WO1992022202A1 - Thrombolytic and perfluorochemical treatment for myocardial infraction - Google Patents

Abstract

A method of treatment for myocardial infarction (MI) in human patients utilizing intravenous administration of a thrombolytic agent and of a perfluorochemical emulsion.

Description

THROMBOLYTIC AND PERFLUOROCHEMICAL TREATMENT

FOR MYOCARDIAL INFARCTION

BACKGROUND OF THE INVENTION

The present invention relates to a method of treatment for human myocardial infarction. In particular, the present invention relates to the use of an intravenously administered perfluorochemical in conjunction with intravenous thrombolytic therapy for the treatment of myocardial infarction.

Coronary artery disease remains a major cause of morbidity and mortality in the United States. Myocardial infarction is an important complication of coronary artery disease and usually results from a critical reduction in coronary blood flow secondary to coronary thrombosis. Coronary angiography performed within four hours of symptoms of acute myocardial infarction has demonstrated infarct vessel occlusion in more than 85% of Patients. See, e.g., DeWood et al. New Engl. J. Med 303:897-9 (1980). The partial elimination of lethal arrhythmias, since the introduction of intensive care units, has resulted in pump failure accounting for the largest mortality in the early post infarction period (see, e.g.. Page et al. New Engl. J. Med 285:133 (1971)). Medically refractory ventricular tachyarrhythmias also tend to occur in patients in whom greater than 40% of the left ventricular mass is irreversibly damaged. It has also been documented that premature ventricular contractions are more frequent in patients with a larger myocardial infarction and that this may be a marker for lethal arrhythmias and sudden death in the postinfarction period. Conversely, patients with smaller infarcts generally have a smoother clinical course and better ejection fractions.

Therefore, any form of therapy directed to reduction of infarct size has important clinical applications. Reperfusion decreases the extent of myocardial necrosis in the animal model if given early in the course of infarction. See, e.g., Jennings and Reimer Circulation 68:1-25 (1983). As reperfusion therapy has been accepted as a routine clinical therapy, efforts have now focused on interventions to improve the outcome within the framework of administration of thrombolytic therapy.

However, a major problem of intravenous reperfusion therapy has been the disappointing degree of improvement in left ventricular function. See, e.g., Califf et al. Circulation 78:11-213 (1988) and Califf et al. J Am Coll. Card 14:1382-1388 (1989). In fact, the total improvement in survival associated with thrombolytic therapy cannot be explained by improvement in systolic left ventricular function. See, e.g, Topol and Califf J Am Coll. Card 13:1477-1480 (1989) and Braunwald Circulation 79:441-444 (1989). Animal studies have indicated that the amount of myocardial salvage after reperfusion may be limited by anatomic and metabolic abnormalities occurring at the time of reperfusion. See, e.g., Braunwald E. J. Clin Invest 76:1713-1719 (1985). This phenomenon has been referred to as reperfusion injury. Although the exact mechanisms of reperfusion injury are not well understood, possibilities include the "no-reflow" phenomenon, endothelial cell damage, free radical production and calcium mobilization. See, e.g., Braunwald E. J. Clin Invest 76:1713-1719 (1985). Kloner et al. (J. Clin Invest 54:1496, 1974) demonstrated that following between 40-90 minutes of occlusion, severe anatomic abnormalities occurred in the microcirculation of a canine model; these workers termed this the no-reflow phenomenon. Possible abnormalities associated with this phenomenon include severe capillary damage, interstitial edema, leukocyte plugging of the microcirculation and endothelial cell dysfunction.

Other problems have also been associated with thrombolytic therapy. Thus, after initial improvement following the administration of anti-thrombolytic agents, some patients have experienced reocclusion of the artery by thrombus. Additionally, many patients have suffered moderate to life-threatening bleeding, apparently caused by the anti-thrombolytic agent. Fluosol^® (20% Intravascular Perfluorochemical Emulsion), an oxygen carrying perfluorochemical emulsion, has been shown to be effective in relieving cardiac ischemia. See, e.g., Mainz ed. PROCEEDINGS V INTERNATIONAL SYMPOSIUM ON PERFLUOROCHEMICAL BLOOD SUBSTITUTES W. Zuckschwerdt Verlag, Munich, 197-203 (1981); Anderson et al. AM Heart J 110:720-6 (1985); Murowski and Peetoom eds. IN TRANSFUSION MEDICINE: RECENT TECHNOLOGICAL ADVANCES Alan R. Liss, N.Y. pp 21- 27 (1986); and Kent et al. (submitted) to Amer. J. Cardiol. (1990). Mainz, supra, reported that oxygenated Fluosol^® administered intracoronarily during angiography reduced myocardial ischemia and resulted in significant decreases in electrocardiographic (ECG) abnormalities in humans (see, e.g., Mainz, supra).

Following clinical trials (see, e.g., Anderson et al. AM Heart J 110:720-6 (1985) and Murowski and

Peetoom, supra), Fluosol^® has been approved for use in angioplasty to reduce intraprocedural ischemia in patients at high risk of ischemic complications.

Additionally, the use of intravenous thrombolytic therapy, which has recently been approved for human treatment, has not been suggested to be used in conjunction with intravenous Fluosol^® treatment, since Fluosol^® has only been used intracoronarily in conjunction with angioplasty and intracoronary thrombolytic therapy. See, Virmani et al. Circulation 81:IV-57-IV-68 (1990); Forman et al. J. Amer. Coll. Cardiol. 9:1082-1090 (1987); and Forman et al. Circulation 71:1060-1068 (1986). The use of intravenous thrombolytic therapy has advantages over intracoronary thrombolytic therapy, the most obvious of which is lack of the need for surgery to administer a thrombolytic agent intracoronarily.

