JPWO2004007714A1 - Human prothrombin analog and human mesothrombin analog - Google Patents

Abstract

A human mesothrombin analog having a high ability to activate human protein C and exhibiting an anticoagulant action, and a human prothrombin derivative capable of producing the human mesothrombin derivative. Human prothrombin or an analog of human mesothrombin, comprising a peptide in which Arg at positions 155, 271 and 284 and amino acids at positions 466 to 473 in the human pro (or meso) thrombin sequence are altered in whole or in part. In the case of human mesothrombin, the peptide consists of a double chain, has substantially no procoagulant activity, has a higher ability to activate protein C than human thrombin, and does not substantially convert to human thrombin. . In the case of a human prothrombin analog, when the peptide is a single chain and Arg at position 320 is cleaved and activated, the above properties are exhibited and a human mesothrombin analog can be generated. Useful as an anticoagulant.

Description

  The present invention relates to a human mesothrombin analog, a human prothrombin analog, a DNA encoding the same, a plasmid containing the DNA, and a host cell containing the plasmid. The invention further relates to pharmaceutical compositions comprising such analogs.

Thrombin is the procoagulant that plays the most important role in thrombus formation (Jackson CM, Haemostasis & Thrombosis 3rd Edition, edited by AL Bloom et al., Churchill Livingstone). The most characteristic action of thrombin is the action of converting fibrinogen into fibrin and coagulating blood. It also has the effect of activating blood coagulation factor XIII to crosslink fibrin, the effect of activating blood coagulation factor V and factor VIII, and the effect of activating and aggregating platelets (Michio Matsuda, Koji Suzuki, 7th edition). 2 factors (prothrombin), hemostasis, thrombus, fibrinolysis Chugai Medical). Thrombin also acts as an anticoagulant by binding to thrombomodulin and activating protein C (Sadler JE, Thromb Haemost 78 392-395). Furthermore, the resulting activated protein C exhibits strong anticoagulant activity, and activated protein C itself has been used clinically as a useful pharmaceutical (Kanji S et al., Pharmacotherapy 21, 1389-1402).
By the way, prothrombin is converted into thrombin via mesothrombin, and mesothrombin itself has enzyme activity including coagulation promoting activity as well as thrombin (Walker RK et al., J Biol Chem 269, 27441). -27450). Human prothrombin is produced as a precursor of 622 amino acids (for example, SEQ ID NO: 2 in the sequence listing), and 43 amino acids from the N-terminal are processed into mature 579 amino acids (for example, SEQ ID NO: SEQ ID NO: 1 is exemplified), and is usually present in the blood at about 100 to 150 mg / L (edited by Michio Matsuda, Koji Suzuki, 7th factor (prothrombin), hemostasis / thrombosis / fibrinolysis Chugai Medical, 1994). Prothrombin is cleaved from Arg ³²⁰ by an enzyme such as a prothrombinase complex (coagulation activation factor X, blood coagulation activation factor V, a complex consisting of phospholipid and Ca 2+) or snake venom. An amino acid A chain and a 259 amino acid B chain are converted to mesothrombin consisting of two chains that are cross-linked by an SS bond, and further, by means of a prothrombinase complex, thrombin or mesothrombin itself, etc. Ag ²⁸⁴ of the A chain is cleaved (in addition, Arg ¹⁵⁵ and Arg ^{271 of the} A chain are cleaved) to generate thrombin.
Prothrombin is a vitamin K-dependent protein, usually 10 (Gla ⁶ , Gla ⁷ , Gla ¹⁴ , Gla ¹⁶ , Gla ¹⁹ , Gla ²⁰ , Gla ²⁵ , Gla ²⁶ , Gla ²⁹ , Gla ³² : amino acid position number is It has a Gla (gamma carboxyglutamate) residue (shown as Glu in the sequence listing). Gla in the above-mentioned prothrombin binds to phospholipid in the prothrombinase complex via Ca ²⁺ , and as a result, prothrombin is cleaved by the prothrombinase complex and converted to thrombin via mesothrombin. (Same as above) The thrombin produced by cleaving in this way usually consists of a double chain in which an A chain of 36 amino acids and a B chain of 259 amino acids are cross-linked by an SS bond (Wolfram B et al., Chapter 1, (Thrombin Structures And Function Berliner LJ, Plenum Press 1992).
Although mesothrombin has a higher protein C activation ability than thrombin, it is easily converted to thrombin under physiological conditions, so its physiological half-life is short, and it is converted to thrombin to promote clotting. Since mesothrombin itself exhibits coagulation promoting activity as well as activity, it was difficult to use mesothrombin itself as an anticoagulant. Incidentally, there is a mesothrombin-like polypeptide by Cote HCF et al. (USP 5,510,248) as a prior document similar to the present invention. Cote et al. Show that when a polypeptide in which the prothrombin sequences Arg ¹⁵⁵ , Arg ²⁷¹ , Arg ²⁸⁴ are changed to alanine is activated, a mesothrombin analog is produced, which is indicated by the fibrinogen clotting time of this mesothrombin analog It shows that the procoagulant activity of 6.8% is 6.8% compared to human thrombin. Cote et al. Suggest that this mesothrombin analog can be a protein C activator, although this mesothrombin analog is weaker than human thrombin, but is still a clinical problem in procoagulation It must be said that it has activity and cannot be used practically as an anticoagulant.
Therefore, there has been a need for a pharmaceutical composition that is useful and highly safe, particularly an anticoagulant.

In order to solve the above problems, the present inventors made various human prothrombin analogs and studied their activities. As a result, human prothrombin analogs in which amino acids forming Arg ¹⁵⁵ , Arg ²⁷¹ , Arg ²⁸⁴ and Glu ⁴⁶⁶ -Gly ⁴⁷³ of the human prothrombin sequence have been changed to human mezothrombin analogs under physiological conditions such as the body. Is converted but not substantially converted to thrombin. Furthermore, the human mesothrombin analog itself has substantially no procoagulant activity, and strong protein C activation. It was confirmed that the property has the ability. In other words, this human mesothrombin analog can be a useful anticoagulant, and human prothrombin analog, which is a precursor of this human mesothrombin analog, can be used in a physiological environment. As a result, it was confirmed that it could be a useful anticoagulant and the present invention was completed.
That is, in the present invention, the amino acids Arg ¹⁵⁵ , Arg ²⁷¹ , and Arg ^{284 in} the human mesothrombin sequence have been changed, and all or all of the amino acids forming Glu ⁴⁶⁶ -Gly ⁴⁷³ of the human mesothrombin sequence It is a human mesothrombin analog consisting of a double-chain peptide that is partially modified, and has substantially no procoagulant activity and has a higher ability to activate protein C than human thrombin. And further relates to a human mesothrombin analog characterized in that it is not substantially converted to thrombin.
In the present invention, the amino acids Arg ¹⁵⁵ , Arg ^{27 1} , and Arg ^{284 in} the human prothrombin sequence have been changed, and all or one of the amino acids that form Glu ⁴⁶⁶ -Gly ⁴⁷³ in the human prothrombin sequence. A human prothrombin analog consisting of a single-chain peptide that has been modified in part, and has substantially no procoagulant activity when Arg ³²⁰ is cleaved and activated, and its protein C It is a human prothrombin analog that is capable of producing human mesothrombin analogs that are more active than human thrombin and are not substantially converted to thrombin.

FIG. 1 is an explanatory diagram of the structure, modified sites and cleavage activation of human prothrombin and the prothrombin analogs and mesothrombin analogs of the present invention or comparative examples. A represents the A chain of thrombin, and B represents the B chain of thrombin. F1 represents Fragment 1, F2 represents Fragment 2, and human prothrombin is composed of F1, F2, Arg ²⁷¹ -Arg ²⁸⁴ fragments, A and B.
FIG. 2 is an electrophoretic diagram (SDS-PAGE) for confirming the expression of the prothrombin analog of the present invention or a comparative example.
FIG. 3 is an electrophoretic diagram (SDS-PAGE) for confirming the state and structure of the prothrombin analog of the present invention or comparative example after activation. (A) shows reducing conditions, and (B) shows non-reducing conditions.

