NL8701113A - NEW PHARMACEUTICAL APPLICATION. - Google Patents

Abstract

Tissue plasminogen activator (t-PA), optionally together with superoxide dismutase (SOD), is used to manufacture a medicament for use in the inhibition of damage to jeopardised tissue during blood reperfusion. The medicament is for preventing damage to tissue following ischaemic attack, e.g. in myocardial tissue, when blood supply is restored.

Description

f '- VO 9155

Title: New pharmaceutical application

The present invention relates to tissue plasminogen activator, to its combination with superoxide dismutase, to pharmaceutical formulations thereof, and to their use in human and animal medicine.

5 There is a dynamic balance between the enzyme system capable of forming blood clots, the coagulation system and the enzyme system capable of dissolving blood clots, the fibrinolytic system, which maintains an intact, latent blood vessel bed. In order to prevent blood loss from injuries, blood clots are formed in the damaged vessel. After natural healing of the wound, the superfluous blood clots are dissolved by the action of the fibrinolytic system. Occasionally, blood clots form without traumatic injury and can become lodged in important blood vessels, resulting in partial or even complete obstruction of blood flow. If this occurs in the heart, lung, or brain, the result may be a myocardial infarction, a pulmonary embolism, or a stroke.

Together, these conditions are the leading cause of illness and death in industrialized countries.

Blood clots consist of a fibrous network that can be dissolved by the proteolytic enzyme piasmine. The enzyme is derived from the inactive proenzyme plasminogen, a component of blood plasma, through the action of a plasminogen activator. There are two immunologically different plasminogen activators for mammals. Intrinsically plasminogen activator, also known as urokinase, is an enzyme produced by the kidney that can be isolated from urine. It can also be prepared from a number of tissue culture sources. Extrinsically plasminogen activator, also known as vascular plasminogen activator and tissue plasminogen activator (t-PA), can be isolated from a large number of tissue homogenates, more particularly the human uterus, the cell wall of blood vessels and from certain cell cultures. * In addition to these two types of plasminogen activator, there is also a bacterial product, 35 8701113 4 Λ * -2-streptokinase, prepared from beta-haemolytic streptococci. A major drawback of both urokinase and streptokinase is that they are active throughout the circulation, not just at the site of a blood clot. For example, these can destroy other blood proteins, such as fibrinogen, prothrombin, factor V and factor VIII, which reduces blood clotting capacity and increases the risk of bleeding. In contrast, the biological activity of t-PA depends on the presence of fibrin to which it binds and where it is activated. Maximum activity is therefore only developed at the site of a blood clot, i.e. in the presence of the fibrin network to be resolved, and this largely avoids the risk of bleeding.

The interruption of blood flow in a vessel generally results in an ischemic situation. In this state, the tissue becomes oxygen deficient and endangered, a condition in which the tissue is damaged but potentially still viable. However, if this condition persists for a period of 3 or more hours, the tissue becomes necrotic and, once in this condition, cannot recover. It is therefore important that reperfusion, that is, the restoration of blood flow. take place as soon as possible to save the tissue before permanent damage. Ironically, however, the reperfusion itself, even if performed before the tissue becomes necrotic, results in a complex group of phenomena, including the putative formation of the superoxide radical, which is one. adversely affects oxygen deficient tissue. For that reason, reperfusion can only lead to the partial repair of compromised tissue, while the remainder is permanently damaged by the occurrence of one or more of the aforementioned phenomena.

It has now been found that t-PA prevents damage to compromised tissue during reperfusion by protecting it from one or more of the aforementioned phenomena. The mechanism of action of t-PA in providing such protection is not clear, but it is independent of thrombolytic activity. This newly discovered property 870 1 1 1 3.

Therefore, it makes it possible to use t-PA or a pharmaceutical formulation thereof as an inhibitor of damage to compromised tissue under the conditions described herein. Accordingly, the present invention provides: (a) a method of preventing damage from danger

introduced tissue during reperfusion in a mammal, comprising administering to the mammal an effective amount of t-PA

(B) use of t-PA in preventing damage to compromised tissue during reperfusion in a mammal (c) use of t-PA for the manufacture of a medicament for preventing damage to compromised tissue during reperfusion in a mammal or (d) a pharmaceutical formulation for use in preventing damage to compromised tissue during reperfusion in a mammal comprising t-PA and a pharmaceutically acceptable carrier.

While the present invention can be used to protect any compromised tissue, it is more particularly useful for preventing damage to compromised myocardial tissue.

