roles of various regions of the molecule in its interaction with thrombin, and (ii) making modifications to the molecule to increase the binding affinity between hirudin and thrombin and thereby reduce the necessary dose in therapeutic applications. Some research has focussed on prolonging the activity or half-life of hirudin in vivo, and other research has been in the area of immobilizing hirudin on surfaces used in medical devices which come in contact with blood to provide a non-thrombogenic surface. a. Modifications to Increase Binding Affinity or to
  • PCT Application WO 85/04418 discloses recombinant HV2 where Lys 24 , Asn 33 , Lys 35 , Gly 36 , Asn 47 , Glu 49 , and Asn 53 are replaced by Gln, Asp, Glu, Lys, Lys, Gln, and Asp respectively.
  • European Patent Application No. 87402696.6 discloses recombinant variants 1, 2, and 3 where Tyr 63 or 64 is replaced by Glu or Asp and Lys 47 or Asn 47 is replaced by Arg or His.
  • European Patent Application No. 89400621.2 also discloses amino acid sequence modifications, including those at positions 1, 2, 33, 35, 36, 47, and 63, which increase the in vivo half life of the molecule, increase the specificity of the molecule's interaction with cell surface receptors and increase resistance to carboxypeptidase degradation. Arg is placed in the 33 position, Thr or Ser or Asp are placed at positions 35, and Ser is placed at position 36.
  • European Patent Application No. 89810521.8 describes mutations at the 53, 57, 58, 61, 62, and 63 positions which, depending upon the analog selected, provide increased or decreased antithrombogenic activity.
  • U.S. Patent No. 4,179,337 discloses the attachment of mass-increasing molecules such as polyethylene glycol to proteins.
  • Lazar, et al. describe mutations at position 3 in recombinant hirudin variant 1 (rHV1) where antithrombin activity was increased by replacing Tyr with Phe or Trp, and markedly decreased by replacement with Thr (J.B. Lazar, R.C. Winant & P.H. Johnson. J. Biol. Chem. 266 pp. 685-688 (1991)).
  • European Patent Application No. 89810676.0 describes mutations at positions 1, 2, 27, 36, 47, 57, 58, 61, and 66 where the position 27 substitution is Gln, position 36 substitution is Gln and position 47 substitution is Arg.
  • U.S. Patent No. 4,791,100 discloses mutations of hirudin in positions corresponding to, inter alia, 35 and 36, where at 35 the substitution is Glu and at 36 the substitution is Lys. It also discloses analogs having a greater number of amino acids than native hirudin and others having fewer amino acids than native hirudin.
  • COOH-terminal polypeptide fragments of hirudin are known to bind to thrombin, thereby inhibiting the binding and cleavage of fibrinogen by thrombin.
  • the minimum length polypeptide required to exert inhibitory activity has been reported as Phe 56 -Gln 65 (J.L. Krstenansky, T.J. Owen, M.T. Yates & S.J.T. Mao. J. Med Chem 30 pp. 1688-1691 (1987)).
  • bivalent thrombin inhibitors there are many variations possible on this model of bivalent thrombin inhibitors and in general, bivalent protease inhibitors. For example see J.M. Maraganore, P. Bourdon, J. Jablonski, K.L. Ramachandran and J.W. Fenton,II. Biochemistry 29 pp. 7095-7101 (1990); J. DiMaio, B. Gibbs, D. Munn, J. Lefebvre, F. Ni, and Y. Konishi. J. Biol. Chem. 265 pp. 21698-21703 (1990).
  • European Application No. 89302160.0 discloses peptides of about 8 to 26 amino acids having the biological activity of hirudin.
  • European Application No. 89302159.2 discloses cyclicized synthetic fragments of hirudin having antithrombogenic activity.
  • the analogs can be bound to a surface via a spacer molecule rendering the surface nonthrombogenic.
  • the analogs can be bound to a polymer via a spacer molecule to increase the analogs' in vivo half life.