  Natural human prothrombin is produced by Arg by proteolytic enzymes under physiological conditions such as in blood or blood vessels.³²⁰Cleaved and activated in to produce human mesothrombin. Human mesothrombin thus has human prothrombin as Arg^{To 320}In which each polypeptide is disulfide bond (A chain Cys).²⁹³And B chain Cys⁴³⁹).
  Examples of normal proteolytic enzymes that can substantially cleave prothrombin in a physiological coagulation process include a prothrombinase complex, an on-membrane prothrombin activator, thrombin, and mesothrombin.
  Since many mutations are usually observed in humans, even in the present invention, the sequence of human prothrombin is not particularly limited as long as it is obtained from humans. For example, as an amino acid sequence of mature human prothrombin, For example, the amino acid sequence of 1-579 of SEQ ID NO: 1 is exemplified. Moreover, as the base sequence, the base sequence of 1-1737 of Sequence Listing SEQ ID NO: 3 is exemplified.
  The numbering of amino acids shown in the present invention is numbering with Ala in the amino acid sequence of the mature human prothrombin being 1, and even in a double-stranded mesothrombin analog, for convenience, the human prothrombin sequence (mature) The numbering based on is used.
  Examples of amino acid changes in the present invention include deletion and substitution.
  In the present invention, the numbering based on the common human prothrombin sequence is used for convenience even if there are amino acid changes, for example, deletions. Further, it is allowed to add additional amino acids to the N-terminus or C-terminus of the peptide, but in this case, it is expressed based on the sequence of human prothrombin.
  The human mesothrombin analog of the present invention has substantially no procoagulant activity and has a higher protein C activation ability than human thrombin and is not substantially converted to thrombin. The human mesothrombin analog is not particularly limited.
  The mesothrombin analog of the present invention is not particularly limited as long as it is not substantially converted to thrombin, particularly under physiological conditions such as in blood. For example, a detectable amount of thrombin (human It is also preferable to understand that it is not converted into thrombin or human thrombin-like substance). As a detectable amount, the mesothrombin analog of the present invention is added to a buffer solution, and allowed to stand at a constant temperature, preferably 1 hour at 37 ° C., and then subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE). It can be understood that the amount can be detected as a band when visualized with a dye such as Comassie blue. At this time, the amount of protein applied to SDS-PAGE can be 1 to 100 μg, but a more preferable amount is 10 μg.
  The mesothrombin analog of the present invention itself has substantially no procoagulant activity. For example, using a coagulometer, a test compound is added to a buffer containing 2 mg / ml fibrinogen to increase the coagulation time. What is necessary is just to measure by the method of converting into thrombin activity using the calibration curve with the coagulation time when adding human thrombin. When the mesothrombin analog of the present invention is added, the coagulation promoting activity obtained from the coagulation time may be almost negligible for the same amount of human thrombin. The coagulation promoting activity of the mesothrombin analog of the present invention is preferably 2% or less of the same amount of human thrombin, more preferably 1% or less, and even more preferably 0.1% or less. As mentioned. From another viewpoint, for example, 1.5% or less is preferable, 1.0% or less is more preferable, and 0.1% or less is more preferable. In addition, when a mesothrombin analog in an amount capable of exhibiting a medicinal effect as an anticoagulant is intravenously administered to a human or animal, a marker indicating that a coagulation reaction, that is, a thrombus has occurred, such as D-dimer (Matsuo T et al. (Semi Thromb Hemost 26 101-107, 2000) showing a numerical value within the normal range can be understood as a more preferable specific example of the property of “substantially free of procoagulant activity” in the present invention. The normal range described above is a range that is determined to be a normal state, and indicates a normal range that is determined by a number of healthy human samples. In a commercially available measurement kit, the normal range is usually disclosed as a reference value. For example, in the D-Di test (manufactured by Diagnostica Stago), it is 0.5 μg / ml, although it varies depending on the measurement kit or measurement method used.
  The protein C activation ability of the mesothrombin analog of the present invention is not particularly limited as long as the protein C activation ability is higher than that of human thrombin under biological conditions. A preferred example of the protein C activation ability of the mesothrombin analog of the present invention is 5 times or more that of human thrombin, and more preferably 10 times or more. An example of a simple test method in addition to biological conditions is an example in which normal protein C activation ability measurement is performed under conditions where thrombomodulin coexists with a test compound. That is, this soluble thrombomodulin and the test compound to be measured are mixed, allowed to stand for a certain period of time, preferably 30 seconds, added with protein C, and further allowed to stand for a certain period of time, preferably 5 minutes, to inhibit thrombin. It can be measured by adding an agent and allowing to stand for a predetermined time, preferably 5 minutes, and then adding a chromogenic substrate for activated protein C.
  Examples of conditions in which thrombomodulin is usually present at a molar ratio of 10 times or more with respect to a mesothrombin analog or human thrombin are exemplified. As the thrombomodulin used in the measurement, it is preferable to use soluble thrombomodulin. For example, the examples of JP-A No. 64-6219 or JP-A-2766473, particularly the soluble thrombomodulin of Example 20, should be used. Is preferred. Argatroban, hirudin, antithrombin, etc. can be used as thrombin inhibitors. As a chromogenic substrate, Glu-Pro-Arg-pNA or the like is often used.
  As a modification in the human mesothrombin analog of the present invention, Arg in the sequence of human mesothrombin (although based on the sequence of human prothrombin)^{15 5}, Arg²⁷¹, Arg²⁸⁴And the amino acid sequence Glu of human mesothrombin⁴⁶⁶-Gly⁴⁷³In the normal human mesothrombin sequence consisting of a double-chain peptide, Arg is changed in all or part of the amino acids forming¹⁵⁵, Arg²⁷¹, Arg²⁸⁴Belongs to the A chain and Glu⁴⁶⁶-Gly⁴⁷³The amino acids that form are in the B chain.
  The human mesothrombin analog described above is a human prothrombin analog Arg consisting of a single chain peptide.³²⁰Can be cut off. Similar to natural human prothrombin, the human prothrombin analog of the present invention is capable of reacting with Arg by proteolytic enzyme under physiological conditions or artificial conditions.³²⁰Has the property of being cleaved and activated to yield mesothrombin analogs. Examples of physiological conditions include environmental conditions in blood or blood vessels, and examples of artificial conditions include the measurement conditions described above. Proteolytic enzymes include proteolytic enzymes that can substantially cleave prothrombin in the physiological coagulation process, such as prothrombinase complex, on-membrane prothrombin activator, thrombin and mesothrombin, and similar to the mesothrombin of the present invention. Take the body itself as an example. Furthermore, snake venoms such as the enzymes and ecarin can be reacted in a buffer solution for the purpose of producing a mesothrombin analog in vitro.
  Arg¹⁵⁵, Arg²⁷¹, Arg²⁸⁴Is a site that is cleaved when human prothrombin is converted to thrombin, and is a site that is not cleaved in human mesothrombin. The human mesothrombin analogs and human prothrombin analogs of the present invention have been altered in this part, and this change is particularly limited as long as it does not affect the defining characteristics of human mesothrombin analogs and human prothrombin analogs. Not. Usually, these sites need not be cleaved enzymatically, and may be altered by deletion of Arg, substitution with an amino acid other than Arg, or a combination thereof. The most preferred example is substitution with Ala. In addition, examples in which these Arg are deleted are also included. Arg¹⁵⁵, Arg²⁷¹, Arg²⁸⁴Examples in which all of these are substituted with Ala are preferred.
  In the human prothrombin analog and human mesothrombin analog of the present invention, Glu⁴⁶⁶-Gly⁴⁷³Has changed, and any change at that point may be anywhere as long as it does not affect its defining characteristics. This Glu⁴⁶⁶-Gly⁴⁷³As a modification of, deletion of all 8 amino acids is preferred. Moreover, the deletion of arbitrary 7 amino acids is preferable, and the deletion of arbitrary 6 amino acids is also preferable. Alternatively, a deletion of any 5 amino acids is preferred. Furthermore, a deletion of any 4 amino acids is preferred, and a deletion of any 3 amino acids is also preferred. Furthermore, a deletion of any two amino acids is also preferable, and a deletion of any one amino acid is also preferable. Deletion of any 7 amino acids includes, for example, Glu⁴⁶⁶-Val⁴⁷²Deletion of is preferred. Deletions of any six amino acids include, for example, Glu⁴⁶⁶-Asn⁴⁷¹Deletion of is preferred. Examples of the deletion of any five amino acids include, for example, Glu⁴⁶⁶-Ala⁴⁷⁰Deletion of is preferred. Examples of the deletion of any four amino acids include, for example, Glu⁴⁶⁶-Ala⁴⁶⁹Deletion of is preferred. Examples of the deletion of any three amino acids include, for example, Glu⁴⁶⁶-Ala⁴⁶⁸Deletion of is preferred. Examples of the deletion of any two amino acids include, for example, Glu⁴⁶⁶-Thr⁴⁶⁷Or Thr⁴⁶⁷-Trp⁴⁶⁸Deletion of is preferred. Moreover, examples of deletion of any one amino acid include, for example, Glu⁴⁶⁶, Thr⁴⁶⁷Or Trp⁴⁶⁸Deletion of is preferred.
  In addition, Glu⁴⁶⁶-Thr⁴⁷³Deletion of any one to eight amino acids and / or substitution with other amino acids is also preferred. In particular, Glu⁴⁶⁶-Thr⁴⁶⁹Deletion of any two amino acids and / or substitution with other amino acids is preferred,⁴⁶⁶-Thr⁴⁶⁸Further preferred are deletions of any two amino acids and / or substitutions with other amino acids.
  Glu⁴⁶⁶-Thr⁴⁶⁹Substitution of any one amino acid with another amino acid is preferred,⁴⁶⁶-Thr⁴⁶⁸More preferred is the substitution of any one amino acid with another amino acid. As a particularly preferable change position, Glu⁴⁶⁶-Thr⁴⁶⁷, Thr⁴⁶⁷-Trp⁴⁶⁸, Glu⁴⁶⁶, Thr⁴⁶⁷, Trp⁴⁶⁸Is mentioned.
  Glu⁴⁶⁶-Gly⁴⁷³In connection with Glu⁴⁶⁶-Lys⁴⁷⁴Is a structure called thrombin loop 2 or Autolysis loop, which is the site where thrombin interacts with sodium. Thrombin completely lacking this part has its physiological activity, ie procoagulant activity, and ability to activate protein C Are both reported to be lower than in the wild (QC Dang et al. J Biol Chem 272, 19649-19651, 1997).
  That is, if loop 2 is deleted in thrombin, not only the procoagulant activity but also the protein C activation ability is reduced, so that the human mesothrombin analog that has no coagulation promotion activity and high protein C activation ability, the human It is clear that the effect of the present invention to provide a pro-human mesothrombin analog that changes to a mesothrombin analog is not simply sought.
  In fact, we have determined that Arg in the sequence of human mesothrombin.¹⁵⁵, Arg²⁷¹, Arg²⁸⁴Each amino acid of Glu was changed to Ala and the human mesothrombin sequence Glu⁴⁶⁶-Gly⁴⁷⁴A human mesothrombin analog lacking all of the amino acids forming the protein was prepared, and its ability to activate protein C was confirmed to be about 1/5 that of human thrombin.
    The human mesothrombin analogs and human prothrombin analogs of the present invention include Arg¹⁵⁵, Arg²⁷¹, Arg²⁸⁴Are all replaced by Ala and Glu⁴⁶⁶-Gly⁴⁷³Or Glu⁴⁶⁶-Val⁴⁷²Or Glu⁴⁶⁶-Asn⁴⁷¹Or Glu⁴⁶⁶-Ala⁴⁷⁰Or Glu⁴⁶⁶-Thr⁴⁶⁹Or Glu⁴⁶⁶-Trp⁴⁶⁸Or Glu⁴⁶⁶-Thr⁴⁶⁷Or Thr⁴⁶⁷-Trp⁴⁶⁸Or Glu⁴⁶⁶Or Thr⁴⁶⁷Or Trp⁴⁶⁸Preferred examples include those in which Glu is deleted.⁴⁶⁶-Ala⁴⁷⁰Or Glu⁴⁶⁶-Thr⁴⁶⁹Or Glu⁴⁶⁶-Trp⁴⁶⁸A particularly preferable example is one in which is deleted.
  In the human prothrombin analog and mesothrombin analog of the present invention, Gla (γ carboxyl glutamic acid) may be included in the sequence in the same manner as human prothrombin and mesothrombin. The number and location of Gla are not particularly limited as long as they do not affect the defining characteristics, but are preferably the same as wild-type human prothrombin and mesothrombin.
  The human mesothrombin analogs and human prothrombin analogs of the present invention substantially comprise human prothrombin or a sequence similar to that of human mesothrombin, and have substantially no definitional characteristic thereof, ie, procoagulant activity. As long as the ability to activate Protein C is higher than that of human thrombin and it is not substantially converted to thrombin, or a human mesothrombin analog having that property can be produced, it has no special effect. A chain may be added, or it may have one or more amino acids at the N-terminus or C-terminus. For example, examples of the sugar chain include an N-type sugar chain, an O-type sugar chain, and polyethylene glycol (PEG). The sugar chain to be added varies depending on the type of host cell, and can also be added by a chemical method. As a method for chemically attaching PEG, for example, a method described in JP-A-2001-64300 is exemplified. One or a plurality of amino acids may be added. Examples of the amino acid and amino acid sequence that may be added include Tag. When Tag is attached, a conventionally known purification step, which will be described later, can be adopted, which is convenient and preferable. As Tag, HIS-Tag is generally used, which can be purified using a Ni chelate column. In addition, GST-Tag is also generally used, and purification using a glutathione-fixed column is possible. It becomes possible.
  When Tag is attached to the C-terminus of the prothrombin analog of the present invention, the Tag sequence is usually connected after several amino acid linkers. For example, when HIS-Tag is attached, several amino acid linkers are connected to the C terminus of the human prothrombin-like sequence, and several His, usually about 6, are connected to the C terminus. When adding HIS-Tag, it is simple and preferable to use an invitrogen kit (see page 300 of the 2000-2001 edition catalog of Invitrogen). When GST-Tag is used, several amino acid linkers are connected to the C terminus of the human prothrombin-like sequence, and Glutathation Transferase is connected to the C terminus. When GST-Tag is added, it is simple and preferable to use a Takara kit (see Takara 2002-2003 catalog page C59-C65). Any known linker can be selected as the linker, but can be selected depending on the vector to be used and how to insert the DNA sequence. It is also preferable to select a vector in which an enterokinase recognition site is inserted in the linker portion in advance. For example, when using the pET19b vector (see Takara 2002-2003 catalog C59-C65) manufactured by Takara, it is possible to easily cleave Tag by contacting with enterokinase after purification. . In the present invention, a peptide with Tag and a peptide containing a small amount of amino acid sequence remaining after removal of Tag and the like are included in the human mesothrombin analog and human prothrombin analog of the present invention.
  The present invention further relates to a DNA encoding the above-mentioned human prothrombin analog. Further, in some cases, DNA capable of giving the above-mentioned human mesothrombin analog may be included.
  That is, Arg in the sequence of human prothrombin¹⁵⁵, Arg²⁷¹, Arg²⁸⁴And the amino acid sequence Glu of human prothrombin⁴⁶⁶-Gly⁴⁷³A human prothrombin analog consisting of a single-chain peptide in which all or part of the amino acids forming³²⁰When cleaved and activated, it has substantially no procoagulant activity, and its protein C activation ability is higher than that of human thrombin, and is not substantially converted to thrombin. DNA encoding a human prothrombin analog capable of producing a human mesothrombin analog.
  As a method for obtaining DNA encoding the human prothrombin analog of the present invention, for example, with respect to the base sequence encoding human prothrombin, Arg^{15 5}, Arg²⁷¹, Arg²⁸⁴Is replaced by a codon encoding an amino acid other than Arg or deleted, and Glu⁴⁶⁶-Gly⁴⁷³Can be carried out using various known genetic manipulation techniques so as to substitute or delete all or part of the amino acids that form. Examples of the base sequence encoding human prothrombin include the base sequence of 1-1737 in SEQ ID NO: 3 in the sequence listing. It can be produced as a precursor to which an appropriate signal peptide is connected, and DNA sequences encoding various signal peptides can be added. Preferably, 43 natural human prothrombin precursors are added. An example is a signal peptide encoding an amino acid sequence. Specifically, the base sequence of 1-129 in SEQ ID NO: 4 is exemplified.
