WO1993004082A1

WO1993004082A1 - Analogs of hirudin

Info

Publication number: WO1993004082A1
Application number: PCT/US1992/006910
Authority: WO
Inventors: Cecilia S. L. Ku; Richard Johnson; Julian Breillatt
Original assignee: Baxter International Inc.
Priority date: 1991-08-20
Filing date: 1992-08-17
Publication date: 1993-03-04

Abstract

The present invention provides analogs of hirudin having at least one amino acid in positions 30 to 37 substituted with Tyr and the native Tyr3 and Tyr63or64 residues substituted with a first and a second functional non-reactive amino acid.

Description

ANALOGS OF HIRUDIN

Technical Field

This invention relates to analogs of hirudin and in particular relates to analogs of hirudin which have ant1thrombogenic activity and which can be covalently bound to polymers.

Background Art

Natural hirudin is a mixture of closely related polypeptldes each containing approximately 64 to 66 amino acids and having a molecular weight of approximately 6900. At least 20 natural varlents of hirudin have been Identified. Scharf et al., Febs Letters 255 pp. 105 to 110 (September 1989). It is produced by the European medicinal leech Hirudo medicianlis. It prevents blood from dotting by forming an inhibitory 1:1 molecular complex with activated thrombin (approximately K_iApp=10^-11 to 10^-14M). Hirudin forms a very tight complex with thrombin, wherein over 40% of the hirudin structure intimately contacts the thrombin molecule and covers both the fibrlnogen recognition site of thrombin and the fibrlnogen 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 binding and cleavage of fibrlnogen by thrombin.

It is impractical to prepare natural hirudin in quantities necessary for therapeutic use. At least three recombinant hlrudlns are now available Identical to native variants except that the recomblnants lack the sulphate residue on the tyrosine at position 63 found in native variants. These recomblnant hirudins show pharmacological properties very similar to native hirudin (Markwardt, Sem. Thromb. Hemostas. 15 pp. 269 to 282 (1989)). European Patent Application 87402696.6 shows the amino acid sequence of hirudin variants 1, 2, and 3 (HV1, HV2, and HV3).

Three regions of the hirudin molecule are now believed to be essential to the thrombin-h1rudin high affinity interaction based on X-ray crystallography (Rydel, T. J. et al., Science 249 (1990) pp. 277-280) and structure-activity studies (Krstenansky, J. L. et al., J. Hed. Chem. 30 (1987) pp. 1688 to 1691). First, the three NHj-termlnal amino acid residues at positions 1, 2, and 3 of hirudin form a parallel beta strand with Ser²¹⁴ to Glu²¹⁷ of thrombin and participate in several dozen non-polar interactions with side chains of amino acids in and around the active site of thrombin. Second, the NH₂-terminal amino acid domain of hirudin from approximately Thr⁴ to Pro⁴⁸ is a compactly folded structure composed of four loops stabilized by three dlsulflde bridges and antlparallel beta structures. The main function of this domain is to position and facilitate the interaction of hlrudln's NH₂-terminal trlpeptlde at the thrombin active site. Third, the COOH-termlnal tall of hirudin (Glu⁴⁹-Pro⁶⁰) binds in the an1on-binding exoslte of thrombin and terminates in a hydrophobic helical turn Glu⁶¹-Leu⁶⁴. The exoslte constitutes the fibrlnogen binding recognition site of thrombin and is dominated by numerous polar and non-polar interactions. The presence of hirudin in the exoslte prevents fibrlnogen from being recognized.

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 into contact with blood to provide a nonthrombogenic surface. a. Modifications to Increase Binding Affinity or to Prolong in Vivo Half-Life of Hirudin

PCT Application WO 85/04418 discloses recomblnant 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 recomblnant 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 carboxypeptldase degradation. Arg is placed in the 33 position, Thr or Ser or Asp are placed at position 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 antlthrombogenic activity.

U.S. Patent No. 4,179,337 discloses the attachment of mass-increasing molecules such as polyethylene glycol to proteins.

Lazar, J. B. et al. describe mutations at position 3 in recomblnant hirudin variant 1 (rHVl) where antlthrombin activity was increased by replacing Tyr with Phe or Trp, and markedly decreased by replacement with Thr (J. B. Lazar, R. C. Wlnant, and

P. H. Johnson, J. Biol. Chem. 266, pp. 685-688 (1991)).

Johnson, P. H. et al. in "Biochemistry and Genetic Engineering of Hirudin," Seminars in Thrombosis and Homeostasls, Volume 15, No. 13 (1989) at pp. 309 describes hirudin fragments having antlthrombogenic activity. These fragments correspond to residues

42 to 65 and 51 to 65. European Patent Application No. 89810522.6 describes mutations at positions 1, 27, 36, 47, 48, 49, 51, and 52. The substitution at 36 is Lys, Arg, Asn, Val, Leu or Gin. The substitution at 27 is Gln, Asn, Leu, Arg or Val. The substitution at 49 is Asn or Met. The substitution at 47 is Arg, Asn, Val or Leu.

