JPS6322026A - Medicinal composition - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to tissue plasminogen activators, their combinations with superoxide dismutase, pharmaceutical combinations containing them, and their use in medicine and veterinary medicine.

A dynamic equilibrium exists between the enzyme system capable of forming blood clots, the coagulant system, and the enzyme system capable of dissolving blood clots, the fibril system, which maintains a healthy, well-organized vascular bed. To limit blood loss during injury, blood clots form within injured blood vessels. After the injury has healed naturally, the fibrinolytic system activates and dissolves excess blood clots. Blood clots can form in the absence of traumatic injury and can lodge in vital blood vessels, causing partial obstruction or even complete occlusion of blood flow. If this occurs in the heart, lungs, or brain, it can result in myocardial infarction, pulmonary embolism, or stroke, respectively. Together these diseases account for the leading cause of morbidity and death in industrialized countries.

Blood clots! Consists of a network of IN elements, proteolytic enzymes,
Can be dissolved by plasmin. This enzyme is derived from the inactive enzyme plasminogen, a plasma component, by plasminogen activators. There are two immunologically identifiable plasminogen activators in mammals.
There are seeds. Endogenous plasminogen activator, also known as urokinase, is an enzyme produced by the kidneys and can be isolated from urine. Exogenous plasminogen activator, known as vascular plasminogen activator and tissue plasminogen activator (t-PA), is present in many tissue homogenates (particularly human uterus), blood vessel wall cells and certain It can be isolated from the cell culture medium of the species. In addition to these two plasminogen activators, there is also a bacterial product, streptokinase, which is produced from beta-hemolytic streptococci. A major drawback of both urokinase and streptokinase is that they are active throughout the circulation, not just at the site of clotting. They can also destroy other blood proteins such as, for example, fibrinogen, prothrogon, factor 5 and factor 8, thus leading to a decrease in hematometic capacity and an increased risk of hemolysis. . On the other hand, t-PA
The biological activity of t-PA is dependent on the presence of fibrin, and t-PA binds to and is activated there. That is, maximum activity occurs only at the site of blood clots, ie due to the presence of a fibrin network to be dissolved, so that the risk of hemolysis is largely avoided.

Blockage of blood flow within a blood vessel generally results in the development of an ischemic condition. Under these conditions, the tissue becomes oxygen deprived and becomes in a dangerous state, ie, the tissue is injured but still has a chance of survival.

However, if such conditions persist, for example, for three hours or more, the tissue dies and once this condition occurs, recovery is not possible. Therefore, it is important to restore flow or blood flow as soon as possible to save tissue before it suffers permanent damage. Ironically, however, reperfusion itself, even if performed before tissue necrosis, produces a complex set of phenomena, including the potential for generation of superoxide radicals, which have deleterious effects on hypoxic tissue. As a result, reperfusion can only lead to partial recovery of the exposed tissue, while the remaining tissue is permanently damaged by the manifestation of one or more of the above-mentioned phenomena.

The present invention allows tissue to undergo one or more of the above-mentioned phenomena during reperfusion.
It was discovered and perfected that t-PA prevents damage to at-risk tissue by protecting against more than one species. The mechanism of action by which t-PA confers this protection is unknown, but is independent of its action as a thrombolytic agent. This newly discovered property allows
1-PA or a pharmaceutical composition containing it can be used as an inhibitor of damage to tissues exposed to hazards in environments such as those outlined herein.

That is, the present invention provides a method for inhibiting damage to tissue exposed to danger during reperfusion in a mammal, characterized by administering an effective amount of t-PA to the mammal υ Use of t-PA for the manufacture of a medicament to prevent damage to tissues exposed to hazards during reperfusion in mammals - Provides a pharmaceutical composition for inhibiting damage to tissues exposed at risk during reperfusion of a mammal, comprising PA and a pharmaceutically acceptable carrier.

Although the present invention can be used to protect any exposed tissue, it is particularly useful in preventing damage to exposed myocardial tissue.

The t-PA used in the present invention may be any biologically active protein corresponding to mammalian, particularly human t-PA, including both glycosylated and non-glycosylated forms. EP-A-112122
Single chain t-PA, double chain t-PA, as described in
PA, or mixtures thereof, fully glycosylated human t-PA has an apparent molecular weight of about 70,000 on a polyacrylamide gel and an isoelectric point of 7.5.
~8.0. The specific activity of t-PA is approximately 500.0OO
Preferably IU/η (IU/■ = international unit/
III. The international unit is WHO.

