Patent 6,500,646

Drugs in which peptides having affinity specific for phospholipids, preferably those which are contained in the constituents of lipid bilayers forming the surface layers of cells and of which the proportion in the outer part of each lipid bilayer increases when the cell is not normal, for example, in the case where it is damaged, denatured or activated, and biologically active substances bind to each other, deoxyribonucleic acid (DNA) which codes for the amino acid sequence of the drug in the case where the biologically active substance is a peptide, and processes for producing such drugs. The drugs and novel peptides are useful as preventives and therapeutics of diseases involving coagulopathy, inflammations and immune response.

BEST MODE FOR CARRYING OUT THE INVENTION

The "phospholipid" as used in the present invention is a desired phospholipid that is intended for targeting a biological substance to a specified site and which is contained in the constituent components of the lipid bilayers forming the surface layers of cells such that the proportion of its content in the outer part of each lipid bilayer will increase when the cells are not normal, for example, in the case where they are damaged, denatured or activated. More specifically, the phospholipid is such that its content increases in such cases as where a blood coagulation is in progress, the so-called immune response reactions of cells such as their activation, impairment and/or apotosis due to inflammation or immunocytes are in progress, a cell impairing reaction due to active oxygen is in progress or where a cell activating and/or impairing reaction due to proteases is in progress. As typical examples of such phospholipid, phosphatidylserine and phosphatidylethanolamine (Alan J. Schroit et al., Biochim. Biophys. Acta, Vol. 1071, 313 (1991)) may be mentioned, and phosphatidylserine is preferred.

The term "having affinity" as used in the present invention refers to the performance of a certain interaction. The term "interaction" includes mutual binding, formation of a complex, mutual recognition of molecules, tendency to move and/or aggregate in a specified direction, causing the shape of molecules to change, and mutual reaction. In the case of mutual binding, the mode of binding is in no way limited and it may be non-covalent bonding typified by electrostatic bonding and hydrophobic bonding or covalent bonds typified by a disulfide bond, an ester bond, an ether bond and a peptide bond.

Thus, the first aspect of the present invention is a substance or a composition, preferably a drug, that contain both a substance having affinity for a phospholipid and a biologically active substance, and which are characterized in that their action and efficacy are enhanced under such a condition that a specified phospholipid is present.

The term "under such a condition that a phospholipid is present" as used herein includes not only artificially created environments but also natural environments. Artificially created environments include the state where phospholipids, blood, cells, living tissues and their disrupted products are contained in vitro or within containers such as test tube whereas natural environments include all in vivo parts such as blood vessels, brain and other organs.

It should also be noted that the "peptide" as used in the present invention is in no way limited in the length of its amino acid sequence and covers the range from so-called dipeptides consisting of two amino acids to polypeptides consisting of 1,000 or more amino acids.

The substance having affinity for a phospholipid as used in the present invention is in no way limited in the molecules it is composed of or its shape as long as it has affinity for a specified phospholipid. Preferably, it is a peptide, more preferably a peptide consisting of the following amino acid sequence:

(A1)n₁ -(A2)n₂ -(A3)n₃

where A1 is the amino acid sequence denoted by SEQ ID NO:1 or 10, and A2 and A3 are the amino acid sequences denoted by SEQ ID NOS:2 and 3, respectively. It should be noted that A1 may contain all or part of the amino acid sequence denoted by SEQ ID NO:1. For example, A1 may contain the amino acid sequence denoted by SEQ ID NO:10 but it preferably contains all of the amino acid sequence of SEQ ID NO:1. Symbol Xaa in SEQ ID NO:2 is Thr or Leu. It should also be noted that A3 may contain part or all of the amino acid sequence of SEQ ID NO:3. Referring to n₁, n₂ and n₃, they are any numbers representing the repeating numbers of the amino acid sequence A1, A2 and A3, respectively; n₁ is preferably 5 or less, more preferably 0 or 1, n₂ is preferably at least 1, more preferably 1, 2 or 3, and n₃ is preferably 5 or less, more preferably, 0 or 1.

The method of combining the sequences set forth above is not limited in any way as long as the stated peptide has affinity for a specified phospholipid. Advantageous examples to be combined include the sequences denoted by SEQ ID NOS:5-9 and 22.