European Application No. 89810676.0 describes mutations at positions 1, 2, 27, 36, 47, 57, 58, 61, 62, and 66 where the position 27 substitution is Gln, position 36 substitution is Gln and position 47 substitution is Arg.

The following references disclose modifications at the C-termlnal and N-terminal ends of the hirudin molecule: European Patent No. 142860; U.S. Patent No. 4,801,576; U.S. Patent No. 4,745,177; U.S. Patent No. 4,767,742, and European Application No. 86102462.8.

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.

b. Immobilization of Hirudin on Surfaces

European Application No. 89311022.1, European Application No.

89307922.8 to Ito, and references cited therein disclose the attachment of hirudin to surfaces. The data disclosed in the Ito application shows substantial loss of antlthrombogenic activity occurred when the molecule was immobilized on the surface.

c. Fragments

Various COOH-terminal polypeptlde fragments of hirudin are known to bind to thrombin, thereby inhibiting the binding and cleavage of fibrlnogen by thrombin. The minimum length polypeptlde required to exert inhibitory activity has been reported as Phe⁵⁶-Gln⁶⁵ (J. L. Krstenansky, T. J. Owen, M. T. Yates, and S. J. T. Mao, J. Med. Chem. 30, pp. 1688-1691 (1987)). Addition of amino acid residues to this polypeptlde to increase its length and provide the amino acid sequences found in the several hirudin variants up to and including the complete sequences Glu⁴⁹-Gln⁶⁵ augments the thrombin inhibitory activity of the fragments; and, the sequence may be extended to include Gly⁴²-Gln⁶⁵ without compromising the efficacy of the inhibitor. Moreover, the deletion of Gln⁶⁵ from the polypeptldes of these series provides an analogous series of useful thrombin inhibitory peptldes.

in a further extension of this reasoning, numerous synthetic variations of the polypeptlde sequences found in the natural hirudin variants (pept1dom1met1c analogs of hirudin peptldes) have been prepared and found to possess thrombin inhibitory activity. Notable among them are those described in European Patent Application No. 89302159.2 and others utilizing non-protein amino acids (European Patent Application No. 89122451.1).

Maraganore, J. M. et al. in an abstract presented at a symposium entitled HB1omed1cal Horizons of the Leech" on October 24 - 28, 1990 disclose synthetic peptldes which bind to both the anlon binding exoslte and the active site. The peptldes are called "hlrulogs" and consist of (i) an active-site specificity sequence, (ii) a poly-gly linker, and (iii) an anlon binding exoslte recognition sequence.

The principal hlrulog is: (D-Phe)-Pro-Arg-Pro-(Gly)_4-8-Asn- Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu in which the dodecapeptlde Asn...Leu mimics the hirudin peptlde Asn⁵³-Gln⁶⁵ of HV2 and binds to the fibrlnogen recognition site on thrombin. The pentapeptlde (D-Phe)-Pro-Arg-Pro-Gly binds in the active site of thrombin, and the oligo-Gly serves to connect the two binding moieties (J. M. Maraganore, P. Bourdon, J. Jablonski, K. L. Ramachandran, and J. W. Fenton, II, Blochem. 29, pp.7095-7101 [1990]). There are many variations possible on this model of bivalent thrombin inhibitors (and in general, bivalent protease inhibitors. For example, see J. DiMaio, B. Gibbs, D. Munn, J. Lefebvre, F. Ni, and Y. Konishi, J. Boil. Chem. 265 pp. 21698 to 21703 (1990).

European Application No. 89302160.0 discloses peptldes of about 8 to 26 amino acids having the biological activity of hirudin.

European Application No. 89302159.2 discloses cycllclzed synthetic fragments of hirudin having antlthrombogenic activity.

It is a primary object of this invention to provide antlthrombogenic hirudin analogs having amino acids available for attachment of spacer molecules. The analogs can be bound to a surface via a spacer molecule rendering the surface to nonthrombogenlc. Alternatively, the analogs can be bound to a polymer via a spacer molecule to increase the analogs' in vivo half life. It is a further object of this invention to provide nonthrombogenlc materials comprising such analogs attached to surfaces. It is a further object of this invention to provide analogs attached to polymers. Summary of the Invention

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 Tyr³ and Tyr^63or64 residues substituted with a first and a second functional nonreactlve 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 Tyr³ substituted with Phe, lle or Leu, and Tyr^63or64 substituted with Asp or Glu.