National In5titut ror B+
0+00iCal 5standardSand Con
Trol, 1lolly Hill, Hampst
ead, London.

MW 3b f6RB, U, is an activity unit defined by).

Preferably, the amino acid sequence of t-PA substantially corresponds to the sequence shown in FIG.

That is, is the arrangement the same as the arrangement in Figure 1?
or allelic origin or other one or two
containing deletions, substitutions, insertions, inversions, or additions of one or more amino acids, the resulting sequence has at least 80%, preferably 90% homology with the sequence in Figure 1, and the biological and It retains its immunological properties. In particular, the sequence shown in FIG. 1 or the same amino acid sequence except that the 245th amino acid from the serine N-terminus is valine instead of methionine, or any of them may contain the polypeptide N-terminal sequence GJ! V-AJ! a
Examples include sequences to which -Arg is added.

The amino acid sequence shown in FIG. 1 has 35 cysteine residues and therefore has the characteristics of 17 disulfide bridges. Figure 2 shows the predicted structure (due to the formation of disulfide bonds) of the sequence between the 90th amino acid and the proline C-terminus, based on analogy with other proteins whose structures have been determined in detail. Shown below. Although the structure of the N-terminal region is more uncertain, several proposals have been made (Progress in Fibrino
lysis, 1983.

6.269-273; and Proc, Natl.
AcadSci, 1984.81.5355-53
59).

The most important structural feature of t-PA is the two kringle regions (9) that are responsible for the protein's binding to fibrin.
(between the 2nd and 173th amino acids and between the 180th and 261st amino acids), and the serine protease region that constitutes the main region of the B chain and is responsible for the activation of plasminogen. The particularly important amino acids in serine proteases are the catalytic triad, His/
ASI)/Ser. In key PA these are 32
They are located at the 21st, 371st and 463rd positions.

The disulfide bridge between cysteine amino acid residues 264 and 395 is also important in holding A and 81 together in double-chain t-PA.

In Figures 1 and 2, the following conventional one-letter and three-letter codes are used to indicate amino acid residues.

ASp D Aspartic acid Thr T Threonine Ser S Serine Gj! Lj E Glutamic acid pro P Proline GIV G Glycine Ala A Alanine Cys C cysteine ■aj! ■ Valine 11e I Isoleucine LeuL Leucine Tyr Y Tyrosine Phe F Phenylalanine) 1is H Histidine Ar(] R Arginine Lys K Lysine Trp W Tryptophan Gin Q Glutamine Met M Methionine Asn N Asparagine t-PA has been reported and known in this technical field. can be obtained by any operation described.

For example, Biochim, Biophys, Acta
, 1979, 580.140-153: EP-A
-41766 or from normal or neoplastic cell lines of the type described in EP-Δ-113319. However, t-PA, for example EP-A-
93619:EP-A-117059:EP-A-11
7060: EP-A-173522: EP-A-174
835:EP-△-174 835:EP-A-178
105:WO86/○1538; WO86105514
Alternatively, those obtained from transformed or transfected cultured cell lines induced using recombinant DNA techniques as described in WO 86105807 are preferred. For the production of t-PA, 111 Chinese hamster ovaries (CHO) were used, and Ho1. Ce1
1. Biol, 1985.5 (7), 1750~
Particularly preferred is the method described in 1759.

In this method, the cloned gene is used together with the gene encoding dihydrofolate reductase (dhfr).
Co-transfect into r-cHo cells. Transformants expressing dhfr on media lacking nucleosides are selected and exposed to high concentrations of methotrexate. In this way, the dhfr and t-PA genes are simultaneously amplified, leading to a stable cell line capable of expressing high levels of t-PA.

The t-PA is preferably prepared by any procedure reported and known in the art, such as Biochim, B
iophys, Acta, 1979. 580.
140-153; J, Biol, Chcm,
1979.254(6), 1998-2003: 1
bid, 1981.

256 (13), 7035~A041; Eur,
J.

Biol, 1983. 132. 681-686:
EP-A-41766; EP-A-113319 or GB-A-2122219.

In the method of the present invention, t-PA is used alone or in combination with other therapeutic agents that can also prevent damage to tissues exposed during reperfusion. A particularly useful example of such a combination is superoxide dismuter-lx' (SOD), an enzyme known to scavenge and destroy superoxide radicals, a phenomenon that causes tissue damage. There is a combination of actual,
When t-PA and SOD are combined, t-PA or SOD
The blocking effect was significantly enhanced compared to that by itself.
It was also revealed that he would be elevated. Accordingly, the present invention also provides blends of t-PA and SOD.