The t-PA for use in the present invention can be any bioactive protein that substantially corresponds to mammal, and especially human, t-PA and includes forms with and without glucosylation. It can be single or double chain t-PA or a mixture thereof, as described in EP-A-112 122 and, in the case of fully glucosylated human t-PA, has an apparent molecular weight on polyacrylamide gel of about 70,000 and an isoelectric point between 7.5 and 8.0. Preferably, the 35 t-PA has a specific activity of about 500,000 Xü / mg (International units / mg, the International unit being a unit of activity as determined by WHO, National Institute for Biological 870 1 Hl

* V

-4-

Standards and Control, Holly Hill, Hampstead, London, NW3 6RB, UK).

Preferably, the amino acid sequence of t-PA substantially corresponds to that shown in FIG. 1. The sequence is therefore identical to that in Fig. 1 or contains one or more amino acid deletions, substitutions, insertions, inversions or additions of allelic origin or otherwise, wherein the resulting sequence has at least 80 and preferably 90% homology to the sequence in FIG. 1 and 10 retain substantially the same biological and immunological properties of the protein. In particular, the sequence is identical to that in Fig. 1, or has the same sequence except that the amino acid is in the 245th position of the serine N-terminus valine, rather than methionine, in both cases the sequence is equally 15 has an additional polypeptide N-terminal presequence from Gly-Ala-Arg.

The amino acid sequence of Fig. 1 has 35 cysteine residues and therefore the ability to form 17 disulfide bridges.

Based on analogy with other proteins, the structure of which has been determined in more detail, the postulated structure for the sequence (based on disulfide bonding) between the amino acid in the 90th position and the proline C-terminus is shown in FIG. 2.

The structure of the N-terminal region is less certain, although a number of proposals have been published (Progress in Fibrynolysis, 1983, 6, 269-273; and Proc.Natl.Acad.Sci., 1984, 81, 5355-5359 ).

The most important properties of the structure of t-PA are the two 'kringle' regions (between the 92nd and 173rd amino acids and between the 180th and 261st amino acids) which are responsible for the binding of the protein to fibrin and the serine protease region which contains the major part of the B chain, and which is responsible for the activation of plasminogen. The amino acids of particular interest in serine proteases are the catalyst triads, His / Asp / Ser. In t-PA these are present at the 322nd, 371st and 35th 463rd positions. The disulfide bridge between the 264th and 395set cysteine amino acid residues is also important, as it holds the A and B chains together in the double chain form of t-PA.

8701113 —5— -

The Figs. 1 and 2 are used for the amino acid residues in the following one and three letter codes:

Asp D Asparagic Acid Cys C Cysteine Arg R Arginine 5 Thr T Threonine Val V Valine Lys K Lysine

Ser S Serine lie I Isoleucine Trp. W Tryptophan

Glu E Glutamic acid Leu L Leucine Gin Q Glutamine

Pro P Proline Tyr Y Tyrosine With M Methionine

Gly G Glycine Phe F Phenylalanine Asn N Asparagine 10 Ala A Alanine His H Histidine

The t-PA can be obtained using any of the methods described or known in the art. For example, it can be obtained from a normal or neoplastic cell line of the type described in Biochimica et Biophysica Acta, 1979, 580, 140-153? EP-A-41 766 or EP-A-113 319. However, it is preferred that t-PA is obtained from a cultured, transformed or transfected cell line obtained by recombinant DNA technique as described in eg EP-A-93 619; EP-A-117 059? EP-A-117 060? EP-A-173 552; EP-A-174 835; 20 EP-A-178 105? WO 86/01538? WO 86/05514 or WO 86/05807. More particularly, it is preferred that Chinese hamster ovari (CHO) cells be used for the production of t-PA and obtained in the manner described in Molecular and Cellular Biology, 1985, 5 (7), 1750-1759.

In this way, the cloned gene is co-transfected with the gene encoding dihydrofolate reductase (dhfr) in dhfr "CHQ cells. Transformers expressing dhfr are chosen on media that do not contain nucleosides and are exposed to increasing concentrations of methotrexate. Thus, the dhfr and t-PA genes are co-amplified, leading to a stable cell line capable of delivering high levels of t-PA.

30

The t-PA is preferably purified using any of the procedures described or known in the art, such as the procedures described in Biochimica et Biophysica Acta, 1979m 580, 140-153; J. Biol. Chem., 1979, 254 (6), 1998-2003; ibid, 1981, 35 256 (13), 7035-7041? Bur. J. Biochem., 1983, 132, 681-686? EP-A-41 766; EP-A-113 319; or GB-A-2 122 219.

t-PA can be used in the manner of the present invention either 8701113 v-6- alone or in combination with another therapeutic agent that also prevents damage to compromised tissue during reperfusion. A particularly useful example of such a combination is with superoxide dismutase (SOD), an enzyme known to capture and destroy superoxide radicals. Superoxide radicals are one of the phenomena that are capable of causing tissue damage. Indeed, it has also been found that the combination of t-PA and SOD provides a significantly potentiated level of inhibition, compared to that provided by t-PA or SOD as such. Consequently, the present invention also provides a combination of t-PA and SOD.

