RS20050031A - Method for treatment of indwelling catheter occlusion using fibrinolytic metalloproteinases - Google Patents

Abstract

A method is provided for the localized intravascular administration of a fibrinolytic metalloproteinase to a human subject in amounts that are both safe and effective to lyse an occluding fibrin-containing blood clot, while also avoiding the neutralizing effects of α2- macroglobulin in the circulating blood. A method is also provided for the treatment of a blood clot in, around or attached to an indwelling vascular access device. A method for restoring patency and function of an indwelling vascular access device is also provided.

Description

PROCEDURE FOR TREATMENT OF OCCLUSIONS

USING A PREPARED CATHETER

OF FIBRINOLYTIC METALLOPROTEINASES

This application is a continuation-in-part of U.S. Pat. Application no. 09/466,276, filed Dec. 17, 1999, which is incorporated herein by reference.

This invention relates to the therapeutic administration of fubrinolytic metalloproteinases, more specifically to the procedure for administering these agents in vivo, via local release into vasculature thrombi, in order to achieve clot lysis. The present invention also relates to a method for dissolving blood clots in a prepared catheter, shunt, or other vascular access device in order to restore the function of that vascular access device.

Vascular occlusions caused by blood clots, such as thrombi and emboli, are serious medical problems that, if not treated promptly, can be limb- or life-threatening. Devices and procedures have been developed for the treatment and removal of vascular blood clots. For illustration, see U.S.

Patent No. 4,447,236 (Quinn), issued May 8, 1984; U.S. Patent No. 4,692,139

(Stiles), admitted September 8, 1987; U.S. Patent No. 4,755,167 (Thistle et al.), issued July 5, 1988; U.S. Patent No. 5,167,628 (Boyles), issued Dec. 1, 1992; U.S. Patent No. 5,222,941 (Don Michael), issued June 29, 1993; U.S.

Patent No. 5,250,034 (Appling et al), issued October 5, 1993; U.S. Patent No. 5,370,653 (Cragg), issued Dec. 6, 1994; U.S. Patent No. 5,380,273 (Dubrul et al.), issued Jan. 10, 1995; U.S. Patent No. 5,498,236 (Dubrul et al.), issued March 12, 1996; U.S. Patent No. 5,626,564 (Zhan et al.), granted May 6, 1997; U.S. Patent No. 5,709,676 (Alt), granted Jan. 20, 1998; U.S. Patent No. 5,865,178 (Yock), granted Feb. 2, 1999; and WO 90/07352 (published July 12, 1990). Those methods and devices include infusion catheters to deliver thrombolytic and fibrinolytic agents into the blood clot and dissolve it. Infusion catheters are typically used in conjunction with enzymatically active agents, which are able to break down the fibrin in the clot and thereby effectively dissolve the clot. These enzymes are typically referred to as thrombolytic or fibrinolytic agents.

Fibrolase is a known fibrinolytic zinc-metalloproteinase, which was first isolated from the venom of the southern copperhead snake (Agkistrodon contorthx contortrix). See Guan et al., Archives of Biochemistry and Biophysics, 1991, 289(2). 197-207; Randolph et al., "Protein Science", Cambridge University Press, 1992, pages 590-600; European Patent Application No. 0 323 722 (Valenzuela et al ), published July 12, 1989; and the U.S. Patent No. 4,610,879 (Markland et al.), assigned September 6, 1986. Fibrolase has been shown to be fibrinolytic, and this metalloproteinase has been documented to have proteolytic activity against the Aa-chain of fibrinogen, with reduced proteolytic activity for the Bp-chain and no activity for the γ-chain of fibrinogen; Ahmed et al., Hemostasis, 1990, 20, 147-154. Since fibrin is a major component of blood clots, the fibrinolytic properties of fibrolase indicate its potential as a clot-dissolving agent for thrombolytic use in vivo; see Markland et al., Circulation, 1994, 9(5), 2448-2456, and Ahmed et al., supra.