  As an amino acid changing method, for example, CGC, CGT, and ACG that are codons representing Arg may be changed to any one of GCT, GCC, and GCG that are codons indicating Ala. A mutation method can be used, and a method using a commercially available kit (for example, Takara LA PCR in vitro mutation Kit) is more practical. For example, Arg¹⁵⁵In order to substitute Ala with Ala, it is possible to prepare 5'-ACT GTA GCG ATG ACT CCA GCT AGC GAA G-3 'and use the above-mentioned commercially available kit. Arg^{15 5}Can be prepared using a mutation primer having GCT as GTC in the above-mentioned mutation primer, and if a codon indicating another amino acid is used, all amino acids can be changed. Is possible. In addition, in order to prevent misread by PCR, it is sometimes necessary to introduce a codon having the same amino acid to be translated but a different sequence before and after the codon to be mutated, It is also a preferred example.
  As a deletion method, a mutation method using PCR can be used as in the substitution. That is, an oligonucleoside from which the codon of the amino acid to be deleted is removed may be used, for example, Glu⁴⁶⁶-Thr⁴⁶⁹In order to delete this, an oligonucleoside (5′-TGG GGC AAC CTG AAG GCC AAC GTT GGT AAG-3 ′) from which the codon of 4 amino acids has been removed can be prepared and prepared using the aforementioned commercial kit. Is possible. In addition, the DNA sequence at the 5 ′ end of human prothrombin and the DNA sequence before the amino acid to be deleted, and the oligonucleoside of the DNA sequence after the amino acid to be deleted and the DNA sequence at the 3 ′ end of human prothrombin, respectively It is also possible to use PCR to create two DNAs and connect them. For example, Glu⁴⁶⁶-Thr⁴⁶⁹5′-TTTTATTATTACCCATGGCGCACGTCCCGAGGGC-3 ′ and 5′-CGG GTG ACA GGC TGG GGC AAC CTG AAG-3 ′ and 5′-GCC AAC GTT GGG AAG CGG GAG AAG CGG And 5′-CATTGATCAGTTTGGAGAGCGGCCGCGTT-3 ′, using human prothrombin cDNA as a template DNA, can be prepared by connecting two DNAs. Using a similar technique, Glu⁴⁶⁶-Lys⁴⁷⁴It is possible to delete any amino acid of the amino acids constituting
  Further, the human prothrombin analog or human mesothrombin analog of the present invention may have Tag attached thereto, and purification is facilitated by the added Tag. However, when Tag is added, A DNA sequence encoding the Tag sequence may be added. Specifically, HIS Tag can be added using a pSECTag2 kit (manufactured by Invitrogen), and protein purification is simplified by a Ni chelate column. Further, T7Tag, GST-Tag, etc. are generally used. For example, T7Tag is introduced using a pET3 vector (Takara Biomedical) and purified using an antibody column against T7 (T7 Tagafinity purification Kit, manufactured by Novagen), or a pET41 vector (Takara Biomedical) is used. Examples thereof include a method of introducing GST-Tag and purifying using glutathione immobilization resin (GST Bind Resin, manufactured by Novagen).
  In the present invention, a DNA encoding the human prothrombin analog of the present invention is contained by binding the above-described DNA to a known plasmid that can function in the host, and the human prothrombin analog or human mesothrombin analog is A plasmid that can be used for expression is obtained.
  The present invention also provides a transformant obtained by transferring the above plasmid into an appropriate host cell and performing transformation.
  As a method for producing the human prothrombin analog or human mesothrombin analog of the present invention, for example, the following method is mentioned as a preferred example, but other methods are not particularly limited as long as the present invention is achieved.
(A) ligating a DNA that encodes the peptide and a replicable plasmid to obtain a replicable recombinant DNA comprising the DNA and the replicable plasmid;
(B) transforming a host cell with the replicable recombinant DNA to form a transformant;
(C) selecting the transformant from the parent cell of the cell;
(D) culturing the transformant to proliferate cells containing the DNA and producing the peptide; and
(E) The peptide produced by the cell is isolated from the cultured cell.
  In order to produce the peptide of the present invention by expressing the DNA of the present invention, it is usually preferable to use eukaryotic cells such as vertebrate cells as host cells, and known host-vectors for those hosts. A system can be used. Examples of eukaryotic cells are preferably animal cells such as VERO (ATCC CCL-81) cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, WI38, BHK, COS-7 and MDCK cell lines. . In order to express the DNA of the present invention in the aforementioned eukaryotic cells, the recombinant DNA of the present invention should generally be located at a functional sequence for controlling gene expression, eg, origin of replication, upstream of the DNA of the present invention. It contains a promoter, ribosome binding site, polyadenylation site and transcription termination sequence. Such functional sequences that can be used to express the DNA of the present invention in eukaryotic cells can be obtained from viruses and viral substances.
  For example, promoters that can be used in the present invention can be obtained from adenovirus 2, polyoma virus, simian virus 40 (SV40), cytomegalovirus (CMV), and the like. In particular, adenovirus 2 main late promoter, SV40 early and late promoter, and CMV promoter can be mentioned as preferable examples.
  For the origin of replication, exogenous origins such as those derived from viruses such as adenovirus, polyoma, SV40, varicella stomatitis virus (VSV), bovine papilloma virus (BPV) can be used. In addition, when using a plasmid having the property of being integrated into the host chromosome, the origin of replication of the host chromosome can be used.
  As described above, cells transformed with the replicable recombinant DNA of the present invention are selected from the parent cells remaining untransformed according to at least one phenotype given to the recombinant DNA. . A phenotype can be provided by inserting at least one marker gene into the recombinant DNA. In addition, a marker gene inherent in a replicable expression vector can be used. Examples of marker genes include drug resistance genes such as neomycin and Zeocin resistance and genes encoding dihydrofolate reductase (hereinafter referred to as “DHFR”).
  The transformed cells contain the DNA encoding the peptide of the present invention by culturing in an ordinary known method using an ordinary nutrient medium, and the cells producing the peptide can be proliferated. The cells can produce the peptide of the present invention by expressing the DNA encoding the peptide of the present invention. After culturing, the peptide of the present invention can be isolated from the culture of the transformant using an ordinary known method such as column chromatography.
  The scope of the present invention includes cells that contain the DNA encoding the peptide of the present invention and that produce the peptide, wherein the transformant has been cultured and expanded in this manner.
  For purification, a known purification method can be employed. For example, it can be performed according to a known purification method of human prothrombin, and a method of combining barium precipitation, ammonium sulfate fractionation, and DEAE-Sephadex chromatography by Bajaj SP et al. (Prep Biochem 11 397-412, 1981).
  Further, for example, a purification method using an anti-human prothrombin antibody can be used. As the anti-human prothrombin antibody, any of a monoclonal antibody and a polyclonal antibody may be used, and examples of the antibody species include mouse, rabbit, sheep, goat, etc. Any animal species can be used. An example of such an antibody is goat anti-human prothrombin polyclonal antibody ICN-6801001 manufactured by Cosmo Bio. The carrier for binding the antibody used for purification may be any known gel or bead-like carrier such as a sepharose carrier, agarose carrier, polystyrene carrier, and glass carrier. A carrier activated in advance for binding the antibody is more preferable. Examples of such carriers include NHS-activated Sepharose carrier (NHS-activated Sepharose 4 Fast Flow Lab Packs, manufactured by Amersham) and CNBr-activated Sepharose carrier (CNBr-activated Sepharose 4B Lab Packs).
  For example, when an amino acid sequence that can be used for purification, such as Tag, is added, a purification method specific to each amino acid sequence can be applied. For example, when the HIS-Tag is attached, it can be purified using a Ni chelate resin, and is usually performed using a purification kit. As such a kit, ProBond nickel chelate resin (Invitrogen 2000-2001 edition catalog page 395) manufactured by Invitrogen can be exemplified. Moreover, when GST-Tag is attached, it is possible to refine | purify using glutathione solidification resin, and it is normally performed using a purification kit. An example of such a kit is Glutathione Superflow Resin (see page 229 of Clontech 2002 edition catalog) manufactured by Clontech. When it is necessary to delete Tag etc. after purification, a method suitable for each can be adopted as appropriate. For example, when an enterokinase sequence is incorporated, it is treated with enterokinase, Tag can be excised by removing. Examples of such kits include enterokinase Max and EK-Away resin (Invitrogen 2000-2001 edition catalog, pages 403 and 404, respectively) manufactured by Invitrogen. it can. In order to obtain a human mesothrombin analog, for example, as described above, the human prothrombin analog is expressed, and then in any process, the human prothrombin analog is cleaved and activated under conditions where it is activated. It is preferable to use an analog. In the purification, it is preferable to substantially purify to such an extent that it can be used as the pharmaceutical composition of the present invention.
  Furthermore, the present invention relates to a pharmaceutical composition comprising these human prothrombin analogs or human mesothrombin analogs as active ingredients.
  The pharmaceutical composition of the present invention can be prepared using a therapeutically effective amount of a human prothrombin analog or a human mesothrombin analog alone, or both, and admixed with a pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carrier include excipients, binders such as carboxymethylcellulose, additives, stabilizers, isotonic agents, buffers, and the like, and a solvent such as distilled water may be contained. .
  When the pharmaceutical composition of the present invention is administered to humans, it can be administered by parenteral routes such as intravenous, subcutaneous, intramuscular, pulmonary, and nasal routes. In some cases, it can be administered orally. Usually, fine powders, microcapsules, injections and the like are exemplified. Furthermore, it is possible to release the drug slowly by formulation. The dose of the compound of the present invention varies depending on the administration method, the age, weight and symptom of the patient, but is generally 0.001 to 1000 mg, more preferably 0.1 to 100 mg per day for an adult. It is exemplified that the dose is divided into several or several times. The administration period is generally daily for several days to several months, but both the daily dose and the administration period can be increased or decreased depending on the symptoms of the patient.
  The compound which is an active ingredient of the pharmaceutical composition of the present invention is a biological protein and is an analog of human prothrombin which is present in a large amount (100-200 mg / dl) in blood. From this, it is thought that it can be safely administered to a living body. Actually, when 10 mg / kg of the pharmaceutical composition of the present invention was administered to 5 mice, all cases survived and no particularly problematic symptoms were confirmed.
  The pharmaceutical composition of the present invention is effective as an anticoagulant. Therefore, the pharmaceutical composition of the present invention comprises thrombosis, endotoxemia, sepsis, disseminated intravascular coagulation syndrome (DIC), multiple organ failure (MOF), adult respiratory failure syndrome (ARDS), systemic hyperinflammatory syndrome. It can be used in treating or preventing diseases such as (SIRS). Thrombosis is a general term for diseases in which a thrombus occurs in a blood vessel of any organ in the living body and damages the organ. Specifically, myocardial infarction, unstable angina, Occlusion, embolism after atrial membrane abnormality or atrial surgery, Kawasaki disease, cerebral infarction, cerebral embolism, cerebral thrombosis, spinal cord injury, transient ischemic attack (TIA), post-transplant hepatic vein occlusion (VOD) ), Hepatitis, thrombotic peripheral arterial occlusion (Burger disease, TAO), arteriosclerotic peripheral arterial occlusive disease (ASO), deep vein thrombosis (DVT) such as post-cerebral infarction, head injury, spinal cord injury, and surgery , Economy class syndrome, pulmonary embolism, peripheral arterial thrombosis, peripheral arterial embolism, nephritis, pregnancy toxemia, systemic lupus erythematosus (SLE), Sjogren's syndrome, lupus anticoagulant, Behcet's disease Give Can.
  Moreover, the pharmaceutical composition of this invention has the effect of suppressing the inflammatory reaction accompanying the coagulation reaction with suppression of the coagulation reaction. Similarly, it can be used for diseases in which thrombomodulin provided with activated protein C is used. Specifically, diseases such as sepsis, endotoxemia, multiple organ dysfunction (MOF), adult respiratory failure syndrome (ARDS), and systemic hyperinflammatory syndrome (SIRS) are exemplified.
  The effectiveness of the pharmaceutical composition of the present invention can be easily confirmed by using animal experiments using a disease state model. That is, as a pathological model for showing the effectiveness of the pharmaceutical composition of the present invention as an anticoagulant, a model in which the abdominal vena cava is stagnated, a model in which the abdominal vena cava is treated with TF or iron chloride, abdominal vena cava A model in which a coil is inserted is also exemplified. Specifically, for example, the method of Solis MM et al. (Thromb 73, 385-394 1994) can be mentioned. In this model, the effectiveness of venous thrombosis such as DVT is particularly effective. I can judge. Further, a model for ballooning the carotid artery, an AV shunt model, and a Colts model are also exemplified. Specifically, for example, the method of Gruber A et al. In this model, the effectiveness of arterial thrombosis such as myocardial infarction, cerebral infarction, unstable angina pectoris, and peripheral arterial occlusion can be determined. Further, a model for administering TF is exemplified, and specifically, for example, the method of Mohri M et al. (Am J Hematol 45, 298-303 1994) is exemplified. In this model, in particular, the effectiveness of DIC is judged. it can. Examples of the pathological model for confirming the anti-inflammatory action of the pharmaceutical composition of the present invention include a model in which a Gram-negative bacterium or endotoxin is administered. Specifically, the method of Uchiba M et al. (Am J Physiol 271, L470- 475 1996), Gonda Y et al. (Thromb 71, 325-335, 1993), and in this model, in particular, sepsis, endotoxemia, multiple organ failure (MOF), adult respiratory failure syndrome ( ARDS), effectiveness of systemic hyperinflammatory syndrome (SIRS) can be judged.
  The pharmaceutical composition of the present invention may contain a compound having a function of further enhancing the production of a human mesothrombin analog from the human prothrombin analog and the anticoagulant action of the human mesothrombin analog. Any compound can be used as long as it has an anticoagulant action, but it is preferably a compound related to the protein C activation pathway, more preferably human protein C, human active protein C, PROTAC, human thrombomodulin. Soluble thrombomodulin can be mentioned as an example. Examples of the soluble thrombomodulin include thrombomodulin in each of Examples in JP-A No. 64-6219, and thrombomodulin in JP-B-7-98840 and WO92 / 00325. These proteins are preferably produced by a production method that can be used as a pharmaceutical composition, regardless of gene production, fractionation from blood or urine, and purification. Moreover, as a method for administering these pharmaceutical compositions, it is desirable to administer them simultaneously in the same formulation, but administration in different formulations or administration over time is also possible. For example, the day after the administration of protein C or human soluble thrombomodulin, the human prothrombin analog or human mesothrombin analog can be administered, or vice versa.

