US 20080311 1 05A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0311105 A1 Zimmerman et al. (43) Pub. Date: Dec. 18, 2008

(54) REVERSIBLY INACTIVATED ACIDIFIED PCT/US00/31090, filed on Nov. 13, 2000, which is a PLASMN continuation-in-part of application No. 09/438.331, filed on Nov. 13, 1999, now Pat. No. 6,355.243. (76) Inventors: Thomas P. Zimmerman, Raleigh, NC (US); Valery Novokhatny, Publication Classification Raleigh, NC (US); Shan Jiang, Cary, NC (US); James Colandene, (51) Int. Cl. Raleigh, NC (US) A638/48 (2006.01) (52) U.S. Cl...... 424/94.64 Correspondence Address: WOMBLE CARLYLE SANDRIDGE & RICE, (57) ABSTRACT PLLC ATTN: PATENT DOCKETING 32ND FLOOR, The present invention provides a fibrinolytic composition P.O. BOX 7037 useful as a therapeutic for administration to a patient having a ATLANTA, GA 30357-0037 (US) thrombotic occlusion. In one aspect of the present invention, the fibrinolytic composition comprises a reversibly inacti (21) Appl. No.: 12/197,617 vated acidified serine substantially free of a plasmi nogen activator, a low buffering capacity buffer, and option (22) Filed: Aug. 25, 2008 ally, a stabilizing agent. In another aspect of the invention, the fibrinolytic composition of the present invention comprises a Related U.S. Application Data reversibly inactivated acidified substantially free of a (60) Division of application No. 10/143,112, filed on May , a low buffering capacity buffer, and 10, 2002, which is a continuation of application No. optionally, a stabilizing agent.

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REVERSIBLY INACTIVATED ACIDIFIED . The heavy chain (60 kDa) consists of five triple PLASMN loop kringle structures with highly similar amino acid sequences. Some of these kringles contain so-called lysine REFERENCE TO RELATED APPLICATION binding sites that are responsible for plasminogen and plas min interaction with fibrin, C2-antiplasmin or other proteins. 0001. This application is a division of U.S. patent appli Variant forms of truncated plasmin, including variants lack cation Ser. No. 10/143,112, filed May 10, 2002, incorporated ing some or all of the kringle regions of the plasmin heavy herein fully by reference, which in turn is a continuation of chain, are disclosed by Wu et al. in U.S. Pat. No. 4,774,087, International Application PCT/US00/31090, filed Nov. 13, incorporated herein by reference in its entirety. SK and sta 2000, and published in English on May 25, 2001, which in phylokinase activate plasminogen indirectly by forming a turn is a continuation-in-part of U.S. patent application Ser. complex with plasminogen, which Subsequently behaves as a No. 09/438,331, filed 13 Nov. 1999. plasminogen activator to activate other plasminogen mol FIELD OF THE INVENTION ecules by cleaving the arginyl-valine bond. 0007 Plasmin is a different mechanistic class of throm 0002 The present invention relates generally to composi bolytic agent that does not activate plasminogen. Plasmin tions useful in thrombolytic therapy. More particularly, the directly cleaves fibrin in a thrombus, resulting in lysis. This present invention is directed to novel compositions compris avoids the requirement for plasminogen or plasminogen acti ing reversibly inactivated acidified composi vators to be present in a thrombus. Many clots that are defi tions useful in clot dissolution therapy wherever directed cient in plasminogen due to thrombus contraction triggered delivery to a thrombus is feasible. by platelets and by Factor VIII. 0008. Although tha, SK and UK have been successfully BACKGROUND employed clinically to reduce a thrombotic occlusion, serious 0003. The blood clotting process, as a mechanism of limitations persist with their use in current thrombolytic hemostasis or in the generation of a pathological condition therapy. For example, because the systemic administration of involving thrombi, requires two cooperating pathways: 1) tPA is not specifically targeted to the thrombus, it can result in following activation, triggered by the , cir significant systemic hemorrhage. Other limitations associ culating platelets adhere to one another accompanied by the ated with plasminogen activators impact their overall useful release of factors like thromboxane A2 and the subsequent ness. At best, the use of current thrombolytic therapy results formation of a plug created from the aggregated platelets, and in restored vascular blood flow within 90 minutes in only 2) the activation of a cascade of proteolytic and about 50% of patients, while acute coronary re-occlusion cofactors, most of which are plasma glycoproteins synthe occurs in roughly 10% of the patients. Coronary recannuli sized in the liver, to produce a thrombus. Thrombi are com Zation requires on average 45 minutes or more, and intracere posed mainly of an insoluble fibrin network, which entraps bral hemorrhage occurs in 0.3% to 0.7% of patients. Residual circulating blood cells, platelets, and plasma proteins to form mortality is still about 50% of the mortality level in the a thrombus. absence of thrombolysis treatment. 0004 Thromboembolic disease, i.e., the pathological 0009. A different approach that avoids many of the prob blockage of a blood vessel by a blood clot, is a significant lems associated with the systemic administration of a plas cause of mortality and morbidity. Most spontaneously devel minogen activator is to generate plasmin at the site of the oping vascular obstructions are due to the formation of intra thrombus or to directly administer the plasmin either into or vascular blood clots, or thrombi. Small fragments of a clot proximally to the thrombus. Reich et al. in U.S. Pat. No. may also detach from the body of a clot and travel through the 5,288.489 discloses a fibrinolytic treatment that includes circulatory system to lodge in distant organs and initiate parenteral administration of plasmin into the body of a further clot formation. Myocardial infarction, occlusive patient. The concentration and time of treatment were suffi stroke, deep venous thrombosis (DVT) and peripheral arterial cient to allow active plasmin to attain a concentration at the disease are well-known consequences of thromboembolic site of an intravascular thrombus that is sufficient to lyse the phenomena. thrombus or to reduce circulating fibrinogen levels. Reich et 0005 Plasminogen activators are currently the favored al. require generation of the plasmin from plasminogen agents employed in thrombolytic therapy, all of which con immediately prior to its introduction into the body. Vert plasminogen to plasmin and promote by 0010. In contrast, Jenson in U.S. Pat. No. 3,950,513 dis disrupting the fibrin matrix (Creager M. A. & Dzau V. J., closes a porcine plasmin preparation that is asserted to be Vascular Diseases of the Extremities, ppgs. 1398-1406 in stabilized at low pH. However, such plasmin solution must be Harrison's Principles of Internal Medicine, 14" ed., Fauci et neutralized before systemic administration to humans for al, editors, McGraw-Hill Co., New York, 1998; the contents thrombolytic therapy. of which is incorporated herein by reference in its entirety). (0011 Yago et al. in U.S. Pat. No. 5,879,923 discloses The most widely used plasminogen activators include a plasmin compositions employed as a diagnostic reagent. The recombinant form of tissue-type plasminogenactivator (tPA), compositions of Yago et al. consist low concentrations of (UK) and (SK), as well as a new plasminata neutral pH and an additional component that may generation of plasminogen activators selected for improved be 1) an oligopeptide consisting of at least two amino acids, or pharmacokinetics and fibrin-binding properties. All of these 2) at least two amino acids, or 3) a single amino acid and a plasminogen activators, however, act indirectly to effect lysis polyhydric alcohol, and the amino acids are specifically iden and require an adequate Supply of their common Substrate, tified. plasminogen, at the site of the thrombus. 