JP2004534842A - Regulatory peptide of human tissue plasminogen activator - Google Patents

Abstract

The present invention relates to polypeptides and their use in the prevention and / or treatment of side effects induced by plasminogen activators, in particular tPA or uPA. The present invention further relates to compositions and / or therapies in which the polypeptides of the invention are combined with one or more currently used plasminogen activators, and furthermore, fibrin with reduced side effects. It relates to a method of achieving an improved dissolution effect.

Description

【Technical field】
[0001]
The present invention discloses a peptide comprising six amino acids (EEIIMD) that have the property of binding to a "docking" site outside the active site of urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA). . The present invention also relates to the control of tPA and uPA activity, particularly when tPA or uPA is administered in the treatment of ischemic stroke, particularly the ability of tPA to induce hemorrhage (ICH) in the cerebrum.
[Background Art]
[0002]
Tissue-type plasminogen activator is the only treatment for acute thromboembolic attacks and has been approved by the Food and Drug Administration (FDA). However, there are reasons to be concerned that the use of tPA for the treatment of ischemic stroke may cause secondary intracerebral hemorrhage in patients. See Wardlaw JC et al, Lancet 1997, 350: 607-614. Subsequent predictive intracerebral hemorrhage occurs at an incidence of about 6% and about 50% of patients die. The occurrence of cerebral hemorrhage after treatment with TPA is due to its ability to disrupt normal vasoactivity of cerebral blood vessels. tPA has been shown to have a dose-dependent vasoactive or vasodilator effect in addition to promoting plasminogen activity.
Tissue-type plasminogen activator is a natural molecule released from vascular endothelial cells, and elimination or clearance in the liver rapidly removes t-PA from blood. Hepatocytes express low-density lipoprotein receptor-related proteins or d2-macroglobulin receptors that bind to tPA or a complex of tPA and tcuPA with a plasminogen activator inhibitor (PAI-1). Separately, endothelial cells, which are also involved in the rapid clearance of tPA, alternately express the 170Kda mannose-dependent receptor.
[0003]
Plasminogen activator inhibitor type 1 interacts with both tPA and uPA and inhibits the catalytic activity of both proteins. PAI-1, which binds with high affinity to tPA and uPA, is present in high concentrations in the circulation of hypertensive patients. Also, lowering blood pressure with medical treatment reduces PAI-1 levels. The underlying mechanism for the effect of increasing PAI-1 under certain physiological conditions is not well understood. However, the inverse relationship with tPA and / or uPA suggests that PAI-1 neutralizes the vasoactive effects of tPA and / or uPA in some way. Simmons M, Cardiol. Clin 1995, 13: 339-345; Cipolla M et al., Stroke, 2000, 31: 940-945; of PAI-1; and Higazi, AA-R et al., J. Biol. Chem. ., 1997, 272: 27053-27057.
Whether there is a relationship between increased levels of PAI-1 and naturally produced tPA under certain physiological conditions, or due to the use of PAI-1 and commercially produced tPA Whether it has a relationship with intracerebral hemorrhage has not been previously assessed. The present invention provides a better understanding of the regulation of PAI-1, or tPA, or uPA, if any, and also provides compositions or products that are preferably effective in controlling the activity of tPA or uPA, Thereby reducing the risk of intracerebral hemorrhage in patients receiving thrombolytic treatments such as tPA and / or uPA.
DISCLOSURE OF THE INVENTION
[Problems to be solved by the invention]
[0004]
(Summary of the Invention)
The present invention relates to a composition of a polypeptide composed of six amino acids having an inhibitory activity on vasoactivity of tPA and uPA, and use thereof.
More specifically, the polypeptides are useful in preventing and / or treating bleeding disorders associated with tPA treatment administered for treating thromboembolic disorders.
The present invention also relates to a method of reducing the occurrence of intracerebral hemorrhage in patients receiving tPA or uPA as a fibrinolytic treatment, as an additional treatment.
In yet another aspect, the invention relates to a pharmaceutical kit for the treatment of thromboembolism in a mammal, said kit comprising a sterile container of tPA in commercially available form and a sterile container for each of the agents or a mixture thereof. And a container, both of which are effective in treating thromboembolism, while preventing the occurrence of the side effects of tPA in the same therapy.
[0005]
Such kits may, if desired, include a therapeutically effective amount of uPA or tPA to treat thromboembolism while preventing side effects.
It is also within the scope of the present invention to provide tPA or uPA kits in combination therapy with other fibrinolytic agents. Conjunctive treatments include tPA, uPA, tcuPA, streptokinase, rt-PA, or alteplase, rt-PA derivatives (eg, reteplase, lanoteplase, and TNK-rt-PA), anisoylated plasminogen streptokinase complex (APSC It is a further object of the present invention to provide a method of treating thromboembolic disease using a combination with one or more fibrinolytic agents, including anistreptase or a streptokinase derivative.
[Means for Solving the Problems]
[0006]
(Detailed description of the invention)
Advantages and features of the present invention will become immediately apparent with reference to the following detailed description and drawings.
According to the present invention, there is provided a pharmaceutical composition of a peptide, which composition has an inhibitory effect on bleeding diseases associated with tPA and / or uPA, which occur as a serious side effect of fibrinolytic agents. Also, a method of reducing the occurrence of intracerebral hemorrhage in a patient receiving tPA or uPA in the treatment of a thromboembolic disease is contemplated by the present invention.