  The following examples further illustrate the invention, but the invention is not limited thereto.

Method for preparing cDNA of human prothrombin analog Human prothrombin cDNA was obtained from human liver cDNA library (Clontech) by using two kinds of oligonucleotide primers, namely, 5 ′ primer (5′-TTTTATTATTACCATGGCGCACGTCCCAGGC-3 ′ and 3 ′. It was obtained by PCR using the primer (5'-CATTGATCAGTTTGGAGAGCGGCCCCGGGTT-3 '. The DNA sequence of pUC / hPT prepared by introducing this cDNA (1.9 kb) into the multicloning site of the pUC19 plasmid, SmaI cleavage site is This was confirmed using a DSQ2000L DNA sequencer (manufactured by Shimadzu Corp.) The cDNA fragment inserted into this pUC / hPT contains human of SEQ ID NO: 4 in the sequence listing. It was confirmed that the nucleotide sequence of rothrombin (precursor) was contained, pUC / hPT was cleaved with EcoRI and NotI, and the obtained cDNA fragment was separated by agarose electrophoresis, and a DNA purification kit (QIA quick DNA purification kit). And QIAGEN (Germany).
To obtain His-Tag-labeled human prothrombin (PT-Tag), a 1.9 kb cDNA fragment obtained by cleaving pUC / hPT with EcoRI and NotI was used as a secretory expression vector (pSecTag, manufactured by Invitrogen, CA USA). ) In the EcoRI-NotI site.
In addition, human prothrombin mutants were prepared by preparing the following various human prothrombin analog cDNAs by PCR using mutant oligonucleotides having mutant codons for the above-mentioned pUC / hPT. Into a secretory expression vector (pSecTag). That is, in order to replace Arg ¹⁵⁵ with Ala, a mutant oligonucleotide consisting of 5′-ACT GTA GCG ATG ACT CCA GCT AGC GAA G-3 ′ (amino acid sequence Thr Val Ala Met Thr Pro Ala ¹⁵⁵ Ser Glu) is used. The codon CGC representing Arg ¹⁵⁵ was changed to GCT. Hereinafter, similarly, “Native terminal codon sequence of 6 amino acids on the N-terminal side of the amino acid to be changed−Codon sequence of amino acid to be changed−Base sequence consisting of a native codon sequence of 2 amino acids on the C-terminal side of the changed amino acid” The codon CGT representing Arg ²⁷¹ was changed to GCC and the codon AGG representing Arg ²⁸⁴ was changed to GCG.
Further, in order to delete the 4 amino acid sequence from Glu ⁴⁶⁶ to Thr ⁴⁶⁹ , an oligonucleotide deleted from this 4 amino acid codon, GAGACGTGGACA, 5′-CGG GTG ACA GGC TGG GGC AAC CTG AAG GCC AAC GTT It was prepared using GGT AAG GGG CAG CAG CCC AGT-3 ′.
Furthermore, in order to delete the 5 amino acid sequence from Glu ⁴⁶⁶ to Ala ⁴⁷⁰ , this 5 amino acid codon, an oligonucleotide from which GAGACGTGGACAGCCC has been deleted, 5′-CGG GTG ACA GGC TGG GGC AAC CTG AAG AAC GTT GGT In order to delete the 3 amino acid sequence from Glu ⁴⁶⁶ to Trp ⁴⁶⁸ using AAG GGG CAG CCC AGT GTC-3 ′, an oligonucleotide having the 3 amino acid codon, GAGAGTGGGG deleted, 5′-CGG GTG ACA GGC TGG GGC AAC CTG AAG ACA GCC AAC GTT GGT AAG GGG CAG CCC-3 ′.
Furthermore, in order to delete the 6 amino acid sequence from Glu ⁴⁶⁶ to Ala471, this 6 amino acid codon, an oligonucleotide from which GAGACGTGGACAGCCAAC has been deleted, 5′-CGG GTG ACA GGC TGG GGC AAC CTG AAG GTT GGT AAG In order to delete the 7 amino acid sequence from Glu ⁴⁶⁶ to Ala 472 using GGG CAG CCC AGT GTC CTG-3 ′, an oligonucleotide 5′-CGG GTG ACA in which this 7 amino acid codon, GAGACGTGGACAGCCAACGTT, was deleted using GGC TGG GGC AAC CTG AAG GGT AAG GGG CAG CCC AGT GTC CTG CAG-3 ', the 8 amino acid sequence from ^{Glu 466} to ^{Ala 473} In order to lose the oligonucleotide, the oligonucleotide prepared by deleting this 8-amino acid codon, GAGACGTGGACAGCCACAGTTGGT, 5′-CGG GTG ACA GGC TGG GGC AAC CTG AAG AAG GGG CAG CCC AGT GTC CTG CTG GTG .
In addition, in order to delete the two amino acid sequences from Glu ⁴⁶⁶ to Thr ⁴⁶⁷ , an oligonucleotide deleted from this two amino acid codon, GAGACG, 5′-CGG GTG ACA GGC TGG GGC AAC CTG AAG TGG ACA GCC In order to delete the 2 amino acid sequence from Thr ⁴⁶⁷ to Trp ⁴⁶⁸ using AAC GTT GGT AAG GGG CAG-3 ′, this 2 amino acid codon, oligonucleotide deleted with ACGTGGG, 5′-GTG ACA In order to delete Glu ⁴⁶⁶ using GGC TGG GGC AAC CTG AAG GAG ACA GCC AAC GTT GGT AAG GGG CAG CCC-3 ′, the codon of this amino acid, GAG-deleted oligonucleotide 5′-CGG G G ACA GGC TGG GGC AAC CTG AAG ACG TGG ACA using GCC AAC GTT GGT AAG GGG-3 ', in order to delete the ^{Thr 467} is codon for the amino acids, oligonucleotides were deleted ACG, 5' GTG ACA GGC TGG GGC AAC CTG AAG GAG TGG ACA GCC AAC GTT GGT AAG GGG CAG-3 ′ was used to delete Trp ^468, and the codon of this amino acid, TGG-deleted oligonucleotide 5 ′ -Prepared using ACA GGC TGG GGC AAC CTG AAG GAG ACG ACA GCC AAC GTT GGT AAG GGG CAG-3 '.
In addition, in order to delete the 9 amino acid sequence from Glu ⁴⁶⁶ to Lys ⁴⁷⁴ , an oligonucleotide lacking the 9 amino acid codon, GAGACGTGGACAGCCAACGTTGGTAAG, 5'-CGG GTG ACA GGC TGG GGC AAC CTG AAG GGG CAG Prepared using AGT GTC CTG CAG GTG GTG-3 ′.
PT-RA155; human prothrombin analog cDNA of a comparative example in which Arg ¹⁵⁵ was changed to Ala.
PT-RA271: Comparative human prothrombin analog cDNA in which Arg ²⁷¹ and Arg ²⁸⁴ were replaced with Ala.
PT-MT; Comparative human prothrombin analog cDNA in which Arg ¹⁵⁵ , Arg ²⁷¹ and Arg ²⁸⁴ were replaced with Ala (polypeptide described in USP 5,510,248).
^PTDA554; Arg ^155, Arg 271, human prothrombin analogue cDNA of the comparative example of Asp554 in addition to ^{Arg 284} was replaced with Ala.
PTDL554; Comparative human prothrombin analog cDNAs in which Arg ¹⁵⁵ , Arg ²⁷¹ and Arg ²⁸⁴ were replaced with Ala and Asp 554 was replaced with Leu were prepared.
Also, PT-delta ⁴⁶⁹ ; human prothrombin analog cDNA of the present invention in which Arg ¹⁵⁵ , Arg ²⁷¹ and Arg ²⁸⁴ are replaced with Ala, and Glu ⁴⁶⁶ to Thr ⁴⁶⁹ are deleted.
PT-delta (466-470); A human prothrombin analog cDNA of the present invention in which Arg ¹⁵⁵ , Arg ²⁷¹ and Arg ^{2 84} are replaced with Ala, and Ala ⁴⁷⁰ is deleted from Glu ⁴⁶⁶ .
PT-delta (466-468); human prothrombin analog cDNA of the present invention in which Arg ¹⁵⁵ , Arg ²⁷¹ and Arg ^{2 84} are replaced with Ala and Tlu ⁴⁶⁸ is deleted from Glu ⁴⁶⁶ .
PT-delta (466-467); human prothrombin analog cDNA of the present invention in which Arg ¹⁵⁵ , Arg ²⁷¹ and Arg ^{2 84} are replaced with Ala and Glu ⁴⁶⁶ to Thr ⁴⁶⁷ are deleted.
PT-delta (467-468); human prothrombin analog cDNA of the present invention in which Arg ¹⁵⁵ , Arg ²⁷¹ and Arg ^{2 84} are replaced with Ala, and Thr ⁴⁶⁷ to Trp ⁴⁶⁸ are deleted.
PT-delta (466); human prothrombin analog cDNA of the present invention in which Arg ¹⁵⁵ , Arg ²⁷¹ and Arg ²⁸⁴ are replaced with Ala and Glu ⁴⁶⁶ is deleted.
PT-delta (467); human prothrombin analog cDNA of the present invention in which Arg ¹⁵⁵ , Arg ²⁷¹ and Arg ²⁸⁴ are replaced with Ala and Thr ⁴⁶⁷ is deleted.
PT-delta (468); human prothrombin analog cDNA of the present invention in which Arg ¹⁵⁵ , Arg ²⁷¹ and Arg ²⁸⁴ are replaced with Ala and Trp ⁴⁶⁸ is deleted.
PT-delta (466-471); A human prothrombin analog cDNA of the present invention in which Arg ¹⁵⁵ , Arg ²⁷¹ and Arg ^{2 84} are replaced with Ala, and Asn471 is deleted from Glu ⁴⁶⁶ .
PT-delta (466-472); A human prothrombin analog cDNA of the present invention in which Arg ¹⁵⁵ , Arg ²⁷¹ and Arg ^{2 84} are replaced by Ala, and Val472 is deleted from Glu ⁴⁶⁶ .
PT-delta (466-473); human prothrombin analog cDNA of the present invention in which Arg ¹⁵⁵ , Arg ²⁷¹ and Arg ^{2 84} are replaced with Ala and Glu ⁴⁶⁶ to Gly ⁴⁷³ are deleted.
PT-delta (466-474); human prothrombin analog cDNA in which Arg ¹⁵⁵ , Arg ²⁷¹ and Arg ^{2 84} are replaced with Ala, and Lys ⁴⁷⁴ is deleted from Glu ⁴⁶⁶ .
It was created.
A schematic representation of the mutation site and the resulting human mesothrombin analog after activation by the enzyme is shown in FIG. 1 for reference. PT-delta 469 was shown as a representative example of the human prothrombin analog of the present invention.

