IL93973A - Pharmaceutical compoaition and package containing a modified t-pa having a relatively slow rate of disappearance from the blood and agent for enhancing its absorption in the bloodstream - Google Patents

Description

93973/3 ,Π-ΌΠ1 3ΐέϊ23 ητπ PHARMACEUTICAL COMPOSITION AND PACKAGE CONTAINING A MODIFIED t-PA HAVING A RELATIVELY SLOW RATE OF DISAPPEARANCE FROM THE BLOOD, AND AGENT FOR ENHANCING ITS ABSORPTION IN THE BLOODSTREAM SURVIVAL TECHNOLOGY, INC.

C: 10434 93973/2 1 The present application .is -an application for a patent of addition to IL 76,120, which corresponds to U.S. Patent No. ,4,661,469.

The present invention relates, in general, to a pharmaceutical composition for use in rapidly inducing thrombolysis in a mammal using a modified form of wild-type t-PA and, in particular, to such a composition which enables the same to be accomplished in a manner that both increases the overall bioavailability of the modified t-PA and avoids the undesirable effects, including bleeding, associated with the prolonged elevation of blood levels of such modified t-PA.

The rapid dissolution of a blood clot in a~ patient can be accomplished by intravenously administering to that patient a protein thrombolytic agent. Clinically available protein thrombolytic agents act by directly or indirectly activating plasminogen, a circulating glycoprotein with a high affinity for fibrin. The product of plasminogen activation is plasmin, a serine protease capable of degrading, in addition to fibrin, fibrinogen and other coagulation factors.

Tissue-type plasminogen activator (t-PA ) is one such protein thrombolytic agent. A major pharmacological attribute of t-PA is its relatively high affinity for fibrin. This property facilitates the interaction of t-PA with fibrin-bound plasminogen (Km = 65ji for free plasminogen and 0.14 y for fibrin bound plasminogen; Sobel et al., Eds. Tissue Plasminogen Activator in Thrombolytic Therapy ( 1987 ) p. 25-39 ) . The comparatively low characterizing the interaction of t-PA with fibrin- bound plasminogen is associated with localized formation of plasmin within and on nascent thrombi. The "clot-selectivity" of t-PA thus reflects the intensity of activation of plasminogen to form plasmin upon exposure to the activator in the domain of fibrin as compared with activation of 93973/3. plasminogen In the systemic circulation. (For recent reviews see Pannekoek et ai Fibrinolysis 2:123 (1988) and Krause Fibrinolysis 2:123(1988)).

Combining t-PA with absorption enhancers such as hydroxy1amine has been disclosed in U.S. Patent Nos. 4,658,830, 4,661,469 and 4,772,585, as well as in Sobel et al. P.N.A.S. 82:4258-62 (1985) and Sobel et al. Circulation 75(6) : 1261-72 (1987) . In addition, modified forms of t-PA have been disclosed in such publications as the following: Larsen et al. Biol . Chem. 253(2) :1023-9 (1988); Collen et al . Blood 71(l):216-9 (1988) ; Lucore et al . Circulation 77(4):906-14 (1988); Pannekoek et al. Fibrinolysis 2:123-32 (1988); and Krause Fibrinolysis 2:133-42 (1988).

Although t-PA in combination with absorption enhancers such as hydroxylamine has been disclosed, and the existence of modified forms of t-PA are known, their combination would not have been obvious. In fact, the literature nowhere suggests that using a modified t-PA having a long half-life would significantly improve the therapeutic viability of intramuscularly injected t-PA. Moreover, the literature does not suggest that the therapeutic value of intramuscularly injected t-PA could be stabilized by using a modified t-PA having a slower rate of disappearance from the circulatory system, or that any modified protein could be used for such a purpose.

Although t-PA Is" present only in small quantities in the body, recombinant DNA techniques have resulted in the wide spread availability of a · ~ clinically useful form of t-PA having many properties in common with wild-type t-FA (Ny et ai.