It should, however, be noted that the aforementioned amino acid sequences are illustrative only and the peptide having affinity for a specified phospholipid according to the present invention is in no way limited in its amino acid sequence as long as it has affinity for a specified phospholipid and depending on the need, the aforementioned amino acid sequences may be subjected to substitution, deletion, insertion, addition and so forth. Further, they may be modified as required. Alternatively, peptides having affinity for phosphatidylserine that consist of entirely different amino acid sequences from the aforementioned ones may be substituted, for example, a peptide derived from a Gla region (characterized by .gamma.-carboxyglutamic acid residue) which has affinity for the phosphatidylserine contained in a coagulation-related factor such as factor Xa (Mann K. G. et al., Blood, Vol. 76, 1 (1990)), a peptide having affinity for the phosphatidylserine derived from factor V [Thomas L. O. et al., J. Biol. Chem., Vol. 267, 4189 (1992)], a peptide having affinity for the phosphatidylserine derived from Annexin V (M. A. Swairjo et al., Nature Struct. Biol., Vol. 2, 968 (1995)), and so forth or derivatives thereof.

The "biologically active substance" in the drug of the present invention may be of any substance that exhibits a pharmacological action in vivo and it may include a peptide having a biological activity, a chemical substance exhibiting a pharmacological action, as well as their aggregates, encapsulations and so forth, a peptide with being preferred. The "peptide having biological activity" mentioned herein may be any peptide that is involved in in vivo reactions and it may be modified, as required. Preferably, the biologically active substance is a peptide which inherently makes a certain mutual interaction with cell membranes by itself or a peptide that interacts with substances that occur within cells, on cell membranes, in the surface layers of cells or their surroundings; modified versions, variants and derivatives of such peptides are also included. If the biologically active substance is an inherently non-water-soluble peptide, as exemplified by some membrane proteins, it may optionally be modified to a water-soluble form. Advantageous examples of such peptides include a factor involved in a blood coagulation system, a factor involved in a fibrinolytic system, a factor involved in immune response, a factor suppressing cytopathy, a factor inhibiting the activity of proteases and so forth, more preferably, a factor suppressing a blood coagulation system, a factor enhancing a fibrinolytic system, a factor suppressing a complement activating reaction, a factor suppressing the cytopathy due to active hydrogen, a factor inhibiting the activity or proteases and so forth. To illustrate in greater detail, examples of the factor involved in a blood coagulation system and/or the factor suppressing a blood coagulation system include TM, the second region of UTI, antithrombin III (ATIII), a tissue factor pathway inhibitor (TFPI) and so forth, as well as modified versions, variants and derivatives of these factors. Examples of the factor involved in a fibrinolytic system and/or the factor enhancing a fibrinolytic system include tPA, urokinase (UK) and so forth, as well as modified versions, variants and derivatives of these factors. Examples of the factor involved in immune response reactions include complement regulatory proteins such as MCP and a decay-accelerating factor (DAF) which are factors that suppress a complement activating reaction, UTI which inhibits immunocyte-derived proteases and the second region of UTI, as well as modified versions, variants and derivatives of these factors. Examples of the factor suppressing the cytopathy due to active oxygen and/or the factor inhibiting the activity of proteases include UTI, the second region of UTI, elafin, a secretory leukoprotease inhibitor (SLPI) and so forth, as well as modified versions, variants and derivatives of these factors. Other examples of the factor suppressing the cytopathy due to active oxygen include SOD, catalase and so forth, as well as their modified versions, variants and derivatives (i.e., factors having an active oxygen scavenging action). Among these advantageous examples, TM, the second region of UTI, MCP and UTI, as well as their modified versions, variants and derivatives are preferred, and those peptides which are represented by SEQ ID NOS:4 and 23-25 are particularly preferred. These are illustrative only and will in no way limit the biologically active substance to be used in the present invention.

In the drug according to the first aspect of the present invention, the "chemical substance exhibiting a pharmacological action" is a substance selected from among all substances that exhibit pharmacological actions but which exclude peptides having biological activity and as long as it exhibits a pharmacological action in vivo, it will in no way be limited by the molecular formula with which it is denoted. Specifically, examples include artificially synthesized compounds, chemical substances obtained by separation from natural products and microorganism-produced substances, nucleic acids, saccharides, lipids and so forth, as well as their modified versions. To illustrate in greater detail, cyclophosphamide which is an immunosuppressive substance, actinomycin D which is an anti-cancer agent, an antisense oligonucleotide, hyaluronic acid and lecithin may be mentioned; it should be noted that these are merely intended for illustrative purposes and will in no way limit the biologically active substance to be used in the present invention.