  • nonthrombogenic materials comprising such analogs attached to surfaces.
  • analogs attached to polymers are examples of polymers.
  • FIG. 1 shows the amino acid sequence of HV2-Phe 3 Gln 33 Tyr 35
  • Figure 2 depicts the expression vector for the sequence in Figure 1.
  • the present invention provides an analog of hirudin having at least one amino acid in positions 30 to 37 substituted with Tyr, and the native Tyr 3 and Tyr 63or64 residues substituted with a first and a second functional nonreactive amino acid.
  • a preferred embodiment of the invention is an analog of hirudin having at least one amino acid in positions 32 to 36 substituted with Tyr, the native Tyr 3 substituted with Phe, Ile or Leu, and Tyr 63or64 substituted with Asp or Glu.
  • analog includes fragments and analogs of hirudin wherein a tyrosine residue is attached to the NH 2 -terminal position of such analogs, and the residue equivalent to Tyr 63 , when present, is substituted with either Glu or Asp.
  • analog includes peptidomimetic analogs of hirudin which are bivalent inhibitors of thrombin, where a tyrosine residue is inserted in or near the oligomer that links the COOH-terminal hirudin peptide mimic to the peptide that binds in the active site of thrombin, and where a functional nonreactive amino acid, preferably Asp or Glu, replaces the residue equivalent to Tyr 63 .
  • the present invention also overcomes the disadvantages of the prior art by providing an antithrombogenic hirudin analog covalently attached to a spacer molecule at a reactive nonfunctional Tyr residue of the analog.
  • the present invention further provides Applicant's novel analogs attached to surfaces rendering such surfaces nonthrombogenic.
  • the present invention further provides Applicant's novel analogs attached to mass-increasing molecules, which will have a prolonged half-life in vivo.
  • the present invention provides analogs of hirudin having at least one "reactive" amino acid in positions 30 to 37 and having a “functional" but “nonreactive” amino acid at positions 3 and 63.
  • a prominent loop or finger region extends out away from the hirudin-thrombin interface and contains at its tip the sequence Leu 30 Gly 31 Ser 32 Asn 33 Gly 34 Lys 35 Gly 36 Asn 37 (for hirudin variant 2). Amino acid substitutions in this region are believed not to affect the interaction of hirudin with thrombin. See European Patent Application No. 89400621.2 and Rydel, et al., Science 249 pp. 277 to 280 (1991).
  • this loop is a preferred site for substitution with Tyr to allow the attachment of a spacer molecule for surface immobilization of a hirudin analog to render the surface nonthrombogenic.
  • the novel hirudin analog may be bound to an oligomer, a polymer, a macromolecule, or other mass-increasing molecule, thereby increasing the effective molecular weight of hirudin and prolonging its in vivo half-life and its anticoagulative effect in the circulation when administered therapeutically.
  • nonreactive shall mean an amino acid which, due to its nature and/or position within the analog, will not form a covalent bond with certain mass-increasing and spacer molecules described below.
  • functional shall mean an amino acid in a particular position necessary for the analog to have antithrombogenic activity.
  • Hirudin includes a Tyr at positions 3 and 63 or 64, which are functional in the sense that they are believed to be necessary for the molecule to have antithrombogenic activity (See European Patent Application No. 87402696.6 and Lazar et al., op. cit.). However, the native Tyr at 3 and 63 are also reactive. If not substituted these amino acids will react with the spacer or mass-increasing molecule rendering the product ineffective as an antithrombogenic agent. European Patent Application Nos. 87402696.6 and 89810521.8 suggest that the substitution of Asp or Glu for the native Tyr at position 63 will not destroy the antithrombogenic activity of the analog.