Proc. Natl. Acad. 5ci . U.S.A. 81:5355 (1984); including amino acid sequence and clearance rate (that is, the in vivo half-life is approximately the. same as that of wild-type t-PA) (which form is hereinafter designated wild-type-like t-PA) . In addition, a variety of modified forms of t-PA have been genetically engineered which display properties potentially advantageous in a ciini-;;aJ setting including increased specificity for fibrin-bound plasminogen and increased In vivo half life. 93973/1 The wlld-type-like_t-PA which is currently in clinical use (alteplase, recombinant) is administered intravenously to hospitalized patients to achieve therapeutic blood levels. Because the in vivo half-life of this form of t-PA, like wild-type t-PA, is so short, continuous intravenous infusion ia required to avoid rapid disappearance of the protein from the circulation with resulting subtherapeutic concentrations, In addition, intravenous administration has been deemed necessary because of the urgent need for rapid clot lysis to interrupt the heart attack in progress before death of a substantial amount of heart muscle. Blood levels of this thrombolytic agent can be rapidly adjusted so as to avoid the disadvantageous -2a- 3 depletion of blood proteins, including fibrinogen, that results when elevated levels of the agent are present in the bloodstream for prolonged periods.

The benefit conferred upon the heart by coronary thrombolysis is directly dependent on the rapidity with which thrombolysis is initiated after the onset of coronary occlusion (Bergmann et al. Am. J. Med. 73:573 (1982)). It has been demonstrated in dogs that salvage of heart muscle, reflected by its ability to sustain intermediary metabolism, is prominent when thrombolysis is induced within one hour after the onset of occlusion but minimal or absent when thrombolysis is delayed for three hours or more. Similar conclusions have been reached in studies of preservation of ventricular function in patients treated with fibrinolytic agents (Koren et al. N. Engl. J. Med. 313:1384 (1985); Mathey et al.

Am. Coll. Cardiol. 6:518 (1985)). Furthermore, the magnitude of reduction of mortality associated with pharmacologically induced coronary thrombolysis is directly dependent upon the rapidity of its implementation following the onset of symptoms (GISSI, Lancet 1:397 (1986)).

In view of the desirability of inducing thrombolysis as rapidly as possible, a self- administration procedure for wild-type (or wild- type-like) t-PA for pre-hospital use has recently been developed and is described in United States t Patent No. 4,772,585 issued September 9, 1988.

The procedure involves the intramuscular injection of a solution of the drug, in combination with a solubilizer (i.e., arginine) and absorption enhancers (i.e., hydroxylamina and methyla ine ) , This procedure results in the rapid attainment of a thrombolytic level o£ t-PA in the blood (i.e. within about 15 min.) followed by a modest decline with significant levels persisting in the blood for as long as 6 hours or more (see Figure 1; Figure 2 shows the results obtained in the absence of an enhancer) (see also Sobel et al Proc. Natl. Acad.

Scl. USA 82 $4258 (1985) and Sobel et al Circulation 75.1261 (1987)).

It has been postulated that following intramuscular injection of the t-PA-containing composition, the enhancer and soiubilizer (being small molecules) diffuse out of the injection site more rapidly than does the t-PA itself/ resulting in the lack of rapid absorption of a portion of the t-. PA dose at the injection site. That portion of the administered dose is not only unavailable for inducing the desired therapeutic effect, but i addition, by its slow release from the injection site, can complicate subsequent therapeutic interventions and increase the likelihood of internal bleeding.

The development of a method of rapidly inducing thrombolysis that results in the therapeutically effective utilization of intramuscularly injected protein-thrombolytic agent would increase the desirability of self- administrable treatment procedures by reducing the quantity (and, therefore, potentially the cost) of thrombolytic agent required to induce thrombolysis, and by diminishing the risk of blood protein degradation and internal bleeding accompanying same. It is an object of the present invention to provide such a method.