0012) Numerous technical problems, such as the difficulty 0006 UK and tRA convert plasminogen to plasmin by of preparing plasmin free of contaminating plasminogen acti cleaving the Arg-Val peptide bond. The resulting two vators, have prevented clinical use of plasmin. Plasmin prepa polypeptide chains of plasmin remain joined by two inter rations were typically extensively contaminated by the plas chain disulfide bridges. The light chain of 25kDa carries the minogen activators streptokinase and urokinase, resulting in catalytic center and is homologous to and other serine the attribution of thrombolytic activity to the contaminating US 2008/0311 1 05 A1 Dec. 18, 2008 plasminogen activators rather than to plasmin itself. The con include serine, threonine, methionine, glutamine, glycine, taminating plasminogen activators can also trigger systemic isoleucine, Valine, aspartate, and alanine. Other low buffering bleeding at sites other than the targeted thrombosis. One capacity acids may be employed and include formic acid, factor limiting clinical use of plasmin is that plasmin, as a acetic acid, citric acid, hydrochloric acid, lactic acid, malic serine protease with broad specificity, is highly prone to auto acid, tartaric acid, benzoic acid, derivatives thereof, and com degradation and loss of activity at physiological pH when binations thereof. The amino acids and the other low buffer prepared as a highly purified and highly concentrated solu ing capacity acids may be combined in any desired combina tion. This provides severe challenges to the production of tion as well. high-quality plasmin, to the stable formulation of this active 0020 Stabilizing agents which may be employed in the protease for prolonged periods of storage prior to use, and to present invention include pharmaceutically acceptable carbo safe and localized administration of plasmin to human hydrates, salts, glucosamine, thiamine, niacinamide, citrul patients Suffering from occlusive thrombi. line, and combinations thereof. 0013 Thus, there is a need for a therapeutic composition 0021. Thus, a unique fibrinolytic composition is now pro comprising a stabilized serine protease capable of cleaving vided that Successfully addresses the shortcomings of exist fibrin and a pharmaceutically acceptable carrier with a pH ing compositions and provides distinct advantages over Such range sufficiently low to reversibly inactivate the serine pro compositions. Additional objects, features, and advantages of tease, yet sufficiently high to limit acid hydrolysis of peptide the invention will become more apparent upon review of the bonds within the serine protease. Further, there is a need for detailed description set forth below when taken in conjunc Such therapeutic composition to have a low buffer capacity to tion with the accompanying drawing figures, which are maintain low pH during storage, yet permit plasminto rapidly briefly described as follows. revert to its active form at the pH in the local environment of the clot. BRIEF DESCRIPTION OF THE FIGURES 0014. There is also a need for a therapeutic composition 0022 FIG. 1 illustrates the pH dependence of plasmin comprising a reversibly inactivated acidified serine protease activity as measured with the chromogenic substrate S2251. stabilized by at least one pharmaceutically acceptable stabi 0023 FIG. 2 illustrates plasmin stability in acidified saline lizing agent and a pharmaceutically acceptable carrier. (pH 3.7) as measured by a caseinolytic assay. 0015 These and other objectives and advantages of the 0024 FIG. 3 illustrates that pH stability of plasmin does invention will become fully apparent from the description and not depend on the buffering agent nor concentration of buff claims that follow or may be learned by the practice of the ering agent. invention. 0025 FIG. 4 illustrates the effectiveness of plasmin ortRA plus plasminogen in thrombolysis. SUMMARY OF THE INVENTION (0026 FIG. 5 illustrates the stability at 37° C. of a revers 0016. This invention overcomes the disadvantages of the ibly inactivated acidified plasmin at pH of 3.7, with carbohy prior art by providing a fibrinolytic composition which can be drate stabilizers. therapeutically administered directly at or proximal to a site (0027 FIG. 6 illustrates the stability at 37° C. of a revers of a thrombotic occlusion. Further, the fibrinolytic composi ibly inactivated acidified plasmin at a pH of 3.7 with glu tion of the present invention has a Substantially long-term cosamine, niacinamide, thiamine or citrulline as a stabilizing shelf life with respect to the prior art. agent. 0017. In one aspect of the present invention, the fibrin 0028 FIG. 7 illustrates the progressive degradation of a olytic composition comprises a reversibly inactivated acidi plasmin composition at a pH of 2.2, 3.5, or 3.7. fied serine protease Substantially free of a plasminogen acti 0029 FIG. 8 illustrates the cleavage sites generated in vator, a low buffering capacity buffer, and optionally, a plasmin at pH 2.2 and 3.8. stabilizing agent. Such serine proteases include trypsin, chy 0030 FIG. 9 illustrates the titration, with human serum, of motrypsin, pancreatic II, G, prostate-spe plasmin Solutions having various low buffering capacity buff cific antigen, leukocyte elastase, , , , CS. human tissue , and plasmin. Plasmin includes Glu 0031 FIG. 10 compares the thrombolytic potency of plas plasmin or Lys-plasmin, derivatives and modified or trun min in saline at pH 3.7 with plasmin neutralized before injec cated variants thereof, including, but not limited to, midi tion into the clot. plasmin, mini-plasmin, or micro-plasmin. 0018. In another aspect of the invention, the fibrinolytic DETAILED DESCRIPTION composition of the present invention comprises a reversibly 0032. A full and enabling disclosure of the present inven inactivated acidified plasmin Substantially free of a plasmi nogen activator, a low buffering capacity buffer, and option tion, including the best mode known to the inventors of car ally, a stabilizing agent. Plasmin includes Glu-plasmin or rying out the invention is set forth more particularly in the Lys-plasmin, derivatives and modified or truncated variants remainder of the specification, including reference to the thereof, including, but not limited to, midi-plasmin, mini Examples. This description is made for the purpose of illus plasmin, or micro-plasmin. trating the general principles of the invention and should not 0019 Buffers employed in the present invention include be taken in the limiting sense. such low buffering capacity buffers which are present in the 0033. The present invention addresses the need for a composition at a concentration at which the pH of the com fibrinolytic composition that is stable on storage and can be position is rapidly raised to a neutral pH by adding no more therapeutically administered to a patient having a thrombotic than about an equal Volume of serum to the composition. In occlusion. Therefore, in one aspect the present invention pro one aspect of the invention, the buffer comprises at least one vides a fibrinolytic composition comprising a reversibly inac pharmaceutically acceptable acid, Such as an amino acid, a tivated acidified serine protease substantially free of a plas derivative of the at least one amino acid, a dipeptide, an minogen activator, a low buffering capacity buffer, and oligopeptide which includes the at least one amino acid, and optionally, a stabilizing agent. Such serine proteases include combinations thereof. Amino acids employable as the buffer trypsin, , II, , US 2008/0311 1 05 A1 Dec. 18, 2008 prostate-specific antigen, leukocyte elastase, chymase, tial spillage of plasmin from the immediate vicinity of the tryptase, acrosin, human tissue kallikrein, and plasmin. Plas thrombus site will be quickly neutralized by circulating min includes Glu-plasmin or Lys-plasmin, derivatives and C2-antiplasmin. modified or truncated variants thereof, including, but not 0039. In the past, there have been several technical chal limited to, midi-plasmin, mini-plasmin, or micro-plasmin. lenges associated with plasmin purification, and storage, as well as with its therapeutic use and delivery. Plasmin is an 0034. The present invention further provides a fibrinolytic active serine protease and is Subject to autodigestion and composition comprising a reversibly inactivated acidified inactivation at a physiological pH. Plasmin degradation, serine protease Substantially free of plasminogen activator unfortunately, is also most evident in the pH range required and a pharmaceutically acceptable acidified carrier, further for in vivo thrombolysis. comprising a pharmaceutically acceptable stabilizing agent. 