The term "aggregate" of the substance having biological activity or the compound exhibiting a pharmacological action covers the substance having biological activity or the compound exhibiting a pharmacological action which have been assembled in a quantity greater than a specified level due, for example, to chemical bonding or physical adhesion, and the term "encapsulation" of the substance having biological activity or the compound exhibiting a pharmacological action covers the substance having biological activity or the chemical compound exhibiting a pharmacological action which are incorporated within liposomes, microcapsules or high-molecular weight matrices and so forth.

The mode in which both the substance having affinity for a phospholipid and the biologically active substance are contained according to the first aspect of the present invention is not limited in any particular way. A preferred mode is such that the substance having affinity for a phospholipid is substantially integral with the biologically active substance to form the drug of the present invention without causing a complete compromise in the affinity for a specified phospholipid which is possessed by the substance having affinity for a phospholipid or in the activity of the biologically active substance. As long as the substance having affinity for a phospholipid is substantially integral with the biologically active substance to form the drug of the present invention without causing a complete compromise in the affinity for a specified phospholipid which is possessed by the substance having affinity for a phospholipid or in the activity of the biologically active substance, all possible modes such as a mixture, a composition, a complex, a bound form and so forth are included. Thus, the two substances may be simply mixed with each other or they may be contained in a composition or, alternatively, they may perform interaction with each other. The term "interaction" covers mutual binding, formation of a complex, mutual recognition of molecules, tendency to move and/or aggregate in a specified direction, causing the state of molecules to change and mutual reaction, with direct or indirect bonding in either of these forms being preferred. In the case of mutual binding, the mode of binding is in no way limited and it may be non-covalent bonding typified by electrostatic bonding and hydrophobic bonding or covalent bonding typified by a disulfide bond, an ester bond, an ether bond and a peptide bond. Depending on the need, a suitable linker and an adapter such as a peptide having any amino acid sequence or any compound may be interposed, as exemplified by a biotin-avidin bond, an antibody-antigen bond or a bond formed by a receptor and its ligand; further, modifications may be applied as required. If at least one of the biologically active substance and the substance having affinity for a phospholipid is a peptide, examples of the binding site in the peptide include an amino group, a carboxyl group and a thiol group in a cysteine residue that are present at the N terminus, C terminus or inside chains, with the N- or C terminus being preferred. In a particular case where both the biologically active substance and the substance having affinity for a phospholipid are peptides, it is preferred to contain a bond established by a peptide bond and a more preferred mode is such that the N terminus of either one of the biologically active substance and the substance having affinity for a phospholipid is bound to the C terminus of the other peptide via a peptide bond. Thus, chimeric proteins and fused proteins may be mentioned as typical examples. Among these cases, the one in which the N terminus of the substance having affinity for a phospholipid is linked to the C terminus of the biologically active substance via a peptide bond is particularly preferred. To give a specific preferred example, a drug according to the present invention may be mentioned that contains a peptide composed of an amino acid sequence in which an amino acid sequence selected from among SEQ ID NOS:5-9 and 22 is linked to the C terminus of an amino acid sequence selected from among SEQ ID NOS:4 and 23-25.

The present invention also provides a novel peptide having affinity for phosphatidylserine which consists of or contains the amino acid sequences denoted by SEQ ID NOS:6, 7 and 22, as well as a pharmaceutical that contains said peptide as a component. Said pharmaceutical includes one in which the novel peptide having affinity for phosphatidylserine according to the present invention is mixed with or bound to a biologically active substance or its aggregate or encapsulation. The biologically active substance with or to which the novel peptide having affinity for phosphatidylserine has mixed or bound, or the aggregate or encapsulation of the substance has such a nature that it will be delivered selectively on the surface of cells that are not normal, as exemplified by damaged, denatured or activated cells. States more specifically, a biologically active substance to which the novel peptide having affinity for phosphatidylserine according to the present invention has bound, optionally via a suitable linker, has such a nature that it will be delivered selectively on the surface of cells that are not normal, as exemplified by damaged, denatured or activated cells. The novel peptide having affinity for phosphatidylserine according to the present invention may be modified with a substance having affinity for the constituent components of the skeleton of an encapsulation of a biologically active substance and subsequently mixed or reacted with the encapsulation of the biologically active substance to produce an encapsulation containing the biologically active substance to the surface of which the novel peptide having affinity for phosphatidylserine has bound and this encapsulation similarly has such a nature that it will be delivered selectively on the surface of cells that are not normal, as exemplified by damaged, denatured or activated cells. To illustrate, if the biologically active substance is contained in liposomes, the novel peptide having affinity for phosphatidylserine according to the present invention is modified with a suitable phospholipid such as phosphatidylethanolamine and thereafter mixed with liposomes to produce a drug in which the novel peptide having affinity for phosphatidylserine according to the present invention has bound to the surface layers of the liposomes and the drug has such a nature that it will be delivered selectively on the surface of cells that are not normal, as exemplified by damaged, denatured or activated cells.