Novel Acting Thrombolvtic (NAT) is a modified form of fibrolase that differs from fibrolase in that NAT contains 201 amino acids with the N-terminal sequence SFPOR, while the N-terminal sequence of native fibrolase begins with EQRFPQR and is 203 amino acids long. Amino-end modification is designed to prevent chemical reactions of amino acid residues, which are able to form a variable amount of cyclized glutamine (proglutamic acid), which has the potential to create site-to-site variety in product quality and uniformity. Thus, NAT can be viewed as a more stable molecule.

Despite these structural differences, NAT and fibrolase are similar in terms of enzymatic (fibrinolytic) activity. This similarity in biological activity is consistent with data indicating that the active site in the fibrolase molecule extends between amino acids 139-159, as described by Manning in Toxicon, 1995, 33, 1189-1200, and its predicted location in three-dimensional space is away from the amino terminus. The active site for fibrolase and NAT molecules contains a zinc atom, which is complexed with three histidine residues.

Published literature on fibrolase derived from venom, showed its proteolytic activity against fibrogen at Lys413-Leu<4>14, against oxidized p-chain of insulin at Ala<14->Leu<15>; Retzios and Markland, Thrombosis Research, 1994, 74, 355-367; Pretzer et al., Pharmaceutical Research, 1991, 8, 1103-1112; and Pretzer et al., Pharmaceutical Research, 1992, 9, 870-877. It was also determined that NAT has proteolytic activity on these substrates, at the same cleavage sites.

In contrast to fibrinolytic metalloproteinases, such as fibrolase and NAT, clot lysis agents such as streptokinase, urokinase, and tissue-type plasminogen activator (tPA) are plasminogen activators that promote thrombolysis by activating the endogenous fibrinolytic system. More specifically, plasminogen activators catalyze the conversion of plasminogen to plasmin, a serine protease. Plasmin is able to cleave fibrinogen and fibrin at arginyl-lysyl bonds, and this is through the generation of plasmin, so plasminogen activators ultimately affect fibrin degradation and clot lysis. Today's commercially available agents are plasminogen activators, such as urokinase, streptokinase, or tPA.

Unlike the plasminogen activator class of thrombolytic drugs, fibrinolytic metalloproteinases, such as fibrolase and NAT, do not rely on the endogenous fibrinolytic system (conversion of plasminogen to plasmin). So this class

clot lysis agents can be distinguished from plasminogen activators by their unique mode of action, so they are defined as "direct" fibrinolytic agents.

Alpha2-macroglobulin, as the dominant proteinase inhibitor present in mammalian serum, is one of the largest serum proteins (it has a molecular weight of 725 kilodaltons). The specificity of ci2-macroglobulin for proteinases is broad, including serine, cysteine, aspartic acid and classes of metalloproteinases. The a2-macroglobulin molecule is a tetramer of identical subunits, linked in pairs via disulfide and by non-covalent linking of the halves of the molecule. Thus, under reducing conditions, natural α2-macroglobulin can dissociate into its four monomeric subunits.

Each α2-macroglobulin subunit possesses a region that is highly sensitive to proteolytic cleavage (the "bite" region). Proteolysis of the bite region causes a conformational strand in α2-macroglobulin, which traps the proteinase within the α2-macroglobulin molecular structure. This process is described in the literature as the "venus fly-trap" interaction (flytrap plant, Dionaea muscipula). When the proteinase is attacked, it is stemmed, and therefore cannot approach its macromolecular substrate.

In addition, a covalent bond can form between α2-macroglobulin and many of the proteinases it attacks. As mentioned, proteinase attack causes a conformational kink in the α2-macroglobulin molecule. It is assumed that after this conformational change, the thioester bond inside the a2-macroglobulin molecule becomes reactive, so it can form a covalent bond with nucleophilic residues (such as lysine) of the attacked proteinase. Thus, within the bloodstream, a2-macroglobulin can effectively neutralize various proteinases.