~~ It may be noted that portions of the present application which do not fall within the Scope of the claims are not relevant to the invention. 5 IN THE DRAWINGS: Figure 1 - Plasma levels of wild-type-like t-PA administered intramuscularly to a dog in the presence of enhancers. A lightly anesthetized dog was given two simultaneous intramuscular injections of 2 ml containing wild-type-like t-PA in a dose of 10 mg/kg body weight, 0.079 M hydroxylamine 0.63 M methylaraine, 0.2 M arginine phosphate, and 0.4 M sodium acetate at a final pH of 3.0.

Figure 2 - Plasma levels of wild-type-like t-PA administered intramuscularly to a dog in the absence of enhancers. A lightly anesthetized dog was given two simultaneous intramuscular injections of 2 ml containing wild-type-like t-PA in a dose of 10 mg/kg body weight, 0.2M arginine phosphate, and 0.4 M sodium acetate at a final pH of 3.0.

Figure 3 - Half-life in the circulation of a rabbit of wild-type-like t-PA and of the enzymatic deglycosylation product thereof (o-o = deglycosylated; ·-· = untreated).

Figure 4 - Variant-1 t-PA plasma time-concentration curve (60 min.) {·-· = variant-1; o-o = wild-type-like).

Figure 5 - Half-life in the circulation of a rabbit of variant-1 t-PA.

Figure 6 - Variant-2 t-PA plasma time-concentration curve (50 min.) (o-o = variant-2; ·-· = wild-type-like).

Figure 7 - Half-life in the circulation of a dog of variant-2 t-PA (o-o = ELISA (ng/ml); ·-· = SOFIA ( IU/ml ) ) .

Figure 8 - Variant-1 t-PA plasma time-concentration curve (300 minutes ) (o-o = wild-type-like; ·-· = variant-1).- 6 In accordance with the principles of the invention, the above-noted objective (that of providing a method of rapidly inducing thrombolysis in a manner that results in the therapeutically effective utilization of intramuscularly injected protein-thrombolytic agent) is accomplished by intramuscularly administering to a mammal a composition that includes: 1) a protein-thrombolytic agent having a rate of disappearance from the blood slower than that of wild-type t-PA, in an amount sufficient to produce a thrombolytically effective level of the agent in the bloodstream of the mammal for a period of time sufficient to induce and complete thrombolysis , wherein the amount is such that significant bleeding resulting from the presence of and persistence of the agent in the bloodstream is avoided, and 2) an enhancer in an amount sufficient to increase the rapidity of absorption of the thrombolytic agent into the bloodstream of the mammal. A preferred protein thrombolytic agent is a t-PA molecule modified in a manner such that the half-life thereof is at least twice that of wild-type t-PA.

In a preferred embodiment, the protein thrombolytic agent, administered in an amount as described above, has a half-life such that the initial, therapeutically critical, peak concentration observed following intramuscular injection persists for a sufficiently long period of time (at least 3 hours after injection) and at a sufficiently high level (a therapeutically acceptable level) to avoid the likelihood of reocclusion. That is, the relatively rapid decline in the plasma level of the thrombolytic agent to a subtherapeutic level (prior to the appearance of the second concentration peak (see Figure 1)), observed with intramuscularly administered wild-type (or wild-type-like) t-PA, is avoided.

In the method of the instant invention, the ratio of: 1) that portion of the intramuscularly administered dose of modified t-PA that is present in the blood stream of the mammal during the time period (approximately 1-60 minutes) immediately following intramuscular injection (and that is thus available for inducing and completing clot lysis) to 2) that portion of the dose that remains at the injection site only to be released into the bloodstream after that initial (approximately 1-60 minutes) period, is significantly greater than the corresponding ratio resulting from the intramuscular administration of wild-type (or wild-type-like) t-PA. As a consequence, a smaller quantity of modified t-PA is required to be administered to achieve a rapid thrombolytic effect than is required when wild-type (or wild-type-like) t-PA is used. As a result of the lower administered dose, the amount of thrombolytic agent present in the circulation after the first hour (approximately) is small enough and persists for a sufficiently abbreviated period, to reduce the risk of predisposing the individual receiving the intramuscular injection to internal bleeding. In a preferred embodiment, the amount present in the circulation after the, initial period is, however, sufficient to inhibit reocclusion.