The novel peptide having affinity for phosphatidylserine according to the present invention can also be used as a drug with which a biologically active factor that is involved in the progress of a blood coagulation is inhibited from binding to the target site of action by a competitive reaction.

The DNA of the present invention may be of any kind as long as it has, in effect, a DNA sequence coding for the peptides of the substance having affinity for a phospholipid and/or the biologically active substance which compose the drug of the present invention. More specifically, the present invention provides DNA which contains, preferably consists of, the following sequences:

(J1)-(D1)m-(D2)m₂ -(D3)m₃ -(J1)

where J1 is a DNA sequence coding for the amino acid sequence of a peptide having biological activity and at least either one of 5'- or 3' terminus will be sufficient; D1, D2 and D3 are DNA sequences coding for the peptides represented by A1, A2 and A3, respectively, with D1 being the DNA sequence denoted by SEQ ID NO:11 or 12, D2 the DNA sequence denoted by SEQ ID NO:13 or 14, and D3 the DNA sequence denoted by SEQ ID NO:15. It should be noted that D1 may contain all or part of the DNA sequence of SEQ ID NO:11 to such an extent that there will be no change in its translational frame. For instance, D1 may contain the DNA sequence of SEQ ID NO:12 and it preferably contains all of the DNA sequence of SEQ ID NO:11. Symbol M in SEQ ID NOS:13 and 14 signifies A or C. It should also be noted that D3 may contain part or all of the DNA sequence of SEQ ID NO:15 to such an extent that there will be no change in its translational frame. Symbols ml, m₂ and m₃ are any numbers that represent the repeating numbers of the DNA sequences, D1, D2 and D3, respectively; m₁ is preferably 5 or less, more preferably 0 to 1, m₂ is preferably at least 1, more preferably 1, 2 or 3, and m₃ is preferably 5 or less, more preferably 0 to 1. The method of combining the stated sequences is in no way limited as long as the peptides composed of the amino acid sequences translated from the stated DNA provide the drug of the present invention. To give advantageous examples, J1 may be DNA coding for a factor involved in a blood coagulation, a factor involved in a fibrinolytic system, a factor involved in immune response, a factor suppressing cytopathy, a factor inhibiting the activity of proteases and so forth, more preferably, a factor suppressing a blood clotting reaction, a factor enhancing a fibrinolytic system, a factor suppressing a complement activating reaction, a factor suppressing the cytopathy due to active oxygen, a factor inhibiting the activity of proteases and so forth. To illustrate in greater detail, examples of the factor involved in a coagulation system reaction and/or the factor suppressing a blood coagulation include TM, the second region of UTI, ATIII, TFPI and so forth, as well as modified versions, variants and derivatives of these factors. Examples of the factor involved in a fibrinolytic system and/or the factor enhancing a fibrinolytic system include tPA, UK and so forth, as well as modified versions, variants and derivatives of these factors. Examples of the factor involved in an immune response reactions include complement regulatory proteins such as MCP and DAF which are factors suppressing a complement activating reaction, UTI which inhibits immunocyte-derived proteases and the second region of UTI, as well as modified versions, variants and derivatives of these factors. Examples of the factor suppressing the cytopathy due to active oxygen and/or the factor inhibiting the activity of proteases include UTI, the second region of UTI, SLPI and so forth, as well as modified versions, variants and derivatives of these factors. Other examples of the factor suppressing the cytopathy due to active oxygen include SOD, catalase and so forth, as well as their modified versions, variants and derivatives (i.e., factors act as active oxygen scavenger). A typical example of a more preferred combination of sequences may be a DNA sequence consisting of nucleotide sequences such that a nucleotide sequence of SEQ ID NOS:16-20 or No. 26 is linked to the 3' end of a nucleotide sequence of SEQ ID NO:21 and SEQ ID NOS:27-29.