  • the functional Tyr 3 and Tyr 63 are replaced with functional yet nonreactive amino acids to prevent reaction of the spacer or mass-increasing molecule at the functional positions.
  • the preferred functional, nonreactive amino acids for position 3 in hirudin are Phe, Leu and Ile.
  • the preferred functional, nonreactive amino acids for position 63 or 64 are Asp and Glu.
  • Tyr is placed at position 35 in hirudin. Tyr may also preferably be placed at position 33.
  • hirudin variant 2 native Asn at 47 may be substituted with Lys to enhance the binding affinity of the molecule to thrombin.
  • the present invention requires at least one nonfunctional amino acid available for reaction.
  • additional nonfunctional reactive Tyr substitutions may enhance the usefulness of the analog for chemical attachment to surfaces or in promoting increased in vivo half life of the analog.
  • Those skilled in the art using routine experimentation will be able to determine whether the introduction of too many of the disclosed Tyr substitutions in the analog will impair its usefulness in chemical attachment to surfaces or to mass-increasing macromolecules, due to, for example, steric hindrance of the portions of the analog which bind to thrombin.
  • the analogs of the present invention may be prepared using recombinant DNA techniques known to those skilled in the art, for example, by subjecting the gene that codes for hirudin to site-specific mutagenesis and expressing the mutated gene in a suitable host such as a yeast or bacterium.
  • European Patent No. 200655 discloses an expression system for hirudin in yeast.
  • the plasmid used to transform the yeast may be altered by methods known to those skilled in the art to create the novel mutations described herein.
  • European Patent Application Nos. 89810521.8 and 89810522.6 of Ciba Geigy AG and patent applications cited therein disclose microbial hosts for vectors containing hirudin DNA sequences.
  • the analog described in Example 1 below was made by the methods disclosed in European Patent Applications No. 87401649.6 and 89400621.2.
  • fragments of the hirudin molecule, COOH-terminal polypeptide fragments, peptidomimetic analogs, and bivalent inhibitors may also be modified accorc ⁇ ng to the criteria of the present invention provided that such "fragment” has at least one site where a Tyr may be substituted without eliminating the antithrombin activity of the "fragment", and further provided that the functional groups on the "fragment” are either nonreactive or can be substituted with a functional nonreactive amino acid. Fragments or peptides having such activity and modifications thereof fall within the intent and scope of the present invention.
  • analog as used herein shall include a fragment of the hirudin molecule, peptidomimetic analogs, and bivalent inhibitors having antithrombogenic activity.
  • analog as used herein shall include a synthetic peptide having antithrombogenic activity by virtue of an amino acid sequence analogous to that of the functional portions of the native hirudin molecule.
  • the NH 2 -terminal end of the hirudin peptides and peptidomimetic analogs known to inhibit thrombin activity has a lesser influence on their effectiveness than the COOH- terminal residues. See Johnson, P. H. et al. in "Biochemistry and Genetic Engineering of Hirudin", Seminars in Thrombosis and Hemostasis, Volume 15, No 13 (1989) and J. L. Krstenansky, T. J. Owen, M. T. Yates, and S. J. T. Mao, J. Med. Chem. 30, PP. 1688-1691 (1987).
  • bivalent thrombin inhibitors described by Maraganore et al., Biochem. 29, pp. 7095 to 7101 (1990) offer design flexibility in the placement of a reactive amino acid residue for attachment of spacer or mass increasing molecules.