In accordance with the foregoing, the protein thrombolytic agents of the instant invention are those having a half-life longer than that of wild-type t-PA, preferably at least twice as long. The longer half-life translates into a faster rate 8 of accumulation early after injection and a slower rate of disappearance (clearance) of thrombolytic activity from the bloodstream.

Suitable protein thrombolytic agents include modified forms of t-PA having an altered carbohydrate structure at one or more amino acids. The altered carbohydrate structure may result, for example, 1) from a substitution, deletion or addition mutation at or near the glycosylation sites of wild-type t-PA, 2) from the deletion of a portion of the wild-type sequence containing one or more glycosylation sites, 3) from the chemical or enzymatic removal of carbohydrate moieties from naturally occurring or genetically engineered t-PA, or 4) from the growth of organisms or cells which are transformed or transfected with a recombinant DNA molecule containing a sequence encoding wild-type t-PA under conditions such that a pattern of glycosylation distinct from that of wild-type t-PA (or wild-type-like t-PA) results. A number of such modified forms of t-PA are known in the art.

For example, a form of t-PA modified so as to be devoid of functional carbohydrate structure at amino acid 117 (but otherwise with functionally unmodified carbohydrate structures and with full biological activity) is disclosed in EP 238304 (Genentech). Mutant t-PA proteins having a slower physiological clearance rate than wild-type t-PA and a prolonged in vivo stability are also disclosed in EP 225286 (Ciba Geigy) . Specifically, these proteins differ from wild-type t-PA in that at least one Ser (or Thr) residue occurring at the N-glycosylation sites is replaced by another amino acid which prevents N-glycosylation. Another modified form of t-PA suitable for use in the 9 present invention is produced by treating wild-type t-PA with endoglycosidase-H, as described by Lucore et al. (Circulation 77: 906 (1988)).

Other modified forms of t-PA suitable for use in the present invention that are known in the art include the following. Deletion mutants of human t-PA having a specific thrombolytic activity and fibrin specificity comparable to that of t-PA obtained from melanoma cell culture but having a longer half-life, are described by Collen et al (Blood 71:216 (1988)). A degraded species of t-PA having a reduced in vivo clearance rate and comprising a fibrolytically intact B chain of wild type t-PA linked to kringle 2 as the only functionally and structurally intact domain of wild-type t-PA A chain (Mr - 38,000/40,000) is disclosed in EP 196920 (Beecham). A variant of wild-type t-PA lacking finger and epidermal growth factor domains as well as asparagine-linked glycosylation has been described by Larsen et al. (Thromb. Haemost . 58:491 (1987)) as having a monoexponential clearance rate that is markedly longer than that of wild-type t-PA.

Protein thrombolytic agents suitable for use in the present invention also include hybrid plasminogen activators containing plural, heterologous polypeptide kringles such as are disclosed in EP 213794 (American Home Products Corp.). The tris-kringle plasminogen activators described, constructed from appropriate coding sequences of urokinase and t-PA clones, display a longer half-life in vivo when compared with either of the wild-type activators. In addition, analogs of t-PA that have had the native domain regions either rearranged, deleted, or added, or a 10 combination thereof, are disclosed in E? 231624 (Upjohn Co.)* These analogs are described as having a longer half-life and increased fibrin affinity.

Also within the scope of the invention are modified forms of t-PA having the same primary structure as wild-type t-PA except that the amino acids that form the epidermal growth factor domain are at least partially missing (at least one of loops I and II, the binding region, the catalytic region and the fibrin finger being present), such as are disclosed in E? 242836 (3oehringer Mannheim GM3E) and are described as having the same fibrolytic activity as wild-type t-PA but with a much longer in vivo half-life.

