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WO1993020194A1 - Analogue d'activateur tissulaire du plasminogene - Google Patents

Analogue d'activateur tissulaire du plasminogene Download PDF

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Publication number
WO1993020194A1
WO1993020194A1 PCT/JP1992/000415 JP9200415W WO9320194A1 WO 1993020194 A1 WO1993020194 A1 WO 1993020194A1 JP 9200415 W JP9200415 W JP 9200415W WO 9320194 A1 WO9320194 A1 WO 9320194A1
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WO
WIPO (PCT)
Prior art keywords
amino acid
acid sequence
analog
plasminogen activator
natural
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP1992/000415
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English (en)
Japanese (ja)
Inventor
Hideshi Sato
Sene Takahashi
Takaharu Negoro
Yoshiaki Sudo
Hideo Agui
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sumitomo Pharma Co Ltd
Original Assignee
Sumitomo Pharmaceuticals Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sumitomo Pharmaceuticals Co Ltd filed Critical Sumitomo Pharmaceuticals Co Ltd
Priority to PCT/JP1992/000415 priority Critical patent/WO1993020194A1/fr
Publication of WO1993020194A1 publication Critical patent/WO1993020194A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel t-PA analogs. More specifically, in the amino acid sequence of the tissue plasminogen activator (hereinafter referred to as t-PA), an amino acid sequence corresponding to positions 276 to 3006 of natural t-PA. By replacing at least part of the protein with the corresponding amino acid sequence of urinary plasminogen activator (hereafter, UK), the fibrin specificity has been improved.
  • t-PA tissue plasminogen activator
  • t-PA is known to degrade thrombus formed in blood vessels by converting plasminogen, an inactive zymogen, to plasmin, an active enzyme.
  • T-PA has relatively high fibrin specificity, whereas existing thrombolytics, such as UK-strain tokinase, do not have any fibrin specificity. have. For this reason, it has the property of specifically dissolving fibrin and, consequently, thrombus, and has attracted attention as a thrombolytic agent with less side effects such as bleeding.
  • t-PA in clinical practice requires much higher doses than expected for reopening the occluded coronary artery. I got it.
  • t-PA fibrin-specific, but only slightly in the blood. It is still a non-negligible problem that it also degrades blinogen, resulting in a generalized bleeding tendency. From this point of view, t-PA variants have also been produced for the purpose of imparting higher thrombus specificity, and numerous reports have been made (references 2 to 12). Few have achieved.
  • thrombus specificity was achieved by replacing all four amino acids from positions 296 to 299 of natural t-PA with alanine.
  • modified t-PA with improved fibrin specificity 4.4-fold, and plasma clot specificity about 3-fold. Disclosure of the invention
  • the present invention provides a novel t-PA analog with improved fibrin specificity.
  • plasminogen activating activity is very low in the presence of fibrinogen, but close to or higher than natural t-PA in the presence of fibrin.
  • the present invention provides a novel t-PA analog which exhibits a gene activating activity and therefore has improved fibrin specificity.
  • the t-PA analogs of the present invention are less likely to cause activation of plasminogen and subsequent degradation of fipurinogen when circulating in the blood. Since it expresses a plasminogen-activating activity close to that of natural t-PA and dissolves thrombus only when it reaches the local thrombus where Lin is present, it is more than natural t-PA.
  • the fibrin specificity is higher, and therefore the thrombus specificity is higher, and the tendency of systemic blood bleeding is high. It is expected that the side effect is unlikely to occur. Accordingly, the present invention provides a highly safe and novel thrombolytic agent as a therapeutic thrombolytic agent.
  • the t-PA analog of the present invention inhibits the inhibition of t-PA by plasminogen activator inhibitor 1 (hereinafter abbreviated as PAI-1), which is an immediate inhibitor of t-PA. Resistant to it. Therefore, it is expected that the protein will reach the thrombus site while maintaining its activity without being inhibited by PAI-1. Therefore, it is expected that the utility as the therapeutic agent for thrombosis of the present invention is further enhanced in addition to the above-mentioned fibrin specificity.