As used herein the term analog includes fragments and analogs of hirudin wherein a tyroslne residue is attached to the NH₂ terminal position of such analogs, and the residue equivalent to Tyr⁶³, when present, is substituted with either Glu or Asp.

As used herein the term analog includes peptidomimetic analogs of hirudin which are bivalent inhibitors of thrombin, where a tyroslne residue is substituted in or near the ollgomer that links the COOH-terminal hirudin peptlde mimic to the peptide that binds in the active site of thrombin, and where a functional nonreacti ve amino acid, preferably Asp or Glu, replaces the residue equivalent to Tyr⁶³.

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 of the analog.

The present invention further provides Applicants' novel analogs attached to surfaces rendering such surfaces nonthrombogenic.

The present invention further provides Applicants' novel analogs attached to mass-increasing molecules which will have a prolonged half life in vivo.

Detailed Description of the Invention

The present invention provides analogs of hirudin having a 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 hirudln-thrombin interface and contains at its tip the sequence Leu³⁰ Gly³¹ Ser³² Asn³³ Gly³⁴ Lys³⁵ Gly³⁶ Asn³⁷ (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. 277-280 (1991). in accordance with the present invention 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. Alternatively, in accordance with the present invention, 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 antlcoagulatlve effect in the circulation when administered therapeutlcally.

To avoid reaction of the spacer molecules or mass-increasing molecule with Tyr in positions outside the finger region, where attachment to a molecule might destroy antlthrombogenic activity, Tyr residues outside the finer region are substituted with amino acids that will not react with the molecule. As used herein the term "nonreactlve" shall mean an amino acid which, due to its nature and/or its position within the analog, will not form a covalent bond with certain mass-increasing and spacer molecules described below. As used herein the term "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 antlthrombogenic 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 antlthrombogenic activity of the analog. X-ray crystallographlc studies have shown that in a hirudln-thrombin complex, Tyr³ of hirudin occupies a hydrophobic cleft adjacent to the active site of thrombin that is occupied in a similar manner by the Phe residue of the thrombin inhibitor PPACK (Phe-Pro-Arg-chloromethylketone) (Rydel, et al., op. cit.). This suggests that substitution of Phe for Tyr³ would not significantly reduce the antlthrombogenic activity of the analog. Accordingly, in the analog of the present invention, the functional Tyr³ and Tyr⁶³ are replaced with functional yet nonreactlve amino acids to prevent reaction of the spacer or mass-increasing molecule at the functional positions. The preferred functional nonreactlve amino acids for position 3 in hirudin are Phe, Leu, and lle. The preferred functional, nonreactlve amino acids for position 63 or 64 in hirudin are Asp and Glu. When both the finger region substitution and these terminal substitutions are made the result is an analog having Tyr available for reaction with a spacer or mass-increasing molecule in the nonfunctional finger region and functional yet nonreactlve amino acids in positions 3 and 63 or 64.

in a preferred embodiment Tyr is placed at position 35 in hirudin. Tyr may also preferably be placed at position 33.

As disclosed in European Application No. 87402696.6 in 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 reactive Tyr available for reaction. The inclusion of 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 use of too many of the disclosed Tyr substitutions in the analog will impair its usefulness in chemical attachment to surfaces 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 recomblnant DNA techniques known to those skilled in the art for example, by subjecting the gene coding for the hirudin to site specific mutagenlsls and expressing the mutated gene in a suitable host such 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 using 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 Gelgy 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 Application Nos. 87401649.6 and 89400621.2.

Those skilled in the art will understand that fragments of the hirudin molecule, COOH-terminal polypeptlde fragments, peptidomimetic analogs, and bivalent inhibitors (as described above in the section entitled "Fragements") may also be modified according 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 antithrombln activity of the "fragment", and further provided that the functional groups on the "fragment" necessary for antlthrombogenic activity are either nonreactlve or can be substituted with a functional nonreactlve amino acid. Fragments or peptldes having such activity and modifications thereof fall within the intent and scope of the present invention. Accordingly, the term analog as used herein shall include a fragment of the hirudin molecule, peptldomimetic analogs, and bivalent inhibitors having antlthrombogenic activity. The term analog as used herein shall also include a synthetic peptide having antlthrombogenic activity by virtue of an amino acid sequence analogous to that of the functional protein of the native hirudin molecule.

For example, the NH2-terminal end of the hirudin peptldes and peptldomimetic 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 Homeostasis, 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). Attachment of a Tyr residue to the NH₂-terminal end or equivalent region of these various inhibitory polypeptldes, and peptldomimetic analogs, accompanied by substitution of the Tyr⁶³ equivalent residue by Asp or glu, provides a unique attachment site for an immobilizing spacer molecule or mass-increasing molecule according to the methods of this invention.