Using dog models, Hirooka and Cohn (PROCEEDINGS IV INTERNATIONAL SYMPOSIUM ON PERFLUOROCHEMICAL BLOOD SUBSTITUTES Kyoto, Amersterdam, Excerpta Medica. pp 285-293 (1978)) and Nunn et al. (Am J Cardiol 52:203-205 (1983)) demonstrated that preservation of the myocardium following experimental ligation of a coronary artery was markedly improved by the additional intracoronary administration of Fluosol^®.

In separate studies, Forman, using the 24 hour occlusion-reperfusion canine model, demonstrated that administration of only Fluosol^® intracoronarily (15 ml/kg) or intravenously (25 ml/kg) resulted in a reduction in infarct size of 55% and 40%, respectively; Forman et al. Circulation 71:1060-1068 (1985); Bajaj et al. Circulation 79:645-656 (1989). Additionally, a small but significant improvement in regional ventricular function at 24 hours post reperfusion was observed. See, e.g., Forman et al. Circulation 71:1060- 1068 (1985)

Therefore there exists a need to provide an alternative means to intracoronary thrombolytic therapy that not only substantially reduces the need for surgery to perform reperfusion, which can include thrombolytic therapy, but also enhances the effectiveness of thrombolytic therapy by reducing the problems associated with reperfusion injury existent with thrombolytic therapy, by reducing the likelihood of reocclusion of the affected artery by thrombus, and by reducing the likelihood of bleeding events.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a method of treatment for myocardial infarction (MI) in human patients utilizing intravenous administration of a perfluorochemical emulsion in conjunction with thrombolytic therapy, such as intravenous administration of at least one thrombolytic agent in conjunction with intravenously administered Fluosol^®. It is also an object of the present invention to provide a kit comprising a perfluorochemical emulsion and at least one thrombolytic agent for use in treating a patient for a myocardial infarction. Such a treatment can be utilized to alleviate problems associated with, and improve the effectiveness of, thrombolytic therapy for myocardial infarction, thereby, e.g., to increase ventricular function and to reduce one or more of the sequellae of ischemic events: infarct size, ventricular arrythmias, no-reflow phenomena, capillary damage, interstitial edema, leukocyte plugging of the microcirculation, free radical production, calcium mobilization and endothelial cell dysfunction. Such a treatment also has the effect of reducing the likelihood of reocclusion of the affected artery by thrombus and the likelihood of bleeding events.

According to one aspect of the present invention, a method is provided for treating a human patient for a myocardial infarction which comprises the steps of (A) administering intravenously a therapeutically effective amount of a thrombolytic agent; and (B) administering intravenously a therapeutically effective amount of a perfluorochemical emulsion; wherein an improved ventricular function and a decreased infarct size is effected as compared to the administration of the thrombolytic agent without the perfluorochemical emulsion.

In another aspect of the invention, the likelihood of occurrence of re-occlusion of the affected artery by thrombus is reduced as compared to the administration of a thrombolytic agent without a perfluorochemical emulsion. In a further aspect of the invention, the likelihood of occurrence of post-thrombolytic administration bleeding is reduced as compared to the administration of a thrombolytic agent without a perfluorochemical emulsion.

In a preferred embodiment, the perfluorochemical emulsion is Fluosol^®, used as a 20% perfluorochemical emulsion, the 20% perfluorochemical emulsion being prepared as described herein below from a stem emulsion containing perfluorodecalin in an amount of about 17.5% weight per volume; perfluorotri-n-propylamine in an amount of about 7.5% weight per volume; Poloxamer 188^® in an amount of about 3.4% weight per volume; egg yolk phospholipids in an amount of about 0.5% weight per volume; potassium oleate in an amount of about 0.040% weight per volume; glycerol in an amount of about 1.0% weight per volume; and water as the remaining portion to make a total of about 100%.

In another preferred embodiment, the thrombolytic agent comprises tissue plasminogen activator. Alternatively, it is preferred that the thrombolytic agent comprises at least one of tissue plasminogen activator, streptokinase, prourokinase, urokinase, and anistreplase. In still another preferred embodiment, a method is provided wherein the therapeutically effective amount of the perfluorochemical emulsion is administered in the range of about 5 to 22 ml per kg body weight at a rate of about 5 to 25 ml/min. In a more preferred embodiment, the perfluorochemical emulsion is administered in the range of about 10 to 20 ml per kg body weight at a rate of about 10 to 20 ml/min. In a particularly preferred embodiment, the perfluorochemical emulsion is administered at about 15 ml per kg body weight at a rate of about 15 to 20 ml/min. The above doses of perfluorochemical emulsion are preferably given over a time period of from about 30 to 120 minutes depending on the weight of a patient.

For example, an 80 kg patient would receive a dose of 15 ml/kg of Fluosol^® or a total dose of 1200 ml Fluosol^®. The initial five minutes administration at a rate of about 1 ml/minute and then at a rate of about 10 ml/min for five minutes would provide 55 ml of the 1200 ml total Fluosol^® dose. Then if the rate was increased to 20 ml/min the patient would receive the remainder of the 1200 ml dose in about 57 minutes.

Another object of the present invention is to provide a kit that can be used for the intravenous treatment of a human patient for a myocardial infarction, the kit comprising (A) a receptacle containing at least one dose of a therapeutically effective amount of a thrombolytic agent; and (B) a receptacle containing at least one dose of a therapeutically effective amount of a perfluorochemical emulsion.

In a preferred kit embodiment, the perfluorochemical emulsion is Fluosol^®, used as a 20% perfluorochemical emulsion, the 20% perfluorochemical emulsion being prepared as described herein below from a stem emulsion containing perfluorodecalin in an amount of about 17.5% weight per volume; perfluorotri-n-propylamine in an amount of about 7.5% weight per volume; Poloxamer 188^® in an amount of about 3.4% weight per volume; egg yolk phospholipids in an amount of about 0.5% weight per volume; potassium oleate in an amount of about 0.040% weight per volume; glycerol in an amount of about 1.0% weight per volume; and water as the remaining portion to make a total of about 100%.