(1) Expression and purification of human prothrombin analog An expression vector containing the human prothrombin analog cDNA including the above-mentioned comparative example was introduced into COS-7 cells using a transfection reagent (FuGENE6, Roche Diagnostics, IN USA). did. The cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) (GIBCO) and 10 μg / ml vitamin K (manufactured by SIGMA) for 24 hours, and then further containing 10 μg / ml vitamin K. The cells were cultured in serum DMEM medium for 48 hours. The medium was collected, equilibrated with Buffer N (50 mM TrisHCl, pH 8.0, 0.5 M NaCl), and connected to a high performance liquid chromatography for protein purification (AKTA explorer 10S, manufactured by Pharmacia) (Ni-NTA resin column). Column SF, manufactured by QIAGEN; 8 ml). The column was washed with 5 volumes of Buffer N and eluted with a two step gradient (Buffer N containing 0-40 mM and 40-250 mM imidazole, flow rate 2 ml / min) to collect fractions containing human prothrombin analogs. .
The fraction was dialyzed with TBS (25 mM TrisHCl, pH 8.0, 150 mM NaCl, 5 mM CaCl 2), and the protein concentration was determined with a protein quantification reagent (DC Protein Assay kit, manufactured by Bio-Rad, CA USA). Fractions were diluted 2 × with 2 × SDS Buffer (125 mM TrisHCl, pH 6.8, 30% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.2% bromophenol blue (BPB, Sigma), 10% -SDS polyacrylamide gel electrophoresis (SDS-PAGE) The protein was transferred to a polyvinylidin fluoride (PVDF) membrane (FineTrap NT-32, manufactured by Nippon Kogyo Co., Ltd.) and anti-myc antibody (1000-fold diluted, Invitrogen) The product was treated overnight at 4 ° C. After washing the PVDF membrane, a protein staining kit (VECTASTAIN Elite ABC kit, Vector Lab, CA USA) and a peroxidase protein staining kit (POD immunostain set, Japanese) (Fig. 2) It was confirmed that a band appeared in almost the same place as PT-Tag, and that a human prothrombin analog could be made. PT-delta 469 was shown as a representative example of the analog, and it was confirmed that other human prothrombin analogs of the present invention and PT-delta (466-474) human prothrombin analogs could be similarly prepared. .
(2) N-terminal amino acid analysis of human prothrombin analog The human prothrombin analog fraction containing the comparative example purified by the above-mentioned Ni-NTA column SF was diluted 2-fold with 2 × SDS Buffer, and 10% -SDS polyacrylamide gel electrolysis was performed. After electrophoresis, the protein was transferred to a PVDF membrane (ProBlott, Perkin Elmer). After staining the PVDF membrane with a 50% ethanol-10% acetic acid solution containing 0.1% amide black (manufactured by Sigma), the band was cut out and washed, and then the amino acid sequence was converted into an amino acid sequencer (model 477A-120A, Applied Biosystems). Manufactured by CA USA). The N-terminus of all human prothrombin analogs showed Ala-Asn-Thr-Phe-Leu, confirming that human prothrombin analogs could be made.