Advantageous examples of the DNA according to the fourth aspect of the present invention which codes for the amino acid of the novel peptide having affinity for phosphatidylserine are the DNA sequences of SEQ ID NOS:17, 18 and 26 (which code for the peptides of SEQ ID NO:6, 7 and 22, respectively).

The present invention also provides a recombinant DNA, such as a plasmid or expression vector, that contains the DNA of the invention.

As is well known in the art, in accordance with the degeneracy of genetic codes, at least one base in the DNA gene sequence which encodes polypeptide can be replaced by another base without changing its amino acid sequence. Hence, the DNA of the present invention may have base sequences having one or more base replacements based on the degeneracy of genetic codes. Particularly in the case of producing the peptide of the present invention by genetic engineering techniques, the DNA may have sequences having one or more base replacements in order to provide codons that will be used with high frequency in specified host cells. It should be noted that in the present invention, the sequence of DNA is described starting from the 5' terminal end. In the present invention, A, G, C and T stand for deoxyadenylic acid, deoxyguanylic acid, deoxycytidylic acid and thymidylic acid, respectively.

The drug of the present invention can be obtained by a process in which the substance having affinity for a specified phospholipid and the biologically active substance are prepared individually and then mixed or bound to each other. Herein, the substance having affinity for a specified phospholipid may be mixed with or bound to the biologically active substance by any method as long as the substance having affinity for a specified phospholipid is substantially integral with the biologically active substance to form the drug of the present invention without causing a complete compromise in the affinity for a specified phospholipid which is possessed by the substance having affinity for a specified phospholipid or in the activity of the biologically active substance.

The substance having affinity for a specified phospholipid according to the present invention is preferably a peptide and can be produced by a process characterized in that at least one of the following steps is performed:

a) the step of obtaining said peptide by chemical synthesis;

b) the step of obtaining DNA having a sequence coding for the amino acid sequence of said peptide;

c) the step of incorporating said DNA into a vector so as to give a replicable recombinant DNA containing said DNA;

d) the step of transforming a host cell with said recombinant DNA to give a transformant capable of expressing said peptide; and

e) the step of culturing said transformant such as to produce said peptide and recovering said peptide from the culture mixture.

The chemical synthesis method for producing a peptide having a specified amino acid sequence according to the present invention may typically be implemented by using an automatic peptide synthesizer.

The DNA having a sequence coding for the peptide of the present invention may typically be prepared in the following manner. Unless otherwise expressly stated, general genetic engineering techniques can be implemented on the basis of the procedures described in literature (such as "Molecular Cloning, A LABORATORY MANUAL", Second Edition, T. Maniatis et al., Cold Spring Harbor Laboratory Press (1989)). To begin with, cDNA prepared on the basis of mRNA extracted from human cells or organs or, alternatively, a commercially available human cDNA library or human chromosomal DNA is used as template DNA. Then, by referring to the known DNA sequence (e.g., the sequence of human factor VIII) and using a DNA probe chemically synthesized with an automatic DNA synthesizer, the template DNA is screened to obtain DNA coding for the desired polypeptide.

The DNA coding for a polypeptide having affinity for a specified phospholipid can also be obtained solely by chemical synthesis using an automatic DNA synthesizer. Another preferred method for obtaining the DNA coding for a peptide having affinity for a specified phospholipid is one utilizing a polymerase chain reaction (hereinafter designated as PCR). Briefly, by referring to the known DNA sequence (e.g., the sequence of human factor VIII), a DNA primer is chemically synthesized with optional base sequences and restriction enzyme recognizing sites being attached as required and PCR is performed using the above-mentioned cDNA as a template DNA such that the desired DNA is obtained. It should be noted that PCR can be performed by making reference to literature (e.g., "PCR Protocols, A Guide to Methods and Applications", Michael A. I. et al., Academic Press (1990)).