[0007]
The invention also includes pharmaceutical compositions and kits comprising the polypeptides in combination with one or more fibrinolytic agents. The fibrinolytic agent may be tPA, uPA, scuPA, tcuPA, streptokinase, rt-PA, or alteplase, rt-PA derivatives (eg, reteplase, lanoteplase, and TNK-rt-PA), anisoylated plasminogen streptokinase complex (APSC), or anistreplase, or a streptokinase derivative.
The present invention further provides cerebral hemorrhage and vascular abnormalities by providing a combination alone or in combination in an effective amount to prevent and / or inhibit the side effects associated with fibrinolytic agents such as tPA and uPA. And other methods for preventing and / or treating side effects.
tPA is a single-chain serine protease composed of 530 amino acids, but 527 were originally identified. The t-PA enzyme is composed of several domains with homology to other proteins.
A finger domain composed of 4 to 50 residues, a growth factor domain composed of 50 to 87 residues, two Krungl structures composed of residues 87-176 and 176-262, and a catalytic triad. Protease domain composed of residues 276-527 including the structure (triad). Initial binding of t-PA to fibrin is determined by the finger domain and kringle 2, where t-PA binds to the exposed carboxyl terminus of lysine residues.
[0008]
tPA has a weak affinity for plasminogen in the absence of fibrin (Km = 76 uM), but has a higher affinity in the presence of fibrin (K between 0.5-1.5 OM). In this reaction, plasminogen mainly binds to fibrin via a specific structure called the "lysine binding site". Thus, one way to control fibrinolysis is at the level of plasminogen activation located on the fibrin surface.
Plasminogen activation inhibitors, particularly PAI-1 and PAI-2, physiologically inhibit plasminogen activators, for example PAI-1 is the major inhibitor of t-PA and u-PA in plasma. PAI-1, a serine protease inhibitor, is a single-chain glycoprotein from endothelial cells and other cell types. PAI-1 inhibits tPA by forming a complex between the active site of tPA and the "bait" residue of PAI-1 (Arg346-Met347).
[0009]
PAI-1 concentrations in plasma are increased in several diseases, including venous thromboembolism, obesity, sepsis, and coronary artery disease. High PAI-1 activity constitutes an independent risk factor for myocardial infarction in young patients within three years of the first attack. There is a clear correlation between the 24-hour cycle change in the onset time of myocardial infarction, the highest incidence at 8:00 am, and the highest 24-hour rhythm of plasma PAI-1 activity early in the morning.
Plasminogen activation inhibition type 1 interacts with both tPA and uPA and inhibits the catalytic activity of both proteins. PAI-1 binds with high affinity to tPA and uPA (see Heckman CM, Archires of Biochem Biophysics, 1988, 262: 199-210) and is present at high concentrations in the circulation of hypertensive patients. . As medical treatment lowers blood pressure, PAI-1 levels decrease. See Erden YC et al. AmJ Hypertens, 1999, 12: 1071-1076. Under certain physiological conditions, the action of the underlying mechanism that explains the increase in PAI-1 is not understood.
PAI-1 reacts with single-stranded tPA, double-stranded tPA and tcuPA. The second-order rate constant for inhibition of single-chain tPA by PAI-1 is about 10 ⁷ M- ¹ _s , while inhibition of double-chain tPA and tcuPA is somewhat faster. Positively charged regions in TPA (residues 296-304) and uPA (9 residues 179-184) are involved in this early reaction. PAI activity is cleared very quickly from the liver circulation. Except for platelets containing functional, inactive PAI-1, PAI-1 is not accumulated intracellularly, but is secreted rapidly and constitutively after synthesis.
[0010]
PAI-1 binds to tPA and uPA via two independent epitopes, one of which binds to the active site. The other epitope is composed of six amino acid residues EEIMD corresponding to 350 to 355 amino acid residues of PAI-1. This second PAI-1 epitope interacts with the "docking" binding site of uPA and tPA outside the active site. See Adams DS et al., J. Biol. Chem, 1999, 266: 8476-8482.
The present invention describes the effect of the peptide on the vasoactivity of tPA and uPA, and shows that this peptide counteracts the promoting effect of tPA on phenylephrine-induced vasoconstriction in aorta ring culture. Similarly, the peptides of the present invention eliminate the promoting effect of uPA on phenylfurin-induced vasoconstriction. These observations are described in detail in the Examples section.
The peptides of the present invention are useful for clot lysis during the treatment of thrombolysis in myocardial infarction, stroke, and related complications, while preventing and / or inhibiting the side effects of tPA or uPA on blood vessels. Has no effect on fibrinolytic activity.
Commercially available tPA is produced in two forms by recombinant DNA technology (recombinant t-PA, rt-PA, etc.). That is, a single-stranded preparation (alteplase) and a double-stranded preparation (deuteplase). Other tPA types include reteplase (r-PA0, mutant rt-PA, TNK-rt-PA).
[0011]
Fibrin-selective alteplase preferred dose therapy is weight-adjusted and front-loaded for 90 minutes of therapy (15 mg bolus, 0.75 mg / kg [not more than 50 mg] for 30 minutes and 60 minutes) 0.05 mg / kg [not to exceed 35 mg]).
The preferred dosage regimen of the peptide consists of an effective amount to prevent the adverse vasoactive effects of tPA on a case-by-case basis. The peptide may constitute a sequence element of varying numbers of amino acids, or the peptide may have one or more amino acid modifications in its sequence.
The peptides of the present invention are useful in treating sepsis when administered in effective doses alone or in combination with traditional anticoagulant treatments. Under physiological conditions, several antithrombotic mechanisms work in concert with preventing clotting and protecting blood fluidity. Thrombin, which escapes the monitoring of this physiological antithrombotic system, is used to convert fibrinogen to fibrin. Fibrin then stimulates the fibrinolytic system.