その結果、本発明のすべてのヒトプロトロンビン類似体はエカリンによる活性化を受け、上述の発色基質の切断活性（表１におけるＣｈｒｏｍｏｇｅｎｉｃ Ａｃｔｉｖｉｔｙ）を示すことが確認され、ヒトプロトロンビン類似体がさらに何等の活性体（ヒトメゾトロンビン類似体またはトロンビンと推測される）に変化したことが確認できた。一方、ＰＴ−ｄｅｌｔａ（４６６−４７４）ヒトプロトロンビン類似体はエカリンによる活性化を受けず、上述の発色基質の切断活性が１％以下であることが確認された。このことから、ＰＴ−ｄｅｌｔａ（４６６−４７４）ヒトプロトロンビン類似体は酵素としての能力が欠落しており、ＰＴ−ｄｅｌｔａ（４６６−４７４）ヒトプロトロンビン類似体から誘導されるヒトメゾトロンビン類似体は、メゾトロンビン化によるプロテインＣ活性化能力の増強（２０倍弱）があったとしても、その能力は、ヒトトロンビンに比べ、１／５以下であることが予測された。
なお、エカリンを添加する前のプロトロンビン類似体や、エカリン自体は、特別に上記の発色基質の切断活性を示さない。
さらに３０分後、２−メルカプトエタノールを含む（還元条件下）あるいは含まない（非還元条件下）状態において２ｘＳＤＳ Ｂｕｆｆｅｒで２倍に希釈し、１０％−ＳＤＳポリアクリルアミドゲル電気泳動にかけ、前述の方法を用いて、可視化した（図３（Ａ）は還元条件下、同図（Ｂ）は非還元条件下）。その結果、エカリンにより全てのヒトプロトロンビン類似体は切断を受け、ヒトトロンビンＢ鎖を生じていることが確認された（図３（Ａ）；還元条件下）。さらに、ＰＴ−ＴａｇおよびＰＴ−ＲＡ１５５はヒトトロンビンを、ＰＴ−ＲＡ２７１はヒトメゾトロンビンＤｅｓＦ１を示すバンドが生じるのに対し、ＰＴ−ＭＴ、ＰＴ−ＤＥＬＴＡ４６９、ＰＴ−ＤＡ５５４およびＰＴＤＬ５５４は元のそれぞれのヒトプロトロンビン類似体を示すバンドと同一の場所にバンドが生じることが確認された（図３（Ｂ））。すなわち、ＰＴ−ＭＴ、ＰＴ−ＤＥＬＴＡ４６９、ＰＴ−ＤＡ５５４およびＰＴＤＬ５５４は、酵素により活性化を受けるものの、トロンビンにさらに変換されることがないことを示している（図１参照のこと）。
本発明のヒトプロトロンビン類似体であるＰＴ−ＤＥＬＴＡ４６９は、エカリンにより活性化されて本発明のヒトメゾトロンビン類似体が作成され、図３（Ｂ）において、これに対応するバンドが検出されている。これらのバンドを切り取ることにより、精製された本発明のヒトメゾトロンビン類似体が取得出来る。なお、ＰＴ−ＤＥＬＴＡ４６９以外の本発明のヒトプロトロンビン類似体及びＰＴ−ｄｅｌｔａ（４６６−４７４）ヒトプロトロンビン類似体においても、同様に精製されたヒトメゾトロンビン類似体が取得出来る。
（２）活性化されたヒトプロトロンビン類似体の凝固活性
前述のＮｉ−ＮＴＡカラムで精製したヒトプロトロンビン類似体画分およびヒトプロトロンビン（ｎａｔｉｖｅ）を０．１％のＢＳＡを含むＴＢＳ ｂｕｆｆｅｒで同一の酵素活性（発色基質の切断活性）を示す濃度（およそ５００ｍＡｂｓ／ｍｉｎ）に調製し、終濃度０．５ｎＭのエカリンを加えて３７℃にて３０分間、活性化した。１／１０容量の２０ｍｇ／ｍｌフィブリノーゲン（シグマ社製）を加え、凝固するまでの時間をｃｏａｇｕｌｏｍｅｔｅｒ（Ｂａｘｔｅｒ，Ｇｅｒｍａｎｙ）を用いて測定した。凝固活性はヒトトロンビンの検量線を用いて算出し、ヒトプロトロンビン（ｎａｔｉｖｅ）に１００％とする相対活性で表した（表１の凝固活性）。ＰＴ−ＲＡ２７１およびＰＴ−ＭＴ（比較例）が、それぞれ１６％および１２％の相対活性を示すのに対し、ＰＴ−ＤＥＬＴＡ４６９、ＰＴ−ＤＥＬＴＡ（４６６−４７０）、ＰＴ−ＤＥＬＴＡ（４６６−４６８）は、いずれも１％以下の相対活性を示した。したがって、ＰＴ−ＤＥＬＴＡ４６９、ＰＴ−ＤＥＬＴＡ（４６６−４７０）、およびＰＴ−ＤＥＬＴＡ（４６６−４６８）はほぼ完全に凝固促進活性を有しないことが認められた。なお、上記以外の本発明のヒトプロトロンビン類似体においても、同様な結果が得られた。
（３）活性化されたヒトプロトロンビン類似体のプロテインＣ活性化能力
前述のＮｉ−ＮＴＡカラムで精製したヒトプロトロンビン類似体画分およびヒトプロトロンビン（ｎａｔｉｖｅ）を０．１％のＢＳＡを含むＴＢＳ ｂｕｆｆｅｒで終濃度２ｎＭに調製し、終濃度０．２ｎＭのエカリンを加えて３７℃にて３０分間、活性化した。終濃度８０ｎＭのヒトプロテインＣ（コスモバイオ社製）、同じく４０ｎＭの可溶性トロンボモジュリン（旭化成社製；特開昭６４−６２１９号公報または特許第２７６６４７３号公報の実施例２０の可溶性トロンボモジュリン）および同じく１００μＭのリン脂質（ｐｈｏｓｐｈａｔｉｄｙｌｃｈｏｌｉｎｅ：ｐｈｏｓｐａｔｉｄｙｌｓｅｒｉｎｅ＝３：１）を含む等容量の０．１％ＢＳＡを含むＴＢＳ ｂｕｆｆｅｒを加え３７℃で６０分間、活性化した。反応後、等容量の終濃度１ｍＭの発色基質ＬｙｓＳｅｒＴｈｒＡｒｇ−ＭＣＡ（ペプチド研究所製）を加え、発生するアミノメチルクマリン（ＭＣＡ）量を蛍光プレートリーダー（Ｇｅｎｉｏｓ、ＴＥＣＡＮ、Ｓｗｉｔｚｅｒｌａｎｄ）を用いて測定し、ヒトプロトロンビン（ｎａｔｉｖｅ）を１００％とする相対活性として表した（表１のプロテインＣ活性化能力）。ＰＴ−ＲＡ２７１が１０８％の相対活性を示すのに対し、ＰＴ−ＤＥＬＴＡ４６９、ＰＴ−ＤＥＬＴＡ（４６６−４７０）、ＰＴ−ＤＥＬＴＡ（４６６−４６８）は、いずれも１０００％以上の相対活性を示した。なお、ＰＴ−ＭＴ、ＰＴ−ＤＬ５５４も１０００％以上の相対活性を示した。なお、上記以外の本発明のヒトプロトロンビン類似体においても、同様な結果が得られた。
ＰＴ−ＤＥＬＴＡ４６９、ＰＴ−ＤＥＬＴＡ（４６６−４７０）、ＰＴ−ＤＥＬＴＡ（４６６−４６８）および本発明のヒトプロトロンビン類似体は活性化されると、トロンボモジュリンの存在下、プロテインＣ活性化能力を有し、このプロテインＣ活性化能力がヒトトロンビン（ｎａｔｉｖｅ）に比べ高いことを確認した。
（４）ヒトプロトロンビン類似体の血液における抗凝固作用（トロンビン産生抑制作用）
トロンビン産生抑制作用は、多くの抗凝固薬の抗凝固作用を評価する際に用いられている作用の一つであり、トロンビン産生に抑制作用を示す物質は臨床においても有効であると考えられている（Ｍｏｈｒｉ ｅｔ ａｌ．，Ｔｈｒｏｍｂ Ｈａｅｍｏｓｔ ８２，１６８７−１６９３ １９９９）。すなわち、正常人ボランティアから得た１６０μｌのフィブリノーゲン除去クエン酸ヒト血しょうに対して、２０μｌのヒトプロトロンビン（ｎａｔｉｖｅ）、ＰＴ−ＭＴ（比較例）あるいはヒトプロトロンビン類似体の例としてＰＴ−ＤＥＬＴＡ４６９、ＰＴ−ＤＥＬＴＡ（４６６−４７０）、あるいはＰＴ−ＤＥＬＴＡ（４６６−４６８）を含むＴＢＳ ｂｕｆｆｅｒを、それぞれ、終濃度１μｇ／ｍｌとなるように加えた。さらに２０μｌの６２５０倍希釈したウサギ脳ティッシュファクター（国際試薬社製）、０．２ｍｇのリン脂質（フォスファティディルコリン：フォスファティディルセリン：フォスファティディルエタノールアミン＝２：１：２ シグマ社製）および１２０ｍＭのＣａＣｌ２、０．１％のＢＳＡを含むＴＢＳ ｂｕｆｆｅｒを加え、凝固反応を開始させた。反応開始５分後に、反応液を５μｌ採取し、３００μｌの１ｍＭの発色基質Ｇｌｕ−Ｐｒｏ−Ａｒｇ−ｐＮＡ（Ｓ−２３６６、第一化学薬品製）を含むＴＢＳ ｂｕｆｆｅｒに加え３．５分間反応させた。３０μｌの氷酢酸を添加し、反応を停止させ、４０５ｎＭの吸光度をプレートリーダー（ＴＨＥＲＭＯｍａｘ モレキュラーディヴァイス社製）を用いて測定した。その結果、ヒトプロトロンビン（ｎａｔｉｖｅ）はトロンビン産生を助長したのに対して、ＰＴ−ＭＴおよびＰＴ−ＤＥＬＴＡ４６９、ＰＴ−ＤＥＬＴＡ（４６６−４７０）、あるいはＰＴ−ＤＥＬＴＡ（４６６−４６８）はトロンビン産生を抑制した。この結果、本発明のヒトプロトロンビン類似体はトロンビン産生抑制作用を示し、抗凝固活性を有することが確認された。
（５）活性化されたヒトプロトロンビン類似体のマウスにおける作用
ヒトプロトロンビン（ｎａｔｉｖｅ）およびＰＴ−ＭＴ、ＰＴ−ＤＥＬＴＡ４６９、ＰＴ−ＤＥＬＴＡ（４６６−４７０）、あるいはＰＴ−ＤＥＬＴＡ（４６６−４６８）を０．１％のＢＳＡを含むＴＢＳ ｂｕｆｆｅｒで終濃度１μｇ／ｍｌに調製し、終濃度１ｎＭのエカリンを加えて３７℃にて３０分間、活性化した。これらの溶液を、そのままそれぞれ２匹の雄マウス（Ｂａｌｂ／ｃ 日本チャールズリバー社製）に投与し、３０分後エーテル麻酔下、クエン酸採血し、血しょうを得た。血漿中のＤ−ｄｉｍｅｒをキット（Ｄ−Ｄｉテスト、ダイアグノスティカ社製）を用いて測定した。
その結果、ヒトプロトロンビンおよびＰＴ−ＭＴにおいて、通常有り得る濃度（１μｇ／ｍｌ）を超えるＤ−ｄｉｍｅｒが検出されたのに対して、ＰＴ−ＤＥＬＴＡ４６９、ＰＴ−ＤＥＬＴＡ（４６６−４７０）、あるいはＰＴ−ＤＥＬＴＡ（４６６−４６８）では正常レベルであった。
本発明の活性化されたプロトロンビン類似体ＰＴ−ＤＥＬＴＡ４６９、ＰＴ−ＤＥＬＴＡ（４６６−４７０）、あるいはＰＴ−ＤＥＬＴＡ（４６６−４６８）（すなわち本発明のメゾトロンビン類似体）は、生体内において血栓を生ずることがないのに対して、従来公知のＵＳＰ５，５１０，２４８に記載されたヒトプロトロンビンＰＴ−ＭＴの活性化物では、生体内においては血栓の形成が無視できないことが確認された。
したがって、本発明の活性化されたプロトロンビン類似体は、凝固促進活性を実質的に有さず且つそのプロテインＣ活性化能力がヒトトロンビンに比較して高く、さらにトロンビンに実質的に変換されないことが明確であり、生体内において血栓を形成することがなく、抗凝固薬として使用し得ることが示された。
（６）ヒトプロトロンビン類似体およびヒトメゾトロンビン類似体の毒性
ヒトプロトロンビン（ｎａｔｉｖｅ）および本発明のヒトプロトロンビン類似体の例としてＰＴ−ＤＥＬＴＡ４６９、ＰＴ−ＤＥＬＴＡ（４６６−４７０）、あるいはＰＴ−ＤＥＬＴＡ（４６６−４６８）、本発明のヒトメゾトロンビン類似体の例として、前述の方法で活性化したＰＴ−ＤＥＬＴＡ４６９、ＰＴ−ＤＥＬＴＡ（４６６−４７０）、あるいはＰＴ−ＤＥＬＴＡ（４６６−４６８）のそれぞれ１０ｍｇ／ｋｇをそれぞれ４匹の雄Ｂａｌｂ／ｃマウス（日本チャールズリバー社製）に静脈内投与し、投与後１週間観察した。その結果、いずれの投与群に於いても致死例は認められず、本発明のヒトプロトロンビン類似体およびメゾトロンビン類似体は安全に投与できることが示された。なお、上記以外の本発明のヒトプロトロンビン類似体においても、同様な結果が得られた。(1) Evaluation of thrombin activity of activated human prothrombin analog Human prothrombin analog fraction containing comparative example purified by Ni-NTA column described above and human prothrombin (native, manufactured by Sigma) 0.1% Using a 96-well plate with a final concentration of 0.5 nM snake venom ecarin (purified by Yamada et al., J Biol Chem 271, 5200-5207) with a TBS buffer containing BSA of Activated at 37 ° C. for 15 minutes. 1/10 volume of 2.5 mM chromogenic substrate ValProArg-pNA (manufactured by Peptide Institute) was added, and the amount of paranitroaniline (pNA) generated was determined using Kinetic Plate Reader (Well Reader, Seikagaku Corporation). Measured (Table 1).