The step of incorporating the DNA of interest into a vector can be implemented in accordance with the general genetic engineering techniques described in the stated literature. Briefly, cloning sites in the vector are digested with suitable restriction enzymes and the DNA of interest, optionally after digestion with restriction enzymes, is inserted between the digested cloning sites, optionally with linkers and so forth. The vector to be used herein may be of any kind as long as it is capable of replication within the host to be used but it is preferred to select a vector that contains a promoter, a ribosome binding site, sequence of a signal peptide and/or a poly(A)⁺ signal, all being necessary for expressing the peptide of interest within a host, and which is replicable within the host to be used. As the promoter, ribosome binding site, sequence of signal peptide and poly(A)⁺ signal that are to be used, all promoters, ribosome binding sites, sequence of signal peptide and poly(A)⁺ signals that will function within the host to be used are applicable and these can be synthesized chemically or are available from any cells, host to be used, virus, plasmid or phage and so forth.

The step of introducing the resulting recombinant DNA into a host cell can be implemented in accordance with the methods customary in the art concerned and which are described in literature (e.g. "Shin Saibo Kogaku Jikken Purotokol", edited by Department of Oncology, Institute of Medical Science, The University of Tokyo, published by Shujunsha, 1991), as exemplified by a competent cell procedure, a calcium phosphate procedure, a DEAE dextran procedure or electroporation and so forth. The host cell into which the resulting recombinant DNA is to be introduced may be eukaryotic as typified by Hela cell, COS cell, CHO cell, yeasts and insect cells or prokaryotic as typified by Escherichia coli and Bacillus subtilis and any cell that is suitable for expressing the peptide of the present invention may be selected as appropriate and subsequently used. It should also be noted that the host cell and the vector are advantageously used in such a combination that they are mutually functional to be capable of expressing DNA coding for the peptide of interest. Examples of the preferred vector-host combination include the combination of COS cell or CHO cell with a vector containing the early promoter of simian virus 40 (SV40), a vector containing EF-1.alpha. promoter (EF promoter) or a vector containing SR.alpha. promoter and so forth, the combination of yeast Saccharomyces cerevisiae with a vector containing the promoter of a 3-phosphoglycerate kinase gene and so forth, as well as the combination of E. coli HB101 with a vector containing tryptophan promoter derived E. Coli.

The host transformed with the expression vector can be cultured using nutrient media in accordance with known general methods for culturing microorganisms, animal cells or insect cells. The peptide of interest produced by the transformed host can be purified, isolated and recovered from the culture broth by making reference to many articles and literature (e.g., "Shin Seikagaku Jikken Koza 1. Tanpakushitsu I", edited by The Japanese Biochemical Society, published by Tokyo Kagaku Dojin, 1990)). Briefly, the peptide of interest can be obtained in a pure form using at least one method selected from among desalting, concentration, salting out, ultrafiltration, ion-exchange chromatography, reverse-phase chromatography, isoelectric chromatography, affinity chromatography, and gel filtration.

A peptide having biological activity can be obtained by the same procedures as those described above for obtaining the peptide having affinity for a specified phospholipid. Briefly, human TM, for instance, can be obtained by chemical synthesis or using genetic engineering or by the combination of both.

A chemical substance exhibiting a pharmacological action can be obtained by chemical synthesis. If it is difficult to obtain by chemical synthesis or if the cost for chemical synthesis is exorbitant, it may be extracted, separated or purified from natural products or it may be separated and purified from the supernatant of the culture broth of a microorganism.

If the drug of the present invention is such that both the biologically active substance and the substance having affinity for a phospholipid are peptides and that the N terminus of either one of the peptides and the C terminus of the other are joined linearly by a peptide bond, with a peptide of a given length being optionally interposed as a linker, the drug can be directly obtained by a process characterized in that at least one of the following steps is performed:

a) the step of obtaining a peptide having the amino acid sequence of the drug of interest by chemical synthesis;

b) the step of obtaining DNA having a sequence coding for the amino acid sequence of the drug of interest;

c) the step of incorporating said DNA into a vector so as to give a replicable recombinant DNA containing said DNA;

d) the step of transforming a host cell with said recombinant DNA to give a transformant capable of expressing the peptide of interest;

e) the step of culturing said transformant to produce the peptide of interest and recovering said peptide from the culture broth.

The peptide having the amino acid sequence of the drug of interest can be obtained by chemical synthesis, typically using an automatic peptide synthesizer.