The bivalent thrombin inhibitors described by Maraganore et al., Blochem. 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 tyroslne residue inserted in or near the oligoglycine connecting link that joins the active site binding moiety with the longer peptlde that binds in the fibrinogen recognition site provides a unique site for attaching a spacer, when in accordance with this invention, the Tyr⁶³ 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 an amino group into the aromatic ring. Many reagents developed for derivatization and immobilization of proteins are designed to react with primary amino groups. However, the antlthrombin activity of hirudin is significantly impaired by attachment of spacer molecules to its NH₂-terminal amine or to certain of its lysyl epsllon-amino groups. An aryl amine on Tyr avoids these drawbacks by reacting with amine reactive agents under conditions that virtually exclude alkyl amine, e.g., Lys, reactivity. An aryl amine of Tyr has a pK_a of about 4.8, I.e., 1t is 50% protonated at pH 4.8. Since the protonated form of a primary amine is unreactive to the commonly used spacer attachment chemistries, the tyrosyl amine residue can react effectively at pH 5.0. However, at pH 5.0, less than 0.1% of the a-amine of the NH₂-terminal residue (pK_a about 8.0), and less than 0.001% of the epsllon-amine of Lys (pK_a about 10.0) would be reactive with such spacers. Chemical methods that insert the aryl amine into Tyr may also react with Trp and free Cys; however, hirudin has no Trp residues and all 6 Cys residues are engaged in unreactlve dlsulfide bridges. Therefore, specific reactivity with Tyr is obtained.

A less preferred means to attach spacers to Tyr uses spacers activated with diazonlum 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 hlrudin'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, benzoqulnones, vinyl sulfones, hydrazldes and imidazolyl carbonyls. Typically such spacer molecules are blfunctional, wherein one end of the spacer contains an amine-reactive chemical mciety, while the other end contains the same or a different reactive species for attachment to the surface.

Whereas the spacer molecule is covalently bound to hirudin or its analogs in accordance with the present invention, 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 non-thrombogenic 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 the surface. Alternately the hlrudin-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. Examples of such reactive pairs include, but are not limited to, thiol-maleimide, thlol- iodoacetate, and hydrazide-aldehyde (from oxidized sugar cis-diols). Alternately, the hirudin analog and its spacer may be attached to the surface by noncovalent binding means, which may include, for example, those that operate predominantly by hydrophobic binding mechanisms, or by fl uorophi lic 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 fluorophllic moiety that will bind directly to a similarly hydrophobic or fluorophllic surface. Or, the said spacer may have at its free end a chemical mciety 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. in another embodiment, the hlrudin-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. Or, where the high affinity receptor molecule has multiple binding sites for its Ugand, the receptor molecule may be attached to the surface by binding to one of its specific Hgands 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 attched to materials which are useful in the production and use of medical products, systems and devices. Such 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, include homo- and co-polymers derived from one or more, of the following: 1-olefins, such as ethylene, propylene, tetrafluoroethylene, hexafluoro- propyiene, 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.; saccharldes, such as glucose, glucosamine, guluronic acid, sulfated glycoseaminoglycans, agarose, alginic acid, etc.; siloxanes, such as dimethyl siloxane, 3-aminopropyl siloxane, etc. Useful polymers include biodegradable, partially biodegradable and non-biodegradable polymers. Other useful materials are 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.

It is understood that the mechanisms described for attachment of hirudin and its analogs to surfaces in accordance with the present invention are equally applicable to their attachment to mass-increasing molecules for the purpose of prolonging their in vivo half-life. Examples of such mass-increasing molecules include polymers such as polyethylene oxide, polypropylene oxide, polyvinylpyrrolidone or the polyglucoses; and macromolecules including polypeptldes such as serum albumin and gelatin, and polysaccharides such as heparin, or hydroxyethyl starch.

EXAMPLE 1

HV2 has Tyr at positions 3 and 63. In accordance with the present invention the reactive functional Tyr at position 3 is replaced with nonreactive functional Phe, and the reactive functional Tyr at 63 is replaced with nonreactlve functional Asp. These replacements do not eliminate the thrombin binding activity of the molecule. Reactive nonfucntlonal Tyr is then substituted for the nonreactlve 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 acti vity of the antithrombogenic analog.

