Link:  Pharm/Biotech Resources

Title:  Antithrombin nucleotides and proteins from horn fly

United States Patent:  6,927,279

Issued:  August 9, 2005

Inventors:  Cupp; Eddie Wayne (Auburn, AL); Cupp; Mary Smith (Auburn, AL)

Assignee:  Auburn University (Auburn, AL)

Appl. No.:  200659

Filed:  July 22, 2002

Compositions and methods for preventing hematophagous infestation of cattle are provided, directed at isolated proteins with antithrombin activity and nucleotide sequences encoding the proteins. The protein named thrombostasin is isolated from the salivary glands of Haematobia irritans. The compositions are useful as veterinary vaccines in prevention of blood-feeding in cattle by the infesting horn fly. The proteins of the invention are also useful in treatment of thrombosis.

SUMMARY OF THE INVENTION

Isolated proteins with antithrombin activity and nucleotide sequences encoding the proteins are provided. The protein named thrombostasin is isolated from the salivary glands of Haematobia irritans, the blood-feeding horn fly. The provided proteins and nucleotides are particularly useful as veterinary vaccines in prevention of blood-feeding in cattle by the infesting horn fly.

The proteins of the invention are also useful in treatment of thrombosis.

Methods of administering the proteins and nucleotide sequences of the invention are also provided.

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions for preventing hematophagy (blood-feeding) in cattle, and treatment of thrombosis in a mammal are provided. The compositions comprise protein from the salivary gland of the hematophagous horn fly Haematobia irritans which, as described in Yeates et al. (1999) Annu. Rev. Entemol. 44: 397-428, belong to the suborder Cyclorrhapha of the order Diptera. Nucleotide sequences encoding the antithrombin protein are additionally provided. The protein has been designated thrombostasin. The major function of the protein is to prevent coagulation by inhibiting the activity of thrombin (factor II).

By "hematophagy" is intended feeding on the blood of a host organism by another organism. By "hematophagous infestation" is intended a host-parasite relationship comprising feeding on the blood of the host by the parasite. By "thrombosis" is intended the formation, development or presence of a thrombus. By "antithrombin activity" is intended a biological activity that reduces or eliminates the procoagulant action of thrombin; and/or inhibits thrombosis.

It is recognized that methods are available in the art to obtain the complete coding sequence for the antithrombin protein of the invention. Such methods are disclosed for example in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

Substantially purified preparations of thrombostasin are provided. Such substantially purified preparations include proteins substantially free of any compound normally associated with the protein in its natural state. Such proteins can be assessed for purity by SDS-PAGE, chromatography, electrophoresis or other methods. See, M. P. Deutscher (ed.), Guide to Protein Purification, Academic Press, Inc. (1990).

The terms "substantially pure" or "substantially purified" are not meant to exclude artificial or synthetic mixtures of the protein with other compounds. It is recognized that the antithrombin proteins of the present invention include those proteins homologous to, and having essentially the same biological properties as, the antithrombin protein described herein, and particularly the protein disclosed herein in SEQ ID NO: 2, SEQ ID NO:5, or SEQ ID NO:7. This definition is intended to encompass natural allelic variations in the genes. It is also recognized that "substantially purified" proteins of the present invention as described herein can be of other species of origin, including but not limited to other species of the suborder Cyclorrhapha.

The invention also provides fragments of the antithrombin protein and nucleotide sequence disclosed in SEQ ID NOs: 1, 2, 4, 5, 6, and 7. Fragments of the protein may range in size from at least 10, 20, 30 or more amino acids. Such fragments may retain biological activity or comprise active regions of the protein.

Polynucleotide fragments may also range in size from at least 15, 20, 30 or more contiguous nucleotides. The sequences find use as hybridization process or molecular markers.

Such fragments can be readily made by chemical methods including commercially available automated methods or by recombinant DNA methods known to the ordinarily skilled artisan, and described below. It is recognized that biological functions of anti-hemostasis, including those related to antithrombin anticoagulant activity and/or modulation of immune response may be carried out by the described fragments.

The invention additionally encompasses the nucleotide sequences which encode the proteins of the invention. The nucleotide sequence of the PCR-cloned coding sequence from H. irritans is provided in SEQ ID NO: 1; however, it is recognized that cloned genes of the present invention can be of other species of origin, including but not limited to other species of the suborder Cyclorrhapha.