  • PAI-1 plasminogen activator inhibitor 1
  • FIG. 1 is a schematic diagram showing a restriction enzyme cut map of a plasmid pTZB-000.
  • FIG. 2A shows a schematic diagram of the construction of plasmid pHSGB and a restriction enzyme recognition site.
  • FIG. 2B is a schematic diagram of a synthetic DNA linker for preparing plasmid pHSGB and its assembly.
  • FIG. 3 is a schematic diagram of the assembly of plasmid pHSGB—O00.
  • FIG. 4 is a schematic diagram of the assembly of plasmid pHSGB-0000NA.
  • FIG. 5 shows the sequence of the synthetic oligonucleotide used for site-directed mutagenesis.
  • FIG. 6 shows a sequence of a synthetic oligonucleotide having a partial sequence of UK, a single PA, or both, which was used for partial recombination of the t-PA sequence.
  • FIG. 7 is a schematic diagram of the assembly of the plus pH S GB B—C S 1 ⁇
  • FIG. 8 is an assembly schematic diagram of the plasmid pCDM8—CS1. BEST MODE FOR CARRYING OUT THE INVENTION
  • the present inventors have found that in the amino acid sequence of t — ⁇ ⁇ , the amino acid corresponding to positions 276 to 306 of natural t — PA (the position immediately after the single-stranded Z double-strand cleavage site). Replacing at least part of the amino acid sequence with the corresponding amino acid sequence of the UK will result in a novel t-PA analog with improved fibrin specificity Was found.
  • the object of the present invention is a
  • a therapeutic agent for thrombosis comprising the t-PA analog according to 1) above as an active ingredient.
  • Preferred examples of the t-PA analog according to 1) above include, for example,
  • the position 276 of the natural t — PA referred to here is the position immediately after the single-stranded Z double-strand cleavage site, and corresponds to positions 276 to 306 of the natural t — PA.
  • the amino acid sequence region of K corresponds to the amino acid sequence from 159 to 188, which is the position immediately after the cleavage site of pro-UK (—strand) to UK (double-strand). That is what you do.
  • the corresponding amino acid sequence of UK is the amino acid sequence region of natural t-PA.
  • the amino acid sequence of the natural t-PA is less than that of the natural amino acid corresponding to the natural t-PA, and the corresponding amino acid at position 295 of the natural t-PA in the UK Amino acids are treated as if they do not exist.
  • the preparation of the t-PA analogs of the present invention can be performed, for example, using at least some amino acid residues of the amino acid sequence corresponding to positions 276 to 306 of natural t-PA.
  • a cDNA encoding the amino acid sequence of the corresponding region of t-PA is replaced with a cDNA encoding the corresponding amino acid sequence in the UK or a synthetic oligonucleotide.
  • linking it with an appropriate expression vector, introducing it into the host, and expressing and producing the t-PA analog protein as the final target substance of the present invention. It can be. The details are described below.
  • cDNA that normally encodes natural t-PA can be used.
  • CDNA encoding native t-PA has already been obtained, for example, as described in Reference 13 by cloning from paused melanoma cells. Therefore, its cDNA sequence and amino acid sequence are also known.
  • the cDNA sequence and amino acid sequence of a modified t-PA that increases the half-life for example, References 14 and 15
  • References 14 and 15 which are modifications of natural t-PA
  • a part of the amino acid sequence corresponding to positions 276 to 306 of natural t-PA can be easily replaced with the corresponding sequence of UK by the same operation. Can be.
  • Nsp V TTCG AA
  • a at the position of amino acid at position 6 TTCGCA
  • GCATAC GCATAC
  • GTATAC restriction enzyme recognition site
  • the DNA fragment containing the UK sequence to be replaced is prepared by double-stranded oligonucleotides synthesized by the phosphoamidite method (Reference 18). It is convenient to prepare it in the form. In addition, if necessary, a method in which a plurality of short synthetic oligonucleotides are prepared and these are joined together can be employed.
  • the method for replacing the t-PA sequence with the UK sequence is to cut the t-PA sequence by cleaving the introduced NspV and AccI restriction enzyme recognition sites with the restriction enzyme, as described above. Instead, a DNA fragment containing the UK sequence is ligated to this site (Ligea —Shion) is convenient.