In another preferred kit embodiment, the thrombolytic agent comprises tissue plasminogen activator. Alternatively, it is preferred that the thrombolytic agent comprises at least one of tissue plasminogen activator, streptokinase, prourokinase, anistreplase and urokinase. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS It has been discovered that the intravenous administration of a therapeutically effective amount of a perfluorochemical emulsion and at least one thrombolytic agent is useful as a method of treating a human patient for myocardial infarction. This method is to be utilized to alleviate problems associated with, and improve the effectiveness of, intravenous thrombolytic therapy for myocardial infarction.

According to one aspect of the present invention, intravenous perfluorochemical and thrombolytic therapy are administered concurrently as soon as possible after the onset of symptoms of a myocardial infarction, in order to improve, e.g., ventricular function and reduce one or more of ischemic events, infarct size, ventricular arrythmias, no-reflow phenomena, capillary damage, interstitial edema, leukocyte plugging of the microcirculation, free radical production, calcium mobilization and endothelial cell dysfunction, as compared to thrombolytic therapy without perfluorochemical emulsion intravenous administration.

As used herein, "perfluorochemical emulsion" refers to an aqueous emulsion of an oxygen-transferring perfluorocarbon compound, preferably having a particle size of less than about 0.3 microns. Suitable emulsions have good oxygen transferability to ischemic, hypoxic and anoxic tissues, a favorable vapor pressure range to allow reasonable expiration of the perfluorocarbon compounds used in the emulsion and clinically acceptable toxicity; the emulsion may be transparent or opaque.

A perfluorocarbon compound emulsion, used in the context of a method of the present invention, comprises at least one perfluorocarbon compound, an emulsifier, and, to the extent needed, physiological salts and/or monoglycerides thereof. Such perfluorocarbon compound emulsions are described in U.S. Patent Nos. 3,911,138 to Clark, Jr., 3,962,329 to Yokoyama et al and 4,252,827 to

Yokoyama et al, Yokoyama, K. et al: "A Perfluorochemical

Emulsion as an Oxygen Carrier", Artificial Organs 8:34- 40, 1984, and Yokoyama, D. et al: "Selection of 53 PFC

Substances for Better Stability of Emulsion and Improved Artificial Blood Substances", Advances in Blood

Substitute Research, New York: Liss, Inc., 1983, all of which are incorporated herein by reference.

Preferred fluorocarbon compound emulsions comprise at least one perfluorocarbon compound having about 9-11 carbon atoms selected from the group consisting of perfluorodecalin, perfluoromethyldecalin, perfluoro alkylcyclohexanes having 4 to 6 carbon atoms in the alkyl, perfluoroalkyltetrahydrofurans having 5 to 7 carbon atoms in the alkyl and perfluoroalkyltetra-hydropyrans having 4 to 6 carbon atoms in the alkyl and perfluoroalkanes having 9 to 11 carbon atoms; at least one perfluoro tert-amine having 9 to 11 carbon atoms selected from the group consisting of perfluoro tert-alkylamines having 9 to 11 carbon atoms, perfluoro N-alkylpiperidines having 4 to 6 carbon atoms in the alkyl and perfluoro N-alkylmorpholines having 5 to 7 carbon atoms in the alkyl; a high-molecular-weight nonionic surfactant having a molecular weight of about 2,000 to 20,000; a phospholipid; and at least one fatty acid compound selected from the group consisting of fatty acids having 8 to 22 carbon atoms; and physiologically acceptable salts and monoglycerides thereof. The ratio of the perfluorocarbon compound and the said perfluoro-tert-amine is 95-50 to 5-50 by weight.

The "high-molecular-weight nonionic surfactant" has a molecular weight of 2,000 to 20,000 and includes polyoxyethylene-polyoxypropylene copolymers, polyoxyethylene alkyl ethers, and polyoxyethylene alkyl aryl ethers. The concentration of the surfactant in the emulsion is about 2.0 to about 5.0%, preferably 3.0 to 3.5% (W/V).

The symbol "% (W/V)" means the amount proportion of a material by weight (in grams) based on 100 ml of the resulting emulsion.

The following perfluoro compounds can be used alone or in combination to practice the present invention and can be contained in the emulsion in an amount of about 10 to 50% (W/V):

Perfluorocarbons useful herein include those having about 9 to 11 carbon atoms, such as a perfluorocycloalkane or perfluoroalkylcycloalkane which includes, for e x a m p l e , perfluoromethylpropylcyclohexane, perfluoro-butylcyclohexane, perfluorotrimethylcyclohexane, perfluoroethylpropylcyclohexane, perfluorodecalin and

perfluoromethyldecalin; a perfluoro C_4-7-alkyltetrahydropyran such as perfluorohexyltetrahydropyran, perfluoro pentyltetrahydrofuran, perfluoro hexyltetrahydrofuran and perfluoro heptyltetrahydrofuran; and a

perfluoroalkane having about 9-11 carbon atoms such as perfluorononane and perfluorodecane. Other perfluorocarbons useful herein include, e.g., perfluoro C_9-18 polycyclic compounds such as bicyclononanes, methyl and dimethyl bicyclooctanes, pinane, camphane, adamantane and alkyl(C_1-6) adamantanes such as dimethyladamantane, ethyl adamantane, and the like. Other heterocyclic perfluorocarbons useful herein include perfluoro saturated and N-alkyl-substituted quinolines, quinolizines, quinolidines, pyrrolidines and morpholines. Other useful perfluorocarbons include perfluoro tert-amines having about 9 to 11 carbon atoms such as perfluoro tert-alkylamines having 9 to 11 carbon atoms which includes, for example, perfluorotrialkylamines such as perfluoro N, N-diethylpentylamine, perfluoro N,N-diethylhexylamine, perfluoro N, N-dipropylbutylamine and perfluorotripropylamine; a perfluoro N, N-dialkylcyclohexylamine having 9-11 carbon atoms such as perfluoro N,N-diethylcyclohexylamine; a perfluoro N-C₄_₆-alkylpiperidine such as perfluoro N-pentylpiperidine, perfluoro N-hexylpiperidine and perfluoro N-butylpiperidine; and a perfluoro N-C_5-7-alkylmorpholine such as perfluoro N-pentylmorpholine, perfluoro N-hexylmorpholine and perfluoro N-heptylmorpholine.