The hirudin analog HV2 Phe3 Gln33 Tyr35 Lys47 Asp63 was prepared by the following methods: A. Starting Material

Starting material was phage M13TG4892. This phage (a derivative of Ml3TGl 3l ) contains an expression block consisting of:

(a) a slightly modified version of the MF α al promoter (the 5' EcoRI site was converted to a SphI site, and the internal

Bglll site was destroyed by a treatment with the Klenow fragment of E. coll DNA polymerase I);

(b) a variant of the yeast BGL2 signal peptide (BGL2-Val⁷); and

(c) the rHV2-Lys⁴⁷ coding sequence.

B. rHV2-Lys⁴⁷Asp⁶³

By site directed mutagenesis (oligonucleotide OTG2942; Amersham site directed mutagenesis kit #RNP 1523) on single stranded DNA (ssDNA) of Ml3TG4892 the codon TAT (Tyr⁶³) was exchanged for GAC (Asp) resulting in Ml3TG5884. The mutation was verified by sequencing the entire hirudin coding sequence (sequencing primer: OTG2387). rHV2-Lys⁴⁷Asp⁶³ is encoded in Ml3TG5884.

C. rHV2-Phe³Gln³³Tyr³⁵Lys⁴⁷Asp⁶³

By site directed mutagenesis (oligonucleotides OTG2993:Tyr³—> Phe and OTG2994:Asn³³—>Gln, Lys³⁵—>Tyr) on ssDNA of Ml3TG5884 the condons TAT (Tyr³) were exchanged for TTC (Phe), AAT (Asn³³) for CAA (Gln), and AAG (Lys³⁵) for TAC (Tyr) at the same time resulting in M13TG6844. The mutations were verified by sequencing the entire hirudin coding sequence (Figure 1). rHV2-Phe³Gln³³Tyr³⁵ Lys⁴⁷Asp⁶³ is encoded in M13TG6844.

D. Assembly of the Expression Vector pTG6864

The yeast basic expression vector pTG3828 (pBR322, 2 micron, URA3-d, PGKl transc ri pti onal terminator) was used to assemble the expression plasmid. Vector pTG3828 and M13TG6844 (dsDNA) were digested with SphI and Sall 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 was 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 Sphl+Sall.

PTG6864: the yeast rHV2-Phe³Gln³³Tyr³⁵Lys⁴⁷Asp⁶³ production plasmid (Figure 2) is an E. coli-yeast shuttle vector with the following elements:

i. a bacterial segment, which is derived from E. coli plasmid pBR322, harboring a bacterial origin of replication (ori), and the bacterial selection marker for amplcillin resistance (Ap^R);

ii. a segment of the yeast 2 micron eplsome 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 MRαal promoter; v. a sequence coding for a variant form of the yeast BGL2 derived signal peptlde serving as a secretion signal fused in frame to;

vi. the rHV2-Phe³Gln³³Tyr³⁵Lys⁴⁷Asp⁶³ coding sequence; and vii. a segment of the yeast PGK gene serving as a transcriptional terminator.

Thus, pTG6864 confers ampicillin resistance to transformed E. coll cells; and it renders transformed yeast ura3 auxotrophic strains prototrophlc for uracil (Ura+).

The DNA sequence encoding rHV2-Phe³Gln³³Tyr³⁵Lys⁴⁷Asp⁶³ (Figure 1) has been verified after site directed mutagenesis, and is contained in M13TG6844 and pTG6864, respectively.

E. Transformation of yeast

Plasmid pTG6864 has been used to transform Saccharomyces cerevlslae strain MATα ura3 251-373, 328, Leu2-3, -112, his3, pep4-3 to uracil prototrophy (lithium acetate protocol; 5.5 ug of plasmid DNA per 1.3 x 10⁸ cells). This is a haplold strain of mating type α (MATα ) with a nonrevertlng allele of the URA3 gene (ura3- Δ 5) as selectable marker. After four days' incubation at 30°C three Ura+ clones were obtained. Clone N°l was further analyzed.

F. Hirudin production

Ura+ prototrophy of clone N°l was verified. Clone N°l was grown at 30°C for 48 hours to a final cell density (measured as absorbance at 600 nm, where A₆₀₀ = 1 corresponds to 10⁷ cells/mL) of 10 to 12. Cells were centrifuged out, and culture supernatants were tested for thrombin inhibition in a kinetic assay using the chromogenic substrate, Tos-Gly-Pro-Arg-4-nitroanilide acetate (Chromozym TH, Boehringer Mannheim, Germany). Hirudin production is the anti-thrombin activity of yeast culture supernatant (ATU/mL) normalized to the A₆₀₀ or the culture.

EXAMPLE 2

Once an analog is prepared having a nonfunctional reactive Tyr available, known methods may be employed to bind that Tyr to a spacer.