0040. The fibrinolytic composition, as incorporated into 0035. In another aspect of the present invention, the fibrin the present invention, includes the maintenance of the plas olytic composition of the present invention comprises a min in an acidic buffer during purification, as well as its reversibly inactivated acidified plasmin substantially free of a formulation in an acidified carrier having a pharmaceutically plasminogen activator, a low buffering capacity buffer, and acceptable low buffering capacity buffer, thereby providing a optionally, a stabilizing agent. Again, plasmin includes Glu reversibly inactivated acidified plasmin-containing fibrin plasmin or Lys-plasmin, derivatives and modified or trun olytic composition Substantially free of plasminogen activa cated variants thereof, including, but not limited to, midi tor. It is contemplated to be within the scope of the present plasmin, mini-plasmin, or micro-plasmin. invention for the fibrinolytic composition to be a lyophilized composition that may be reconstituted by the addition of a 0036 Buffers employed in the present invention include pharmaceutically acceptable carries such as, but not limited such low buffering capacity buffers which are present in the to, water, physiological saline or any other solvent that will composition at a concentration which would allow the pH of allow administration of the composition to a human or ani the composition to be changed to a physiological pH by mal. Its efficacy in restoring vascular patency was demon contacting body fluids. In an aspect of the invention, the strated in in vitro assays and in an in vivo rabbit jugular vein buffer comprises at least one pharmaceutically acceptable thrombolysis model. acid, such as an amino acid, a derivative of the at least one 0041. The term “reversibly inactivated as used herein amino acid, a dipeptide, an oligopeptide which includes the at refers to an enzymatic activity that is substantially free of least one amino acid, and combinations thereof. Amino acids activity under a specific set of conditions but will revert to an employable as the buffer include serine, threonine, methion active form when transferred to another set of conditions. ine, glutamine, alanine, glycine, isoleucine, Valine, aspartate, 0042. The term “pharmaceutically acceptable carrier as alanine, and combinations thereof. Other low buffering used herein refers to any carrier that is physiologically toler capacity acids may be employed and include formic acid, ated by a recipient human or animal, including, but not lim acetic acid, citric acid, hydrochloric acid, lactic acid, malic ited to, water, salt solutions, physiological saline, or any other acid, tartaric acid, benzoic acid, derivatives thereof, and com liquid or gel in which a fibrinolytic agent such as plasmin may binations thereof. The amino acids and the other low buffer be dissolved or suspended. The “pharmaceutically acceptable ing capacity acids may be combined in any desired combina carrier may include any pharmaceutically acceptable com tion as well. pound that will give a plasmin solution having a pH below 0037 Stabilizing agents which may be employed in the about 4.0 and which has low or Zero buffering capacity. present invention include pharmaceutically acceptable carbo 0043. The term “physiological pH as used herein refers to hydrates, salts, glucosamine, thiamine, niacinamide, citrul a pH between about pH 6.5 and about 7.5, ore typically line, and combinations thereof. Further stabilizing agents between about pH 7.1 and about 7.5. include, but are not limited to, monosacchrides, disacchrides, 0044) The term “body fluid as used herein refers to any polysacchrides, polyhydric alcohols, or combinations body fluid including, but not limited to, blood, serum, plasma, thereof. For example, Such stabilizing agents include Sugars semen and urine. or Sugar alcohols, such as glucose, maltose, mannitol, Sorbi 0045. The term “low pH buffering capacity buffer” or tol. Sucrose, lactose, trehalose, or combinations thereof. Salts, “low buffering capacity buffer as used herein refers to the Such as sodium chloride, potassium chloride, magnesium amount of acid or base that a buffer can neutralize before the chloride, calcium chloride, or combinations thereof, are pH begins to change to an appreciable degree. As used herein employable as stabilizing agents in the present invention. a low buffering capacity buffer will be significantly pH 0038. With the escalating use of arterial and venous cath adjusted by the addition of a small volume of an acid or base eters in the clinics, local delivery of an active plasmin in close relative to the volume of the low buffering capacity buffer proximity to, or actually into, a thrombus offers an attractive solution. For example, in the present invention, the buffer is therapeutic opportunity in thrombolytic therapy. Being an present in the compositionata concentration that would allow active serine protease, plasmin is a direct thrombus-dissolv the pH of the composition to be changed by contacting a body ing agent, in contrast to plasminogen activators that require fluid. This term is meant to include solutions acidified by the presence of the Zymogen plasminogen in the vicinity of strong acids including, but not limited to, hydrochloric acid, the thrombus. Local catheter-directed thrombolytic therapy nitric acid and sulfuric acid, and which have no buffering with active plasmin can be regulated to achieve total throm capacity. bolysis, and plasmin has the potential to be a safer throm 0046. The term “thrombus' as used herein refers to a bolytic agent because the lower dosage required for local thrombus in a blood vessel or device contacting blood (e.g. delivery may significantly reduce bleeding complications fre catheter devices or shunts). A thrombus may comprise fibrin quently associated with high dose thrombolytic therapy and may further comprise, but is not limited to, platelets, induced by plasminogen activators. Furthermore, any poten erythrocytes, lymphocytes, lipid or any combination thereof. US 2008/0311 1 05 A1 Dec. 18, 2008

A “thrombus' may be, but is not limited to, an annular throm refers to a plasmin under conditions where the plasmin is bus, ball thrombus, hyaline thrombus, mural thrombus, strati capable of proteolytically cleaving fibrin. The term “plasmin' fied thrombus or white thrombus. includes, but is not limited to Glu-plasmin, Lys-plasmin, 0047. The term “thrombotic occlusion” as used herein derivatives, modified or truncated variants thereof. The term refers to a partial or total blockage of a vessel due to the “truncated variants' includes, but is not limited to, midi formation of a thrombotic clot, wherein the thrombus com plasmin, mini-plasmin or the micro-plasmin as disclosed in prises at least fibrin. The vascular vessel occluded may be, but U.S. Pat. No. 4,774,087 incorporated herein by reference in is not limited to, a vein, artery, Venule, arteriole, capillary, its entirety. vascular bed or the heart and may be Within any vascularized 0054 The term “anti-coagulant’ as used herein refers to organ or tissue of the human or animal body. The thrombotic any compound capable of inhibiting the formation of a throm occlusion may also be of a catheter or other implant includ bus including, but not limited to, hiruidin, heparin, thrombin ing, but not limited to, prosthetic vessels and grafts of Syn inhibitors, platelet inhibitors, and any derivatives or combi thetic, human or animal origin and effectively blocked by an nations thereof. occlusion comprising fibrin. 0055. The term “serine protease” as used herein refers to 0.048. The term “catheter device' as used herein refers to any serine protease capable of proteoytically cleaving fibrin any catheter or tube-like device that may enter the body, and including, but not limited to, plasmin, trypsin, chymotrypsin, includes but is not limited to, an arterial catheter, cardiac pancreatic elastase II, cathepsin G, prostate-specific antigen, catheter, central catheter, central venous catheter, intravenous leukocyte elastase, chymase, tryptase, acrosin and human catheter, peripherally inserted central catheter, pulmonary tissue kallikrein. artery catheter or tunneled central venous catheter and arte 0056. One limitation of current thrombolytic therapy with rio-Venal shunts. plasminogen activators is plasminogen availability Surround 0049. The term “pharmaceutically acceptable acidified ing or within a thrombus. The local delivery of a fibrinolytic carrier as used herein refers to any pharmaceutically accept agent to a thrombus now allows plasmin itself to be a potent able carrier that has been acidified to a pH below about 4.0. therapeutic agent directly administered to a thrombus. In 0050. The “pharmaceutically acceptable acidified carrier' contrast to various plasminogen activators that are currently may comprise a low or Zero buffering capacity buffer Such as used as thrombolytics, direct localized thrombolytic therapy a carboxylic acid such as, but not limited to, formic acid, with plasmin can be intensified to whatever level is required acetic, proprionic, butyric, citric, Succinic, lactic or malic to achieve clot lysis. This is because plasmin acts directly acids acidified to a pH below about 4.0 by the addition of an upon the fibrin polymer. Also, plasmin, when delivered inorganic acid; or at least one amino acid such as, but not directly into or adjacent to a thrombus, allows a lower effec limited to, glycine, alanine, Valine, isoleucine, threonine or tive dose to be administered with a concomitant reduction in glutamine, methionine, serine, aspartic acid or at least one the systemic hemorrhage typically associated with conven inorganic acid such as, but not limited to, Sulfuric acid, hydro tional thrombolytic therapy. Excess plasmin can also be rap chloric acid, nitric acid or phosphoric acid or any combina idly inactivated by circulating C2-antiplasmin. tion thereof. It is contemplated to be within the scope of the 