When a perfluoro combination including a tertiary amine is used, the ratio of the (non-tertiary amine) perfluorocarbon compound to the perfluoro tert-amine to be used is 50:95 to 50:5 by weight and the total amount of perfluorocarbon compound and perfluoro tert-amine contained in the emulsion is about 10 to 50% (W/V).

The phospholipids used as emulsifier adjuvant in the invention are ones commonly used in the art, and those comprising yolk phospholipid or soybean phospholipid are preferable. The amount present in the emulsion ranges from about 0.1 to abour 1.0% (W/V), and preferably about 0.4 to about 0.6% (W/V). The fatty acid compound used as emulsifying adjuvant is a fatty acid having 8 to 22 carbon atoms, a physiologically acceptable salt such as sodium or potassium salt or a monoglyceride thereof, which includes, for example, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, palmitoleic acid, oleic acid, linoleic acid, arachidonic acid and sodium or potassium salt and monoglyceride thereof. These fatty acid compounds may be used alone or as a mixture of two or more kinds thereof in such a minor amount of 0.004 to 0.1% (W/V), and preferably about 0.02 to 0.04% (W/V). Among these fatty acid compounds, the preferred ones are those having 14 to 20 carbon atoms and their physiologically acceptable salts, and the most preferred are potassium palmitate and potassium oleate, taking into consideration of their good solubility and ease of the preparation of the emulsion.

An example of a perfluorochemical emulsion that can be used according to a method of the present invention is Fluosol^® (Green Cross Corporation, Osaka, Japan), which is a sterile, isotonic perfluorochemical emulsion consisting of perfluorodecalin and perfluorotri-n-propylamine. The emulsion must be stored in a frozen state (-5°C to -30°C) prior to use. Fluosol^® is prepared from a perfluorochemical stem emulsion which contains the following compounds: perfluorodecalin in an amount of about 17.5% weight per volume; perfluorotri-n-propylamine in an amount of about 7.5% weight per volume; Poloxamer 188^® in an amount of about 3.4% weight per volume; egg yolk phospholipids in an amount of about 0.5% weight per volume; potassium oleate in an amount of about 0.040% weight per volume; glycerol in an amount of about 1.0% weight per volume; and water as the remaining portion to make a total of about 100%. The average particle diameter of Fluosol^® emulsified perfluorochemical particles as determined by a laser light scattering method is less than 270 nanometers.

The structure of perfluorodecalin is:

The structure of perfluorotri-n-propylamine is:

CF₃CF₂CF₂ CF₂CF₂CF₃

N CF₂CF₂CF₃

Poloxamer 188^® is a polyoxyethylene-polyoxypropylene copolymer surfactant of about 8350 molecular weight. The structure of Poloxamer 188^® is:

HO - (CH₂CH₂O)_a - [CH(CH3)(CH-O)]_b - (CH₂CH₂O)_c - H where values for a, b and c are approximately 74, 31 and 74, respectively. The structures of remaining constituents are well known.

Additional solution(s) can be added to the stem emulsion to make a final 20% emulsion and which serve to adjust pH, ionic strength and osmotic pressure. Examples of suitable additive solutions include the following additive solutions I and II, in combination:

ADDITIVE SOLUTION I

Ingredient Amount (% w/v)

Sodium Bicarbonate 3.5

Potassium Chloride 0.56

Water q.s.

ADDITIVE SOLUTION II

Ingredient Amount (% w/v)

Sodium Chloride 4.29

Dextrose 1.29

Magnesium Chloride 0.305

Calcium Chloride 0.254 Water q.s.

As used herein, a "thrombolytic agent" refers to an enzyme or compound that is pharmacologically effective in helping to dissolve a thrombus. Thrombolytic agents that can be used in a method of the present invention include one or more of tissue plasminogen activator, streptokinase, urokinase, prourokinase, anistreplase or other known fibrinolytic enzymes or compounds, such as activated human protein C. According to a method of the present invention, a thrombolytic agent can be one or a combination of more than one known thrombolytic agent.

For example, a tissue plasminogen activator (tPA) can be used (also known as fibrinokinase; extrinsic plasminogen activator; t-PA; TPA; Activase^®). tPA has a molecular weight of about 70,000, is a serine protease catalyzing the enzymatic conversion of plasminogen to plasmin through the hydrolysis of a single Arg-Val bond and is a component of the mammalian fibrinolytic system responsible for the specific activation of plasminogen associated with fibrin clots. See, e.g., for a clinical study in coronary occlusion of myocardial infarction: The Thrombolysis in Myocardial Infarction (TIMI) Study Group, N. Engl. J. Med. 312:932 (1985); Verstraete et al. Lancet 1:842 (1985); for a comparative clinical study with urokinase in acute pulmonary embolism: Goldhaber et al. Lancet 2:293 (1988), the contents of which are herein incorporated by reference.

A commercially available form of tPA is exemplified by Activase^®, alteplase (available from Genentech, South San Francisco, California), as a thrombolytic agent used in a method of the present invention. When tPA is administered intravenously with a perfluorochemical emulsion according to a method of the present invention, a therapeutically effective amount of tPA is in the range of about 10 to 150 mg administered intravenously over a three hour period. It is preferred that a dose of 100 mg be administered as 60 mg in the first hour (of which 6 to 10 mg is administered as a bolus over 1 to 2 minutes), 20 mg over the second hour and 20 mg over the third hour. For smaller patients (less than 65 Kg), a dose of 1.25 mg/Kg administered over 3 hours may be used. See, e.g., PHYSICIAN'S DESK REFERENCE, Medical Economics Company Inc., Oradell, New Jersey, 988-989 (1989), the contents of which are herein incorporated by reference.

