JPH05368B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides a tissue plasminogen activating substance (t-
PA) and oxypurinol or a pharmaceutically acceptable salt thereof as active ingredients,
The present invention relates to pharmaceutical compositions for preventing damage to ischemic heart tissue during blood reperfusion in mammals, including humans. A dynamic equilibrium exists between the enzyme systems that can form clots, the coagulation system, and the fibrinolytic system, which can dissolve clots, maintaining a completely open vascular bed. Blood clots form in injured blood vessels to limit blood loss from the injury. After natural healing of the injury, excess blood clots are dissolved by the action of the fibrinolytic system. In some cases, blood clots form even in the absence of traumatic injury and can become lodged within major blood vessels, resulting in partial or even total occlusion of blood flow.
If this occurs in the heart, lungs or brain, myocardial infarction,
May cause pulmonary embolism or stroke. The combination of these symptoms is a major cause of morbidity and mortality in industrialized countries. A blood clot consists of a fibrous network, which can be dissolved by the proteolytic enzyme plasmin. This enzyme is derived from an inactive proenzyme, plasminogen (a component of plasma), by the action of a plasminogen activator. There are two immunologically distinct mammalian plasminogen activators. Endogenous plasminogen activator, also known as urokinase, is an enzyme produced by the kidneys and can be isolated from urine. It can also be prepared from many tissue culture sources. Exogenous plasminogen activators, also known as vascular plasminogen activators and tissue plasminogen activators (t-PA), are found in significant tissue homogenates (particularly the human uterus), vascular cell walls and some can be isolated from cell cultures of species. In addition to these two plasminogen activators, there is a bacterial product, streptokinase, produced from beta-hemolytic streptococci. The major drawback of both urokinase and streptokinase is that they are active throughout the circulation and not only at the site of the clot. For example, they destroy fibrinogen, prothrombin, factor and other blood proteins such as factor, reduce blood clotting ability and increase the risk of bleeding. In contrast, the biological activity of t-PA is dependent on the presence of fibrin, to which t-PA binds and is activated. Maximum activity is therefore developed only at the site of the clot, ie at the site where there is a fibrin network to be lysed, which greatly avoids the risk of bleeding. Disruption of blood flow within a blood vessel generally leads to an ischemic condition. In this condition, tissues are deprived of oxygen and become at risk. The tissue in this state is damaged but still potentially alive. However, if this condition is maintained for a period of time, ie, 3 hours or more, the tissue becomes necrotic, and once this condition is reached, there is no recovery.
Therefore, it is important that reperfusion, or recovery, of blood flow occurs as soon as possible to save the tissue before it suffers permanent damage. Ironically, even if blood flow reperfusion occurs prior to tissue necrosis, this reperfusion itself can result in the expected production of superoxide groups that have deleterious effects on hypoxic tissue. This results in complex phenomena including Therefore, while reperfusion may lead to partial recovery of the tissue at risk, the remaining portions of the tissue are permanently damaged due to one or more of these events occurring. Under normal conditions, xanthine and hypoxanthine are present in low concentrations in muscle cells and are enzymatically converted to uric acid by xanthine dehydrogenase, which uses NAD (nicotinamide-adenine dinucleotide) as an electron acceptor. however,
In ischemic conditions, xanthine and hypoxanthine concentrations generally increase to an enormous or even greater extent. Findings have been submitted that the enzyme xanthine dehydrogenase is itself converted to xanthine oxidase when blood reperfusion occurs. The production of xanthine oxidase in turn metabolizes xanthine and hypoxanthine to uric acid with oxygen used as an electron acceptor instead of NAD. In this case, superoxide groups are produced as a by-product [The New England
Journal of Medicine, 1985, 312(3), 159~
163 pages]. It has now been found that the combination of t-PA and oxypurinol or a pharmaceutically acceptable salt thereof prevents damage to tissue at risk during the period of blood reperfusion. It has been found that the combination of t-PA and oxypurinol produces significantly more potent levels of inhibition compared to that provided by t-PA or oxypurinol each by itself. Therefore, the present invention provides t-
The present invention relates to a pharmaceutical composition for inhibiting damage to ischemic heart tissue during blood reperfusion in mammals including humans, containing PA and oxypurinol or a pharmaceutically acceptable salt thereof as active ingredients. The present invention particularly provides an advantageous means for both eliminating blood clots and preventing damage to tissue at risk during the subsequent period of blood reperfusion. That is, t-
PA and oxypurinol were first administered at t-
Clearance of the clot occurs due to the known thrombolytic action of PA, followed by inhibition of tissue damage due to the combined action of t-PA and oxypurinol.