Hirudin containing an aryl amine on tyroslne 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 x 10^-6M) with a ten-fold molar excess of ethanollc tetranitromethane. The reaction was monitored by absorbance at 428 nanometers for 2 hours, then terminated by gel filtration on on Bio-Gel P-6DG pre-equilibrated with the sodium phosphate buffer. Hirudin-Tyr-NO₂ was reduced to Hirudin-Tyr-NH₂ by adding a ten-fold molar excess of sodium dithionite in the sodium phosphate buffer and incubating until the nitrophenol absorbance at 428 nanmeters 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.

EXAMPLE 3

Attachment of SPDP to Hirudin-Tyr-NH₂

Attachment of Sulfo-LC-SPDP to Hirudin-Tyr-NH₂ 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-NH₂ (0.143 x 1-^-6M) 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 two hours at room temperature. Excess Sulfo-LC-SPDP was removed by desalting on B1o-Gel P-6DG pre-equi librated with 0.01 M sodium phosphate, pH 7.0, and the SPDP-hirudin concentrated and stored at -20°C. The LC-SPDP-hirudin was incubated before use with a 3-fold molar excess of dithiothreitol in 0.04 M sodium acetate, 0.005 M EDTA, pH 4.5, until absorbance at 343 nanmeters reached a plateau, then the LC-SPDP-hirudin was purified by desalting on Bio-Gel P-6DG. EXAMPLE 4

Binding N-Acetyl-homocysteine to Hirudin-Tyr-NH₂

N-Acetyl-homocysteine was attached to hirudin that contained an aryl amine on tyrosine by the following method: Into a solution of hirudin Tyr-NH₂ (0.143 x 1-^-6M) in 0.04 M sodium acetate, pH 5.0, was mixed a ten-fold molar excess of N-acetyl-homocysteine thlolactone (AHTL) in methanol and reaction continued for two hours with constant agitation at room temperature. Excess AHTL was removed by desalting on Bio-Gel P6-DG pre-equilibrated with 0.01 M sodium phosphate, pH 7.0, and the N-acetyl-homocysteinyl hirudin concentrated and stored at -20°C. All reactions were performed under stream of nitrogen gas. EXAMPLE 5

Surface Immobilization of Hirudin-Spacer Conjugates by Thiol Ethers Sulfo-LC-SPDP or N-Acetyl-homocysteine-hirudin was attached to surfaces by formation of a thlol ether bond. In brief an agarose gel bearing either a long-chain iodoacetyl group (0.5 ml SulfoLink Gel, Pierce) or a malelmide (SulfoSMCC: Sulfosuccinimidyl 4-(maleimidomethyl) cyclohexane-12-carboxylate) was reacted with either of the above hirudin derivatives (1.43 x 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-HCl, 0.005 M EDTA-Na, pH 8.5; incubated with 0.05 M cystelne, 0.05 M Tris-HCl, 0.005 M EDTA-Na, pH 8.5 for 1 hour; washed with 1 M NaCl; then equilibrated with physiological saline, pH 7.2.

EXAMPLE 6

Binding of NHS-LC-Biotin to Hirudin-Tyr-NH₂

NHS-LC-Biotin (Sulfosuccinimidyl-6-(biotinamido) hexanoate) was attached to hirudin containing an aryl amine on tyrosine by the following method: To hirudin-Tyr-NH₂ (0.143 x 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.

EXAMPLE 7

Attachment of Hirudin-Spacer Conjugates by Avidin-Biotin Complexes Hirudin-Tyr-NH2-LC-biotin conjugates were bound to soluble avldin, avidin-coated polystyrene beads (Fluorlcon 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.

EXAMPLE 8

Anti-Thrombin Activity of Attached Hirudin Analogs

The thrombin inhibitory activity of rHV2 Phe³ Gln³³ Tyr³⁵ Lys⁴⁷ Asp⁶³: (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 amldolysls of H-D-Phenylalanyl-L-pipecolyl-L-arginine- p-nitroanilide dlhydro- chloride (Kabi, S-2238), where zero thrombin activity was 100% inhibition. Aliquots containing 0.13 - 1.3 plcomoles of hirudin as (a) the analog, H-Tyr, (b) the analog with an aryl amine on Tyr³⁵: (H-Tyr-NH₂), (c) the analog with LC-biotin attached to the tyrosyl amine: (H-Tyr-B), (d) the biotlnylated analog attached to soluble avldln: (H-Tyr-B-Av), and (e) the biotlnylated analog attached to avidin-coated beads: (H-Tyr-B-Av-Beads); (f) the analog with N-acetyl-homocysteine attached to the tyrosyl amine and to SulfoLink Gel (H-Tyr-AH-SLG); (g) the analog with LC-SPDP attached to the tyrosyl amine and to SulfoLink Gel (H-Tyr-PDP-SLG); were reacted with 1.3 plcomoles of human a-thrombin in 0.05 M Tris-HCl, pH 7.4, 0.1% bovine serum albumin, for 1 hour at 22°C; then centrlfuged. Aliquots of the supernatants were diluted with the Tris-BSA buffer and the reaction initiated by addition of S-2238. Reaction velocities were monitored at 410 nanmeters and the quantity of hirudin analog and its derivatives in each assay was determined by radioiodinated hirudin analog tracer. The analog H-Tyr retained virtually all of its specific thrombin inhibitory activity during insertion of an amine into the tyrosine ring, attachment of LC-blotln, and when bound to soluble avidin or to avidin-coated beads. However, when the analog was attached to a surface via the shorter spacer molecules, diminished thrombin inhibitory activity was observed (Table I).