Protein thrombolytic agents convaiently modified by molecules that increase the circulatory half-life, for example polyethylene ^glycol, are also within the scope of the invention.

Methods for preparing the protein thrombolytic agents of the instant invention are known in the art, as evidenced by the protocols given in the above-indicated references.

A therapeutically acceptable dose of the protein thrombolytic agents can be determined by one skilled in the art using known techniques without undue experimentation. Preferably, the dose should be sufficient to complete thrombolysis within 1 hour of intramuscular administration in approximately 70% of the animals treated, but not sufficient to induce internal bleeding typified by a decrease in the Diasma fibrinogen level of more than 50% over the 11 The protein thrombolytic agents of the invention, for example, modified forms of t-PA, require the presence of at least one absorption enhancer when administered intramuscularly. Such enhancers must be administered with the intramuscular injection of the protein thrombolytic agent to effect prompt absorption of the large molecule into the bloodstream in therapeutically significant quantities.

The absorptio rate of the protein thrombolytic agents of the instant invention in the blood is enhanced by utilizing with the protein thrombolytic agent dosage, a dosage of an absorption enhancing agent for modified t-PA or other protein-thrombolytic agent of the invention, e.g. hydroxylamine hydrochloride. The safety of the absorption- enhancing agents is described in United States Patent No. 4,772,585. Preferably, the absorption enhancing agent, such as hydroxylamine hydrochloride, is mixed in with the protein-thrombolytic agent dosage to form a single mixed dosage which is then injected intramuscularly e.g. as described in U.S. patent 4,658,830. An example of an amount of absorption enhancing agent, such as hydroxylamine hydrochloride, which is added to, for example, the dosage of modified t-PA, as previously described, to form a single mixed dosage is an amount of from 0.1 to 85, e.g. 0.1 to 40 or 1 to 85 milligrams per kilogram of body weight.

The absorption enhancing agent hydroxylamine is preferably employed in the form of a non-toxic water soluble salt. Thus there can be used for example, in place of hydroxylamine, salts such as hydroxylamine hydrochloride, hydroxylamine hydrobromide, hydroxylamine hydroiodide, 12 hydroxylamine sulfate, hydroxylamine nitrate, hydroxylamine acetate, and hydroxylamine proporionate. Most preferably there is employed hydroxylamine hydrochloride.

There is also contemplated as absorption enhancing agents for modified t-PA or other protein-thrombolytic agents in accordance with the invention compounds such as ammonia (ammonium hydroxide), ammonium carbonate and other ammonium salts, e.g. ammonium chloride, ammonium acetate, ammonium bromide and ammonium sulfate, urea, mono and dialkyl ureas, e.g. methyl urea, ethyl urea, propyl urea, butyl urea, Ν,Ν-dimethyl urea, Ν,Ν-diethyl urea, N,N-diisopropyl urea, mono and dialkyl ureas, e.g. phenyl urea, p-tolylurea, Ν,Ν-diphenyl urea, N,N-di-p-tolyl urea, thiourea, hydantoin, 5-substituted hydantoins, e.g. 5-alkyl, 5-aralkyl, and 5-aryl hydantoins and 5,5-dialkyl and 5,5-diaryl hydantoins, e.g. 5-methyl hydantoin, 5-ethyl hydantoin, 5-ethyl hydantoin, 5,5-dimethyl hydantoin, 1, 5-trimethylene hydantoin, 1,5-tetramethylene hydantoin, 5-phenyl hydantoin, 5-p-tolyl-hydantoin, and 5,5-diphenyl hydantoin, guanidine, methyl guanidine, hydrazine, alkyl and aryl hydrazines, e.g. methyl hydrazine, ethyl hydrazine, butyl hydrazine, phenyl hydrazine and diphenyl hydrazine, alkyl and aryl hydroxylamines, e.g. methyl hydroxylamine, ethyl hydroxylamine and phenyl hydroxylamine. The substituted ureas, hydrazine and hydroxylamines likewise can be used in the form of salts, e.g. as hydrochlorides.