0057 The present invention contemplates that plasmin present invention for the acid moiety of the pharmaceutical may be produced from plasminogen using any method that carrier to be at least one physiologically tolerated buffer, will yield a purified active plasmin substantially free of plas oligopeptide, inorganic or organic ion or any combination minogen activator. It is within the scope of the present inven thereof that will maintain a pH in the pharmaceutically tion for the plasminogen to be any recombinant plasminogen acceptable carrier below a value of about 4.0. or a truncated plasminogen such as, but not limited to, the 0051. The term “carbohydrate” as used herein refers to any mini-plasminogen and micro-plasminogen, as disclosed by pharmaceutically acceptable saccharide or disaccharide Such Wu et al. in U.S. Pat. No. 4,774,087 incorporated herein by as, but not limited to, glucose, fructose, maltose, Sucrose, reference in its entirety. For example, Examples 1 and 2 below lactose, trehalose, mannose, Sugar alcohols including, but not disclose a method whereby active plasmin was prepared from limited to, Sorbitol and mannitol, and polysaccharides Such plasminogen purified from Cohn Fraction II+III. The purity as, but not limited to, dextrins, dextrans, glycogen, starches of the plasmin obtained using this method was greater than and celluloses, or any combination or derivative thereof that 95% and the specific activity was in the range of 18-23 are pharmaceutically acceptable to a human or animal. CU/mg. The plasmin preparations were substantially free of 0052. The term “stabilizing agent” as used herein refers to urokinase, or any other plasminogen activator used for con at least one compound Such as, but not limited to, polyhydric version of plasminogen into plasmin. alcohols, glycerol, ascorbate, citrulline, niacinamide, glu 0058. The plasmin of the present invention was purified by cosamine, thiamine, or inorganic salt Such as, but not limited binding to a benzamidine affinity column and the Subse to, sodium chloride, potassium chloride, calcium chloride, quently eluted plasmin was collected and stored in an acidi magnesium chloride or manganese chloride or any combina fied pharmaceutically acceptable carrier. Results showed that tion thereof that will increase the stability of a preparation of a low pH in the range of about 2.5 to about 4.0 greatly plasmin. stabilized the plasmin composition, even when held at room 0053. The term “reversibly inactivated acidified plasmin' temperature or greater. While not bound by any one theory, it as used herein refers to any catalytically active form of plas is believed that at this low pH the plasmin has minimal serine min capable of proteolytically cleaving fibrin when under protease activity that would otherwise lead to autodegrada physiological conditions, but reversibly inactivated when tion, as is seen when plasmin is stored at physiological pH of placed at a pH between about pH 2.5 to about 4.0. The term between about 7.0 to about 7.5. “inactivated as used herein refers to a total or substantial 0059. When the plasmin is administered directly into a reduction in enzymic activity compared to the activity at thrombus, or proximal thereto, the plasmin encounters the physiological pH. The term “active plasmin' as used herein physiological pH of the clot of about 7.4. The acidified phar US 2008/0311 1 05 A1 Dec. 18, 2008

maceutically acceptable carrier attains the pH value of the catheter to the thrombus, the reversibly inactivated acidified thrombus, whereupon the plasmin recovers its serine protease plasmin composition's exposure to serum inhibitors is activity and begins to digest fibrin. Furthermore, the high reduced. Catheter delivery to a thrombus allows precision in concentration of fibrin within the thrombus provides an alter placing the plasmin composition, especially within the native Substrate for the plasminto minimize auto-degradation thrombus. and maximize thrombolysis. 0065. A description of the method of treating a thrombotic occlusion in a patient using atherapeutically effective dose of 0060 Fibrinolytic therapy employing a plasmin prepara the reversibly inactivated acidified plasmin compositions of tion that renders plasmin proteolytically inactive until admin the present invention is disclosed in U.S. patent application istered into, or immediately adjacent to, a thrombus and Ser. No. 10/143,157, entitled “Method of Thrombolysis by which is also Substantially free of any plasminogen activator Local Delivery of Reversibly Inactivated Acidified Plasmin', reduces the likelihood of undesirable systemic hemorrhage. commonly assigned and filed contemporaneously with the Excess administered plasmin is rapidly inactivated by circu instant application, and is incorporated herein by reference in lating serum inhibitors such as C-antiplasmin, and the plas its entirety. minogen activators that would otherwise circulate to induce 0066. Additionally, a process for producing the reversibly distal fibrinolysis are substantially absent. inactivated acidified plasmin composition of the instant 0061 Reversibly inactivated acidified plasmin, of the invention is disclosed in U.S. patent application Ser. No. present invention may be readily stored, even at 37°C., in low 10/143,156, entitled “Process for the Production of a Revers buffering capacity pharmaceutically acceptable carriers such ibly Inactivated Plasmin Composition', commonly assigned as, but not limited to, 2 mM Sodium acetate. Any pharmaceu and filed contemporaneously with the instant application, and tically acceptable moiety may be used, singularly or in com is incorporated herein by reference in its entirety. bination that maintains the compositionata pH in the range of 0067 Thus, the reversibly inactivated acidified plasmin composition of the present invention can be stored without a about 2.5 to about 4.0, especially at a pH of about 3.1 to about significant loss in the activity of the plasmin restored by pH 3.5. Acidic compounds useful in the present invention, adjusting the pH of the composition to physiological pH and either singly or any combination thereof, include but are not safely used as a thrombolytic agent during catheter-assisted limited to formic acid, acetic acid, citric acid, hydrochloric administration to a patient having a thrombotic occlusion. acid, a carboxylic acid Such as, but not limited to, lactic acid, The present invention is a plasmin composition Substantially malic acid, tartaric acid, benzoic acid, serine, threonine, free of plasminogen activators that exhibits at least compa methionine, glutamine, glycine, isoleucine, Valine, alanine, rable fibrinolytic activity to thA and the safety profile appears aspartic acid, derivatives thereof, or combinations thereof at least similar in this animal model of local thrombolytic that will maintain the pH in the pharmaceutically acceptable delivery. It is contemplated to be within the scope of the carrier between about pH 2.5 to about pH 4.0. present invention that the reversibly inactivated acidified 0062. The reversibly inactivated acidified plasmin compo fibrinolytic enzyme may be, but is not limited to, plasmin, sition of the present invention may further comprise at least derivatives of plasmin such as truncated forms thereof includ one stabilizing agent such as a pharmaceutically acceptable ing, but not limited to, mini-plasmin and micro-plasmin as carbohydrate including, but not limited to, monosaccharides, disclosed by Wu et al. in U.S. Pat. No. 4,774,087 incorporated disaccharides, polysaccharides, and polyhydric alcohols. For herein by reference in its entirety. example, pharmaceutically acceptable carbohydrate stabiliz 0068. The present invention is further illustrated by the ers contemplated to be within the scope of the present inven following examples, that are provided by way of illustration tion include Sugars such as, but not limited to. Sucrose, glu and should not be construed as limiting. Even though the cose, fructose, lactose, trehalose, maltose and mannose, and invention has been described with a certain degree of particu Sugar alcohols including, but not limited to, Sorbitol and larity, it is evident that many alternatives, modifications, and mannitol. Contemplated within the scope of the present variations will be apparent to those skilled in the art in light of invention are polysaccharides such as, but not limited to, the present disclosure. Accordingly, it is intended that all Such dextrins, dextrans, glycogen, starches and celluloses, or any alternatives, modifications, and variations that fall within the combination thereofpharmaceutically acceptable to a human spirit and the scope of the invention be embraced by the or animal patient. defined claims. 