  • a tyrosine residue inserted in or near the oligoglycine connecting link that joins the active site binding moiety with the longer peptide that binds in the fibrinogen recognition site provides a unique site for attaching a spacer, when in accordance with this invention, the Tyr equivalent residue is replaced with Glu or Asp.
  • Tyr is used for spacer attachment because it provides for site specific chemical reactions that avoid binding the spacer to other residues that could interfere with hirudin's activity. The specificity of these reactions depends on the altered reactivity of groups inserted into the phenolic ring of Tyr.
  • a preferred means to attach a spacer to a Tyr residue is to insert a primary amine into its phenolic ring.
  • Many reagents developed for derivatization and immobilization of proteins are designed to react with primary amino groups in their neutral, unprotonated state.
  • hirudin Use of these reagents with hirudin under usual derivatization conditions will impair the antithrombin activity of hirudin by attaching spacer molecules to its NH 2 -terminal amine or to certain of its lysyl epsilon-amino groups.
  • An aryl amine on Tyr avoids these drawbacks by reacting with amine reactive agents under conditions that virtually exclude reactivity with the alkyl amines of Lys and the NH 2 -terminal amino acid residue.
  • An aryl amine of Tyr has a pK a of about 4.8, i.e., it is 50% protonated at pH 4.8.
  • the tyrosyl amine residue will react at about 50% efficiency at pH 5.0. However, at pH 5.0, less than 0.1% of the alpha-amine of the NH 2 -terminal residue (pK a about 8.0), and less than 0.001% of the epsilon-amine of Lys (pK a about 10.0) will be reactive with such spacer chemistries. This provides the site-specificity.
  • a less preferred means to attach spacers to Tyr uses spacers activated with diazonium salts, which react directly and efficiently with the phenolic ring of Tyr. However, this reaction is not specific to Tyr, since His residues also react and hirudin's only His is essential to thrombin binding.
  • Other less preferred spacer chemistries used to attach spacers to Tyr residues are photo-oxidation, N-bromosuccinimide and sulfonyl halides which also react with amino acid side chains other than Tyr.
  • Spacers capable of reacting predominantly with aryl amines rather than with alkyl amines at about pH 5.0 include, but are not limited to, those containing N-hydroxysuccinimidyl esters, imidate esters, thiolactones, carboxyanhydrides, sulfonyl halides, isourea esters, benzoquinones, vinyl sulfones, hydrazides and imidazolyl carbonyls.
  • spacer molecules are bifunctional, wherein one end of the spacer contains an amine-reactive chemical moiety, while the other end contains the same or a different reactive species for attachment to the surface.
  • the attachment of the spacer to the surface may occur by any binding means or combination of binding means, that will retain a sufficient concentration of hirudin or its analogs at the surface to provide a nonthrombogenic and anticoagulant surface under the conditions of use.
  • Attachment of the hirudin analog and its spacer to the surface may be by covalent means, reacting the group on the free end of the spacer with a reactive group on the surface.
  • the hirudin-spacer conjugate may be coupled to a reactive group on the free end of a different spacer or on a macromolecule which are themselves covalently bound to the surface.
  • the preferred chemical reactions to attach hirudin analogs to a surface or to a surface-bound spacer are those that occur rapidly and quantitatively under moderate conditions and avoid reaction with reactive amino acid side chains of hirudin and its analogs or denaturation of the molecule.