Although the present invention can be used to protect any tissue at risk, it is particularly useful in preventing damage to at-risk myocardial tissue. The t-PA used in the present invention can be any biologically active protein that substantially corresponds to mammalian, particularly human t-PA, including both those with and without glycosylation. do. The t-PA can be single-chain or double-chain t-PA or mixtures thereof as described in EP-A-112 122 and has an apparent molecular weight based on polyacrylamide gels of about 70,000 and 7.5 It has an isoelectric point of ~8.0. Preferably, the t-PA has a specific activity of about 500,000 IU/mg (IU/mg is International Units/mg; International Units are
WHO, National Institute for Biological Sciences
Standards and Control.Holly Hill,
unit of activity as defined by Hampstead, London, NW3 6RB, United Kingdom). The amino acid sequence of t-PA preferably substantially corresponds to the sequence set forth in FIG. Accordingly, this sequence may be identical to the sequence in Figure 1 or may contain deletions, substitutions, insertions, or omissions of one or more amino acids, whether of allelic origin or otherwise.
including rearrangements or additions, the resulting sequence is at least 80%, preferably 90%, of the sequence in Figure 1.
homology and essentially possess the same biological and immunological properties of the protein. In particular, this sequence is identical to the sequence shown in Figure 1, or from the serine N-terminus.
They have the same sequence except that the amino acid at position 245 is valine instead of methionine,
Both sequences optionally have either the first three amino acids absent or optionally an additional polypeptide N- of Gly-Ala-Arg.
It can have a pre-terminal sequence. The amino acid sequence shown in Figure 1 has 35 cysteine residues and therefore has the potential to form 17 disulfide bridges. Based on analogy with another protein whose structure has been determined in more detail, a hypothetical structure for the sequence between the amino acid present at position 90 and the proline C-terminus (resulting from disulfide bond formation) is proposed. Shown in Figure 2. Although several proposals have been made regarding the structure of this N-terminal region, there is little certainty [Progress in Fidrinolysis, 1983, 6 , 269-
273 pages and Proc. Natl. Acad. Sci., 1984, 81 ,
Pages 5355-5359]. The most important structural features of t-PA are the two Kringle regions (between amino acids 92 and 173 and between amino acids 180 and 261). is involved in the binding of this protein to fibrin, and another important feature is the serine protease region, which contains the main part of the B-chain and is involved in the activation of plasminogen. A particularly important amino acid in serine proteases is the catalytic triad, His/Asp/
Ser. In t-PA these occur at positions 322, 371 and 463. 264
The disulfide bridge between cysteine amino acid residues 395 and 395 is also important, and the t
- Holding the A and B chains of PA together. In Figures 1 and 2, conventional one-letter and three-letter codes are used for amino acid residues as follows: AspD Aspartate IleI Isoleucine ThrT Threonine LeuL Leucine SerS Serine TyrY Tyrosine GluE Glutamate PheF Phenylalanine ProP Proline HisH Histidine GlyG Glycine LysK Lysine AlaA Alanine ArgR Arginine CysC Cysteine TrpW Tryptophan ValV Valine GlnQ Glutamine MetM Methionine AsnN Asparagine t-PA can be obtained by any of the methods disclosed or known in the prior art. As an example, t-PA is Biochimica et al.