While the foregoing has been decrlbed with respect to preferred embodiments and alternatives thereto one skilled in the art should realize that modifications and changes may be made to the foregoing while stm falling within the spirit, intent, and scope of the present invention. All such modifications and changes are considered a part of this invention.

Claims

IN THE CLAIMS:

1. An analog of hirudin having:

(a) at least one amino acid in positions 30 to 37 substituted with Tyr; and

(b) native Tyr at 3 and 63 or 64 substituted respectively with a first and a second functional non-reactive amino acid.

2. An analog of hirudin variant type 2 having:

(a) at least one amino acid in positions 30 to 37 substituted with Tyr;

(b) native Tyr at 3 and 63 substituted respectively with a first and a second functional non-reactive amino acid; and

(c) Asn at 47 substituted with Lys.

3. The analog of Claims 1 and 2 wherein the first and second functional non-reactive amino acid is Phe, He or Leu and the second functional non-reactive amino acid is Glu or Asp. 4. The analog of Claims 1, 2, and 3 attached to a mass-increasing molecule.

5. The analog of Claims 1, 2, and 3 attached via a spacer molecule to a surface functional group.

6. The analog of Claim 5 where the surface functional group is amine, carboxyl, amino-tyrosyl, hydroxyl, mercury chloride, iodoacetyl, malelmide, sulfhydryl, aldehyde, sulfonyl halide or imidazolyl carbonyl; or a hydrophobic, a fluorophi lic or a high affinity ligand receptor binding site.

7. The analog of Claim 5 where the spacer molecule is biotinavidin, Sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido, hexanoate, homocysteme-thiolactone/sulfosuccinimidyl (4-maleimidomethyl) cyclohexane-1-carboxylate; or an alkyl or alkenyl chain interspersed with nitrogen, oxygen, sulfur or phosphorus heteroatoms and having a molecular weight from about 200 to 20,000 daltons.

8. The analog of Claim 4 where the mass-increasing molecule is agarose, cellulose and its derivatives, polyvinylchloride, polyurethanes, polyolefins, polycarbonates, silicones, polysulfones, acrylates, polystyrene or copolymers thereof; or polyethylene oxide, polypropylene oxide, polyvinylpyrrolidone, polyglucoses, polypeptides such as serum albumin and gelatin, or polysaccharides such as heparin and hydroxyethyl starch. 9. An analog of hirudin variant 2 containing the following substitutions in its amino acid sequence: Phe³Gln³³Tyr³⁵Lys⁴⁷Asp⁶³.

10. Peptidomimetic analogs of hirudin having:

(a) a thrombin binding activity analogous to a thrombin binding activity of hirudin;

(b) having an amino acid sequence functionally analogous to the position 42 to 65 region of native hirudin;

(c) having at least one amino acid in said sequence substituted with Tyr;

(d) having an amino acid residue functionally analogous to the Tyr residue at position 63 or 64 of native hirudin; and

(e) having said amino acid residue substituted with a functional non-reactive amino acid. 11. The analog of Claim 10 where said functional non-reactive amino acid is Asp or Glu. AMENDED CLAIMS

[received by the International Bureau on 18 January 1993 (18.01.93); original claims 1-11 amended; new claims 12-28 added (5 pages)]

1. An antithrombogenic molecule comprising an analog of a natural variant of hirudin having an amino acid sequence analogous to Sequence ID No 3, wherein;

(a) at least one amino acid in positions 30 to 37 is Tyr,

(b) position 3 is a first functional non-reactive amino acid, and

(c) position 63 or 64 is a second functional non- reactive amino acid.

2. An antithrombogenic molecule comprising an analog of hirudin variant type 2 , wherein;

(a) at least one amino acid in positions 30 to 37 is Tyr,

(b) position 3 is a first functional non-reactive amino acid, (c) position 63 is a second functional non- reactive amino acid, and

(d) position 47 is Lys.