The combination of t-PA and SOD provides a particularly convenient means of both removing blood clots and preventing injury to tissues exposed to danger during reperfusion. That is, the administration of t-PA and SOD first removes the pseudoplane by the known thrombolytic action of t-PA, and then induces the combination and
It is expected to result in inhibition of f[I2.

The SOD used in combination with t-PA may be any biologically active protein that substantially corresponds to any one or more of the group of enzymes commonly known by this name. good. Those of mammalian origin, particularly bovine or human, are preferred and are generally associated with and classified by metal cations. Examples of metal cations are iron, manganese, copper; combinations of copper with other metals such as zinc, cadmium, cobalt or mercury are preferred, in particular the copper/dilium combination. Both the manganese and copper/subbox forms of SOD are naturally found in humans. Iron J3 and manganese type SOD of bacterial origin
Both have a molecular weight of about 40°000 and are dimers.

On the other hand, true nucleus/! Manganese-type SOD originating from i-products is a tetramer with a molecular weight of about so,ooo. Copper/zinc SoD of eukaryotic origin is a dimer of approximately 32,000 molecules m, with one copper cation and one zinc cation per subunit. The copper cation binds to the four histidine residues of the subunit, and the zinc cation binds between the histidine and aspartate.

There is also a copper/zinc type SOD of eukaryotic biological origin that has a molecular weight of about 130,000 and consists of four subunits. The molecular weight of each type of SOD was determined using sedimentation equilibrium, molecular sieves or polyacrylamide gels. The isoelectric point of each type of SOD ranges from 4 to 6.5, depending on the degree of sulfation and/or deamidation. The specific activity of the copper/zinc type SOD of bovine or human origin is preferably at least 3°000 LI/■ (units of activity are J. Biol. Chew, 1969. 2).
44.6049-6055).

The amino acid sequence of copper/zinc type SOD of bovine or human origin is J. Biol. Chew, 19
Biochemistry of human origin, 1980
, 19.

2310-2316 and FEBS L(!tt,,
1980.

Preferably, the sequences substantially correspond to those shown in 120.53-55. That is, the sequence is identical to the sequences set forth in these documents or contains deletions, substitutions, insertions, inversions or additions of one or more amino acids, whether of allelic origin or otherwise. , the generated sequences have sufficient homology with the reported sequences and have virtually identical biological and immunological properties! 1
It is intended to hold the following.

The amino acid sequence of copper/zinc-type SOD of bovine origin contains 311iil cysteine residues per subunit (
J. Biol. Chem., 1974.249 (2
2>, 7326-7338). The intrachain disulfide bridge is (
, /S occurs between residues 55 and Cys 144, while -, the limiting disulfide bridge occurs between residues Cys6. The amino acid sequence of copper/zinc-type SOD of human origin contains four cysteine residues per subunit (BiOChe
lliStrV, 1980.19.2310-231
6 and FEBS Lett, 1980, 120
．． 53-55). The intrachain disulfide bridge is CyS57 and c
Cys occurs between 146 residues, while a limited disulfide bridge occurs between Cys6 residues. CyS111 residue remains free.

SOD can be obtained by any suitable procedure reported and known in the art.

For example, SOD is obtained from red blood cells or liver by extraction procedures as described in GB-A-1407807 and GB-A-1529890. Also, SOD
For example, Auth 1 to Laria patent application 27461/8
No. 4, WO35101503, EP-A-138111
, EP-A-164566, EP-A-173280 and EP-A-180964
They can also be obtained from cultured transformed or transfected cell lines induced using techniques.

Preferably, the SOD is prepared using any suitable procedure reported and known in the art, such as that described in EP-A-112299.

By the method of the present invention, t-PA or t-PA and SOD
When using a combination of the following, it is preferred to use the active ingredients in a medical formulation.

When using this combination, the active ingredients can be used in separate dosage forms that are administered simultaneously or sequentially. If they are administered sequentially, the t-PA formulation is given first, followed by the SOD'! It is preferable to administer agent j. In any case, the second administration of the two formulations must not be so different that the advantage of the potentiating effect of the combination of active ingredients in preventing tissue damage in vivo is lost. However, it is much more convenient to contain the amphitropic ingredients together in a single combination formulation than to use separate formulations. Thus, the present invention also provides a pharmaceutical composition comprising t-PA and SOD and a pharmaceutically acceptable carrier.