  • the cDNA encoding the t-PA analog thus produced is ligated to a well-known expression vector such as CDM8 (ori-), and then introduced into an appropriate host.
  • a well-known expression vector such as CDM8 (ori-)
  • CDM8 a well-known expression vector
  • the host any of prokaryotic organisms such as bacteria and eukaryotic organisms such as mammalian cells and yeast can be used. Examples of animal cell hosts include COS-1 and CH0 cells.
  • Can be An efficient method for introducing an expression vector into a host is the electric pulse method (Reference 19).
  • the t-PA analogs produced in the culture supernatants include zinc clear agarose, concananol black A agarose, Sephadex G-150 agarose, etc.
  • the t-PA analog thus obtained which can be easily purified by a known method (Reference 20) or the like, has high fibrin specificity Therefore, when administered to a living body as a thrombolytic agent, it can be used as an active ingredient of a pharmaceutical composition having reduced side effects of systemic blood bleeding as compared with natural t-PA. Further, since the t-PA analog of the present invention has PAI-11 resistance, it can be used as an active ingredient of a pharmaceutical composition which is effective at a lower dose than natural t-PA.
  • a prescribed amount can be dispensed into a container by a conventional method and freeze-dried, whereby the preparation can be extremely easily made.
  • This lyophilized product is a pharmaceutically acceptable carrier. It is dissolved in a single substance, for example, physiological saline, and administered by intravenous or intracoronary injection. The dose may be about the same as the dose of natural t-PA currently used in clinical practice, but the t-PA analog can be used more than the dose because it has few side effects. Administration is performed by continuous intravenous injection or by injecting part of the planned dose first intravenously and continuous intravenous infusion of the remainder.
  • Plasmid p TZB (Reference 21) was prepared by removing all the multi-cloning sites other than BamHI of commercially available Plasmid PTZ 18R (Toyobo Co., Ltd. (T0Y0B0)).
  • the plasmid pTZB-0000 (Reference 21) is a BamHI fragment (Ba) that completely contains the t-PA translation region in the BamHI site of pTZB. mHI cassette) was inserted (Reference 21).
  • Fig. 1 shows a schematic diagram of the restriction enzyme of pTZB-1000. Show.
  • the plasmid pHSGB is a multiplex of commercially available plasmid pHSG398 (manufactured by Takara Shuzo Co., Ltd.) (Fig. 2-A), which has the chromium penicol resistance gene as a marker.
  • the entire DNA site was prepared by replacing the synthetic DNA linker (Fig. 2-B).
  • pHSGB-O00 was constructed by inserting the obtained BamHI cassette into the BamHI site of pHSGB (Fig. 3).
  • the synthetic DNA linker was prepared as follows. First, the oligonucleotide of sequence a, which is self-complementary as shown in Fig. 2-B, was synthesized. The synthesis was carried out using a type 381A DNA synthesizer manufactured by Applied Biosystems, and the purification was performed using the company's 0PC cartridge. This synthetic oligonucleotide is converted into a double strand (FIG. 2—B, sequence b), digested with Fokl and HindE, and synthesized DNA linker is synthesized. (Figure 2-B, sequence c) was isolated and purified from a polyacrylamide gel by standard techniques.
  • This synthetic DNA linker and pHSG398 were digested with HindIII and EcoRI, and the 5 'end was dephosphorylated, and ligated. The strain was transformed to obtain the plasmid pHSGB ( Figure 2-A).
  • This pHSGB is digested with BamHI and the 5 'end is removed.
  • the oxidized product is ligated with a fragment (BamHI cassette) obtained by quenching pTZB-0H with BamHI and isolating it from polyacrylamide gel.
  • Escherichia coli HB101 strain manufactured by Takara Shuzo Co., Ltd. was transformed to obtain plasmid pHSGB-0000 (FIG. 3).
  • p HSGB-0.0NA containing the NspV and AccI recognition sequences in the t-PA translation region of pHSGB-000 Made.
  • a site-directed mutagenesis technique using a synthetic oligonucleotide was used.
  • Fig. 4 shows a schematic diagram of the assembly.
  • M13tvl9EE (Reference 21) was obtained as type I for site-directed mutagenesis.
  • This M13tV19EE contains the cDNA for the native t-PA amino acids 205-32.