Embodiment 1
[0012]
(Effect of tPA on phenylephrine-induced contraction)
FIG. 1 shows a graph describing the effect of tPA on phenylephrine-induced contraction of rat aortic vascular rings cultured in vitro. The contraction of the aortic vascular ring was induced by different concentrations of phenylephrine in the absence of tPA (open triangles), in the presence of 1 nM tPA (open squares), or in the presence of 10 nM tPA (open squares). Induced. This experiment was performed according to the method previously described by Haj-Yehia A et al., FASEB J, 2000, 14: 1411-1422.
The results obtained confirmed that tPA has the ability to induce vasodilation.
FIG. 1 shows that the presence of 1 nM tPA inhibits phenylephrene-induced vasoconstriction. Increasing tPA concentration induced the opposite effect. That is, the presence of 1 nM tPA stimulated the vasoconstriction induced by phenylephrene. Similarly, uPA has the ability to induce vasoconstriction. See Haj-Yehia A., et al. FASEB J, 2000, 14: 1411-1422.
(Effect of PAI-1 on uPA and tPA vasoactivity)
FIG. 2 shows bar graphs describing experimental results on the agonistic activity of uPA and tPA in the presence or absence of PAI-1; for example, the effect of 2 nM utPA or 1 nM tPA on phenylephrine-induced vasoconstriction was determined by PAI- It was measured in the presence or absence of 1 equimolar concentration.
[0013]
FIG. 3 is a bar graph showing the results of studies performed on the effect of PAI-1 derived peptides on the vascular effects of tPA. Aortic vasoconstriction was induced by increasing concentrations of phenylephrine in the presence or absence of 1 nMtPA, in the presence of 1 nMtPA and 1 OM, in the presence of 10 nM tPA, in the presence of 10 nM and 1 OM.
The results obtained show that the 1OM promoting effect of tPA on phenylephrene-induced vasoconstriction has disappeared. A similar effect was shown for uPA. PAI-1 alone had no effect on the contractile effect of the aortic vascular ring (FIG. 3). Thus, the mechanism by which PAI-1 affects the vasoactive effects of tPA or uPA is through its interaction with docking sites.
(Effect of PAI-1-derived peptide on tPA-mediated clot lysis)
FIG. 4 is a bar graph showing the experimental results regarding the effect of a PAI-1-derived peptide on tPA-mediated clot lysis. The ability of tPA to induce clot lysis was determined in the presence and absence of 1 OM. In these experiments, blood obtained from volunteers was allowed to stand at room temperature for 1 hour to form a clot, which was separated from plasma, placed on absorbent paper to remove all serum, and divided into several parts. Was. This portion was weighed and placed in PBS buffer alone or in PBS containing 100 nM tPA in the presence or absence of 1 OM. After incubation for 3 hours at room temperature, the thrombus was separated from the medium, dried and weighed.
[0014]
Two methods were used to determine whether a peptide affected the fibrinolytic activity of tPA by inhibiting plasminogen activity. 1) a chromogenic assay described in detail previously (Higazi AA.-R, et al. J. Biol. Chem., 1995, 270: 9472-9477), and 2) a previously described clot lysis test ( Higazi AA-R et al., Blood 1988, 92: 2075-2083).
The results obtained indicated that there was no significant effect on the catalytic activity of tPA (FIG. 4).
Thus, these data show that PAI-1 derived peptides and derivatives neutralize the vasoactivity of tPA or uPA, thereby reducing side effects in blood vessels, and in the case of myocardial infarction, stroke and similar diseases, It shows that complications that occur during thrombotic treatment can be prevented.
The scope of the invention is not limited by the embodiments disclosed in the examples, which are intended as a description of one aspect of the invention, and any functionally equivalent methods are within the scope of the invention. Indeed, various modifications of the invention in addition to those described and shown herein will become apparent to those skilled in the art from the foregoing description. Such improvements would be included in the claims.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, any equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
[Brief description of the drawings]
[0015]
FIG. 1 describes the effect of tPA on isolated rat aortic vascular ring phenylephrine-induced contraction in vitro. Reduction of the aortic vascular ring was induced by changing the phenylephrine concentration in the absence of tPA (open triangles), in the presence of 1 nM tPA (open squares), or in the presence of 10 nM tPA (open squares). . This experiment was performed according to the method previously described in Haj-Yahia A et al., FASEB J, 2000, 14: 1411-1422.
FIG. 2 is a bar graph showing the experimental results on the vasoactivity of uPA and tPA in the presence or absence of PAI-1, for example, phenylephrine-induced vasoconstriction in 2 nM uP or 1 nM tPA was equimolar. Determined in the presence or absence of a concentration of PAI-1.
FIG. 3 is a bar graph showing the results of a study performed on the effect of a PAI-1-derived peptide on the vascular effects of tPA. Aortic vascular ring contraction was induced by increasing phenylephrene concentration in the presence or absence of 1 nM tPA, in the presence of 1 nM tPA + 1 OM, in the presence of 10 mM tPA, in the presence of 10 nM tPA + 1 OM.
FIG. 4 is a bar graph showing the results of experiments on the effect of PAI-1 derived peptides on tPA-mediated clot lysis. The ability of tPA to induce clot lysis was determined in the presence or absence of 1 OM. In these experiments, blood obtained from volunteers was left at room temperature for 1 hour to allow coagulation, clots were separated from plasma, placed on absorbent paper, all serum was removed, and several portions were removed. divided. The parts were weighed and placed in PBS buffer alone or in PBS buffer containing 100 nM tPA in the presence or absence of 1 OM. After a 3 hour incubation at room temperature, the thrombus was separated from the medium, dried and weighed.