  • the hirudin analog and its spacer may be attached to the surface by non-covalent binding means, which may include, for example, those that operate predominantly by hydrophobic binding mechanisms, or by fluorophilic associations, or by high affinity ligand receptor binding.
  • the spacer attached to the hirudin analog may have at its free end, for example, a hydrophobic or a fluorophilic moiety that will bind directly to a similarly hydrophobic or fluorophilic surface.
  • the said spacer may have at its free end a chemical moiety that reacts to produce a covalent bond with the free end of a second spacer that is attached to the surface by non-covalent means.
  • the hirudin-attached spacer may terminate in a high affinity ligand, such as a biotin molecule, which would then bind to its high affinity receptor molecule, such as avidin, that is itself covalently bound to the surface.
  • the receptor molecule may be attached to the surface by binding to one of its specific ligands that is itself attached to the surface by any of the covalent or non-covalent binding means or combination of binding means just described.
  • the hirudin of this invention may be attached to materials which are useful in the production and use of medical products, systems and devices.
  • materials include naturally occurring, genetically derived and synthetic materials.
  • Naturally occurring materials include tissues, membranes, organs and naturally occurring polymers.
  • One example of a genetically derived material is poly-beta-hydroxybutyrate.
  • Such naturally occurring, genetically derived and synthetic polymers homo- and co-polymers derived from one or more of the following: 1-olefins, such as ethylene, propylene, tetrafluoroethylene, hexafluoropropylene, vinylidene difluoride, etc.; vinyl monomers, such as vinyl chloride, styrene, maleic anhydride, methylmethacrylate, acrylonitrile, etc.; ethers, such as ethylene, tetramethylene, etc.; esters, such as ethylene-terephthalate, bisphenol A-terephthalate, etc.; carbonates, such as bisphenol A, 4,4-dihydroxybiphenylene, etc.; amides (including ureas and urethanes), such as nylons, segmented polyurethanes, proteins, etc.; saccharides, such as glucose, glucosamine, guluronic acid, sulfated glycoseaminoglycan
  • Polymers which are useful in this invention may include biodegradable, partially biodegradable and non-biodegradable polymers.
  • Other useful materials include metals, such as aluminum and stainless steel; glass, ceramics, and carbon in its various forms.
  • the choice of the material to which hirudin or its analogs may be attached generally depends on the function of the medical device or product incorporating that material. Given a specific material or combination of materials in a single device, or system of multiple devices, a surface attachment strategy is formulated for hirudin, following principles and logic well known to those skilled in the art. The above considerations ultimately determine the chemical group selected for the free end of the spacer attached to hirudin, and the subsequent members of the chain that retains hirudin at the material surface.
  • mass-increasing molecules examples include, but are not limited to, polymers such as polyethylene glycol or oxide, polyvinylpyrrolidone or the polyglucoses; and macromolecules such as serum albumin, avidin, heparin, or hydroxyethyl starch.
  • globular mass-increasing molecules may be attached to hirudin by means of a long spacer that provides hirudin with sufficient spatial freedom to achieve its inhibitory position on thrombin; or, in other words, steric interference between the mass-increasing molecule and thrombin must not block the presentation of hirudin to its binding sites on thrombin.