As a result, it was confirmed that all the human prothrombin analogs of the present invention were activated by ecarin and exhibited the above-mentioned chromogenic substrate cleavage activity (Chromogenic Activity in Table 1). It was confirmed that the body was changed to a human body (presumed to be human mesothrombin analog or thrombin). On the other hand, it was confirmed that the PT-delta (466-474) human prothrombin analog was not activated by ecarin and the cleavage activity of the chromogenic substrate was 1% or less. From this, PT-delta (466-474) human prothrombin analog lacks the ability as an enzyme, and human mesothrombin analog derived from PT-delta (466-474) human prothrombin analog is Even if there was an enhancement of protein C activation ability by mesothrombinization (a little less than 20 times), the ability was predicted to be 1/5 or less compared to human thrombin.
In addition, the prothrombin analog before the addition of ecarin and ecarin itself do not particularly show the cleavage activity of the chromogenic substrate.
After further 30 minutes, with 2-mercaptoethanol (under reducing conditions) or without (non-reducing conditions), it was diluted 2-fold with 2 × SDS Buffer, and subjected to 10% -SDS polyacrylamide gel electrophoresis. (FIG. 3A is under reducing conditions, and FIG. 3B is non-reducing conditions). As a result, it was confirmed that all the human prothrombin analogs were cleaved by ecarin to generate human thrombin B chain (FIG. 3 (A); under reducing conditions). Furthermore, PT-Tag and PT-RA155 produce human thrombin and PT-RA271 produces human mesothrombin DesF1, whereas PT-MT, PT-DELTA469, PT-DA554 and PTDL554 are the original humans. It was confirmed that a band was generated at the same place as the band showing the prothrombin analog (FIG. 3B). That is, PT-MT, PT-DELTA 469, PT-DA554 and PTDL554 are activated by the enzyme, but are not further converted into thrombin (see FIG. 1).
PT-DELTA 469, which is a human prothrombin analog of the present invention, is activated by ecarin to produce the human mesothrombin analog of the present invention. In FIG. 3B, a band corresponding to this is detected. By cutting out these bands, a purified human mesothrombin analog of the present invention can be obtained. In addition, in the human prothrombin analog of the present invention and PT-delta (466-474) human prothrombin analog other than PT-DELTA 469, a similarly purified human mesothrombin analog can be obtained.
(2) Coagulation activity of activated human prothrombin analogue The same enzyme in the above-described Ni-NTA column purified human prothrombin analogue and human prothrombin (native) in TBS buffer containing 0.1% BSA The concentration (approximately 500 mAbs / min) showing the activity (chromogenic substrate cleavage activity) was prepared, and final concentration of 0.5 nM ecarin was added and activated at 37 ° C. for 30 minutes. 1/10 volume of 20 mg / ml fibrinogen (manufactured by Sigma) was added, and the time until coagulation was measured using a coagulometer (Baxter, Germany). The clotting activity was calculated using a human thrombin calibration curve, and expressed as a relative activity of 100% in human prothrombin (native clotting activity in Table 1). PT-RA271 and PT-MT (comparative example) show relative activities of 16% and 12%, respectively, whereas PT-DELTA 469, PT-DELTA (466-470), and PT-DELTA (466-468) , Both showed a relative activity of 1% or less. Therefore, it was recognized that PT-DELTA 469, PT-DELTA (466-470), and PT-DELTA (466-468) have almost no procoagulant activity. Similar results were obtained with the human prothrombin analogs of the present invention other than those described above.
(3) Protein C activation ability of activated human prothrombin analog The human prothrombin analog fraction purified by the above-mentioned Ni-NTA column and human prothrombin (native) were mixed with TBS buffer containing 0.1% BSA. The final concentration was adjusted to 2 nM, and ecarin with a final concentration of 0.2 nM was added to activate at 37 ° C. for 30 minutes. Human protein C (manufactured by Cosmo Bio) having a final concentration of 80 nM, soluble thrombomodulin of 40 nM (manufactured by Asahi Kasei Corporation; soluble thrombomodulin of Example 20 of JP-A No. 64-6219 or Japanese Patent No. 2766473) and 100 μM An equal volume of TBS buffer containing 0.1% BSA containing phospholipid (phosphopatylylline = 3: 1) was added and activated at 37 ° C. for 60 minutes. After the reaction, an equal volume of 1 mM chromogenic substrate LysSerThrArg-MCA (manufactured by Peptide Institute) was added, and the amount of aminomethylcoumarin (MCA) generated was measured using a fluorescence plate reader (Genios, TECAN, Switzerland) It was expressed as relative activity with 100% human prothrombin (native) (Protein C activation ability in Table 1). PT-RA271 showed a relative activity of 108%, whereas PT-DELTA 469, PT-DELTA (466-470) and PT-DELTA (466-468) all showed a relative activity of 1000% or more. PT-MT and PT-DL554 also showed a relative activity of 1000% or more. Similar results were obtained with the human prothrombin analogs of the present invention other than those described above.
When activated, PT-DELTA 469, PT-DELTA (466-470), PT-DELTA (466-468) and human prothrombin analogs of the invention have the ability to activate protein C in the presence of thrombomodulin; This protein C activation ability was confirmed to be higher than that of human thrombin (native).
(4) Anticoagulant action of human prothrombin analog in blood (thrombin production inhibitory action)
Thrombin production inhibitory action is one of the actions used to evaluate the anticoagulant action of many anticoagulants, and substances that show thrombin production inhibitory action are considered to be effective in clinical practice. (Mohri et al., Thromb Haemost 82, 1687-1693 1999). That is, with respect to 160 μl of fibrinogen-removed citrated human plasma obtained from a normal volunteer, 20 μl of human prothrombin (native), PT-MT (comparative example), or human prothrombin analogs such as PT-DELTA 469, PT- TBS buffer containing DELTA (466-470) or PT-DELTA (466-468) was added to a final concentration of 1 μg / ml, respectively. Furthermore, 20 μl of rabbit brain tissue factor diluted 6250 times (made by Kokusai Reagent Co., Ltd.), 0.2 mg phospholipid (phosphatidylcholine: phosphatidylserine: phosphatidylethanolamine = 2: 1: 2 made by Sigma) TBS buffer containing 120 mM CaCl 2 and 0.1% BSA was added to initiate the coagulation reaction. Five minutes after the start of the reaction, 5 μl of the reaction solution was collected, added to TBS buffer containing 300 μl of 1 mM chromogenic substrate Glu-Pro-Arg-pNA (S-2366, manufactured by Daiichi Chemicals), and reacted for 3.5 minutes. . 30 μl of glacial acetic acid was added to stop the reaction, and the absorbance at 405 nM was measured using a plate reader (THERMOmax Molecular Devices). As a result, human prothrombin (native) promoted thrombin production, whereas PT-MT and PT-DELTA 469, PT-DELTA (466-470), or PT-DELTA (466-468) inhibited thrombin production. did. As a result, it was confirmed that the human prothrombin analog of the present invention has a thrombin production inhibitory action and has anticoagulant activity.
(5) Action of activated human prothrombin analogs in mice Human prothrombin (native) and PT-MT, PT-DELTA 469, PT-DELTA (466-470), or PT-DELTA (466-468) The final concentration was adjusted to 1 μg / ml with TBS buffer containing 1% BSA, and final concentration of 1 nM ecarin was added to activate at 37 ° C. for 30 minutes. Each of these solutions was directly administered to two male mice (Balb / c manufactured by Nippon Charles River Co., Ltd.), and after 30 minutes, citric acid was collected under ether anesthesia to obtain plasma. D-dimer in plasma was measured using a kit (D-Di test, manufactured by Diagnostics Inc.).
As a result, in human prothrombin and PT-MT, D-dimer exceeding the normal concentration (1 μg / ml) was detected, whereas PT-DELTA 469, PT-DELTA (466-470), or PT-DELTA (466-468) was a normal level.
The activated prothrombin analog PT-DELTA 469, PT-DELTA (466-470), or PT-DELTA (466-468) of the present invention (ie, the mesothrombin analog of the present invention) produces a thrombus in vivo. On the other hand, it was confirmed that thrombus formation was not negligible in vivo in the active substance of human prothrombin PT-MT described in USP 5,510,248 known in the art.
Therefore, the activated prothrombin analog of the present invention has substantially no procoagulant activity and its ability to activate protein C is higher than that of human thrombin and is not substantially converted to thrombin. It was clear that it did not form thrombus in vivo and could be used as an anticoagulant.
(6) Toxicity of human prothrombin analogues and human mesothrombin analogues Examples of human prothrombin and human prothrombin analogues of the present invention include PT-DELTA 469, PT-DELTA (466-470), or PT-DELTA (466). -468), as an example of the human mesothrombin analog of the present invention, 10 mg / kg of PT-DELTA 469, PT-DELTA (466-470), or PT-DELTA (466-468) activated by the above-described method, respectively. Were intravenously administered to 4 male Balb / c mice (manufactured by Charles River, Japan), and observed for 1 week after administration. As a result, no lethal cases were observed in any of the administration groups, indicating that the human prothrombin analog and mesothrombin analog of the present invention can be safely administered. Similar results were obtained with the human prothrombin analogs of the present invention other than those described above.

Effect of human prothrombin analog and human mesothrombin analog on rat thrombus model Anticoagulant action was confirmed by rat thrombosis model by the method of Solis et al. (Soris et al. J Vasc Surg 14, 599-604, 1991) It was. That is, male SD rats (Nihon Charles River Co., Ltd.) were anesthetized by administering pentobarbital. The abdomen was opened, the abdominal vena cava was exposed, and branches such as the renal vein were tied. Raw diet (n = 4), PT-DELTA 469, PT-DELTA (466-470), or PT-DELTA (466-468) 1 mg / kg (n = 4), PT-DELTA 469, PT activated by the method described above -DELTA (466-470) or PT-DELTA (466-468) 1 mg / kg (n = 4) was administered from the tail vein, and 3 minutes later, after the abdominal vena cava was tied at the branch of the renal vein, The abdominal vena cava was injured by gently pinching it several times with tweezers. After 15 minutes, ligation was performed again at the ascending branch site, and the abdominal vena cava was cut above and below the ligated site. The excised abdominal vena cava was incised while being immersed in physiological saline, the thrombus generated inside was excised, washed thoroughly with physiological saline, and weighed.
As a result, PT-DELTA 469, PT-DELTA (466-470), and PT-DELTA (466-468) and activated PT-DELTA 469, PT-DELTA (466-470), and PT-DELTA (466-468). ) Suppressed thrombus formation. From these results, it was confirmed that the human prothrombin analog and human mesothrombin analog of the present invention exhibited an anticoagulant action in vivo and were particularly effective against thrombosis.