Biophysica Acta, 1979, 580 , pp. 140-153;
It can be obtained from normal or neoplastic cell lines of the type described in EP-A-41766 or EP-A-113319. However, t-PA is
For example, EP-A-93619, EP-A-117059, EP
-A-117060, EP-A-173552, EP-A-
174835, EP-A-178105, WO86/01538,
Chinese hamster ovary (CHO) cells are preferably derived using recombinant DNA techniques such as those described in WO86/05514 or WO86/05807 to obtain culture-transformed or transfected cell lines. −Used for the production of PA, this cell is used for Molecular and Cellular
It is particularly preferred to induce it by the method described in Biology, 1985, 5 (7), pages 1750-1759. In this method, the cloned gene is cotransfected into dhfr ^- CHO cells with the gene encoding dihydrofolate reductase (dhfr). Transformants expressing dhfr are selected on nucleoside-deficient media and exposed to increasing concentrations of methotrexate. The dhfr and t-PA genes are thus amplified together, resulting in high levels of t-PA.
Stable cell lines capable of exhibiting PA are obtained. t-PA is preferably purified using any of the previously disclosed or previously known methods such as those described in the following references: Biochimica
et Biophysica Acta, 1979, 580 , 140-153
Page, J.Biol.Chem.1979, 254(6), 1998-2003
p. 1981, 256(13), pp. 7035-7041, Eur.J.
Biochem., 1983, 132, pp. 681-686, EP-A
-41766, EP-A-113319, or GB-A-
2122219. Oxypurinol, also known as alloxanthin, is the name for 4,6-dihydroxypyrazolo[3,4-d]pyrimidine and is the major metabolite of allopurinol, the subject of US Pat. No. 3,624,205. It can be prepared by any suitable synthetic method heretofore disclosed or known and may be prepared by Sigma Chemical Co., Ltd. (St. Louis).
(Missouri, USA). Examples of pharmaceutically acceptable salts of oxypurinol include alkali metal and alkaline earth metal salts. Particularly preferred are the sodium salts. When using t-PA and oxypurinol or a pharmaceutically acceptable salt thereof in the present invention, it is preferable to use these components in the form of a pharmaceutical composition. These active ingredients can be used in separate formulations or in the form of a single combination formulation; however, in the case of a single formulation, both active ingredients are Of course, they must be internally stable and compatible with each other. Therefore,
The present invention provides a pharmaceutical composition containing t-PA and oxypurinol or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. Generally, t-PA and oxypurinol or a pharmaceutically acceptable salt thereof are administered by the intravenous route, either by infusion or by injecting the entire amount at once. Therefore, parenteral compositions are needed. It is preferred to provide the physician or veterinarian as a lyophilized composition because of the significant transportation and storage benefits. A physician or veterinarian can reconstitute this lyophilized composition in the appropriate amount of solvent, if necessary. Parenteral lyophilized pharmaceutical compositions containing t-PA are known in the art. Examples of such prior art include EP-A-41766, EP-A-
93619, EP-A-112122, EP-A-113319, EP
-A-123304, EP-A-143081, EP-A-
156169, WO86/01104, Japanese Special Public Service 1982-
No. 120523 (Patent Application 1982-6936) and Japanese Special Publication
No. 58-65218 (patent application No. 56-163145) is included.
Additional examples include GB-A-2176702 and GB-A-
Contains 2176703. Parenteral pharmaceutical compositions containing oxypurinol include aqueous and non-aqueous solutions and suspensions, which also contain antioxidants, buffers, bacteriostatic agents,
It can contain isotonic agents or other ingredients that enhance bioavailability. A lyophilized pharmaceutical composition of oxypurinol can be prepared from a sterile solution by lyophilization in a conventional manner. Parenteral lyophilized compositions containing t-PA and oxypurinol together in a single combination formulation are prepared in a manner analogous to the preparation of compositions suitable for t-PA or oxypurinol per se. can. However, as mentioned above, it is necessary to adapt the compositions, taking into account the stability and mutual compatibility of the active ingredients in a particular composition. Intravenous infusions are normally carried out using parenteral solutions contained in an infusion bag or bottle, or in an electrically operated infusion device. The solution is delivered by gravity feeding or using an infusion pump.