The DNA having a sequence coding for the amino acid sequence of the drug of interest may typically be prepared in the following manner. Unless otherwise expressly stated, general genetic engineering techniques can be implemented on the basis of the procedures described in literature (such as "Molecular Cloning, A LABORATORY MANUAL", Second Edition, T. Maniatis et al., Cold Spring Harbor Laboratory Press (1989)). To begin with, cDNA prepared on the basis of mRNA extracted from human cells or organs, a commercially available human cDNA library or human chromosomal DNA is used as template DNA. Then, by referring to the known DNA sequence of a peptide having biological activity (e.g. human TM DNA), the template DNA is screened using a DNA probe chemically synthesized with an automatic DNA synthesizer to obtain DNA (I) that codes for part or all of the peptide having biological activity. DNA (I) can also be obtained by chemical synthesis alone using an automatic DNA synthesizer. Similarly, DNA (II) can be produced that codes for a peptide having affinity for a specified phospholipid (e.g. the C-terminal region of human factor VIII). Another preferred method for obtaining DNA (I) and DNA (II) is one utilizing PCR. Briefly, by referring to the known DNA sequence (e.g., human TM DNA or human factor VIII DNA), a DNA primer is chemically synthesized with optional sequences and restriction enzyme recognizing sites being attached as required, and PCR is performed using the above-mentioned cDNA as a template DNA such that the desired DNA is obtained. It should be noted that PCR can be performed by making reference to literature (e.g., "PCR Protocols, A Guide to Methods and Applications", Michael A. I. et al., Academic Press (1990)). The thus obtained DNA (I) and DNA (II) are optionally digested with restriction enzymes and bound together, with a chemically synthesized DNA linker being optionally interposed, thereby yielding a DNA fragment containing the DNA coding for the drug of the present invention.

The step of incorporating the stated DNA into a vector so as to give a replicable recombinant DNA containing said DNA, the step of transforming a host cell with said recombinant DNA to give a transformant capable of expressing the drug of interest, and the step of culturing said transformant to produce the drug of interest and recovering said drug from the culture broth can be implemented by the same procedures as those described above for obtaining the peptide having affinity for a specified phospholipid.

If the novel peptide having affinity for phosphatidylserine according to the present invention is bound to a biologically active substance or its aggregate or encapsulation, there is provided a means of enabling the biologically active substance or its aggregate or encapsulation to be delivered selectively on the surface of cells that are not normal, as exemplified by damaged, denatured or activated cells. Take, for example, the case where the biologically active substance is contained within encapsulations, the substance having affinity for a specified phospholipid is modified with a substance having affinity for the components making up the skeleton of the encapsulations and thereafter mixed with the encapsulations containing the biologically active substance, whereby said substance having affinity for a specified phospholipid is bound to the surfaces of the encapsulations to yield the drug of the present invention. To give a preferred example for the case where the biologically active substance is contained within liposomes, the substance having affinity for a specified phospholipid is modified with a suitable phospholipid such as phosphatidylethanolamine and thereafter mixed with the liposomes, whereby the substance having affinity for a specified phospholipid is bound to the surface layer of each liposome to yield the drug of the present invention.

The substances or drug of the present invention, for example, the substances to be described in the Examples were not found to have any significant toxicity.

In addition, the drug of the present invention may appropriately be combined with pharmaceutical carriers and media such as sterilized water, biological saline, vegetable oils, mineral oils, higher alcohols, higher fatty acids and innocuous organic solvents, etc. and further with optional excipients, coloring agents, emulsifiers, suspending agents, surfactants, solubilizers, anti-adsorbents, stabilizers, preservatives, humectants, antioxidants, buffering agents, isotonic solutions, palliatives, etc. so as to take the form of pharmaceutical compositions (e.g., injections and oral drugs) or kits. The drug of the present invention can be administered systemically or topically and either rapidly or in a sustained manner, preferably by peroral routes, for example, by intravenous injection, intracoronary injection, intramuscular injection, intraperitoneal injection or subcutaneous injection, etc. However, the use of the drug of the present invention is by no means limited to these methods of administration. In addition, it may be used in combination with other drugs.

The dose of administration of the drug of the present invention can be adjusted as appropriate for the biologically active substance contained in said drug and depending upon the severity of the disease the patient is suffering from.