The combination of t-PA and SOD provides a particularly suitable means both for the removal of blood clots and for the prevention of damage to compromised tissue during reperfusion. Therefore, the administration of t-PA and SOD will first result in the removal of the blood clot by the known thrombolytic activity of t-PA and then in the inhibition of tissue damage by the combined action of t-PA and SOD.

20

The SOD for use in combination with t-PA can be any bioactive protein that substantially corresponds to one or more of a group of enzymes generally known by this name. It is preferably derived from mammals and more particularly from bovine or human origin, and is generally associated with a metal cation with which it is generally classified. Exemplary metal cations include iron, manga, copper, and preferably combinations of copper and other metals, such as zinc, cadmium, cobalt, or mercury, of which a copper / zinc combination is preferred.

30 Both the manganese and the copper / zinc forms of SOD occur naturally in humans. The iron and manganese forms of SOD of bacterial origin both have a molecular weight of about 40,000 and are dimers.

The manganese form of SOD of eukaryotic origin, on the other hand, has a molecular weight of about 80,000 and is a tetramer. The copper / zinc form of SOD of eukaryotic origin has a molecular weight of about 32,000 and is a dimer with one copper cation and one zinc cation per subunit. The copper cation is ligated to four histidine residues per subunit and the zinc cation is ligated between histidine and aspartic acid. There is also a copper / zinc form of SOD

8701113-7 of eukaryotic origin which has a molecular weight of about 130,000 and which consists of four subunits. The molecular weights of the various forms of SOD were determined using sedimentation equilibrium, molecular sieving or using polyacrylamide gels. The isoelectric points of the various forms of SOD range from 4-6.5 depending on the degree of sulfation and / or deamidation. Preferably, the specific activity of the copper / zinc form of SOD of bovine or human origin is at least 3000 µ / mg (the unit of activity being defined in 10 J. Biol. Chem., 1868, 244, 6049-6055).

Preferably, the amino acid sequence of the copper / zinc form of bovine or human origin SOD essentially corresponds to that described in J. Biol. Chem., 1974, 249 (22), 7326-7338, in the case of bovine origin and Biochemistry, 1980, 19, 2310-2316 and FEBS Letters, 1980, 120, 53-55, in the case of those of human origin. The sequence is therefore identical to that described in these articles and contains one or more amino acid deletions, substitutions, insertions, inversions or additions of allelic ofigine or otherwise, the resulting sequence having sufficient homology to the published sequence so that it is essentially the same biological and immunological properties.

The amino acid sequence of the copper / zinc form of SOD of bovine origin 25 contains three cysteine residues per subunit (J.Biol.Chem., 1974, 249 (22), 7326-7338). The chain disulfide bridge is present between the Cys 44 and Cys 144 residues, while the interchain disulfide bridge is present between the Cys 6 residues. The amino acid sequence of the copper / zinc form of SOD of human origin contains four cysteine residues per 30 subunit (Biochemistry, 1980, 19, 2310-2316 and FEBS Letters, 1980, 120, 53-55). The disulfide bridge in the chain is present between the Cys 57 and the Cys 146 residues, while the disulfide bridge between the chains is also present between the Cys 6 residues. The Cys 111 residue remains free.

The SOD can be obtained by any suitable procedure described or known in the art. For example, it can be obtained from erythrocytes or from liver by an extraction procedure of the type described in 8701113-8-GB-Al 407 807 and GB-A-1 529 890. SOD can also be obtained from a cultured transformed or transfected cell line, obtained using recombinant DNA technology as described in e.g. Australian patent application 27461/84 WO 85/01503, EP-A-138 111, EP-A-164 566, EP-A-173 280 and EP-A-180 964.

The SOD is preferably purified using an appropriate procedure described or known in the art, such as the procedure described in EP-A-112 299.

10

When using t-PA, or a combination of t-PA and SOD, in the manner of the present invention, it is preferable to use the active ingredient or ingredients in the form of a pharmaceutical formulation. In the. In the case of the combination, the active ingredients can be used in separate formulations which can be administered simultaneously or sequentially. When administered consecutively, it is preferable to administer the t-PA formulation first and then the SOD formulation. In any case, the delay in administering the second of the two formulations should not be such as to lose the benefit of a potentiated effect of the combination of the active ingredients in vivo in preventing tissue damage. However, rather than the use of separate formulations, it is. much more convenient to present the active ingredients together in a single combined formulation. Accordingly, the present invention also provides a pharmaceutical formulation comprising t-PA, SOD and a pharmaceutically acceptable carrier.