0063 Stabilizing agents contemplated as within the scope 0069. It should be appreciated by those of skill in the art of the present invention and useful in stabilizing the revers that the techniques disclosed in the examples which follow ibly inactivated acidified plasmin composition of the present represent techniques discovered by the inventors to function invention include, but are not limited to, glycerol, niacina well in the practice of the invention, and thus can be consid mide, glucosamine, thiamine, citrulline and inorganic salts ered to constitute preferred modes for its practice. Those of Such as, but not limited to, sodium chloride, potassium chlo skill in the art should, however, in light of the present disclo ride, magnesium chloride, calcium chloride, or any combina Sure, will appreciate that many changes can be made in the tion thereof. Other stabilizing agents contemplated as within specific embodiments disclosed and still obtain like or similar the scope of the present invention may include, but are not results without departing, again, from the spirit and scope of limited to, pharmaceutically acceptable compounds Such as the present invention. The contents of all references, pub benzyl alcohol or benzoic acid, to retard microbial contami lished patents and patents cited throughout the present appli nation. cation are hereby incorporated by reference in their entirety. 0064. A plasmin composition according to the present EXAMPLES invention can be administered by any method that will deliver the plasmin as a bolus or as a prolonged infusion directly into Example 1 a thrombus, or to a site a short distance proximal to the Sources of Proteins Investigated thrombus whereupon the plasmin composition can rapidly 0070 Plasminogen was purified from Cohn Fraction encounter the thrombus. By minimizing the distance from the II+III paste by affinity chromatography on Lys-Sepharose as US 2008/0311 1 05 A1 Dec. 18, 2008

described by Deutsch & Mertz (1970). Thus, 200 g of the and combined with 5 ml of urokinase-Sepharose. The use of paste was resuspended in 2 liter of 0.15M sodium citrate 50% glycerol reduces autodegradation of plasmin during buffer, pH 7.8. The suspension was incubated overnight at 37° activation. Plasmin is stable in 50% glyceroland can be stored C., centrifuged at 14,000 rpm, filtered through fiberglass and in this solution at -20°C. for an extended period. mixed with 500 ml of Lys-Sepharose 4B (Pharmacia). Bind 0074 The plasminogen activation occurred at room tem ing of plasminogen was at room temperature for 2 hours. The perature for between 2 hours and 24 hours depending on the Lys-Sepharose was then transferred onto a 2 liter glass filter, freshness of the urokinase-Sepharose. With a fresh batch of and washed several times with 0.15M sodium citrate contain urokinase-Sepharose, activation could be completed in 2 ing 0.3M NaCl until the absorbance at 280 nm dropped below hours. It deteriorates, however, and becomes less efficient 0.05. Bound plasminogen was eluted with three 200 ml por after several cycles, necessitating the use of SDS-PAGE tions of 0.2M e-aminocaproic acid. Eluted plasminogen was under reducing conditions to monitor the progress of plasmi precipitated with 0.4 g solid ammonium Sulfate/ml of plas nogen activation. Upon completion of the activation, the plas minogen solution. The precipitate of crude (80-85% pure) min solution was filtered from the urokinase-Sepharose with plasminogen was stored at 4°C. a glass filter, and immediately applied to benzamidine 0071 Low-molecular weight urokinase (LMW-uroki Sepharose. nase) (Abbokinase-Abbott Laboratories, Chicago III.) was further purified by affinity chromatography on benzamidine (ii) Capturing of Plasmin on Benzamidine-Sepharose. Sepharose. The Urokinase was then coupled to CNBr-acti vated Sepharose 4B by mixing 1.3 mg of LMW-urokinase in 0075 Since the plasmin is a serine protease with trypsin 50 mMacetate buffer, pH 4.5, and diluting with 5 ml of the like specificity, benzamidine-Sepharose is an affinity absor coupling buffer, 0.1M sodium bicarbonate, pH 8.0. bent that allowed capture of the active plasmin. A plasmino 0072 This solution was immediately combined with 5 ml gen solution in 50% glycerol was applied to the 50 ml of CNBr-activated Sepharose previously swollen and washed benzamidine-Sepharose column equilibrated with 0.05M in 0.1 MHC1. The coupling occurred for 4 hours on ice with Tris, pH 8.0, containing 0.5M NaCl with a flow rate of 3 shaking. The excess of the CNBr active group was blocked ml/min. The column was run at 3 ml/min at 3-7°C. The front with 0.1M Tris, pH 8.0. Each batch of urokinase-Sepharose portion of the non-bound peak contained high-molecular was used 5 times and stored in 50% glycerol in water at 4°C. weight impurities. The rest of the non-bound peak is repre between the cycles. Tissue plasminogen activator (Activase) sented by residual non-activated plasminogen and by inactive was from Genentech. Plasminogen-free fibrinogen and autodegradation products of plasmin. C-thrombin (3793 U/ml) were from Enzyme Research, Inc. (iii) Elution of the Bound Plasmin with Low pH Buffer. C-Antiplasmin was obtained from Athens Research Tech 0076. To protect plasmin from inactivation at neutral pH nologies. Commercially available plasmin was from Haemo conditions, acidic elution conditions were selected. The plas tologic Technologies, Inc. Chromogenic plasmin Substrate min bound to benzamidine-Sepharose was eluted with 0.2M S2251 was from Chromogenix. ''I-Labeled human fibrino glycine buffer, pH 3.0 containing 0.5M NaCl. The bound gen (150-250 uCi/mg) was from Amersham Pharmacia Bio peak was typically divided into three pools, two front peaks, tech. SDS-polyacrylamide gel electrophoresis was per B1 and B2, and the bulk of the eluted material as B3. formed in the Pharmacia Phast System apparatus using pre 0077. Non-reducing gel analysis showed that all three made 8-25% gradient gels and SDS-buffer strips. The source pools contained highly pure (>95%) plasmin. The gel analy of plasminogen is not limited to purification from a plasma sis, however, in addition to the heavy and light chains of Source. It is contemplated that plasminogen may also be plasmin, revealed some low molecular weight bands in a obtained from a transgenic or recombinant source. range of 10-15 kDa as a result of partial internal cleavage degradation of the plasmin. Example 2 0078. The front portion of peak B1 typically contained most of the low molecular weight impurities. The B2 and B3 Purification of Active Plasmin pools were less degraded. The front portion of the bound peak (i) Activation of Plasminogen to Plasmin Using Urokinase had very little of the plasmin activity and was usually dis Sepharose. carded. The loss of activity in this material may be due to autodegradation during chromatography, because there is no 0073 Plasminogen was cleaved to plasmin yielding plas glycerol present in the eluted material, and the pH of the front min without contamination of the final preparation by using portion is intermediate between the pH of the equilibrating an immobilized plasminogen activator. Urokinase cleaves and eluting buffers, typically in a range of pH 6-6.5. The plasminogen directly. Plasminogen activation by urokinase eluted plasmin, Substantially free of plasminogen activators, does not depend on the presence offibrinas in the case oftBA, was collected in tubes containing 2M glycine buffer, pH 3.0 and urokinase is a human protein. These factors, and its relative low cost, make urokinase the preferred activator, (10% of the collected volume). although this does not preclude the use of tRA, streptokinase or any other cleavage means yielding an active plasmin (iv) Formulation of Eluted Material in Acidified Water (pH capable offibrin degradation. The ammonium Sulfate precipi 3.7). tate of crude plasminogen was centrifuged at 14,000 rpm and 0079 Eluted plasmin was dialyzed with water and acidi resuspended in a minimal volume using 40 mM Tris, contain fied to about pH 3.7 with glacial acetic acid. Any acid pro ing 10 mM lysine, 80 mM. NaCl at pH 9.0 to achieve the final viding a pharmaceutically acceptable acidified carrier having protein concentration of 10-15 mg/ml. The plasminogen solu a low buffering capacity buffer and having a pH between tion was dialyzed overnight against the same buffer to remove about 2.5 to about 4.0 can be used. For example, also con ammonium Sulfate. The dialyzed plasminogen solution (10 templated within the scope of this invention is the use of other 20 ml) was diluted with an equal volume of 100% glycerol acids and amino acids such as, but not limited to, inorganic US 2008/0311 1 05 A1 Dec. 18, 2008 acids, carboxylic acids, aliphatic acids and amino acids hours by reducing SDS-PAGE analysis. Both the heavy chain including, but not limited to, formic acid, acetic acid, citric and light chain degraded rapidly within hours at 22°C. and 4 acid, lactic acid, malic acid, tartaric acid, benzoic acid, serine, C. as shown in Table 1.