DNAs which hybridize to the nucleotide sequence of the antithrombin gene from the horn fly are also an aspect of this invention. Conditions, which will permit other DNAs to hybridize to the DNA disclosed herein, can be determined in accordance with known techniques. For example, hybridization of such sequences may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 35-40% Formamide with 5× Denhardt's solution, 0.5% SDS and 1×SSPE at 37° C.; conditions represented by a wash stringency of 40-45% Formamide with 5×Denhardt's solution, 0.5% SDS, and 1× SSPE at 42EC; and conditions represented by a wash stringency of 50% Formamide with 5× Denhardt's solution, 0.5% SDS and 1×SSPE at 42EC, respectively, to DNA encoding the genes disclosed herein in a standard hybridization assay. See J. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2^nd ed.) (Cold Spring Harbor Laboratory).

In general, sequences which code for the antithrombin protein and hybridize to the nucleotide sequence disclosed herein will be at least 40% homologous, about 60% to 70% homologous, and even about 80%, 85%, 90% homologous or more with the disclosed sequences. Such sequences are substantially homologous to the nucleotide sequences disclosed herein and encompassed by the invention. Further, the amino acid sequences of the antithrombin proteins isolated by hybridization to the DNA's disclosed herein are also an aspect of this invention. The degeneracy of the genetic code, which allows different nucleic acid sequences to code for the same protein or peptide, is well known in the literature. See, e.g., U.S. Pat. No. 4,757,006.

The hybridization probes may be cDNA fragments or oligonucleotides, and may be labeled with a detectable group as known in the art. Pairs of probes which will serve as PCR primers for the antithrombin gene or a protein thereof may be used in accordance with the process described in U.S. Pat. Nos. 4,683,202 and 4,683,195.

The polypeptides of the invention may be subject to one or more post-translational modifications such as sulphation, COOH-amidation, acylation or chemical alteration of the polypeptide chain.

It is recognized that the nucleotide and peptide sequences of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the peptides and proteins can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, T. (1985) Proc. Natl. Acad. Sci. U.S.A 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra (eds.) Techniques in Molecular Biology, MacMillan Publishing Company, NY (1983) and the references cited therein. Thus, the nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant. Likewise, the peptides and proteins of the invention encompass both naturally occurring and modified forms thereof. Such variants will continue to possess the desired activity. It is recognized that the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create sequences deleterious to expression of the gene product. See, EP Patent Application, Publication No. 75,444.

The proteins of the invention include the naturally occurring forms as well as variants thereof. These variants will be substantially homologous and functionally equivalent to the native protein. As used herein, two proteins (or a region of the proteins) are "substantially homologous" when the amino acid sequences are typically at least about 40%, more typically at least about 60%-70%, and most typically at least about 80%, 85%, 90% or more identical. A substantially homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence hybridizing to the nucleic acid sequence, or portion thereof, of the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, or otherwise described herein under stringent conditions as more fully described below.

Thus, a variant of a native protein is "substantially homologous" to the native protein when at least about 40%, more preferably at least about 60%-70%, and most preferably at least about 80%, 85%, 90%, or more of its amino acid sequence is identical to the amino acid sequence of the native protein. A variant may differ by as few as 1, 2, 3, or 4 amino acids. A variant polypeptide can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these.

By "functionally equivalent" is intended that the sequence of the variant defines a chain that produces a protein having substantially the same biological activity as the native protein of interest. Such functionally equivalent variants that comprise substantial sequence variations are also encompassed by the invention. Thus a functionally equivalent variant of the native protein will have a sufficient biological activity to be therapeutically useful. By therapeutically useful is intended effective in achieving a therapeutic goal as discussed below.

Methods are available in the art for determining functional equivalence. Biological activity can be measured using assays specifically designed for measuring activity of the native protein, including assays described in the present invention. Additionally, antibodies raised against the biologically active native protein can be tested for their ability to bind to the functionally equivalent variant, where effective binding is indicative of a protein having conformation similar to that of the native protein.

Variant polypeptides can be fully functional or can lack function in one or more activities. Thus, in the present case, variations can affect the function, for example, of one or more of the modules, domains, or functional subregions of the proteins and polypeptides of the invention.

Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. Functional variants can also contain substitution of similar amino acids, which result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region. As indicated, variants can be naturally-occurring or can be made by recombinant means or chemical synthesis to provide useful and novel characteristics for the polypeptide.

Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity. Sites that are critical can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

The invention further encompasses variant polynucleotides, and fragments thereof, that differ from the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:6, or otherwise described herein, due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:6 or otherwise described herein.

The invention also provides nucleic acid molecules encoding the variant polypeptides described herein. Such polynucleotides may be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions.

Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.

Orthologs, homologs, and allelic variants can be identified using methods well known in the art. These variants comprise a nucleotide sequence encoding a protein that is at least typically about 40%, more typically at least about 60%-70%, and most typically at least about 80%, 85%, 90% or more homologous to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO: 6 or otherwise described herein, or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under stringent conditions, to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO: 6 or otherwise described herein, or a fragment of the sequence. It is understood that stringent hybridization does not indicate substantial homology where it is due to general homology, such as poly A sequences, or sequences common to all or most proteins in an organism or class of proteins.

To determine the percent homology of two amino acid sequences, or of two nucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence, then the molecules are homologous at that position. As used herein, amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity". The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent homology equals the number of identical positions/total number of positions times 100).

The invention also encompasses proteins or polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by the antithrombin proteins described herein. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute the given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

Both identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Preferred computer program methods to determine identify and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J. (1984) Nuc. Acids Res. 12(1):387), BLASTP, BLASTN, and FASTA (Atschul, S. F. (1990) J. Molec. Biol. 215:403); utilizing the default parameters available within the programs. By substantial sequence similarity, identity or homology is intended sequences having at least about 60%, 70%, 75%, 80%, 85%, 90%, 95% or more similarity.

DNA sequences can also be synthesized chemically or modified by site-directed mutagenesis to reflect the codon preference of the host cell and increase the expression efficiency.

The proteins of the invention can be engineered in accordance with the present invention by chemical methods or molecular biology techniques. Molecular biology methods are most convenient since proteins can be engineered by manipulating the DNA sequences encoding them. Genomic DNA, cDNA, synthetic DNA, and any combination thereof may be used for this purpose. Genomic DNA sequences or cDNA sequences encoding proteins can be isolated based on the amino acid sequence of proteins or certain protein properties. Many methods of sequence isolation are known in the art of molecular biology. See particularly Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Plainview, N.Y.), herein incorporated by reference.

To produce an antithrombin polypeptide by recombinant DNA technology, a gene encoding a polypeptide of the invention is prepared. The DNA coding sequence typically does not contain introns. The DNA sequence is isolated and purified, the gene is inserted in an expression vector able to drive expression and production of the recombinant product. The DNA sequence may be a cDNA sequence, or alternatively a synthetic DNA sequence. A synthetic gene is typically prepared by chemically synthesizing oligonucleotides that, in total, correspond to the desired gene. The synthesized oligonucleotides are then assembled to obtain the gene.

If desired, the gene sequence may be modified by site-directed mutagenesis to introduce one or more coding changes. Typically, a gene is constructed with restriction sites at each end to facilitate its subsequent manipulation.

The DNA sequence may be constructed to comprise a leader peptide. The leader peptide is capable of directing secretion of the polypeptide from cells in which the polypeptide is to be expressed. The sequence encoding the leader peptide is typically fused to the 5′-end of the DNA sequence encoding the polypeptide. Leader sequences are known in the art and include the OmpA leader peptide, the leader peptide of vesicular stomatitis virus G protein (VSV G protein). The OmpA leader is useful when expression is in a bacterial host, such as E. coli while the VSVG protein is useful when expression is in insect cells.

The DNA sequence may be constructed to comprise a cleavable site to release the polypeptide of the invention. A DNA sequence may be used which encodes a carrier polypeptide sequence fused via a cleavable linkage to the N-terminus of a polypeptide of the invention. The cleavable linkage may be one cleavable by cyanogen bromide.

For expression of the polypeptides, an expression vector is constructed which comprises a DNA sequence encoding the polypeptide which is capable of expressing the polypeptide in a suitable host. Appropriate transcriptional and translational control elements are provided, including a promoter for the DNA sequence, a transcriptional termination site, and translation start and stop codons. The DNA sequence is provided in the correct frame such as to enable expression of the polypeptide to occur in a host compatible with the vector.

The expression vector typically comprises an origin of replication and, if desired, a selectable marker gene such as antibiotic resistance. The expression vector may be a plasmid, a virus, particularly a baculovirus, and the like.

Once the nucleotide sequences encoding the antithrombin proteins of the invention have been isolated, they can be manipulated and used to express the protein in a variety of hosts including other organisms, including microorganisms.