3. The antithrombogenic molecule of claim 1 or claim 2 wherein the first functional non-reactive amino acid is Phe, lie or Leu, and the second functional non-reactive aminό acid is Glu or Asp.

4. The antithrombogenic molecule of claim l or claim 2 wherein said Tyr in positions 30 to 37 is attached via an aryl amine to a mass increasing molecule.

5. The antithrombogenic molecule of claim 1 or claim 2 wherein said Tyr in positions 30 to 37 is attached via an aryl amine and a spacer molecule to a surface functional group.

6. The antithrombogenic molecule of claim 5 wherein the surface functional group is amine, carboxyl, amino- tyrosyl, hydroxyl, mercury chloride, iodoacetyl,

maleimide, sulfhydryl, aldehyde, sulfonyl halide, or imidazolyl carbonyl.

7. The antithrombogenic molecule of claim 5 wherein the surface functional group is a hydrophobic binding site or a ligand receptor binding site.

8. The antithrombogenic molecule of claim 7 wherein the surface functional group is a ligand receptor binding site having an affinity constant for said ligand of at least about 5 x 10⁴ liters/mole.

9. The antithrombogenic molecule of claim 5 wherein the spacer molecule is avidin, sulfosuccinimidyl 6-(3-(2- pyridyldithio) propionamido) hexanoate, or homocysteine- thiolactone/sulfosuccinimidyl (4-maleimidomethyl)

cyclohexane-1-carboxylate.

10. The antithrombogenic molecule of claim 5 wherein the spacer molecule has a molecular weight of 1000 to 200,000 daltons.

11. The antithrombogenic molecule of claim 4 wherein the mass-increasing molecule is a blood compatible polymer selected from the group consisting of

polyvinylpyrrolidone, polyethyloxazoline, polyethers, polysaccharides and polypeptides.

12. The antithrombogenic molecule of claim 11 wherein the mass-increasing molecule is a polyether, said polyether being polyethylene oxide or polypropylene oxide.

13. The antithrombogenic molecule of claim 11 wherein the mass-increasing molecule is a polysaccharide, said polysaccharide being heparin or hydroxyethyl starch.

14. The antithrombogenic molecule of claim 11 wherein the mass-increasing molecule is a polypeptide, said

polypeptide being either serum albumin or gelatin.

15. An analog of hirudin variant 2 having the amino acid sequence of SEQ ID NO:3.

16. A peptidomimetic analog of hirudin comprising an amino acid sequence analogous to positions 42 to 65 of Sequence ID No 3, wherein;

(a) at least one amino acid within positions 42 to 55 is substituted with Tyr,

(b) position 63 or 64 is a functional, non-reactive amino acid, and

(c) said analog binds to and inhibits the activity of thrombin.

17. The analog of Claim 16 wherein said functional, non- reactive amino acid is Asp or Glu.

18. The analog of claim 16 or claim 17 wherein said Tyr in positions 42 to 55 is attached via an aryl amine to a mass increasing molecule.

19. The analog of claim 18 wherein the mass-increasing molecule is a blood compatible polymer selected from the group consisting of polyvinlylpyrrolidone,

polyethyloxazoline, polyethers, polysaccharides and polypeptides.

20. The analog of claim 19 wherein the mass-increasing molecule is a polyether, said polyether being

polyethylene oxide or polypropylene oxide.

21. The analog of claim 19 wherein the mass-increasing molecule is a polysaccharide, said polysaccharide being heparin or hydroxyethyl starch.

22. The analog of claim 19 wherein the mass-increasing molecule is a polypeptide, said polypeptide being serum albumin or gelatin.

23. The analog of claim 16 or claim 17 wherein said Tyr in positions 42 to 55 is attached via an aryl amine and a spacer molecule to a surface functional group.

24. The analog of Claim 23 wherein the surface functional group is amine, carboxyl, amino-tyrosyl, hydroxyl, mercury chloride, iodoacetyl, maleimide, sulfhydryl, aldyhyde, sulfonyl halide, or imldazolyl carbonyl.

25. The analog of claim 23 wherein the surface functional group is a hydrophobic binding site or a ligand receptor binding site.

26. The analog of claim 25 wherein the surface functional group is a ligand receptor binding site having an

affinity constant for said ligand of at least about 5 x 10⁴ liters/mole.

27. The analog of claim 16 or claim 17 wherein the spacer molecule is avidin, sulfocuccininmidyl 6-(3-(2- pyridyldithio) propionamido) hexanoate, or homocysteine- thiolactone/sulfosuccinimydl (4-maleimidomethyl)

cyclohexane-1-carboxylate.

28. The analog of claim 16 or claim 17 wherein the spacer molecule has a molecular weight of 1000 to 200,000 daltons.