Generally, t-PA or the combination of t-PA and SOD will be administered by the intravascular route, either by infusion or by bolus injection and therefore a parenteral formulation is required. It is preferable to provide a lyophilized to the physician or veterinarian, because of the significant transport and storage benefits it provides. The physician or veterinarian can then reconstitute the lyophilized formulation in an appropriate amount of solvent as and when required.

Parenteral and lyophilized pharmaceutic formulations with t-PA

8701113 t -9- are known in the art. Examples thereof include EP-A-41 766, ΞΡ-Α-93 619? EP-A-112 122; EP-A-113-319? EP-A-123 304; 143-Α-143 081; EP-A-156 169; WO 86/01104? Japanese Patent Publication 57-120523 (Application 56-6936) and Japanese Patent Publication 58265218 (Application 57-5 163145). Additional preferred examples include GB-A-2 176 702 and GB-A-2 176 703. All of these formulations are also suitable for SOD and for the combination of t-PA and SOD.

Intravascular infusions have generally been performed with the parenteral solution contained in an infusion bag or bottle, or in an electrically operated infusion needle. The solution can be administered to the patient from the infusion bag or bottle by gravity feeding or by using an infusion pump. The use of gravity feed infusion systems does not provide sufficient control over the delivery rate of the parenteral solution and therefore the use of an infusion pump is preferred, especially with solutions containing relatively high concentrations of active ingredients. More preferred, however, is the use of an electrically operated infusion needle that provides even greater control over the delivery rate.

20

An effective amount of t-PA, and a combination of t-PA and SOD, for preventing damage to compromised tissue during reperfusion will, of course, depend on a number of factors including, e.g., the age and weight of the mammal , the exact condition requiring treatment and the severity, route of administration, and will ultimately be determined by the treating physician or veterinarian. However, an effective dose in the case of t-PA will generally be from 0.2-4 mg / kg (i.e., 100,000-2,000,000 IU / kg, assuming a specific activity for 30 t-PA of 500,000 IU / mg), preferably from 0.3-2 mg / kg (ie

150,000 to 1,000,000 IU / kg, patient's body weight per hour.

For an adult human weighing 70 kg, an effective amount per hour will generally be from 20-140 mg (i.e. 10,000,000 to 70,000,000 Iu, especially about 70 mg (i.e. 35,000,000 ID When SOD is used in combination with t-PA, an effective dose of SOD is generally from 1000 to 50,000 U / kg, preferably from 720,000 U / kg of patient body weight per hour. 70 kg an effective amount of SOD per hour is preferably 500,000 to 1,5000,000.

8701113 -10- S- *

The following examples are provided to illustrate the present invention and are not to be construed as a limitation thereof.

Example I: preparation of parenteral formulation of t-PA

A parenteral formulation of t-PA was achieved essentially as described in GB-A-2 176 703. The t-PA had a specific activity of about 300,000 IU / mg.

10

Example II: preparation of parenteral formulation of SOP

Bovine SOD in the copper / zinc form was obtained from Sigma Chemical Co.,

St. Louis, Missouri, USA, 63178, as a powder and was dissolved in a substantially neutral physiological saline.

Example III: preparation of parenteral formulation of t-PA and SOD

The formulations of Example I and Example II were mixed and further diluted in physiological saline to achieve the required dosage.

Example IV: Protection of compromised tissue by t-PA and SOD (a) Methodology

Male beagle dogs (10-12 kg) were anesthetized with sodium pentobarbital, incubated and ventilated with room air using a Harvard respirator. Catheters for infusion and arterial blood pressure measurements were implanted in the left jugular vein and left carotid artery. A thoracotomy was performed at the fourth intermediate rib space, the heart was suspended in a pericardial cradle and the left descending anterior artery (LAD) was isolated just below the first major diagonal branch. An electromagnetic flow probe was placed on the LAD. A 90 minute occlusion of the LAD was produced by applying a snare of a 1 / o silk suture distal to the flow probe. Treatment was initiated intra-venously 15 minutes prior to release of this snare occlusion and continued 45 minutes after release. The thoracotomy was closed and animals were allowed to recover from the surgical procedure. 24 hours after the occlusion, the animals were re-anesthetized and the flow in the LAD was reassessed, then the heart was removed for post mortem quantification of the infarct size.