The present invention also provides a novel method for delivery of the biologically active substance in a site-selective manner so as to enhance its action and efficacy by a marked degree. Stated more specifically, the present invention provides a method by which the substance having affinity for a specified phospholipid is bound to the desired biologically active substance so that the latter is delivered on the surface layer of cells to exhibit an enhanced action and efficacy. Herein, the phospholipid which serves as targeted molecules to deliver the biologically active substance consists of molecules that compose cell membranes which are possessed by all cells without exception and, in this respect, it is totally different from the aforementioned specific antigens as the specific receptors, molecules and so forth which are composed of polypeptides. Therefore, all cells, as well as all tissues and organs that are composed of cells can potentially provide sites where the activity of the biologically active substance in the drug of the present invention will increase. In addition, if the affinity conferred on the biologically active substance is for a specified phospholipid, the activity of the biologically active substance can be selectively increased in certain of the cells and in certain of the tissues and organs that are composed of the cells. Thus, the present invention provides a drug delivery method or system based on the entirely new concept that the action and efficacy of a biologically active substance is enhanced by its selective delivery on the surface layers of cells, tissues and organs that are not normal. As will be described in the Examples, the present inventors prepared substances having affinity for a phospholipid, in which a peptide having affinity for a phospholipid was bound to TM, UTI, the second region of UTI or MCP. As it turned out, the TM having affinity for a phospholipid, the UTI having affinity for a phospholipid and the second region of UTI having affinity for a phospholipid were sufficiently increased in their activity and ability of being localized on phospholipid to exhibit an enhanced action and efficacy. The increase in activity and ability of being localized on phospholipid can also be verified for the MCP having affinity for a phospholipid by measuring its action in suppressing complement-dependent hemolysis as described in a literature ("Hotaigaku", Inai M. et al., published by Ishiyaku Shuppan Kabushiki Kaisha, 1982). The Examples provide illustrations of a drug delivery method or system that are based on the aforementioned entirely new concept that the action and efficacy of a biologically active substance is enhanced by selective aggregation of said substance on the surface layers of cells, tissues and organs at various sites such as a site where a blood coagulation is in progress, a site where the so-called immune response reactions of cells such as their activation and impairment due to inflammation or immunocytes are in progress, and a site impaired by active oxygen, and a site where a cell activation and impairing reaction is in progress due to active proteases. In summary, the present invention provides a drug and a novel peptide that are useful as preventives and therapeutics of diseases involving coagulopathy, inflammations and immune response reactions, as well as DNA necessary for producing them and a process for producing said drug. It should be noted that the drug of the present invention is by no means limited to pharmaceuticals and may be used as clinical or research reagents and the like.

The present invention will now be described below more specifically by means of working examples, which are given herein for the mere purpose of illustrating the practice of the invention and are in no way intended to limit the same. The abbreviations used in the following description are based on those which are conventional in the art concerned.

Unless otherwise noted, genetic engineering technology was implemented adopting the protocols described in books such as "Molecular Cloning, A LABORATORY MANUAL", Second Edition, T. Maniatis et al., Cold Spring Harbor Laboratory Press (1989), "A Practical Guide to Molecular Cloning", 2nd Edition, Bernard Perb et al., John Wiley & Sons (1988), "PCR Protocols, A Guide to Methods and Applications", Michael A. I. et al., Academic Press (1990), "Shin Saibo Kogaku Jikken Purotokol", edited by Department of Oncology, Institute of Medical Science, University of Tokyo, published by Shujunsha, 1991, and "Idenshi Kogaku Handobukku", edited by Muramatsu M. et al., published by Yodosha, 1991, as well as the protocols attached to the reagents or equipment used.

The E. coli strains bearing pM1354 and pM1357, respectively, which were expression plasmids for the rsTMTd and rsTMC2 disclosed in the Examples were deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-3, Higashi 1 chome, Tsukuba-shi, Ibaraki-ken, JAPAN under date of Dec. 16, 1996 (under respective Accession Numbers P-16008 and P-16009) and a transfer from the original deposit to an international deposit was effected under date of Dec. 8, 1997 (under respective Accession Numbers FERM BP-6194 and FERM BP-6195). The E. coli strain bearing pM851 which was an expression plasmid for human MCP was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-3, Higashi 1 chome, Tsukuba-shi, Ibaraki-ken, JAPAN under date of Jan. 22, 1992 (under Accession Number P-12715) and a transfer from the original deposit to an international deposit was effected under date of Feb. 18, 1993 (under Accession Number FERM BP-4195).

Claim 1 of 2 Claims

What is claimed is:

1. A peptide comprising an amino acid sequence represented by the general formula selected from the group consisting of:

(A2)n₂ -(A3)₁ and

(A2)n₂,

wherein A2 is the amino acid sequence denoted by SEQ ID NO:2, A3 is the amino acid sequence denoted by SEQ ID NO:3, and n₂ is 2 or 3.

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