  • Ml3mp19E ENA is a single-stranded form of Ml3tv19EE, and the synthetic oligonucleotides P—NspV and P—Acc shown in FIG. I and Anil,
  • oligonucleotides containing the partial sequence of UK were synthesized. The sequence is shown in FIG. These four synthetic oligonucleotides were annealed, and this was combined with the digestion of pHSG-0000NA with NspV and AccI. Thus, E. coli HB101 strain was transformed to obtain a plasmid pHSGB-CS1 (FIG. 7) having UK hybrid type t-PA.
  • Plasmid pCDM8-0000 provided the sequence of the prime motor required to express CS1 in mammalian cells.
  • PCDM8-0000 is the expression vector CDM8
  • the above-mentioned expression plasmid encoding UK hybrid t-PA, pCDM8-CS1 was used to transform COS-1 cells by the electric pulse method described above. After culturing for 24 hours in a DMEM medium containing 10% fetal calf serum and 10 / Ig Zml of lysine chromatographically treated, the medium was serum-free and aprotinin-free DMEM. The medium was replaced and the culture was continued for another 96 hours. After centrifugation of the culture solution, the supernatant was recovered to obtain a t-PA analog CS-1. 90% or more of CS-1 contained in the thus obtained supernatant was present in a single-stranded state. This culture supernatant was used for the subsequent evaluation system.
  • Plasmid ⁇ CDM 8 — CS2 which expresses the UK hybrid t_PA in mammalian cells, is a plasmid p CDM obtained by introducing a Bg1I site into CDM 8 (ori —).
  • Example 2 Instead of CS 3 M, CS 3 R, CS 2 M and CS 2 R in Example 2, CS 4 M, CS 4 R, CS 2 M and CS 2 R shown in FIG. 6 were used. A t-PA analog CS-3 was produced in the same manner as in Example 2 except for the above.
  • Example 2 Instead of CS 3 M, CS 3 R, CS 2 M and CS 2 R in Example 2, CS 4 M, CS 4 R, CS 5 M and CS 5 R shown in FIG. 6 were used. A t-PA analog CS-4 was produced in the same manner as in Example 2 except for the above.
  • Example 2 In place of CS 3 M, CS 3 R, CS 2 M and CS 2 R in Example 2, CS 1 M, CS 1 R, CS 6 M and CS 6 R shown in FIG. 6 were used. Otherwise in the same manner as in Example 2, a t-PA analog CS-5 was produced.
  • Reference example COS-1 cells were transformed using the expression plasmid PCDM 8-0000 described above encoding native t-PA, and the culture supernatant was recovered to obtain native t-PA. The method was in accordance with 5. of Example 1.
  • fibrin cofactors were added to a system for measuring the activation of t-PA using the chromogenic synthetic substrate S-2251, and fibrin was added. The specificity was evaluated according to the method described in Ref.
  • each t-PA sample diluted to 200 ng / ml (but 150 ng / ml for CS-3) in PBS containing 0.01% Tween 80 (protein concentration required for preparation) Is known using polyclonal antibodies against natural t-PA
  • Table 1 shows the relative activity of each t-PA sample to native t-PA.
  • Off Lee Bed Li emissions in addition system reaction 3 0 minutes after full Lee Bed Li Bruno 2 hours after absorbance in one Gen addition system (OD 4 5 -.. ⁇ D 49) corresponding to native t - concentration of PA was determined from the above calibration curve.
  • the fibrin specificity was expressed as a ratio (the activity in the fibrin-added system, the activity in the fibrinogen-added system).
  • each t in plasma and plasma clot The plasminogen activating activity of PA was measured in a reaction system using the chromogenic synthetic substrate S—2251. The method was performed according to the method shown in Ref.
  • the t-PA analog of the present invention has a very low plasminogen activating activity in plasma as compared to natural t-PA, but it has a lower activity than natural t-PA in plasma clots. Close activity It became clear that it had.
  • t-PA analogs of the present invention when circulating in the blood, activate plasminogen followed by fibrinogen. It is unlikely to cause degradation of thrombus, and will express plasminogen activating activity close to natural t-PA and dissolve thrombus only when it reaches the local thrombus where fibrin is present.
  • t Compared to known variants with improved fibrin specificity, as compared to PA, the fibrin specificity is higher, and thus the thrombus specificity is higher. It is expected that the side effect of systemic bleeding tendency is unlikely to occur. Therefore, the t-PA analog of the present invention is extremely significant as a novel thrombolytic agent.