Another thrombolytic agent useful in a method of the present invention is streptokinase (also known as Streptococcal fibrinolysin; plasminokinase; Awelysin^®; Kinalysin^®; Kabikinase^®; and Streptase^®), an enzyme elaborated by hemolytic streptococci which hydrolyzes -CONH- links (peptide bonds) and is an activator of plasminogen, thus producing plasmin, which dissolves fibrin. See, e.g., for isolation from hemolytic Streptococci; Christensen Gen. Physiol. 28:363 (1954); J. Clin. Invest. 38:163 (1949); von Polnitz et al, U.S. patent Nos. 3,063,913; 3,063,914; 3,138,542 (1962, 1962, 1964 all to Behringwerke); Siiteri, Mills, U.S. pat. 3,226,304 (1965 to American Cyanamid); for purification: Singher and Zuckerman, U.S. patent No. 3,016,337 (1962 to Ortho Pharmaceuticals); Siegel et al, Baumgarten, Cole, U.S. patent Nos. 3,042,586 and 3,107,203 (1962, 1963 to Merck & Co.), the contents of which are herein incorporated by reference.

When streptokinase is administered intravenously, patients are treated with streptokinase as soon as possible after onset of symptoms. Streptokinase can be administered intravenously as a total dosage of

1,500,000 IU within 60 min.

Alternatively, Urokinase can be used as a thrombolytic agent according to the present invention (also known as Abbokinase^®; Actosolv^®; Breokinase^®;

Persolv^®; Purochin^®; Ukidan^®; Uronase^®; and Winkinase^®).

Urokinase is serine protease which activates plasminogen to plasmin and is present in mammalian blood and urine.

See, e.g., for isolation from human male urine: H.O. Singher, L. Zuckerman, U.S. pats. 2,961,382 and

2,989,440 (1960, 1961 to Ortho); N.O. Kjeldgaard, J.

Ploug, U.S. pat. 2,983,647 (1961 to Leo Pharm.); J.

Doczi, U.S. pat. 3,081,236 (1963 to Warner-Lambert); for use in thrombolytic therapy: Sherry et al. Ann. Intern. Med. 93:141-144 (1980), the contents of which are herein incorporated by reference.

When urokinase is administered intravenously, it can be administered as soon as possible after onset of symptoms as a bolus of 1 million to 1.5 million units followed by an additional 1 million to 1.5 million units over 60 to 90 minutes.

Alternatively, Prourokinase (enzyme-activating)

(also known as single-chain urokinase-type plasminogen activator; single-chain pro-urokinase; scu-PA; pro-UK; pro u-PA; PUK; Tomieze^®; Sandolase^®) can be used as a thrombolytic agent in a method of the present invention.

Prourokinase is a single-chain proenzyme form of urokinase with intrinsic thrombolytic activity and consists of about 411 amino acid residues, with a molecular weight of about 50,000 daltons. Prourokinase is converted by plasmin to active two-chain urokinase by proteolytic cleavage of the Lys ¹⁵⁸-Ile¹⁵⁹ peptide bond. Prourokinase can be extracted from urine or from kidney tissue culture and purified or can be produced recombinantly. See, e.g., EPA 0139447, published May 2, 1985, the contents of which are herein incorporated by reference.

When prourokinase is administered intravenously, it can be administered as soon as possible after onset of symptoms as a bolus of 9,000 to 36,000 units.

Alternatively, anistreplase (Eminase^®) can be used as a thrombolytic agent in a method of the present invention. When anistreplase is administered intravenously, it can be administered as soon as possible after onset of symptoms as a total dose of 30 units given over 2 to 5 minutes.

Alternatively, a combination of known thrombolytic agents, such as those described above can be used in a method according to the present invention. For example, a combination of tPA and streptokinase can be used.

According to another aspect of the present invention, therapeutically effective amounts of one or more thrombolytic enhancing agents can be used in conjunction with intravenous thrombolytic and perfluorochemical therapy. The term "thrombolytic enhancing agent" as used in the context of the present invention refers to a protein or chemical compound that enhances the tissue sparing potential of a thrombolytic agent or that enhances the prevention of reocclusion in patients undergoing thrombolytic therapy. Examples of thrombolytic enhancing agents of the present invention include recombinant or naturally derived sCR1 (soluble compliment receptor type 1) and HPC (human protein C). sCR1 has been found to have a myocardial protective effect (see, e.g., Weisman et al. Science 249:146-151, 1990). Human protein C (HPC) is therapeutically useful in a form which can be present in vivo or converted in vivo into its activated form (APC), having a potent anticoagulant effect due to its ability to inactivate Factors Va and Villa (see, e.g. Yan et al Biotechnology 8:655-661 (1990) and Esmon J. Biol. Chem. 264:4743-4746 (1989)).

According to another aspect of the present invention, the timing of the initiation of intravenous administration of at least one thrombolytic agent and a perfluorochemical is important in the relative efficacy of a thrombolytic therapy according to the present invention. In particular, the initiation of thrombolytic therapy should occur as soon as possible after the initial symptoms of myocardial infarction (such as acute myocardial infarction) are experienced by the patient to be treated. It is preferred that a method of treatment according to the present invention be initiated within six hours after the onset of symptoms, in order to help assure that an increase of ventricular function is established and to reduce the incidence of congestive heart failure or other possible negative consequences that result from ischemia and myocardial infarction. A patient treated by a method of the present invention can also undergo angiography and angioplasty, if clinically indicated, as well as ECG monitoring. Before discharge a patient can undergo coronary angiography to determine artery status and assessment of infarct size and ventricular function. During and after hospitalization, the patient can be followed for the incidence of complications, including reinfarction, repeat revascularization procedures, clinically significant arrhythmias and episodes of ischemia. Postprocedural status and complications can be evaluated at discharge, six (6) weeks later and six (6) months later.