Generally, t-PA or a blend of t-PA and SOD is
It will be administered via an intravascular route, such as an intravenous drip or a single injection. Therefore, preparations for parenteral administration are required. Preferably, the formulation is provided to the physician or veterinarian in lyophilized form for transportation and storage advantages during supply. The lyophilized dosage form can be reconstituted by a physician or veterinarian with an appropriate amount of solvent as needed at the time of use.

Freeze-dried pharmaceutical compositions containing t-PA for parenteral administration are known in the art. Examples of such techniques are EP-A-41766: EP-A-93619;
-A-112122;EP-A-113319:EP-
A-123304: EP-A-143081; EP-A
-156169: W○ 86101104; Special Publication Showa 57
-120523 (Japanese Patent Application No. 56-6936) and Japanese Patent Publication No. 58-65218 (Japanese Patent Application No. 56-163145). A preferred example is GB-A-2
176702 and GB-A-2176703. Such formulations also contain t-PA in SOD.
and SOD formulations.

Intravascular instillation is typically carried out via a parenteral solution contained within an IV bag or vial or an electrically operated IV syringe. The solution is transported from the IV bag or vial to the patient using gravity feed or an IV pump. Because the rate of administration of parenteral solutions cannot be adequately controlled using gravity-fed infusion systems, the use of infusion pumps is preferred, especially for solutions containing relatively high concentrations of active ingredients. is preferred. However, it is even more preferred to use an electrically operated drip syringe, which allows for more precise control of the rate of administration.

The effective amount of t-PA and the combination of t-PA and SoD to prevent damage to the tissue exposed at risk during reperfusion depends on, for example, age, weight,! 2! Treatment will depend on many factors, including the precise condition being treated, its severity, route of administration, etc., and will ultimately be determined at the discretion of the attending physician or veterinarian. However, effective doses of t-PA generally range from 0.2 to 1 kg per hour per kg of patient body weight.
4Ing (i.e., t-PI3) specific activity to 500.0
OOIU/ri as ioo, ooo~2.000.00
0 IU), preferably in the range of 0.3 to 2 Rg.

Therefore, the effective dose per hour for an adult weighing 70 kg is 20-140 IItg (i.e. 10°00
0.000 to 70,000,0OOIU), especially about 7
0 Itg (or 35,000.000 IU) is preferred. When using SOD in combination with t-PA, SO[
) is generally 1.000 to 50°ooou, preferably 7.00°/kg of patient body weight per hour.
The range is from 000 to 20,000U. Therefore, the effective amount of SOD per hour for an adult weighing 70 degrees is between 500°000 and 1,500. ooou is better.

The following examples further illustrate the invention:
It is not intended to limit the invention in any way.

Example 1: Preparation of a parenteral formulation for t-p A parenteral formulation for t-PA was prepared substantially as described in GB-A-2176703. The specific activity of t-PA was approximately 300,0001 U/■.

Example 2: Production of a parenteral formulation of SOD Copper/zinc type bovine SOD is manufactured by Sigma Chemical Co., Ltd.
Co.

(St. Louis, Hissouri, U.S.
A, 63178) as a powder and dissolved in neutral saline solution.

Example 3: Preparation of t-PA and SOD The formulations of Example 1 and Example 2 were mixed and further diluted with saline solution to achieve the required dosage.

(2) Method Male beagles (10 to 9 in 12) were anesthetized with bentoparbital sodium, tracheally intubated, and ventilated with room air via a Harvard breathing apparatus.

Catheters for infusion and arterial blood pressure measurement were implanted in the left jugular vein and left carotid artery. A thoracotomy was performed at the fourth intercostal space, the heart was suspended in a pericardial rack, and the left anterior descending aorta (LAD) was isolated just below the major oblique branch.

An electromagnetic flow probe was placed on the LAD. 90 LAD with a snare of 110 silk suture distal to the flow probe.
Closed for minutes. Intravenous treatment was started 15 minutes before the release of the snare ef1m and continued until 45 minutes after the release. The thoracotomy was closed and the animals were allowed to recover from the surgical procedure. Twenty-four hours after closure, the animals were re-anesthetized and the flow in the LAD was measured again. Hearts were then removed for post-mortem infarct size quantification.