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Abstract

On obtient un nouvel analogue d'activateur tissulaire du plasminogène (ATP), plus spécifique de la fibrine, en remplaçant au moins une partie de la séquence d'acides aminés présente dans cet ATP. Cette partie correspond à la séquence d'acides aminés située entre la 276ème position (juste après le site de coupure monobrin/double brin) et la 306ème position d'un ATP naturel, et on la remplace par la séquence d'acides aminés correspondante d'un activateur de plasminogène urinaire. Cet analogue n'entraîne que difficilement l'activation du plasminogène et la décomposition consécutive du fibrinogène lorsqu'il circule dans le sang, mais il est à même d'activer un plasminogène de façon comparable à un ATP naturel et entraîne une thrombolyse dès qu'il parvient au site d'un thrombus où de la fibrine est présente. On prévoit ainsi qu'il ne peut guère entraîner de réaction secondaire telle qu'une tendance hémorragique généralisée, ce qui lui offre de bonnes perspectives en tant qu'agent thrombolytique.
PCT/JP1992/000415 1992-04-03 1992-04-03 Analogue d'activateur tissulaire du plasminogene Ceased WO1993020194A1 (fr)

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PCT/JP1992/000415 WO1993020194A1 (fr) 1992-04-03 1992-04-03 Analogue d'activateur tissulaire du plasminogene

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61233630A (ja) * 1985-02-09 1986-10-17 ビ−チヤム・グル−プ・ピ−エルシ− 新規化合物、その製法及びそれを含む医薬組成物
JPH02295483A (ja) * 1989-01-17 1990-12-06 Integrated Genetics Inc Ｔｐａのプロテアーゼ領域中に位置する修飾残基をコードする新しい遺伝子配列

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61233630A (ja) * 1985-02-09 1986-10-17 ビ−チヤム・グル−プ・ピ−エルシ− 新規化合物、その製法及びそれを含む医薬組成物
JPH02295483A (ja) * 1989-01-17 1990-12-06 Integrated Genetics Inc Ｔｐａのプロテアーゼ領域中に位置する修飾残基をコードする新しい遺伝子配列

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANNU. REV. PHARMACOL. TOXICOL., Vol. 30, (1990), D.H. HIGGIMS et al., "Tissue Plasminogen Activator: The Biochemistry and Pharmacology of Variants Produced by Mutagenesis", p. 91-121. *
FIBRINOLYSIS, Vol. 2, (1988), H. PANNEKOEK et al., "Mutants of Human Tissue-Type Plasminogen Activator (t-PA): Structural Aspects and Functional Properties", p. 123-132. *

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