In the practice of the present invention oxygen may be administered along with the perfluorochemical emulsion, i.e., by breathing 100% oxygen. As an alternative or used therewith, the perfluorochemical emulsion can be oxygenated prior to use.

The administration of Fluosol^® has been associated with a very low incidence of adverse reactions. However, as a safety precaution, a patient scheduled for Fluosol^® treatment can receive an intravenous 0.5 ml test dose of Fluosol^® at least five (5) minutes prior to the initiation of the Fluosol^® infusion.

The administration of Fluosol^® may be initiated at lower doses initially to test for tolerance. For example, Fluosol^® can be administered at a rate of about 1 ml per minute for five minutes, followed by about 10 ml per minute for another five minutes, in order to ensure that the patient tolerates the Fluosol^® acceptably. Once such initial doses are given with no contra-indicated effects, intravenous administration can be increased to about 20 ml per minute, or other acceptable level, as determined by the treating physician. Such a level is preferably in the range of about 5 to 25 ml per minute and more preferably in the range of about 15-20 ml per minute.

The total dosing of Fluosol is preferred to be in the range of about 5 to 22 ml per kg of body weight and more preferably in the range of 10 to 20 ml/kg of body weight. For example, 15 ml/kg of body weight can be administered at a rate of 20 ml per minute. Thus, an 80 kg patient would receive a total dose of 15 ml/kg of Fluosol^® or a total of 1200 ml. The initial five minutes administration at a rate of about 1 ml/minute and then at a rate of about 10 ml/min for five minutes would provide 55 ml of the 1200 ml total Fluosol^® dose. Then if the rate was increased to 20 ml/min the patient would receive the remainder of the 1200 ml dose in about 57 minutes.

The doses of other perfluorochemical emulsions will be known to the skilled artisan or can be easily determined using the 5 to 22 ml per kg body weight range as a starting point.

The effectiveness of a method for treating MI according to the present invention can be determined using physiological and clinical endpoints compared to thrombolytic treatment alone. An example of a primary physiological endpoint is the global left ventricular ejection fraction at catheterization prior to hospital discharge after administration of a treatment using a method of the present invention. Another endpoint can be the estimate of infarct size as determined by perfusion deficit on a cardiac scan.

Additional physiological endpoints can include other measures of regional and global cardiac function. Such measures include, e.g., regional left ventricular function and end-systolic and end-diastolic volumes at catheterization prior to hospital discharge.

Additional clinical endpoints can be monitored for efficacy of the primary thrombolytic therapy and for effects of Fluosol^® administration on the clinical course using a method of the present invention. Such clinical endpoints can include the number of episodes of arrhythmias (monitored during the first 24 hours after infusion), the number of patients experiencing reocclusion, the number of bleeding events,

and coronary artery patency rates at catheterization at the time of discharge.

The following are examples of clinical and laboratory measurements which can be used to assess the safety of treatment regimens according to the present invention: Vital signs and status (Day 0, 1, predischarge) 12 lead ECG (Day 0, 1, predischarge)

CBC (complete blood count) (Day 0, 3, predischarge) Prothrombin time, partial

thromboplastin time (Day 0, 3, predischarge)

Serum chemistries (Day 0, 3, predischarge) Cardiac enzymes, (tid-Day 0; bid-Day 1 and

Day 2, qd-Day 3)

As will be apparent to those skilled in the art, the above measurements will be indicative of the safety of treatment using a method according to the present invention, as compared to alternative forms of treatment of myocardial infarction.

The following Examples are presented by way of illustration only and are not intended to limit or narrow in any way the scope of the present invention. Additional embodiments within the spirit and scope of the present invention will be apparent to those skilled in the art.

The following Example of a method of treatment of the present invention provides for a concurrent or overlapping period of intravenous administration of Fluosol^® and at least one thrombolytic agent, initiated as soon as possible after the onset of symptoms caused by a myocardial infarction, preferably within six hours.

EXAMPLE 1: Patient Accession and Timing of Treatment

Patients (evaluated in the inpatient or emergency departments of a suitable hospital) with an acute myocardial infarction of less than six (6) hours duration are considered suitable for treatment. Patients with first infarctions are also considered as candidates for treatment.

EXAMPLE 2: Thrombolytic Therapy Using tPA as a Thrombolytic Agent

Patients are treated with tissue plasminogen activator (t-PA), such as Activase^®, given intravenously at a dose of 100 mg administered as 60 mg in the first hour (of which 6 to 10 mg is administered as a bolus over 1 to 2 minutes), 20 mg over the second hour and 20 mg over the third hour. For smaller patients (less than 65 Kg), a dose of 1.25 mg/Kg administered over 3 hours may be used.

EXAMPLE 3: Thrombolytic Therapy Using Streptokinase as a Thrombolytic Agent

Patients are treated with streptokinase as soon as possible after onset of symptoms. Streptokinase is administered intravenously as a total dosage of 1,500,000 IU within 60 min. EXAMPLE 4: Thrombolytic Therapy Using tPA and Streptokinase as a Thrombolytic Agent

Patients are treated with tPA and streptokinase. tPA is administered in a total dose of 1.0 mg/kg (to a maximum of 90 mg), with 10% of the dose being administered as soon as possible after onset of symptoms as a bolus, and with the remainder being administered over 60 minutes. One million units of streptokinase is administered over 60 minutes concurrently with the tPA.