Once the nucleotide sequence is identified and known, those skilled in the art can produce large quantities of the protein for therapeutic use. Accordingly, recombinant protein and methods for producing the recombinant protein are encompassed by the present invention. In this manner, the nucleotide sequence encoding the antithrombin protein can be utilized in vectors for expression in various types of host cells, including both procaryotes and eucaryotes, to produce large quantities of the protein, or active analogues, or fragments thereof, and other constructs having antithrombin activity.

Generally, methods for the expression of recombinant DNA are known in the art. See, for example, Sambrook et al. (1989) Molecular Cloning, Cold Spring Harbor Laboratory. Additionally, host cells and expression vectors, such as the baculovirus expression described in U.S. Pat. Nos. 4,745,051 and 4,879,236. In general, a baculovirus expression vector comprises a baculovirus genome containing the gene to be expressed inserted into the polyhedron gene at a position ranging from the polyhedron transcriptional start signal to the ATG start site and under the transcriptional control of a baculovirus polyhedron promoter.

A broad variety of suitable procaryotic and microbial vectors are available. Likewise, the promoters and other regulatory agents used in expression of foreign proteins are available in the art. Promoters commonly used in recombinant microbial expression vectors are known in the art and include the beta-lictamase (penicillinase) and lactose promoter systems (Chang et al. (1978) Nature 275:615 and Goeddel et al. (1979) Nature 281:544); A tryptophan (TRP) promoter system (Goeddel et al. (1980) Nucleic Acids Res. 8:4057 and the EPO Application Publication No. 36,776); and the Tac promoter (DeBoer et al. (1983) Proc. Natl. Acad. Sci. U.S.A, 80:21). While these are commonly used, other microbial promoters are available. Details concerning nucleotide sequences of many have been published, enabling a skilled worker to operably ligate them to DNA encoding the protein in plasmid or viral vectors. See, for example, Siedenlist et al. (1980) Cell 20:269.

Eucaryotic host cells such as yeast may be transformed with suitable protein-encoding vectors. See, e.g., U.S. Pat. No. 4,745,057. Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available. Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS), a promoter, DNA encoding the desired protein, sequences for polyadenylation and transcription termination, and a selection gene. An exemplary plasmid is YRp7, (Stinchcomb et al. (1979) Nature 282:9; Kingsman et al. (1979) Gene 7:141; Tschemper et al. (1980) Gene 10:157). This plasmid contains the trp1 gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones (1977) Genetics 85:12). The presence of the trp1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoter sequences for use in yeast vectors include the promoters for metallothionein, alcohol dehydrogenase, adenylate cyclase, 3-phosphoglycerate kinase (Hitzeman et al. (1980) J. Biol. Chem. 255:2073) and other glycolytic enzymes (Hess et al. (1968) J. Adv. Enzyme Reg. 7:149; and Holland et al. (1978) Biochemistry 17:4900) such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al. EPO Publn. No. 73,657.

The invention provides antibody preparations that selectively bind the proteins of the invention, or any variants or fragments thereof as described. An antibody is considered to selectively bind, even if it also binds to other proteins that are not substantially homologous with the antithrombin protein. These other proteins share homology with a fragment or domain of the antithrombin protein giving rise to antibodies that bind to both proteins by virtue of the homologous sequence. In this aspect, it is recognized that antibody binding to the antithrombin protein is still selective.

The preparations encompass monoclonal or polyclonal antibodies, intact antibodies or fragments thereof (e.g. Fab), purified preparations such as affinity-purified preparations, or less pure preparations such as ascites fluid, sera and the like. Methods for raising antibodies are well known in the art and include but are not limited to those described in Harlow and Lane ((1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press), the contents of which are herein incorporated by reference. The invention also embodies antibody preparations which neutralize biological functions of the provided proteins, variants or fragments thereof. Such functions include but are not limited to antithrombin activity. The invention also provides compositions capable of modulating the immune response. By modulating the immune response is intended a determinable change in the immune system of a host organism effected by administering the herein described compositions of the invention to that host. Working examples of such modulation of immune response, as well as methods of making and assessing selectivity of antibody preparations are provided in the Experimental section of this application, and are herein incorporated by reference.