Effects of human prothrombin analogs and human mesothrombin analogs on the rat endoxin model Using the rat endotoxin model (Gonda et al. Thromb 71, 325-335, 1993), the human prothrombin analogs and human mesothrombin of the present invention The inhibitory effect on the inflammatory reaction accompanying the coagulation process of the analog was confirmed. Specifically, endotoxin (derived from E. coli 055: B5, Wako Pure Chemical Industries, Ltd.) was intravenously administered continuously to male SD rats (Nippon Charles River) over 4 hours. Raw diet (n = 4), PT-DELTA 469, PT-DELTA (466-470), or PT-DELTA (466-468) 1 mg / kg (n = 4), PT-DELTA 469, PT activated by the method described above -DELTA (466-470) or PT-DELTA (466-468) 1 mg / kg (n = 4) was administered intravenously before endotoxin administration. Acid blood was collected to obtain blood and plasma, and platelets in blood, fibrinogen in plasma, and tumor necrosis factor (TNF) were respectively obtained with an automatic hemocytometer (Sysmex2000, manufactured by Sysmex), a fibrinogen measurement kit (IAtroset fibrinogen, Made by Diatron) Tsu door TNF ELISA kit (Gibco BRL Co., Ltd.) was used for the measurement.
As a result, the decrease in platelet count and fibrin-gen and the increase in TNF due to endotoxin administration were observed in PT-DELTA 469, PT-DELTA (466-470), and PT-DELTA (466-468) and activated PT-DELTA 469, PT-DELTA (466-470) and PT-DELTA (466-468) were suppressed. That is, from this result, it was confirmed that the human prothrombin analog and the human mesothrombin analog of the present invention have an anticoagulant action and a sufficient inflammation suppressing action. In particular, a model of coagulation abnormality caused by administration of rat endotoxin is frequently used as an animal model such as endotoxemia, DIC, and sepsis, and has been shown to be particularly effective for endotoxemia, DIC, and sepsis.
According to the above examples, the human prothrombin analog of the present invention is activated to produce a human mesothrombin analog but is not substantially converted to thrombin, and the human mesothrombin analog is substantially procoagulant. It was confirmed that it has no activity and has a higher ability to activate protein C than human thrombin. Furthermore, human prothrombin analogs or human mesothrombin analogs can be safely administered, are actually confirmed to have anticoagulant activity in animal experiments, can be used as anticoagulants, and have anti-inflammatory effects. In particular, it was confirmed to be effective against endotoxemia, DIC, sepsis, thrombosis and the like.
Application Examples Hereinafter, application examples of various peptides obtained in the above examples will be described with application examples, but the present invention is not limited to these application examples.
Application example 1
10 mg of the peptide of the present invention
Purified gelatin 20mg
Mannitol 100mg
Sodium chloride 7.8mg
Phosphate (1 and 2) sodium 15.4mg
The above components were dissolved in 2 ml of distilled water for injection, and a near-neutral injection preparation was prepared under normal preparation conditions. That is, the lysate is placed in a sterile vial, pre-frozen at -35 ° C for 2 hours, first dried at -35 ° C at a vacuum of 0.075 Torr for 35 hours, and then at 30 ° C and a vacuum of 0.03 Torr for 5 hours. Secondary dried to produce injection vials. The obtained composition is dissolved in 500 ml of physiological saline or glucose injection immediately before administration, and the solution is used for intravenous infusion. Moreover, the obtained composition is dissolved in 10 ml of physiological saline or glucose injection just before administration, and the solution is used for intravenous injection.
Application example 2
2.5 mg of peptide of the present invention
Albumin 5mg
Mannitol 25mg
Sodium chloride 1.95mg
Sodium phosphate (1 and 2) 3.85 mg
The above components were dissolved in 2 ml of distilled water for injection, and a neutral injection preparation was prepared under normal preparation conditions. That is, the lysate is put in a sterile vial, pre-frozen at −35 ° C. for 2 hours, first dried at −35 ° C. at a vacuum degree of 0.075 Torr for 35 hours, and then at 30 ° C. and a vacuum degree of 0.03 Torr for 5 hours. Secondary dried to produce injection vials. The obtained composition is dissolved in 500 ml of physiological saline or glucose injection immediately before administration, and the solution is used for intravenous infusion. Moreover, the obtained composition is dissolved in 10 ml of physiological saline or glucose injection just before administration, and the solution is used for intravenous injection.

Industrial applicability

  The present invention relates to a human mesothrombin analog, a human prothrombin analog, a DNA encoding the same, a plasmid containing the DNA, and a host cell containing the plasmid. Furthermore, the present invention relates to a pharmaceutical composition comprising such an analog, thereby obtaining a pharmaceutical composition, particularly an anticoagulant, which is more useful and safer than before.

[Sequence Listing]

Claims (32)

Changes have been made to the amino acids Arg ¹⁵⁵ , Arg ²⁷¹ , Arg ^{284 in} the human mesothrombin sequence, and changes have been made in all or part of the amino acids forming Glu ⁴⁶⁶ -Gly ⁴⁷³ in the human mesothrombin sequence. A human mesothrombin analog comprising a double-stranded peptide, which has substantially no procoagulant activity and has a higher ability to activate protein C than human thrombin. Human mesothrombin analog, characterized in that it is not converted in an enzymatic manner. The human mesothrombin analog according to claim 1, wherein the amino acid alterations Arg ¹⁵⁵ , Arg ²⁷¹ , and Arg ²⁸⁴ in the human mesothrombin sequence are substitutions with Ala. 2. The change of all or part of the amino acids forming Glu ^466- Gly ⁴⁷³ of the human mesothrombin sequence is a change of all or part of the amino acids forming Glu ^466- Ala ^470. Or the human mesothrombin analog according to 2. Claim all or part of the amino acid changes that form the ^Glu 466 ^{-Gly 473} of the sequence of human meso ^thrombin, characterized in that all or some of the changes of the amino acids forming Glu 466 ^{-Thr 469} 1 4. The human mesothrombin analog according to any one of items 1 to 3. A change in all or part of the amino acids forming Glu ⁴⁶⁶ -Gly ⁴⁷³ of the human mesothrombin sequence is a deletion of all or part of the amino acids forming Glu ⁴⁶⁶ -Ala ⁴⁷⁰ The human mesothrombin analog according to any one of 1 to 3. A change in all or part of the amino acids forming Glu ⁴⁶⁶ -Gly ⁴⁷³ of the human mesothrombin sequence is a deletion of all or part of the amino acids forming Glu ⁴⁶⁶ -Thr ⁴⁶⁹ The human mesothrombin analog according to any one of 1 to 5. 6. The amino acid sequence forming Glu ^466- Gly ⁴⁷³ in the human mesothrombin sequence is entirely or partially altered by a deletion of an amino acid forming Glu ^466- Ala ^470. Or a human mesothrombin analog according to any of the above. All or part of the amino acid changes that form the ^Glu 466 ^{-Gly 473} of the sequence of human meso ^thrombin, one of claims 1 to 6, characterized in that is a deletion of amino acids forming a Glu 466 ^{-Thr 469} A human mesothrombin analog according to claim 1. All or part of the amino acid changes that form the ^Glu 466 ^{-Gly 473} of the sequence of human meso ^thrombin, one of claims 1 to 6, characterized in that is a deletion of amino acids forming a Glu 466 ^{-Trp 468} A human mesothrombin analog according to claim 1. ^Arg 155 in the sequence of human ^prothrombin, Arg 271, changes to the amino acid ^{Arg 2 84} has been added, and changes in all or part of the amino acids forming ^Glu 466 ^{-Gly 473} of the sequence of human prothrombin is added A single-chain peptide human prothrombin analog which, when Arg ³²⁰ is cleaved and activated, has substantially no procoagulant activity and its ability to activate protein C is human thrombin A human prothrombin analog that is capable of producing a human mesothrombin analog that is higher in nature and is not substantially converted to thrombin. Changing the amino acid ^{Arg 155, Arg 271, Arg 2} 84 in the sequence of human prothrombin, a human prothrombin analog of claim 10 which is a substitution of Ala. The change of all or part of the amino acids forming Glu ⁴⁶⁶ -Gly ⁴⁷³ of the human prothrombin sequence is a change of all or part of the amino acids forming Glu ⁴⁶⁶ -Ala ⁴⁷⁰ 11. The human prothrombin analog according to 11. Claims 10 to all or part of the amino acid changes that form the ^Glu 466 ^{-Gly 473} of the sequence of human ^prothrombin, characterized in that a whole or part of the amino acid changes that form the Glu 466 ^{-Thr 469} 13. The human prothrombin analog according to any one of 12. 11. The modification of all or part of the amino acids forming Glu ^466- Gly ⁴⁷³ of the human prothrombin sequence is a deletion of all or part of the amino acids forming Glu ^466- Ala ^470. 13. The human prothrombin analog according to any one of 1 to 12. Claim all or part of the amino acid changes that form the ^Glu 466 ^{-Gly 473} of the sequence of human ^prothrombin, characterized in that it is a whole or part of the deletion of amino acids forming a Glu 466 ^{-Thr 469} 10 The human prothrombin analog according to any one of 1 to 14. The amino acid sequence forming Glu ⁴⁶⁶ -Gly ⁴⁷³ in the human prothrombin sequence is a deletion of all or part of the amino acids forming Glu ⁴⁶⁶ -Ala ⁴⁷⁰ . A human prothrombin analog according to any of the above. All or part of the amino acid changes that form the ^Glu 466 ^{-Gly 473} of the sequence of human ^prothrombin, claim 10, wherein 15 to be a deletion of amino acids forming a Glu 466 ^{-Thr 469} The human prothrombin analog described in 1. The amino acid sequence forming Glu ⁴⁶⁶ -Gly ⁴⁷³ in the human prothrombin sequence is entirely or partially altered by a deletion of an amino acid forming Glu ⁴⁶⁶ -Trp ⁴⁶⁸ . The human prothrombin analog described in 1. ^Arg 155 in the sequence of human ^prothrombin, Arg 271, changes to the amino acid ^{Arg 2 84} has been added, and changes in all or part of the amino acids forming ^Glu 466 ^{-Gly 473} of the sequence of human prothrombin is added A single-chain peptide human prothrombin analog which, when Arg ³²⁰ is cleaved and activated, has substantially no procoagulant activity and its ability to activate protein C is human thrombin A DNA encoding a human prothrombin analog that is capable of producing a human mesothrombin analog characterized in that it is of a higher nature than that of and is not substantially converted to thrombin. DNA of claim 19 which changes the amino acid ^{Arg 155, Arg 271, Arg 2} 84 , characterized in that a substitution of Ala in the sequence of human prothrombin. The change of all or part of the amino acids forming Glu ⁴⁶⁶ -Gly ⁴⁷³ of the human prothrombin sequence is all or part of the amino acids forming Glu ⁴⁶⁶ -Ala ⁴⁷⁰ 20. DNA according to 20. All or part of the amino acid changes that form the ^Glu 466 ^{-Gly 473} of the sequence of human ^prothrombin, claim 19, characterized in that all or some of the changes of the amino acids forming Glu 466 ^{-Thr 469} The DNA according to any one of 21. 20. The alteration of all or part of the amino acids forming Glu ^466- Gly ⁴⁷³ in the sequence of human prothrombin is a deletion of all or part of the amino acids forming Glu ^466- Ala ^470. To DNA according to any of 21 to 21. 19. all or part of the amino acid changes that form the ^Glu 466 ^{-Gly 473} of the sequence of human ^prothrombin, characterized in that it is a whole or part of the deletion of amino acids forming a Glu 466 ^{-Thr 469} 24. DNA according to any one of. 24. The change of all or part of the amino acids forming Glu ^466- Gly ⁴⁷³ of the human prothrombin sequence is a deletion of amino acids forming Glu ^466- Ala ⁴⁷⁰ DNA according to any one of the above. All or part of the amino acid changes that form the ^Glu 466 ^{-Gly 473} of the sequence of human ^prothrombin, claim 19, wherein 24 to be a deletion of amino acids forming a Glu 466 ^{-Thr 469} DNA described in 1. 25. The amino acid sequence forming Glu ^466- Gly ⁴⁷³ in the human prothrombin sequence is entirely or partially altered by a deletion of an amino acid forming Glu ^466- Trp ⁴⁶⁸ . DNA described in 1. A plasmid containing the DNA according to any one of claims 19 to 27. A cell containing the DNA according to any one of claims 19 to 27 and producing human mesothrombin itself. The pharmaceutical composition which uses the human mesothrombin analog in any one of Claims 1-9 as an active ingredient. The pharmaceutical composition which uses the human prothrombin analog in any one of Claims 10-18 as an active ingredient. The pharmaceutical composition according to claim 30 or 31, wherein the pharmaceutical composition is an anticoagulant.

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