Claims (13)

A polypeptide comprising six amino acids and having an inhibitory effect on vasoactivity induced by a plasminogen activator. 2. The polypeptide of claim 1, which is a component of a variable number of amino acid sequences or has a modification of one or more amino acids in said sequence. The peptide according to claim 1, wherein the plasminogen activator comprises tPA or uPA. The method of claim 1, wherein the plasminogen activator comprises tcuPA, tPA, streptokinase, rt-PA, rt-PA derivative, APSC, recombinant scuPA, pro-urokinase, or a covalently cross-linked scuPA / suPAR complex. Polypeptide. 2. The polypeptide according to claim 1, further comprising tPA, wherein the combination does not cause bleeding and has fibrinolytic activity. 2. The polypeptide of claim 1, further comprising uPA, wherein the combination does not cause bleeding and has fibrinolytic activity. further comprising one or more plasminogen activators, including tcuPA, tPA, streptokinase, rt-PA, rt-PA derivative, APSC, recombinant scuPA, pro-urokinase, or a covalently cross-linked scuPA / suPAR complex; The polypeptide according to claim 1, which has an lytic activity. A method of treating fibrinolysis in a patient in need of fibrinolytic therapy, the method comprising administering to the patient a thrombolytic dose of a thrombolytic agent, followed by effective additional administration of an amount that prevents bleeding or side effects. Administering a dose of EEIIMI, wherein the additional dose of EEIIMI is administered once every 1 to 10 days during the treatment period. 9. The method of treating fibrinolysis according to claim 8, wherein the thrombolytic agent comprises tPA or uPA and is administered in a sufficient dose to a conventional clinical thrombolytic agent. 9. The method of claim 8, wherein the additional dose is a bolus of up to 500 mg. Prevent bleeding experienced by patients, including the administration of a bolus once every 1 to 10 days to the patient, followed by normal clinical doses, inhibiting the vasoactivity of plasminogen activators including tPA or uPA the method of. The method according to claim 8, wherein the thrombolytic agent is tcuPA / PAR or tcuPA. Essentially from one or more plasminogen activators, including tcuPA, tPA, streptokinase, rt-PA, rt-PA derivatives, APSC, recombinant scuPA, pro-urokinase, or covalently cross-linked scuPA / suPAR complex. 9. The method of claim 8, further comprising one or more plasminogen activators.

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