Four groups of dogs were evaluated. Group 1 consisted of saline control. Group II was administered 2.5 mg / kg (750,000 Iü / kg t-PA, group III was administered 16,500 ü / kg bovine SOD, and group IV was administered both 2.5 mg / kg (750,000 IU / kg ( t-PA and 16,500 µ / kg bovine SOD administered The formulations used are as described in Examples I to III.

Myocardial infarct size was quantified by an ex vivo dual reperfusx technique. Cannulas were introduced into the LAD

distai immediately with respect to the site of occlusion and in the • aorta above the heart ostia. The LAD heart bed was perfused with 1.5% triphenyl tetrazolium hydrochloride (TTC) in 0.02M potassium phosphate buffer, pH 7.4. The aorta was perfused in a reflux mode with 0.5% Evans blue dye. Both areas were perfused with their resp. stain at a constant pressure of 100 mm of mercury for 5 minutes. The heart was cut into 8 mm thick slices perpendicular to the apex base axis. The left ventricular region exposed to infarction due to the anatomical dependence of the LAD on blood flow is identified by the lack of Evans blue in this area.

The area of the infarcted myocardium in the area exposed was marked by the lack of tissue staining after perfusion with TTC due to a loss of dehydrogenase enzymes.

30

The transverse ventricular sections were accurately traced onto clear acrylic coatings to provide a permanent record of infart morphology and to allow planimetric confirmation of infarct sizes. Ventricular sections were then depleted of right ventricular muscle, valvular and adipose tissue. The total left ventricular area exposed and the infarct were separated by careful cutting and weighed.

8701113

Claims (17)

Use of t-PA for the manufacture of a medicament for preventing damage to compromised tissue during reperfusion in a mammal. 2. Use of t-PA and SOD to manufacture a medicament for preventing damage to compromised tissue during reperfusion in a mammal. 3. Use of t-PA and SOD for the manufacture of a medicament for removing a blood clot and for preventing damage to compromised tissue during reperfusion in a mammal. Use according to any preceding claim, wherein the tissue is myocardial tissue. Use according to any of the preceding claims, wherein the t-PA is in the single chain form. The use according to any one of claims 1 to 4, wherein the t-PA is in the double chain form. The use of claim 5 or claim 6, wherein the t-PA has the amino acid sequence of FIG. Has 1 or the same amino acid sequence with the amino acid in the 245th place of the serine N-terminus is valine instead of methionine, while the sequences optionally have an additional polypeptide N-terminal presequence of Gly-Ala-Arg. Use according to claim 2 or 3, wherein the SOD is the copper / zinc form of bovine or human origin. 9. Combination of t-PA and SOD. 10. Combination of t-PA and SOD for application in human and veterinary medicine. 11. Combination of t-PA and SOD for use in preventing damage to compromised tissue during reperfusion in a mammal. 12. Combination of t-PA and SOD for use in the removal of a blood clot and for preventing damage to compromised tissue during reperfusion in a mammal. The combination of any one of claims 9 to 12 wherein the t-PA 35 is in the single chain form. 8701113 't' * “• N" '* * -14- The combination of any one of claims 9-12, wherein the t-PA is in the double chain form. The combination lens of claim 13 or 14, wherein the t-PA has the amino acid sequence of Figure 1, or has the same amino acid sequence, wherein the amino acid is in the 245th position of the serine N-terminus valine instead of methionine, while the sequences optionally have an additional polypeptide N-terminal presequence of Gly-Ala-Arg. 15. Salt 5 36.0 ± 8.9 37.3 ± 7.6 II t-PA 6 14.3 ± 11.7 35.7 ± 5.4 III SOD 4 13.0 ± 4.6 30.6 ± 2.6 IV t-PA + 3 2.3 ± 1.3 37.3 ± 9.1 SOD 20 ------- -------------------------------------------------- -------------- * Data are expressed as ± standard error. According to ANOVA, the amount of the left ventricle that is made ischemic. due to mechanical occlusion of the LAD not significantly different between the treatment groups and the control group. s (c) Conclusions The use of t-PA significantly prevented myocardial infarction-30 size, clearly demonstrating its effectiveness in protecting compromised tissue during reperfusion. In addition, the combined use of t-PA and SOD provided a synergistic inhibitory effect in this region, the combination providing a degree of inhibition greater than that provided by either t-PA or SOD alone. 8701113 * * -'v -13- CONCLUSIONS 16. Combination according to any one of claims 9 to 12, wherein the SOD is the copper / zinc form of bovine or human origin. Pharmaceutical formulation, comprising a combination according to any one of claims 9 to 16 and a pharmaceutically acceptable carrier. 15 870 1 1 1 3