TABLE 1 The rapid degradation of plasmin in neutral pH solution at 22 C. and 4 C. % of intact heavy chain % of intact light chain Plasmin Buffer PH Temp Initial 2 hr 4 hr 6 hr Initial 2 hr 4 hr 6 hr 1 mg/ml 0.04M 7.4 22° C. 100%. 27%. 27%. 29%. 100%. 29%. 26%. 28% PO 1 mg/ml 0.04M 7.4 4° C. 100% 32%. 27%. 25%. 100%. 33%. 25%. 22% PO (The intact heavy chain and light chain of plasmin at initial time point were normalized as 100%.) threonine, Valine, glycine, glutamine, isoleucine, P-alanine I0083) Plasmin at 1 mg/ml was incubated at 37° C. for 14 and derivatives thereof, either singly or any combination days under different acidic conditions. The changes in plas thereof, that will maintain the pH in the pharmaceutically min heavy chain and light chain were analyzed by running acceptable carrier of about 2.5 to about 4.0. reducing SDS-PAGE. Plasmin was formulated at 1 mg/ml in 0080 Plasmin-specific activity was measured using an adapted caseinolytic assay as described by Robbins & Sum 0.04M sodium phosphate, pH 7.4 and was also incubated at 4 maria (1970). One ml of 4% casein solution in acidified water C. for six hours. During the incubation, the activity of the and an appropriate Volume of 67 mM Sodium phosphate plasmin sample was measured every two hours by chromoge buffer, pH 7.4 was added to a test polycarbonate tube. The nic potency assay. Plasmin potency was quantitatively mea solutions were vortexed and incubated at 37° C. for 10 min sured using the MLA 1600C analyzer (Pleasantville, N.Y.). utes. Plasmin samples or buffer (blank) were added to each Plasmin hydrolyzed the chromogenic substrate S-2403 tube at 15 second intervals, mixed thoroughly and incubated (D-pyroglutamyl-L-Phenylalanyl-L-Lysine-p-Nitroaniline at 37° C. for 30 minutes. The reaction was stopped with the hydrochloride or abbreviated as pyro-Glu-Phe-Lys-pNA) to addition of 3 ml of 15% trichloroacetic acid and the precipi form peptide and the chromophoric group p-nitroaniline tate was allowed to form for 15 minutes. The tubes were (pNA). The rate of color formation was measured kinetically centrifuged at 3200 rpm for 20 minutes. The supernatants at 405 nm. The amount of substrate hydrolyzed was propor were transferred to cuvettes and the Aso of each sample was tional to the plasmin activity in the sample. A standard curve determined. The specific caseinolytic activity of each sample was generated from the linear regression of the rate of color was determined by the following formula: formation (OD/min) versus the potency of a plasmin stan dard. The linear equation together with the observed rate for an unknown sample was used to calculate the potency of 3.27XA280 (Plasmin Sample) - A280 (Blank) unknowns. The potency of plasmin was reported in units of ug Plasmin in Assay mg/ml. I0084 Plasmin integrity was significantly decreased by The plasmin concentration was determined spectrophoto incubation at a physiological pH, as shown in Table 2. metrically using the extinction coefficient of 1.7 for 0.1% Solution. TABLE 2 Example 3 The rapid decrease of plasmin activity in neutral pH Solution at 4 C. pH-Dependent Stability of Plasmin Chronogenic Potency 0081 Plasmin exhibits abell-shaped pH dependence of its catalytic activity. As shown in FIG. 1, plasmin has maximum Plasmin Buffer pH Initial 2hr 4hr 6hr enzyme activity at pH 7.5-8.0, and its activity rapidly 1 mg/ml PO, 0.04M 7.4 100%. 43.3% 32.6% 26.4% decreases at either more alkaline or more acidic pHs. Plasmin is mostly inactive, and reversibly so, below pH 4.0, due to the (The activity of plasmin Solution at initial time point was normalized as 100%.) protonation of histidine in the catalytic center, as shown by In this neutral pH solution, plasmin activity decreased more than 70% after 6 Robbins & Summaria, (1976) and Castellino & Powell hours at 4°C. (1981). 0082 Plasmin is very unstable at a physiological pH. Both I0085 Plasmin formulated in acidified water at pH 3.7 is the heavy chain and light chains of plasmin degraded dramati stable. It can be kept in this form for months at reduced cally within hours at room temperature and 4°C. Plasmin was temperatures without any loss of activity or the appearance of formulated at 1 mg/ml in 0.04M sodium phosphate, pH 7.4, degradation products of a proteolytic or acidic nature. FIG. 2 and incubated at 22°C. or 4°C. for 6 hours. During the and the data of Table 3 show the stability of plasmin at 4°C. incubation, the plasmin integrity was analyzed every two and at room temperature. US 2008/0311 1 05 A1 Dec. 18, 2008

is formulated in a low buffering capacity solvent and, when TABLE 3 added to a buffered solution Such as plasma, it rapidly adopts the neutral or physiological pH instantly and the precipitation Stability of 1 mg/ml plasmin in the following that usually accompanies the slow passage through the iso acidic conditions at 37°C. electric point, does not occur. % intact % intact heavy chain light chain Example 4 after 14 after 14 Formu- Plasmin days at days at Plasmin has the Same Intrinsic Fibrinolytic Potency lation (mg/ml) Acidic Condition pH 37o C. 37o C. as a Plasminogen/Plasminogen Activator Mixture 1 5 mM HACANaAc 2.5 1996 62% 2 5 mM HACANaAc 3.0 41% 92% I0089 Plasmin has the same intrinsic fibrinolytic potency 3 5 mM HACANaAc 3.4 48% 92% as a plasminogen/plasminogen activator mixture. Fibrin 4 5 mM HACANaAc 3.4 49% 96% 5 5 mM HACANaAc 3.4 SO% 96% olytic potency of plasmin was compared with that of a Lys 6 5 mM HACANaAc 3.7 13% 123% plasminogen and tRA mixture. These experiments were per 7 5 mM HACANaAc 4.0 9.3% 1.07% formed in a defined system consisting of an 'I-radiolabeled 8 5 mM citric 2.27 9.3% 64% fibrin thrombus submersed in PBS. FIG. 4 shows that, in a acid Na citrate 9 5 mM citric 3.1 33% 68% buffered environment, thrombolysis achieved with plasmin is acid Na citrate almost identical to the Lys-plasminogen plus t-A mixture 10 5 mM citric 3.56 46% 88% (curves a and b, respectively). At the same time, no throm acid Na citrate bolysis was observed with thA alone (curve c) or in the 11 5 mM citric 4.0 7.4% 1.04% acid Na citrate absence of any proteins (curved). The data obtained with thA 12 5 mM glycine 2.2 7.3% 1.04% alone shows that its activity is dependent on its Substrate, 13 5 mM glycine 3.1 36% 70% plasminogen, to be an effective thrombolytic. 14 5 mM glycine 3.5 49% 85% 0090. These data indicate that, in the absence of inhibitors 15 5 mM glycine 3.8 12% 85% 16 5 mM glycine 4.1 6% 81% and other protein factors present in plasma, there is no differ 17 5 mM serine 3.4 56% 100% ence in the ability to lyse fibrin thrombi between purified 18 5 mM threonine 3.4 S4% 100% plasminand the combination oftBA and Lys-plasminogen. To 19 5 mM valine 3.4 52% 96% assess the thrombolytic potency of active plasmin, the I 2O 5 mM isoleucine 3.4 51% 100% 21 5 mM B-alanine 3.7 33% 90% fibrin-labeled thrombolysis assay was performed with plasma 22 2 mM benzoic 3.5 42% 93% thrombi in a plasma environment. acid 23 2 mM lactic acid 3.5 45% 91% Example 5 24 2 mM malic acid 3.5 SO% 90% 25 2 mMtartaric acid 3.5 28% 87% Stabilization of Reversibly Inactivated Acidified (The intact heavy chain and light chain in each formulation before incuba Plasmin Composition with a Sugar or Sugar Alcohol tion were normalized as 100%; HAcNaAc— = acetic acidsodium acetate) 0091 Acidified plasmin compositions were formulated I0086. At 4°C., plasmin is stable for at least nine months. according to the present invention, as described in Examples 1 At room temperature, reversibly inactivated acidified plasmin and 2, in 5 mM of acetic acid at pH 3.7 with 0.1M of maltose, is stable for at least two months. To determine the optimal pH mannitol. Sucrose or Sorbitol added as a stabilizer. A plasmin of different buffering agents and the effect of buffer concen composition formulated without any excipient was included tration on plasmin Stability, compositions of 1 mg/ml of plas as a control. All samples were incubated at 37°C. for 7 days min were prepared in 10 mM, 20 mM or 40 mM sodium and the change in plasmin integrity analyzed using SDS acetate or glycine at various pH values. The samples were PAGE under reducing conditions, as described in Example 2 stored at 37° C. for 7 days, and the relative amount of intact above. FIG. 5 demonstrates that the percent degradation of plasmin heavy chain remaining was determined by densito plasmin in the low pH compositions formulated with a Sugar metry of Coomassie stained SDS gels. Shown in FIG. 3 is a or Sugar alcohol are significantly reduced, as compared to the plot of pH versus percent heavy chain relative to total protein control without a Sugar or Sugar alcohol. in each lane of the SDS gels. The results demonstrate a pH stability optimum of about 3.1-3.5, irrespective of the type of Example 6 buffer, or buffer concentration. 