The study was conducted on four groups of dogs. Group 1 served as a saline control. In the second group, t-PA was added at 2.5Rg/phantom (75
0,0001U/Kg), 50016 cows in the third group
．． 500tJ/Ky, t-PA2.5jv/ for the 4th group
To! J (750,0001U/Ng) and bovine 5OD
16.500 LI/Kg was administered.

The formulations used are those described in Examples 1-3.

Myocardial infarct size was quantified by ex vivo double reperfusion method. A catheter was inserted into the LAD and coronary supraoral aorta just distal to the closure site. LAD coronary vascular bed in 0.02M potassium phosphate buffer D117.4.
%triphenyltetrazolium PAFIL salt (TTC>
Irrigated with The aorta was retrogradely perfused with 0.5% Evans blue dye. Both areas were perfused with the respective dye for 5 minutes at a constant pressure of 1100 rn mercury. Hearts were cut into 8 m slices perpendicular to the apical baseline. The left ventricle region at risk for infarction was identified as it lacks Evans blue due to its anatomical dependence on the LAD for blood flow. Myocardial infarction area within the risk area is TTC
It was possible to identify the tissue because it lacks the dehydrogenase enzyme even when perfused with water.

The infarct morphology was recorded by carefully tracing transverse sections of the ventricles on clear acrylic resin paper, and the infarct size was confirmed planimetrically. Then, the right ventricular myocardium, valvular region, and adipose tissue were removed from the ventricular section.

The area at risk and the infarcted area was carefully dissected and weighed from the entire left ventricular area. Infarct size was expressed by the percentage of area at structural risk. Statistical ratios between drug-treated and control groups were determined by Bonferroni
Multiple comparison method (C1rculationRes, 19
80.47.1 to 9) using a one-sided variance test.

A p value of 0.05 or less was considered a significance criterion.

0 Results Table 1 Salt solution 5 36.0±8.9 37.3±7
．． 6II t-PA 6 14.
3±11.7 35.7± 5.4 ・If S
OD 4 13.0± 4.6 30
．． 6± 2.6rV t-PA+SO032,3±
1.3 37°2±9.18 Data are expressed as standard error of the mean.

No significant difference in the proportion of the left ventricle rendered ischemic due to mechanical occlusion of the LAD was found by ANOVA between any of the treatment groups and the control group.

(To) Conclusion: The use of t-PA significantly inhibits the size of myocardial infarction, and does it reduce the size of myocardial infarction? ! The protective ability for tissues exposed to danger during the i-stream was demonstrated. Furthermore, the combination of t-PA and SOD achieved a synergistic inhibition effect, with higher levels of inhibition observed than when using t-PA or SOD alone.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the basic amino acid sequence of t-PA used in the present invention. FIG. 2 is a diagram showing the secondary structure of a part of the basic amino acid sequence of t-PA used in the present invention. ' is the photopic site of the single-chain t-PA, and when cleaved here, gives the second-terminal t-PA. In this case, the A chain contains two kringle regions, and the B chain contains a serine protease region.

Claims (10)

[Claims] (1) A pharmaceutical composition comprising t-PA and a pharmaceutically acceptable carrier for use in inhibiting damage to tissues exposed at risk during reperfusion in mammals. (2) The pharmaceutical composition according to claim 1, wherein t-PA is a single-chain type. (3) The pharmaceutical composition according to claim 1, wherein t-PA is double-stranded. (4) t-PA is the amino acid sequence shown in Figure 1, the same amino acid sequence except that the 245th amino acid from the serine N-terminus is valine instead of methionine, or any of these has a polypeptide N-terminal sequence. Gly-
The pharmaceutical composition according to claim 1 or 2, which has a sequence to which Ala-Arg is added. (5) Blend of t-PA and SOD (6) The formulation according to claim 5, wherein t-PA is single-stranded. (7) The formulation according to claim 5, wherein t-PA is double-stranded. (8) t-PA is the amino acid sequence shown in Figure 1, the same amino acid sequence except that the 245th amino acid from the serine N-terminus is valine instead of methionine, or any of these has a polypeptide N-terminal sequence. Gly-
A formulation according to any one of claims 6 or 7 having an Ala-Arg appended sequence. (9) The formulation according to any of claims 5 to 8, wherein the SOD is of the copper/zinc type of bovine or human origin. (10) A pharmaceutical composition comprising the formulation according to any one of claims 5 to 9 and a pharmaceutically acceptable carrier.