EXAMPLE 5: Thrombolytic Therapy Using Urokinase as a Thrombolytic Agent

Patients are treated with urokinase employing the dosage regimen described hereinbefore as soon as possible after onset of symptoms. Prior to the infusion of urokinase, a bolus dose of heparin ranging from 2500 to 10,000 units is administered intravenously. Prior heparin administration should be considered when calculating the heparin dose for this procedure. EXAMPLE 6: Thrombolytic Therapy Using Prourokinase as a Thrombolytic Agent

Patients are treated with prourokinase as soon as possible after onset of symptoms employing the dosage regimen described hereinbefore. EXAMPLE 7: Fluosol^® Administration in Conjunction with Intravenous Thrombolytic Therapy

Those patients who undergo intravenous thrombolytic therapy, according to Examples 2-6 above, receive a controlled intravenous infusion of Fluosol^® to a total dose of 15.0 ml/kg administered with an infusion pump. Intravenous perfluorochemical and thrombolytic therapy are administered concurrently as soon as possible after the onset of symptoms of a myocardial infarction.

Preparation of Fluosol^® (20%). The Fluosol^® is prepared for use in accordance with an accepted procedure, for example, as set forth below. Briefly, about 20 minutes is required to thaw a bag of Fluosol^® emulsion in a 37ºC water bath with the Fluosol^® bag enclosed in a clear plastic bag. The additive solutions, as described above, are stored at room temperature, and are added sequentially with mixing, under aseptic conditions, to the Fluosol^® emulsion and the complete emulsion is mixed gently by inverting the bag 6 to 10 times.

The administration of Fluosol^® may be facilitated by utilizng a thaw and hold phase prior to compounding with the additive solutions. Essentially, the frozen Fluosol^® emulsion may be thawed at a temperature up to 70°C and stocked at 4ºC for a maximum of one week prior to being compounded with the additive solutions.

Fluosol^® Infusion. Patients receive a total of 15.0 ml/kg Fluosol^® intravenous infusion. In order to minimize any possible adverse responses to the administration of Fluosol^®, the infusion can be controlled by an infusion pump and begin at 1 ml/minute for five (5) minutes. If this initial rate is tolerated, the rate is increased to 10 ml/minute for five (5) minutes and finally increased to a maximum of 20 ml/minute for the remainder of the infusion. For example, an 80 kg patient would receive a total dose of 15 ml/kg of Fluosol^® or a total of 1200 ml. The initial five minutes administration at a rate of about 1 ml/minute and then at a rate of about 10 ml/min for five minutes would provide 55 ml of the 1200 ml total Fluosol^® dose. Then if the rate was increased to 20 ml/min the patient would receive the remainder of the 1200 ml dose in about 57 minutes.

If the patient shows signs of volume overload, the infusion rate is reduced to 10 ml/minute and appropriate treatment administered. Indications of volume overload include new rales in the posterior lung or a new third heart sound during the infusion of Fluosol^®.

Patients must be monitored very closely and diurectics administered promptly if signs of pulmonary edema or congestive heart failure develop. Additionally, a chest x-ray may be taken to confirm any suspected cases of pulmonary edema or conjestive heart failure.

Adverse Response to Fluosol^®. The subject is closely monitored during the infusion of Fluosol^® for clinically relevant changes from baseline values in blood pressure, heart rate, respiratory rate of subjective responses. Due to the complex and variable nature of an evolving MI, the physician's judgement must be used in determining the occurrence of an adverse response to the administration of Fluosol^®. Of particular importance are symptoms indicative of a severe allergic reaction: flushing, labored breathing due to bronchial constriction or atypical pain such as lower back pain.

If the physician determines that the patient has a clinically significant response to the infusion of Fluosol^®, the administration of Fluosol^® can be stopped and the patient withdrawn from the treatment.

The patient may be premedicated with antihistamines for known contrast media sensitivity or at the physician's discretion before initiation of the study and administration of Fluosol^®.

Oxygen Administration. Patients may be administered 100% oxygen (6 to 10 liters per minute) supplied through a non-rebreathing mask. Administration of 100% oxygen, where employed, can be initiated no later than the time the patient starts the Fluosol^® infusion and continues for 8 hours. Prior to and following this requirement, oxygen may be administered at the discretion of the attending physican. Since the major function of Fluosol^® is to deliver oxygen, every effort must be made to maiximize oxygen delivery to the patient. Further, the use of oxygen masks and the delivery of oxygen must be carefully monitored.

As an alternative the Fluosol^® can be oxygenated, for example, with 95% O₂/5% CO₂ above 600 mm Hg partial pressure of oxygen prior to use. Furthermore, such an oxygenated perfluorochemical emulsion can be administered in conjunction with breathing of oxygen as aforementioned.

Further therapy after completion of the initial treatment period can be left to the discretion of the attending physician.

Post-Thrombolvtic, Perfluorochemical Care. Pharmacologic care can include maintenance on aspirin and beta blocker throughout the hospitalization unless contraindicated, with the optional use of nitrates. Rehabilitation care can include a structured educational and activity program prior to discharge. Exercise testing can be used to evaluate each patient for arrhythmia and ischemia and to formulate an exercise prescription prior to discharge. The date of discharge can be determined by the patient's attending physician.

Infarct Size Determination. The size of the infarct can be assessed by tomographic thallium-201 scintigraphy according to known procedures.

Effect of the Treatment on the Likelihood of Reocclusion. The effect of the combined administration of an anti-thrombolytic agent and an oxygen-transferring perfluorochemical, in comparison to the administration of an anti-thrombolytic agent alone, was evaluated through a rabbit model.

First, four rabbits were treated with Activase^®, and four rabbits were treated with Activase^® and

Fluosol^®. The flow of blood through the effected arteries was then measured over a four hour period with the results shown in the chart below.

As can be seen from the above data, the occurrence of reocclusion was less with the treatment comprising the combined administration of the anti-thrombolytic agent and the oxygen-transferring perfluorochemical.