0087 Long-term stability at room temperature is impor Stabilization of Reversibly Inactivated Acidified tant because it would make this formulation compatible with Plasmin Composition with Non-Carbohydrate Stabi long regimens of thrombolytic administration. For example, lizing Agents 36 hour administration of thrombolytics such as tissue plas 0092 Reversibly inactivated acidified compositions were minogen activator or urokinase is common in treatment of formulated at 1 mg/ml in 5 mMacetic acid, pH 3.7, according peripheral arterial occlusions. to the present invention, with 0.1M of glucosamine, niacina 0088. The ability of reversibly inactivated acidified plas mide, citrulline or thiamine added as a non-carbohydrate minto become fully active upon transfer to physiological pH stabilizer. A reversibly inactivated acidified plasmin formu is evidenced by its activity in the caseinolytic assay and also lation without any excipient stabilizing agent was included as in the 125'-fibrin-labeled thrombolysis assays. Both of these a control. All samples were incubated at 37°C. for 7 days and assays are performed at pH 7.4, and there was complete the change in plasmin integrity analyzed using SDS-PAGE recovery of plasmin activity during the change of pH and under non-reducing conditions. Referring now to FIG. 6, all passing through the isoelectric point (pH 5-5.5). The plasmin of the non-Sugar stabilizing agents tested improved the sta US 2008/0311 1 05 A1 Dec. 18, 2008 bility of the reversibly inactivated acidified plasmin compo 0097. Three polypeptides generated by incubation of plas sition at 37° C. over the 7 day test period. min at pH 2.2 began with proline at the N-termini. From the 0093. A reversibly inactivated acidified plasmin composi known amino acid sequence of plasmin, it was determined tions was also formulated at 1 mg/ml in 2 mMacetic acid, pH that these polypeptides were from the heavy chain, starting at 3.4, according to the present invention, with 150 mM sodium positions P63, P155, and P347, as shown in FIG.8. The amino chloride as a stabilizing agent. The same formulation, but acid preceding each proline was an aspartic acid (D62, D154, without sodium chloride, was also prepared and included as a and D346). It is commonly known that aspartyl-prolyl (D-P) control. Samples were incubated at 4° C. for 28 days. The peptide bonds are acid labile. These results demonstrated that change in plasmin integrity was analyzed using SDS-PAGE compositions of plasmin at pH 2.2 were susceptible to acid under non-reducing conditions, as described in Example 3 hydrolysis of peptide bonds. above. The activity was assessed also as described in Example 3. Values were normalized relative to day 0 controls Example 8 that were assigned a value of 100%. The results, as shown in Table 5, demonstrated that plasmin stored at 4°C. was more Low Buffer Capacity of Compositions stable in the low-pH formulation containing sodium chloride. 0.098 Plasmin (1 mg/ml) was formulated in 2 mM acetic, benzoic, lactic, malic or tartaric acid at pH 3.5. The effect of TABLE 5 admixing increasing Volumes of human blood serum to the Stability of reversibly inactivated acidified plasmin pH of 1 ml of the plasmin solution was measured (FIG.9). In composition (2 mM sodium acetate, pH 3.4) with or without all cases, only a small amount of serum, typically 10 to 30% 150 mM sodium chloride, stored at 4°C. of the plasmin Volume, was required to achieve a pH of about 7. In a separate experiment, plasmin compositions contained Sodium chloride % intact heavy % intact light % activity Concentration Plasmin chain after 28 chain after 28 after 28 either 5 or 10 mMacetic acid. The volumes of serum required (mM) (mg/ml) days days days to neutralize the plasmin solution were 30% and 70% of the initial volume. These results demonstrated that low buffering O 1 90 93 81 capacity compositions, of typically around 2 mM, but 150 1 101 95 97 upwards of 100 mM, are readily restored to a pH of about 7. (The intact heavy chain, light and potentcy in each formulation were normal These results also suggest that plasmin would be readily ized before incubation as 100%) neutralized locally within a thrombus and that large volumes (relative to the liquid fraction of the clot) of plasmin could be Example 7 used to affect lysis. Degradation Pattern of Reversibly Inactivated Acidi Example 9 fied Plasmin Composition Characterized by N-Ter Lysis of Thrombi by Reversibly Inactivated Acidified minal Sequencing Plasmin in an in Vitro Thrombus Model 0094. The degradation peptides of plasmin samples were 0099] To compare the efficacy of plasmin and tRA toward characterized by N-terminal sequencing as follows. Plasmin the lysis of long retracted clots, we have developed an in vitro compositions were formulated at low pH values: a pH less model which would mimic parameters of the clots formed in than 2.5 and a pH of 3.5 and 3.8 containing 2 mMacetic acid. patients with PAO. The plasmin samples were analyzed using SDS-PAGE with 0100 InVitro PAO Model. Fresh whole human blood was 4-12% Bis-Tris NuPage gels, as shown in FIG. 7. The protein collected into 30x0.95 cm glass tubes and allowed to clot bands were transferred to a PVDF membrane, stained with spontaneously without additives. Tubes were incubated for Coomassie Blue R-250 (Bio-RAD Laboratories, Hercules, 20 hours at 37° C. to allow full retraction. Retracted clots Calif.) and bands cut out using a scalpel. were separated from serum using USA Standard testing 0095 N-terminal sequence analysis was performed sieves D16 with 14 mesh and their weights were determined. directly from the membrane using a Hewlett Packard 241 Blood clots were transferred into smaller diameterglass tubes Protein Sequencer (Hewlett Packard, Inc., Glen Allen, Va.). that resembled the average size clots in leg arteries (0.6x12 Ten cycles were run for each band so that the corresponding cm). A multi-side port pulse-spray catheter (French size 5 fragment of plasmin could be identified. Molecular weights with the 11 cm spraying tip, Cook, Inc.) was inserted into the for each band were determined with densitometry analysis clot and a thrombolytic reversibly inactivated acidified plas using the Mark 12 marker available from Invitrogen, Inc. (San min composition according to the present invention, or tRA) Diego, Calif.) at 1 mg/ml was delivered in 1 ml increments separated by 1 0096. Three polypeptides generated by incubation of plas hour time intervals. The number of injections corresponds to min at pH 3.8 began at positions (numbering relative to Lys the dose of thrombolytic. The extent of clot lysis was mea plasmin) threonine (T105), glycine (G190) and glutamic acid Sured by the weight of a residual clot and expressed as a (E623). From the known amino acid sequence of plasmin, it percent of clot weight reduction. Although this model is was determined that the first two polypeptides were from the called a PAO model, and mimics the dimensions of the clots heavy chain and the third from the light chain. As shown in found in PAO patients, venous blood was used for clot for FIG. 8, the amino acid preceding the N-terminal amino acid mation. Both thA and the reversibly inactivated acidified plas was either arginine or lysine (K104, R189, and K622). It is min composition according to the present invention were commonly known that plasmin cleaves proteins on the car tested in this model and the results are presented below. boxyl side of lysine and arginine. These results demonstrated 0101 Plasmin is as effective as tha for lysis of fresh clots, that compositions of plasmin at pH 3.8 were susceptible to unlike when tPA and plasmin are used for lysis of retracted autodegradation. clots aged for 20 hours to allow complete cross-linking by US 2008/0311 1 05 A1 Dec. 18, 2008