  • Polyethylene glycol or oxide chains which are generally attached directly to the macromolecule of interest, demonstrate a mass-increasing effect beyond their actual mass because of the larger excluded volume subtended by their highly mobile chains. See: Knauf, M. J. et al., J. Biol. Chem. 263 pp. 15064 to 15070 (1988).
  • Site-directed PEGylation of hirudin at the finger region Tyr positions the mobile polyethylene glycol/oxide chains on the side of the hirudin molecule opposite from its thrombin-binding site.
  • Asp 63 HV2 has Tyr at position 3 and 63.
  • the reactive functional Tyr is replaced with nonreactive functional Phe, and the reactive functional Tyr is replaced with nonreactive functional Asp. These replacements do not reduce the thrombin binding activity of the molecule.
  • Reactive nonfunctional Tyr is then substituted for the native nonreactive nonfunctional Lys at position 35 in the finger region. This change also does not eliminate the thrombin binding activity of the molecule but it does provide a site where Tyr is available for reaction.
  • Asn at position 47 may be changed to Lys as described in European Patent Application No. 87402696.6 to improve the activity of the antithrombogenic analog.
  • the hirudin analog HV2 Phe 3 Gln 33 Tyr 35 Lys 47 Asp 63 was prepared by the following methods:
  • phage M13TG4892 contains an expression block consisting of:
  • yeast BGL2 signal peptide (BGL2-Val 7 )
  • the yeast basic expression vector pTG3828 (pBR322, 2 micron,
  • URA3-d, PGK1 transcriptional terminator was used to assemble the expression plasmid.
  • Vector pTG3828 and M13TG6844 (dsDNA) were digested with SphI and SalI and ligated. The ligation mixture was used to transform E. coli strain BJ5183 to ampicillin resistance (Ap R ). Plasmid DNA was isolated from six Ap R clones, and the PstI restriction profile of each preparation analyzed. Corresponding to the expected restriction profile clone N°l was used for a CsCl purification of pTG6864 (alkaline lysis protocol). Structure of the purified plasmid was verified again by digestion with PstI and SphI+Sall .
  • PTG6864 the yeast rHV2-Phe 3 Gln 33 Tyr 35 Lys 47 Asp 63 production plasmid ( Figure 2) is an E. coli-yeast shuttle vector with the following elements:
  • a bacterial segment which is derived from E. coli plasmid pBR322, harboring a bacterial origin of replication (ori), and the bacterial selection marker for ampicillin resistance (Ap R ) ii. a segment of the yeast 2 micron episome with its origin of replication
  • iii a promoter- and terminator-deleted version of the yeast URA3 gene (URA3-d) serving as a yeast selectable marker iv. a modified version of the yeast MFal promoter v. a sequence coding for a variant form of the yeast BGL2 derived signal peptide serving as a secretion signal fused in frame to
  • yeast PGK gene serving as a transcriptional terminator.
  • pTG6864 confers ampicillin resistance to transformed E. coli cells; and it renders transformed yeast ura3 auxotrophic strains prototrophic for uracil (Ura + ).
  • Plasmid pTG6864 has been used to transform Saccharomyces cerevisiae strain TGY48.1 MAT a ura3 his3 pral prbl prcl cpsl to uracil prototrophy (lithium acetate protocol; 5.5 ⁇ q of plasmid DNA per 1.3 ⁇ 10 cells).
  • TGY48.1 is a haploid strain of mating type a (MAT ⁇ ) with a nonreverting allele of the URA3 gene (ura3- ⁇ 5) as selectable marker. After four days incubation at 30°C three Ura + clones were obtained. Clone N°l was further analyzed.
  • Ura + prototrophy of clone N°l was verified.
  • Cells were centrifuged out, and kinetic assay using the chromogenic substrate, Tos-Gly-Pro-Arg-4-nitroanilide acetate (Chromozym TH, Boehringer Mannheim, Germany).
  • Hirudin production was expressed as the anti-thrombin activity of yeast culture supernatant (ATU/ml) normalized to the A 600 of the culture.