Then, Fluosol^® and one of its components, poloxomer-188, were evaluated as adjuncts to thrombolytic treatment for an occlusive femoral arterial thrombus in a rabbit model. Fluosol^® (n=6), poloxomer188 (2.7% w/v) (n=6), or saline as a control (n=6) were administered in amounts equivalent to 20% blood volume after Activase^® (rt-PA) initiation. No anticoagulants or anti-platelet agents were used in this study. The animals were observed over 3 hours of rt-PA treatment (0.8mg/kg) and an additional one hour recovery period. Thrombolysis and coagulation responses were evaluated in vivo or by ex vivo assays. The mean time to recanalization (>25% of preoccluded flow rate) in the Fluosol^® group was 40% of the mean time in the control group (32 min. vs. 82 min., P<0.05). The mean time in the Poloxamer-188^® group was 77 min. (NS). One control animal never achieved recanalization during the procedure. In addition, arteries in rabbits from the control (3/5) and poloxamer-188 (4/6) treatment groups reoccluded more often than in the Fluosol^® group (2/6). Mean alpha2-antiplasmin levels dropped 30% (P < 0.02) and APTTs were extended by 55% (P < 0.0001) in the Fluosol^® group. Safety problems were limited to moderate bleeding episodes in two rabbits treated with poloxamer^® which also had significant drops in fibrinogen (>25%). In this rabbit model, Fluosol^®, but not poloxamer-188, significantly and safely increased the rate of effective arterial thrombolysis and decreased reocclusions.

Effect of the Treatment on the Likelihood of Bleeding Events. From a pool of 120 patients suffering from acute myocardial infarction, 60 were randomly selected as a control group to be treated with an anti- thrombolytic agent. The other 60 patients were treated with an anti-thrombolytic agent and Fluosol^®, according to the present invention.

Among the 60 patients in the control group, twelve experienced bleeding events (there were 18 bleeding events in total, eleven moderate, four severe and three life-threatening). Among the 60 patients who were treated according to the present invention, only six experienced bleeding events (there were eight bleeding events in total, five moderate, two severe and one life- threatening). Thus, 20% of the control group suffered from such bleeding events, but only 10% of the group treatment according to the present invention.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the above detailed description and above Examples of the present invention. It should be understood, however, that the description and specific Examples, while indicating preferred embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

Claims

WHAT IS CLAIMED IS:

1. A method for treating a human patient for a myocardial infarction comprising the steps of

(A) administering intravenously a therapeutically effective amount of a thrombolytic agent; and

(B) at least during a portion of the treatment time therewith administering intravenously a therapeutically effective amount of an oxygen-transferring perfluorochemical emulsion; to improve patient outcome as compared to the administration of said thrombolytic agent without said perfluorochemical emulsion.

2. The method according to Claim 1, wherein said perfluorochemical emulsion comprises an oxygen-transferring perfluorocarbon compound having a particle size of less than about 0.3 microns.

3. The method according to Claim 1, wherein said perfluorochemical emulsion is a 20% perfluorochemical emulsion, containing perfluorodecalin in an amount of about 14.0% weight per volume and perfluorotri-n-propylamine in an amount of about 6.0% weight per volume

4. The method according to Claim 1, wherein said thrombolytic agent comprises tissue plasminogen activator.

5. The method according to Claim 1, wherein said thrombolytic agent comprises at least one of tissue plasminogen activator, streptokinase, prourokinase, anistreplase and urokinase.

6. The method according to Claim 1, wherein said therapeutically effective amount of said perfluorochemical emulsion is in the range of about 5 to 22 ml per kg body weight.

7. The method according to Claim 6, wherein said therapeutically effective amount of said perfluorochemical emulsion is in the range of about 10 to 20 ml per kg body weight.

8. The method according to Claim 7, wherein said therapeutically effective amount of said perfluorochemical emulsion is about 15 ml per kg body weight.

9. A kit used for the intravenous treatment of a human patient for a myocardial infarction, said kit comprising

(A) a receptacle containing at least one dose of a therapeutically effective amount of a thrombolytic agent; and

(B) a receptacle containing at least one dose of a therapeutically effective amount of a perfluorochemical emulsion.

10. The method according to Claim 1 wherein said thrombolytic agent and said perfluorochemical emulsion are administered concurrently.

11. The method according to Claim 1 wherein said thrombolytic agent and said perfluorochemical emulsion are administered partially sequentially.

12. The method according to Claim 1 wherein said patient is administered oxygen during the administration of said perfluorochemical.

13. The method according to Claim 12 wherein said oxygen is 100% oxygen administered through inhalation.

14. The method according to Claim 12 wherein said oxygen is started at about the time of initiation of said administration of said perfluorochemical emulsion.

15. The method according to Claim 12 wherein said oxygen is administered throughout said administration of said perfluorochemical emulsion.

16. The method according to Claim 12 wherein said oxygen is also administered through oxygenation of said perfluorochemical emulsion prior to administration to said patient.

17. The method of Claim 13 wherein said perfluorochemical emulsion is oxygenated prior to said administration of said emulsion to said patient.

18. A method for treating a human patient for a myocardial infarction comprising the steps of,

(A) administering a therapeutically effective amount of a thrombolytic agent intravenously to lyse a thrombus within a coronary artery; and

(B) at least during a portion of the treatment time therewith administering intravenously a therapeutically effective amount of an oxygen-transferring perfluorochemical emulsion; to reduce the likelihood of occurrence of re-occlusion of the artery by thrombus as compared to the administration of said thrombolytic agent without said perfluorochemical emulsion.

19. The method of claim 18, wherein during the treatment time therewith, heparin is not administered to the patient.

20. A method for treating a human patient for a myocardial infarction comprising the steps of,

(A) administering a therapeutically effective amount of a thrombolytic agent intravenously to lyse a thrombus within a coronary artery; and

(B) at least during a portion of the treatment time therewith administering intravenously a therapeutically effective amount of an oxygen-transferring perfluorochemical emulsion;

to reduce the occurrence of post-thrombolytic administration bleeding as compared to the administration of said thrombolytic agent without said perfluorochemical emulsion.