Factor XIII. tA is unable to lyse such clots. Clot weight for p-nitrophenol at 410 nm and at pH 8.3 (16,595 M), 100 reduction obtained with tEPA-treated clots is similar to the ul of this pooled trypsin column eluate present in the 1.01-ml control, even when the dose is raised to 5 mg per clot. assay corresponded to a concentration of 22.95 uM trypsin 0102 Plasmin, on the other hand, is effective toward both active sites present in the cuvette. Therefore, the original fully retracted and cross-linked clots. There is a dose-depen stock solution of pooled trypsin column eluate contained 231 dence of the lytic effect of plasmin and after five injections (or uM trypsin active sites. This latter value is identical, within 5 mg plasmin in total) the clots are almost completely lysed. experimental error, to the concentration of total trypsin pro In a similar human series of experiments, the same inability to tein present (225uM). These results demonstrate that trypsin dissolve retracted and cross-linked clots was observed with can be adjusted to low pH and then transferred to a higher pH urokinase. Locally delivered plasmin therefore is a more environment with reactivation of its . effective thrombolytic agent than teA and other plasminogen activators. What is claimed is: 0103) These in vitro data show that tha requires the pres 1. A method of treating a subject Suffering from a disorder ence of its Substrate, plasminogen, in the clot to initiate and Susceptible to improvement with plasmin comprising admin maintain clot lysis. Therefore, while plasmin is as effective as istering to the Subject a composition comprising: tPA for lysing fresh or plasminogen-rich clots, plasmin is a reversibly inactivated, acidified plasmin, the plasmin more effective that tA, and other plasminogen activators, for being Substantially free of a plasminogen activator, and lysing of long retracted plasminogen-poor clots. Moreover, a low buffering capacity buffer; the data presented in this example demonstrates that plasmin wherein the composition is a solution Suitable for pharma is effective in its reversibly inactivated acidified form when it ceutical use that can be raised to physiological pH by is injected directly inside the clot. adding no more than about 5 Volumes of serum to the 0104. The PAO model as described above was used to solution relative to a volume of the solution. compare the efficacy of plasmin (1 mg/ml) formulated in a 2. The method of claim 1, wherein the plasmin is selected low pH formulation described in this invention (saline, pH from Glu-plasmin, Lys-plasmin, midi-plasmin, mini-plas 3.7) with a neutral pH plus stabilizer formulation. The results min, or micro-plasmin. are demonstrated in FIG. 10 and show that the low pH for 3. The method of claim 1, wherein the composition further mulation is as efficacious as the neutral pH plus stabilizer comprises an aqueous carrier. formulation. 4. The method of claim 1, wherein the composition further comprises an anticoagulant. Example 10 5. The method of claim 1, wherein the composition has a Trypsin Stabilized at Low pH can be Reactivated by pH between about 2.5 and about 4. Transfer to Higher pH Environment 6. The method of claim 1, wherein the composition is a sterile solution suitable for parenteral administration to 0105 Trypsin (16.4 mg. Sigma Chemical Co. Catalog No. humans. T-1426) was dissolved in 2.28 ml of 50 mM Tris/0.5M NaCl 7. The method of claim 1, wherein the buffer comprises at (pH 8.0). The trypsin solution was loaded onto a 1-ml column least one acid. of Benzamidine-Sepharose (Pharmacia Code No. 17-0568 01) that had been pre-equilibrated with 50 mM Tris/0.5M 8. The method of claim 8, wherein the acid is in the con NaCl (pH 8.0). This column was washed with 4 ml of this centration range of between about 1 mM and about 100 mM. latter buffer, resulting in a decrease in the eluate absorbance 9. The method of claim 1, wherein the buffer comprises a (280 nm) to less than 0.03. Trypsin was eluted from this carboxylic acid, at least one amino acid, a dipeptide, an oli column in an inactivated state with 0.5-ml volumes of 200 gopeptide which includes the at least one amino acid, or a mM glycine/0.5M NaCl (pH 3.0); the third through fifth combination thereof. 0.5-ml fractions eluted from this column contained the peak 10. The method of claim 1, wherein the buffer is selected values of absorbance (280 nm) and were pooled. The absor from formic acid, acetic acid, citric acid, hydrochloric acid, bance (280 nm) of this pooled trypsin eluate was determined lactic acid, malic acid, tartaric acid, benzoic acid, serine, to be 9.22; based upon the extinction coefficient for trypsin threonine, methionine, glutamine, alanine, glycine, isoleu (E for a 1% solution=17.09) and the molecular weight of cine, Valine, aspartic acid, or combinations thereof. trypsin (24,000), the concentration of total trypsin protein in 11. The method of claim 1, wherein the plasmin is in the this pooled column eluate was calculated to be 225 uM. concentration range of between about 0.01 mg/ml to about 50 0106 The concentration of trypsin active sites in this mg/ml. pooled trypsin column eluate was determined by the method 12. The method of claim 1, wherein the composition fur described by Case & Shaw, Biochem. Biophys. Res. Com ther comprises a stabilizing agent. mun. 29, 508 (1967) and incorporated herein by reference in 13. The method of claim 13, wherein the stabilizing agent its entirety, using p-nitrophenylguanidinobenzoate as active is selected from a polyhydric alcohol, a salt, citrulline, or site titrant. This assay was performed at pH 8.3, by diluting a combinations thereof. small volume (100 ul) of the pooled trypsin column eluate 14. The method of claim 13, wherein the stabilizing agent into an assay mixture also containing 700 ul of 50 mM sodium is a pharmaceutically acceptable carbohydrate, salt, glu borate (pH 8.3), 200 ul of 10 mM sodium phosphate/% gly cosamine, thiamine, niacinamide, citrulline, or combinations cine (pH 7.0) plus 10ul of p-nitrophenylguanidininobenzoate thereof. (dissolved in dimethyl formamide); the final pH of this mix 15. The method of claim 13, wherein the stabilizing agent ture composition was determined to be 8.3. The trypsin is a Sugar or Sugar alcohol selected from consisting of glu dependent amount of p-nitrophenol formed in this assay was cose, maltose, mannitol, Sorbitol. Sucrose, lactose, trehalose, monitored at 410 nm. Based upon the extinction coefficient and combinations thereof. US 2008/0311 1 05 A1 Dec. 18, 2008

16. The method of claim 13, wherein the stabilizing agent 19. The method of claim 13, wherein the stabilizing agent is selected from the group consisting of monosacchrides, is selected from the group consisting of a salt, glucosamine, disaccharides, polysaccharides, polyhydric alcohols, and thiamine, niacinamide, and combinations thereof, and the combinations thereof. concentration of the stabilizing agent is in the range of about 0.01M to about 1 M. 17. The method of claim 13, wherein stablizing agent is at 20. The method of claim 1, wherein the buffer is present in carbohydrate having a concentration in the range of about the composition at a concentration Such that the pH of the 0.2% w/v to about 20% w/v. composition can be raised to physiological pH by adding no 18. The method of claim 13, wherein the stabilizing agent more than about one volume of serum to the solution relative is selected from the group consisting of Sodium chloride, to a volume of the solution. potassium chloride, magnesium chloride, calcium chloride, manganese chloride, and combinations thereof. c c c c c