The compositions of the present invention find therapeutic use as veterinary vaccines in treatment of hematophagy in a mammal. The methods comprise administering to the mammal a veterinary vaccine comprising a therapeutically effective amount of the compositions of the invention. In this aspect, a therapeutically effective amount is intended as that amount which effects a determinable reduction, amelioration, elimination or prevention of hematophagous infestation in the mammal to which the vaccine of the present invention was administered. While the vaccines of the invention can be used with any mammal, of particular interest are livestock, more particularly, horses, cattle, and the like. The compositions are useful for vaccination against the hematophagous fly of the suborder Cyclorrhapha, more particularly of the species Haematobia irritans, even more particularly of the subspecies irritans or exigua. However, the invention vaccination against any hematophagous organism where such vaccination using compositions and methods of the present invention is therapeutically effective.

For veterinary applications, the compositions of the invention can be formulated into any acceptable pharmaceutical preparation as described below or any other acceptable preparation for veterinary use. In one embodiment of the invention, the vaccines comprise therapeutically effective amounts of the proteins of the invention, or any variant or fragment thereof as described herein.

In a preferred embodiment, the vaccines comprise the nucleotide compositions of the invention as described herein. As described by Cox et al. (1993) J. Virol. 67:5664-5667; Fynan et al. (1993) Proc. Natl. Acad. Sci. USA 90:11478-11482; and Lewis et al. (1997) Vaccine 15:861-864; and reviewed by Robinson (1997) Vaccine 15:785-787; and Tighe et al. (1998) Immunol. Today 19:89-97, the contents of all of which are herein incorporated by reference, nucleic acid vaccines can be readily constructed and produced. In general, target DNA sequences encoding the protein to be used as an immunogen are cloned into eukaryotic expression vectors. The constructed plasmid is grown in bacteria and purified. The purified plasmid DNA is then directly injected into the animal generally by intramuscular injection, but also by intradermal injection; where its expression by cells in the inoculated host produces the target protein, thereby raising an immune response. See, for example, Cox et al. (1993) J. Virol. 67:5664-5667, herein incorporated by reference. Nanogram levels of DNA-expressed protein may be utilized to stimulate an immune response and protect against infectious agents achieved by skin, muscle and intravenous inoculations of DNA. See, for example, Fynan et al. (1993) Proc. Natl. Acad. Sci. USA 90:11478-11482; Cox et al. (1993) J. Virol. 67:5664-5667, herein incorporated by reference. Such plasmids introduced by intramuscular or intradermal injection stimulate a protective response that abrogates clinical disease following challenge.

The compositions of the present invention can be formulated into pharmaceutical preparations for therapeutic use as antithrombin agents. Such compositions find use in the treatment of venous thrombosis, vascular shunt occlusion and thrombin-included disseminated intravascular coagulation.

The compositions of the invention can be used alone or in combination with other antithrombin and therapeutic agents including veterinary agents such as vaccines. Other agents are known in the art.

The antithrombin compositions can be formulated according to known methods to prepare pharmaceutically useful compositions, such as by admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation are described, for example, in Remington's Pharmaceutical Sciences 19th ed., Osol, A. (ed.), Mack Easton Pa. (1980). In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the antithrombin protein, either alone, or with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved by the use of polymers to complex or absorb the compositions. The controlled delivery may be exercised by selecting appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose, carbosymethylcellulose, or protamine sulfate). The rate of drug release may also be controlled by altering the concentration of such macromolecules.

Another possible method for controlling the duration of action comprises incorporating the therapeutic agents into particles of a polymeric substance such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, it is possible to entrap the therapeutic agents in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethyl cellulose or gelatin-microcapsules or poly(methylmethacrylate) microcapsules, respectively, or in a colloid drug delivery system, for example, liposomes, albumin, microspheres, microemulsions, nanoparticles, nanocapsules, or in macroemulsions. Such teachings are disclosed in Remington's Pharmaceutical Sciences (1980).

In more specific embodiments, a polypeptide of the invention may be converted into a pharmaceutically acceptable salt. It may be converted into an acid additional salt with an organic or inorganic acid. Suitable acids include acetic, succinic and hydrochloric acid. Alternatively, the peptide may be converted into a carboxylic acid salt such as the ammonium salt or an alkali metal salt such as the sodium or potassium salt.