It can be given to the patient from an infusion bag or vial. The use of gravity feed infusion systems does not provide sufficient control over the rate of administration of parenteral solutions. The use of infusion pumps is therefore preferred, especially in the case of solutions containing relatively high concentrations of active ingredient. However, it is particularly preferred to use an electrically operated injector, which allows further overall control of the rate of administration. The present invention relates to t-PA and oxypurinol or their pharmaceutical formulations for use in human and veterinary medicine, particularly for use in preventing damage to tissues at risk during reperfusion periods in mammals. Provide combinations with acceptable salts. The present invention is particularly advantageous in preventing damage to tissue at risk resulting from the development of blood clots, and as noted above, both clot elimination and protection of tissue at risk can be achieved. When using t-PA and oxypurinol or a pharmaceutically acceptable salt thereof in the method of the invention, these active ingredients may be present in separate compositions or in a single combined composition, simultaneously or Can be administered sequentially. When administered sequentially, it is preferred to administer oxypurinol first, eg, by intravenous injection, and then administer t-PA by continuous intravenous infusion. In any event, the administration of t-PA should not be delayed as this would negate the benefit of the potentiating effect of the combination of these two components in vivo in preventing tissue damage. Effective amounts of t-PA and oxypurinol to prevent damage to tissues at risk during the reperfusion period will depend, for example, on the age and weight of the mammal, the exact condition requiring treatment and its This will, of course, vary depending on many factors, including severity and route of administration, and is ultimately at the discretion of the attending physician or veterinarian. However, the effective dose for t-PA is
A range of 150,000 to 1,000,000 IU/Kg of patient's body weight/day, preferably 300,000 to 500,000 IU/Kg of patient's body weight/day is suitable, and in the case of oxypurinol, 0.5 to 10 mg/Kg of patient's body weight/day, preferably teeth
A range of 2.5 to 5 mg/Kg/day is suitable. The following examples are illustrative of the invention and should not be considered as limiting it in any way. Example 1 Production of oxypurinol Pyrazole-3,4-dicarboxylic acid (7.5g)
(this compound is described in Chem. Ber., 1899, 32 , 2292 et seq.), thionyl chloride (150 ml)
Add. The mixture is refluxed for 10 hours. Thionyl chloride was removed under reduced pressure and the resulting powdery residue was
Add portionwise over 1 hour to a cold, stirred solution of t-butanol previously saturated with ammonia at 0°C. Let the mixture stand for 5 hours. The precipitate is separated and boiled for 1 hour with concentrated ammonium hydroxide solution (100 ml). The solution is evaporated to dryness on a steam bath and the residue is crystallized from boiling water. The compound thus produced (pyrazole-3,4
-dicarboxamide) is a colorless branched product,
Melts at 327℃ without decomposition. To a cold solution of 0.4M sodium hypochlorite solution (16.6ml) is added pyrazole-3,4-dicarboxamide (500mg). The reaction mixture turns pink and then pale yellow. After standing for 1 hour at 0°C, the mixture is acidified to PH3 with 2N hydrochloric acid and the flocculent precipitate is separated. The product is recrystallized from boiling water to give rose-like colorless needles of oxypurinol. Example 2 Preparation of injection formulation of oxypurinol Ingredients mg/vial Oxypurinol 150.0 Sodium hydroxide, IN appropriate amount to bring the pH to 12.5 Water for injections Adjust amount to bring the total volume to 15 ml Oxypurinol in sodium hydroxide solution in 10 ml of water Add. Adjust the pH of the solution to 12.5 using sodium hydroxide (IN). After adding additional water, the solution is filtered, sterilized, and then dispensed into sterile vials. The solution is lyophilized to produce a sterile anhydrous cake and the vial is sealed under sterile conditions. The solution is reconstituted with water immediately before injection. Example 3 Preparation of injection preparation of t-PA Freeze-dried injection preparation of t-PA was prepared from GB-A-
2176702. Example 4 t- in the protection of organizations at risk
Demonstration of the synergistic effect of PA and oxypurinol (a) Method Male beagle dogs (10-12 Kg) are anesthetized with sodium pentobarbital, intubated and allowed to breathe on room air via a Haruard respirator. Infusion and arterial blood pressure measurement catheters are implanted in the left jugular vein and left carotid artery. A thoracotomy is performed at the 4th intercostal space, the heart is suspended in a pericardial sheath, and the left descending artery (LAD) is inserted into the first major diagonal branch.