  • Hirudin containing an aryl amine on tyrosine was prepared by nitration followed by reduction (J.F. Riordan and B.L. Vallee. Methods Enzymol. 25 pp. 515-521 (1972)).
  • the nitration reaction was performed at room temperature in 0.01 M sodium phosphate, pH 8.3, by mixing hirudin (0.7 ⁇ 10 -6 M) with a ten-fold molar excess of ethanolic tetranitromethane.
  • the reaction was monitored by absorbance at 428 nanometers for 2 hours, then terminated by gel filtration on Bio-Gel P-6DG pre-equilibrated with the sodium phosphate buffer.
  • Hirudin-Tyr-NO 2 was reduced to hirudin-Tyr-NH 2 by adding a ten-fold molar excess of sodium dithionite in the sodium phosphate buffer and incubating until the nitrophenol absorbance at 428 nanometers disappeared.
  • the aryl amino(tyrosine) hirudin was separated from excess sodium dithionite by desalting on Bio-Gel P-6DG pre-equilibrated with 0.04 M sodium acetate, pH 5.0, concentrated and stored at -20° C.
  • Sulfo-LC-SPDP (Sulfosuccinimidyl 6-[3-(2-pyridyldithio) propionamido] hexanoate) was attached to hirudin containing an aryl amine on tyrosine by the following method: To hirudin-Tyr-NH 2 (0.143 x 10 -6 M) in 0.04 M sodium acetate, pH 5.0, was added a ten-fold molar excess of Sulfo-LC-SPDP and the solution agitated for 2 hours at room temperature.
  • N-Acetyl-homocysteine was attached to hirudin that contained an aryl amine on tyrosine by the following method: Into a solution of hirudin-Tyr-NH 2 (0.143 ⁇ 10 -6 M) in 0.04 M sodium acetate, pH 5.0, was mixed a ten-fold molar excess of N-acetyl-homocysteine thiolactone (AHTL) in methanol and reaction continued for two hours with constant agitation at room temperature.
  • AHTL N-acetyl-homocysteine thiolactone
  • an agarose gel bearing either a long-chain iodoacetyl group (0.5 ml SulfoLink Gel, Pierce) or a maleimide (SulfoSMCC: Sulfosuccinimidyl 4-(maleimidomethyl) cyclohexane-1-carboxylate) was reacted with either of the above hirudin derivatives (1.43 ⁇ 10 -6 M in 0.05 M Tris-HCl, 0.005 M EDTA-Na, pH 8.5) for 1 hour at room temperature.
  • the gel was washed with 0.05 M Tris, 0.005 M EDTA-Na, pH 8.5; incubated with 0.05 M cysteine, 0.05 M Tris, 0.005 M EDTA-Na, pH 8.5 for 1 hour; washed with 1 M NaCl; then equilibrated with physiological saline, pH 7.2.
  • NHS-LC-biotin (Sulfosuccinimidyl-6-(biotinamido)hexanoate) was attached to hirudin containing an aryl amine on tyrosine by the following method: To hirudin-Tyr-NH 2 (0.143 ⁇ 10 -6 M) in 0.04 M sodium acetate, pH 5.0, was added a ten-fold molar excess of NHS-LC-biotin and the solution agitated for 2 hours at room temperature. Excess LC-biotin was removed by desalting on Bio-Gel P-6DG.
  • Hirudin-Spacer Conjugates bv Avidin-Biotin Complexes
  • Hirudin-Tyr-LC-biotin conjugates were bound to soluble avidin, avidin-coated polystyrene beads (Fluoricon particles, Baxter Healthcare) or avidin-coated silicone rubber tubing at a 1:1 molar ratio by incubating in 0.02 M sodium phosphate pH 7.4 for 1 hour at room temperature.
  • thrombin inhibition activity of rHV2 Phe Gln Tyr Lys Asp : (H-Tyr), and its derivatives, including spacer molecules ranging from 200 to 67,000 molecular weight, were determined by incubating them with human thrombin, then measuring the residual thrombin activity as the initial velocity of amidolysis of H-D-Phenylalanyl-L-pipecolyl-L- arginine-p-nitroanalide dihydrochloride (Kabi, S-2238), where zero thrombin activity was 100% inhibition.
  • hirudin-Tyr-PEG adduct Preparation and anti-thrombin activity of hirudin-Tyr-PEG adduct.
  • methoxypolyethylene glycol 5 kD was directly bound to hirudin through an aryl amine on tyrosine by reacting hirudin-Tyr-NH 2 , 0.143 ⁇ 10 -6 M in 0.04 M sodium acetate, pH 5.0, with a fifty-fold molar excess of methoxypolyethylene glycolsuccinimidyl succinate (MPEGSS) for 30 min at room temperature.
  • MPEGSS methoxypolyethylene glycolsuccinimidyl succinate