Likewise there can used as absorption enhancing agents other amines, e.g. alkyl amines and dialkyl amines such as lower alkyl amines and dialkylamines, e.g. methylamine, dimethylamine, 13 ethylamine, diethylamine, isopropylamine, sec-butylamine, diisopropylamine , propylamine, n-butylaraine, aralkylamines, e.g. phenylethylamine, hydroxyaralkylamines, e.g. epinephrine and tyramine, hydroxyalkylamines, e.g. ethanolamine, diethanolamine, triethanolamine, propanolamine, and other amines such as methoxyamine, polyalkylene amines, e.g. ethylene diamine, diethylene triamine. These amines also can be used in the form of salts of non-toxic acids such as salts of the acids mentioned earlier, e.g. as the hydrochlorides. Also there can be used glucoseoxime.

Also while the simultaneous administration of modified t-PA.or other protein-throrabolytic agents and absorption enhancing agents is primarily intended for human use, it is within the scope of the invention that they be administered to other mammals, e.g. dogs, cats, cattle, and horses. In accordance with the teachings of united States Patent No. 4,832,682 issued May 23, 1989, electrical stimulation of the muscle at the injection site can be employed in concert with the inclusion of an absorption-enhancing agent, specifically hydroxylamine hydrochloride, in the injectate using intramuscular injection. Electrical stimulation augments and enhances the absorption of the absorption enhancing agent of the invention.

An automatic injection device suitable for intramuscular self-administration of modified t-PA or other protein thrombolytic agents of the invention can be employed in the method of the 14 present invention. Such a device is described in U.S. patent 4,658,830.

The invention includes packaging the modified t-PA (and/or other protein thrombolytic agents of the invention) and at least one agent enhancing the absorption of the modified t-PA in the blood. Preferably, a combination of hydroxylamine hydrochloride and methylamine is used. There can be used for example a known emergency type automatic injector and the process comprises injecting the two medicament agents into the muscle tissue, e.g. after having received a decision to do so over the telephone from a qualified source and at a time prior to the establishment of direct contact with qualified personal care.

It has also been found that to prevent reocclusions or platelet aggregation it is desirable to administer agents which either: 1. inhibit synthesis of thromboxane A (thromboxane A2) with a thromboxane synthetase inhibitor, e.g. an imidazole such as 4-(2-[lH-imidazol-l-yl]ethoxy) -benzoic acid hydrochloride (dazoxiben) 2. introduce an antagonist of the receptor for the thromboxane A (thromboxane A2) such as [la, 2s (5Z), 38 (IE), 4a]-7-[3-(3-cyclohexyl-3-hydroxyl-1-propenyl ) -7-oxabicyclo [ 2.2.1 ] hept-2-yl ] -5-heptenoic acid) (SQ 27427), or similar compounds such as SQ 29548 and SQ 30741; 3. introduce an antagonist of the receptor for fibrinogen, for example, 7E3 F (ab')2 (Yasuda et al. J. Clin. Investiq. 81:1284 (1988)); or 4. introduce another inhibitor of platelet aggregation, e.g. aspirin, indomethacin, naproxin, sulfinpyrazone, or dipyridamole.

The agent for the prevention of reocclusions or platelet aggregations can be administered simultaneously or sequentially in either order with reference to the modified t-PA and absorption enhancing agent [ e.g. hydroxylamine hydrochloride]. The agent for the prevention of reocclusions or platelet aggregations can be administered in conventional manner, e.g. intramuscularly, intravenously, or even orally.

The receptor antagonist or other agent for prevention of platelet aggregations or reocclusions can be administered for example in an amount of 0.1-10 mg/kg body weight per hour.