Isolate at a position just below. Attach an electromagnetic flow probe to the LAD. 1/0 from flow probe to fast position
A silk suture loop is applied to create a 90 minute occlusion of the LAD. The treatment is 15 minutes before the blockage is released due to this locking.
Begin by intravenous administration at 1 minute and continue for 45 minutes after release. Close the thoracotomy and allow the animal to recover from the surgical situation. Animals are anesthetized again 24 hours after tethering and LAD
Re-evaluate blood flow in The heart is then isolated for post-mortem quantification of infarct size. Five groups of dogs will be evaluated. Group 1 will serve as a saline control. The second group will receive 1.5 mg/Kg of t-PA. Group 3 will receive 3 mg/Kg of t-PA. Fourth
Group received oxypurinol sodium salt 10 mg/Kg
administer. Group 5 receives both 1.5 mg/Kg of t-PA and 10 mg/Kg of the sodium salt of oxypurinol. t-PA is administered as a parenteral formulation obtained by reconstitution with water from the lyophilized formulation of Example 3. Similarly, oxypurinol is reconstituted with water from the lyophilized formulation of Example 2 and brought back to a pH of 9.3 by trituration with hydrochloric acid to administer the resulting parenteral formulation. t-PA had a specific activity of 440000 IU/mg. The size of myocardial infarction is quantified by ex vivo double reperfusion technique. The catheter is inserted into the LAD and into the aorta above the coronary ostium, just away from the locking site. The LAD coronary bed is perfused with 1.5% triphenyltetrazolium hydrochloride (TTC) in 0.02M potassium phosphate buffer (PH7.4). The aorta is perfused with each of these stains for 5 minutes at a constant pressure of 100 mm mercury. The heart is cut into 8 mm thick sections perpendicular to its apical-basal axis. blood flow
Left ventricular regions at risk for infarction due to anatomical dependence on the LAD are identified by the absence of Evans blue in this region. Areas of infarcted myocardium within the risk zone are evidenced by lack of tissue staining when perfused with TTC due to loss of dehydrogenase enzymes. The ventricular cross section is carefully examined through a clear acrylic cover plate to make a permanent record of the infarct morphology and the infarct size is confirmed planometry. The right ventricular muscle, valves and adipose tissue in the ventricular plane are then excised.
All areas of risk and infarction of the left ventricle are carefully excised and separated and weighed. Infarct size is expressed as a percentage of the anatomical area at risk. Statistical comparison of the drug-treated group to the control group was performed using Bonferroni's method of multiple comparisons (Circulation Research, 1980, 47 , pp. 1-9).
One way analysis of variance method using
analysis of variance) (ANOVA). A p-value smaller than 0.05 is considered a criterion for significance. (b) Results

[Table] * This data is expressed as mean value ± standard error.
The proportion of the left ventricle rendered ischemic by mechanical latching of the LAD was between the treatment group and the control group.
There were no significant differences by ANOVA. (c) Conclusion The combination of t-PA and oxypurinol achieved a synergistic effect in preventing damage in tissues at risk during the period of blood flow reperfusion.

[Brief explanation of the drawing]

Figure 1 shows the amino acid sequence of t-PA, and Figure 2 shows the structural features of the two-chain form t-PA.
shows the cleavage site of single-stranded t-PA that gives rise to
The A chain has two circular regions and the B chain has a serine protease region.

Claims (1)

[Claims] 1. Preventing damage to ischemic heart tissue during blood reperfusion in mammals including humans, containing t-PA and oxypurinol or a pharmaceutically acceptable salt thereof as active ingredients. Pharmaceutical composition for. 2. A pharmaceutical composition according to claim 1, wherein the t-PA is in single chain form. 3. A pharmaceutical composition according to claim 1, wherein the t-PA is in dichain form. 4 t-PA has the amino acid sequence described below, or has the amino acid sequence described below except that the amino acid present at the 245th position from the serine N-terminus is valine instead of methionine,
Both sequences may optionally be free of the first three amino acids, or Gly-Ala
Claims 1 to 3 may have an additional polypeptide N-terminal subsequence of -Arg.
The pharmaceutical composition according to any one of paragraphs. [Table] [Table] (5) The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutically acceptable salt of oxypurinol is a sodium salt.