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Un analogue d'hirudine substitué par tyrosine présente une activité antithrombogène inhabituelle. L'invention se rapporte à plusieurs nouvelles stratégies permettant de coupler l'analogue d'hirudine à des surfaces solides tout en conservant simultanément l'activité antithrombogène.
PCT/US1994/010048 1993-09-07 1994-09-07 Analogues d'hirudine WO1995007295A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU77215/94A AU7721594A (en) 1993-09-07 1994-09-07 Analogs of hirudin

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US11693993A 1993-09-07 1993-09-07
US08/116,939 1993-09-07

Publications (1)

Publication Number Publication Date
WO1995007295A1 true WO1995007295A1 (fr) 1995-03-16

Family

ID=22370169

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/010048 WO1995007295A1 (fr) 1993-09-07 1994-09-07 Analogues d'hirudine

Country Status (2)

Country Link
AU (1) AU7721594A (fr)
WO (1) WO1995007295A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001036613A1 (fr) * 1999-11-17 2001-05-25 Haemosys Gmbh Surfaces de polymere hemocompatibles

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0357242A1 (fr) * 1988-08-03 1990-03-07 New England Deaconess Hospital Corporation Substance bio-compatible et thromboréristante revêtue d'hirudine, des ses analoges ou de ces fragments, ainsi que sa méthode de production
US5053453A (en) * 1988-11-01 1991-10-01 Baxter International Inc. Thromboresistant materials and methods for making same
US5112615A (en) * 1988-08-03 1992-05-12 New England Deaconess Hospital Corporation Soluble hirudin conjugates

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0357242A1 (fr) * 1988-08-03 1990-03-07 New England Deaconess Hospital Corporation Substance bio-compatible et thromboréristante revêtue d'hirudine, des ses analoges ou de ces fragments, ainsi que sa méthode de production
US5112615A (en) * 1988-08-03 1992-05-12 New England Deaconess Hospital Corporation Soluble hirudin conjugates
US5053453A (en) * 1988-11-01 1991-10-01 Baxter International Inc. Thromboresistant materials and methods for making same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CELL, Volume 64, issued 22 March 1991, VU et al., "Molecular Cloning of a Functional Thrombin Receptor Reveals a Novel Proteolytic Mechanism of Receptor Activition", pages 1057-1068. *
NATURE, Volume 353, issued 17 October 1991, VU et al., "Domains Specifying Thrombin-receptor Interaction", pages 674-677. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001036613A1 (fr) * 1999-11-17 2001-05-25 Haemosys Gmbh Surfaces de polymere hemocompatibles

Also Published As

Publication number Publication date
AU7721594A (en) 1995-03-27

Similar Documents

Publication Publication Date Title
CA2077691C (fr) Analogues de thrombomoduline presentant une resistance a l'oxydation
US5550213A (en) Inhibitors of urokinase plasminogen activator
WO1991002753A1 (fr) Proteine anticoagulante
CA2065409C (fr) Polypeptides anticoagulants
GB2199582A (en) Analogues of pancreatic secretory trypsin inhibitor
NZ246567A (en) Variant polypeptides of a human kunitz type protease inhibitor domain and pharmaceutical compositions thereof
CA2072375C (fr) Analogue de l'hirudine, methode de fabrication de celle-ci, composition anticoagulante et vecteur de secretion, microorganismes transformes contenant ledit vecteur et methode de fabrication de produits, produite a partir dudit microorganisme
US5118790A (en) Analogs of hirudin
AU617989B2 (en) Variants of hirudin, their use and their preparation
US5837808A (en) Analogs of hirudin
AU685835B2 (en) High molecular weight desulphatohirudin
US5876971A (en) Thrombin inhibitor from the saliva of protostomia
EP0458903A1 (fr) Analogues solubles de la thrombomoduline
WO1995007295A1 (fr) Analogues d'hirudine
Courtney et al. Production and evaluation of recombinant hirudin
CA2063575A1 (fr) Methode servant a la production par recombinaison d'hirudines et de polypeptides de type hirudine
WO1993004082A1 (fr) Analogues de l'hirudine
Wirsching et al. Display of functional thrombin inhibitor hirudin on the surface of phage M13
Wirsching et al. Modular design of a novel chimeric protein with combined thrombin inhibitory activity and plasminogen-activating potential
EP0710285B1 (fr) Analogues d'un polypeptide antithrombine et leur procede de preparation
Shenoy Development of heparin antagonists for clinical use: the design, synthesis, and testing of multiple-helix heparin-binding peptides
GB2242681A (en) Hirudin fragments

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载