The following non-limiting Example describes the invention in more detail.

Example 1 Enzymatically deglycosylated wild-type-like t-PA.

Wild-type-like t-PA (alteplase, recombinant) was deglycosylated with endoglycosidase-H as described by Lucore et al.

(Circulation 77 p.906-914 (1981)) to remove oligosaccharide side chains from residues 117, 184 and 448. The half life in the circulation of the deglycosylated product (designated Endo-H t-PA) was determined and compared to that of wild-type-like t-PA by injecting 0.2 mg/kg body weight intravenously as a bolus in a volume < 1 ml into lightly anesthetized rabbits. Femoral venous blood samples were taken serially over approximately one hour.

As shown in Figure 3, the plasma wild-typelike t-PA time-concentration curve was consistent with biexponential clearance of wild-type-like t-PA 16 from the circulation with a calculated alpha phase half-life of approximately 1.2 minutes and a calculated beta phase half-life of approximately 7.5 minutes. Endo-H t-PA exhibited marked prolongation 5 of the alpha phase half-life to greater than 3.0 (approximately 3.8) minutes and marked prolongation of the beta phase half-life to approximately 25 minutes.

The time-concentration curves of 10 intramuscularly injected wild-type-like and Endo-H t-PA, were also determined in lightly anesthetized rabbits. Two simultaneous intramuscular injections of 1 ml each were administered percutaneously.

Injection media contained selected amounts of wild- I5 type-like or Endo-H t-PA in 0.63 M methylamine, 0.079 M hydroxylamine, 0.4 M sodium acetate, 0.2 M arginine phosphate, at a final pH of 3.0. Serial blood samples were obtained via a jugular venous catheter. 20 Intramuscular injection of a low dose of wild-type-like t-PA (0.86 mg/kg body weight) led to an initial peak of low magnitude followed by a marked decline to essentially zero. In contrast, Endo-H t-PA given intramuscularly at the same dose 25 elicited a substantially greater initial plasma concentration peak followed by sustained elevations of plasma concentrations of Endo-H t-PA throughout the hour of observation.

It is noteworthy that similar observations 30 were obtained with comparisons of wild-type-like and genetically engineered variants of t-PA. The plasma time-concentration curve shown in Figure 4 was 17 obtained when a genetically engineered t-PA variant (variant-1) exhibiting an essentially monoexponential disappearance rate from the circulation with a half-life of approximately 25 minutes (see Figure 5; conditions as described in the Example), was injected intramuscularly into rabbits under conditions as described in the Example at a dose of 4 mg/kg. As will be seen from Figure 4, an initial peak was observed which was substantially greater in magnitude than that observed with wild-type-like t-PA. A marked attenuation of the rate of decline, relative to the wild-type-like t-PA, was also observed. The plasma time-concentration curve generated with the variant was desirable from a therapeutic point of view because of the rapid accumulation of high concentrations of thrombolytic agent in plasma after intramuscular injection, the sustained elevation of concentrations within the therapeutic range over the hour of observation, and the gradual decline of concentrations, compatible with avoidance of predisposition to internal bleeding during an interval corresponding to the one after which clinically effective coronary thrombolysis would have been accomplished.

These same observations were made in additional experiments with another genetically engineered variant of t-PA (variant-2) that exhibited a prolonged half-life as well (Figure 6; the concentration (ELISA) and activity (SOFIA) after an i.v. bolus injection of 0.2 mg/kg are shown in Figure 7). The dose of wild-type-like and variant t-PA used was 2 mg/kg. The magnitude of the peak attained with variant-2 was greater and the rate of decline of the concentration in the plasma was 18 markedly attenuated compared with the corresponding rate associated with wild-type-like t-PA. In these experiments, the injection medium was devoid of 400 mM acetate, constituted with 0.01% Tween 80, and buffered with 0.1 M H3P04 to pH 7.4. The difference in injection media used accounts for the difference in magnitude of peak plasma t-PA concentrations seen after injection of wild-type-like t-PA in Figure 6 compared with values depicted in Figure 4.