A polypeptide or pharmaceutically acceptable salt thereof may be used in a pharmaceutical composition, together with a pharmaceutically acceptable carrier or excipient therefore. Such a formulation is typically for intravenous administration (in which case the carrier is generally sterile saline or water of acceptable purity). A polypeptide can therefore be used for the therapy and prophylaxis of thrombosis and thromboembolisms in a human or other mammal, including the prophylaxis of post-operative thrombosis, for acute shock therapy (for example for septic or polytraumatic shock), for the therapy of consumption coagulopathics, in hemodialyses, haemoseparations and in extracorporeal blood circulation. In one embodiment of the invention, the polypeptide or salt thereof can be coadministered with a plasminogen activator, such as tissue plasminogen activator.

The dosage depends especially on the specific form of administration and on the purpose of the therapy or prophylaxis. The size of the individual doses and the administration regime can best be determined by way of an individual judgment of the particular case of illness; the methods of determining relevant blood factors required for this purpose are familiar to the person skilled in the art. Normally, in the case of an injection the therapeutically effective amount of the compounds according to the invention is in a dosage range of from approximately from 0.005 or 0.01 to approximately 0.05 or 0.1 mg/kg body weight, preferably from approximately 0.01 to approximately 0.05 mg/kg body weight.

The administration is effected by intravenous, intramuscular or subcutaneous injection. Accordingly, pharmaceutical compositions for parenteral administration in single dose form contain per dose, depending on the mode of administration, from approximately 0.4 to approximately 7.5 mg of the compound according to the invention. In addition to the active ingredient these pharmaceutical compositions usually also contain a buffer, for example a phosphate buffer, which is intended to keep the pH value between approximately 3.5 and 7, and also sodium chloride, mannitol or sorbitol for adjusting the isotonicity. The preparations may be freeze-dried or dissolved. An antibacterially active preservative may be included, for example from 0.2 to 0.3% 4-hydroxybenzoic acid methyl ester or ethyl ester.

A composition for topical application can be in the form of an aqueous solution, lotion or gel, an oily solution or suspension or a fat-containing or, especially, emulsified ointment. A composition in the form of an aqueous solution is obtained, for example, by dissolving the active ingredients according to the invention, or a therapeutically acceptable salt thereof, in an aqueous buffer solution of from e.g., pH 4 to pH 6.5 and, if desired, adding a further active ingredient, for example an anti-inflammatory agent, and/or a polymeric binder, for example polyvinylpyrrolidone, and/or a preservative. The concentration of active ingredients is from approximately 0.1 to approximately 1.5 mg, preferably from 0.25 to 1.0 mg, in 10 ml of a solution or 10 g of a gel.

An oily form of administration for topical application is obtained, for example, by suspending the active ingredient according to the invention, or a therapeutically acceptable salt thereof, in an oil, optionally with the addition of swelling agents, such as aluminum stearate, and/or surfactants (tensides) having an HLB value ("hydrophilic-lipophilic balance") of below 10, such as fatty acid monomers of polyhydric alcohols, for example glycerin monostearate, sorbitan monolaurate, sorbitan monostearate or sorbitan monooleate. A fat-containing ointment is obtained, for example, by suspending the active ingredient according to the invention, or a salt thereof, in a spreadable fatty base, optionally with the addition of a tenside having an HLB value of below 10. An emulsified ointment is obtained by triturating an aqueous solution of the active ingredient according to the invention, or a salt thereof, in a soft, spreadable fatty base with the addition of a tenside having an HLB value of below 10. All these forms for topical application can also contain preservatives. The concentration of active ingredient is from approximately 0.1 to approximately 1.5 mg, preferably from 0.25 to 1.0 mg, in approximately 10 g of base.

In addition to the compositions described above and pharmaceutical compositions analogous thereto that are intended for direct medicinal use in the body of a human or a mammal, the present invention relates also to pharmaceutical compositions and preparations for medicinal use outside the living body of humans or mammals. Such compositions and preparations are used especially as anticoagulant additives to blood that is being subjected to circulation or treatment outside the body (for example haemoseparation). Such preparations, such as stock solutions or alternatively preparations in single dose form, are similar in composition to the injection preparations described above; however, the amount of concentration of active ingredient is advantageously based on the volume of blood to be treated or, more precisely, on its thrombin content. Depending on the specific purpose, the suitable dose is from approximately 0.01 to approximately 1.0 mg of the active ingredient/liter of blood, although the upper limit may still be exceeded without risk as the agent is harmless even in relatively high amounts.
 

Claim 1 of 13 Claims

1. A purified protein having antithrombin activity, wherein said protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:7.

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