The dichotomy between plasma time- concentration curves elicited with intramuscular injections compared with wild-type-like t-PA was even more striking when the interval of observation was prolonged. As shown in Figure 8, the concentrations of t-PA sufficient to preclude reocclusion after coronary thrombolysis persisted through the 300 minutes of observation after intramuscular injection of variant-1 t-PA. In contrast, the initial peak after intramuscular injection of wild-type-like t-PA at the same dose (2 mg/kg; as compared to 10 mg/kg used in the experiments the results of which are shown in Figure 1) was of lesser magnitude, and the decline of concentration of wild-type-like t-PA in plasma was so abrupt and extreme that values fell to within the normal physiologic range within one hour and were only minimally elevated subsequently.

The foregoing invention has been described in some detail for purposes of clarity and understanding. It will also be obvious that various combinations in form and detail can be made without departing from the scope of the invention. 93973/2

Claims (17)

1. A pharmaceutical composition for use in rapidly inducin thrombolysis in a mammal comprising (1) a modified form of wild-type t-PA and (2) an enhancer in an amount sufficient to increase the rate of absorption of said modified t-PA into the bloodstream of said mammal, whereby said modified t-PA has a rate of disappearance from the blood lower than that of wild-type t- PA, when used in an amount sufficient to produce a thrombolytically effective level of said modified t-PA in the bloodstream of the mammal for a period of time sufficient to induce and complete thrombolysis, said amount also being such that significant bleeding resulting from the presence and persistence of said modified t-PA in the bloodstream is avoided.

2. ■2. The composition according to claim 1 wherein said modified t-PA has a carbohydrate structure distinct from that of wild-type t-PA.

3. · The composition according to claim 2 wherein said modified t-PA consists essentially of wild-type t-PA having a substitution mutation at or near a site .of N-glycosylation in wild-type t-PA.

4. The composition according to claim 3 wherein said substitution mutation is at amino acid 117·

5. The composition according to claim 1 where said modified t-PA consists essentially of wild-type t-PA from which kringle 1 is deleted. 19 93973/2

6. The composition according to claim 1 further comprising an effective amount of a compound that inhibits platelet aggregation.

7. The composition according to claim 6 wherein said compound is an antagonist for the thromboxane A receptor.

8. The composition according to claim 1 further comprising an effective amount of a compound that inhibits reocclusion.

9. The composition according to claim 1 wherein said amount of said modified t-PA is such that sufficient levels thereof remain in the bloodstream for a period of time adequate to inhibit reocclusion.

10. The composition according to claim 1 wherein the enhancer comprises hydroxylamine or a non-toxic salt thereof.

11. . The composition according to claim 1G wherein the enhancer comprises hydroxylamine hydrochloride.

12. The composition according to claim 11 wherein the enhancer further comprises methylamine.

13. The composition according to claim 1 wherein the enhancer comprises a lower alkylamine or a non-toxic salt thereof.

14. The composition according to claim 13 wherein the' lower alkylamine is methylamine. 2G 93973/4

15. The composition according to claim 1 wherein said period of time sufficient to induce and complete thrombolysis is about 15 to 60 minutes.

16. The composition according to claim 1 wherein said mammal is human.

17. A package containing (1) a modified form of wild-type t-PA and (2) an enhancer capable of being used in an amount sufficient to increase the rate of absorption of said modified t-PA into the bloodstream of a mammal, whereby said modified t-PA has a rate of disappearance from the blood lower than that of wild-type t-PA, when used in an amount sufficient to produce a thrombolytically effective level of said modified t-PA in the bloodstream of the mammal for a period of time sufficient to induce and complete thrombolysis, said amount producing a thrombolytically effective level also being such that significant bleeding resulting from the presence and persistence of said modified t-PA in the bloodstream is avoided. . 21