In addition, the conformational strand in a.2-macroglobulin that occurs upon proteinase attack leads to a form recognized by the reticuloendothelial system. The disappearance of the α2-macroglobulin-attacked proteinase combination is usually described by half-time values in minutes, and is believed to occur via the low-density lipoprotein (LDL) receptor, associated with a secreted protein on macrophages, hepatocytes, and fibroblasts. For more data on a2-macroglobulin, see "Methods in Enzymology", edited by A.J. Barrett, Academic Press, Inc., Philadelphia, 1981, p. 737-754.

Alpha-pharmacroglobulin is able to form a macromolecular complex with fibrolase, NAT or other proteinases. Unlike some proteinases that can form a complex with α2-macroglobulin that can dissociate, fibrolase and NAT are two examples of fibrinolytic metalloproteinases that form a complex that cannot dissociate from α2-macroglobulin under physiological conditions. When purified, for example human a2-macroglobulin and NAT, are incubated together, complex formation begins within seconds and is completely completed within minutes. This phenomenon shows that complex formation in vitro can be rapid and indicates the potential speed of complex formation between a2-macroglobulin and NAT, or other fibrinolytic metalloproteinases in vivo.

Although a2-macroglobulin is one of the major plasma proteins, there is still a finite amount of a2-macroglobulin in the bloodstream, which could be available for binding and neutralization of fibrinolytic metalloproteinase. Therefore, the binding capacity of α2-macroglobulin reaches saturation. When the binding capacity of a2-macroglobulin is exceeded, the concentration of unbound fibrinolytic metalloproteinase increases proportionally, if more fibrinolytic metalloproteinase is added to the sample.

The presence of a2-macroglobulin in the general bloodstream of a patient presents a challenge for systemic (eg, intravenous) administration of fibrolase, NAT, or other fibrinolytic metalloproteinases, which bind to a2-macroglobulin in the general bloodstream. If the saturation level of innate a2-macroglobulin is exceeded by systemic administration of such a fibrinolytic metalloproteinase dose, the latter will be effectively neutralized and rendered ineffective for therapeutic purposes.

In one in vivo study performed on rabbits, the biological efficacy of venom-derived fibrolase was tested after systemic intravenous administration. Ahmed et al., Haemostasis, see above. The dose of fibrolase used was 3.7 mg/kg, estimated to give a final blood concentration of approximately 60 µg/mL, in 3.0 kg rabbits. This amount was chosen based on enzyme inactivation tests in the presence of blood or plasma, presumably due to a2-macroglobulin (see pages 336 and 339).

In the next in vivo study, the biological effect of recombinant fibrolase on clot lysis was investigated in dogs. Markland et al., Circulation, see above. An infusion of 4 mg of this material per kg (animal weight) is given via the catheter, for 5 min, in front of the previously induced thrombus in the left carotid artery (see page 2450). Here again it was observed that the inactivation of fibrolase takes place in the general blood stream, presumably due to the presence of a2-macroglobulin (see page 2454, second column, last paragraph).

As these two studies show, the deactivating effects of α2-macroglobulin can be overcome either by administration or systemic dosing of fibrinolytic metalloproteinase, overcoming the saturation level of α2-macroglobulin (in rabbit studies), or by local release of the enzyme at the site of the clot.

(testing on dogs), while avoiding systemic administration. On the other hand, doses of fibrinolytic proteinase above the α2-macroglobulin saturation level, whether delivered systemically or locally, may exceed levels that are safe and well tolerated by the subject. It should be noted that fibrinolytic metalloproteinases are able to destroy not only fibrin, but can also degrade other structural proteins, and are therefore potentially toxic in vivo, when present in large amounts that exceed the saturation level of a2-macroglobulin.

The formation of a blood clot or other fibrin-based occlusion is also important when using a prepared catheter or other vascular access device. There are many situations that require repeated or prolonged use of a vascular access device, such as a catheter, access graft, cuff, needle, arteriovenous fistula, or shunt. For example, various procedures associated with hemodialysis, chemotherapy, blood infusion or exchange, or other procedures involving repeated intravenous or intra-arterial drug delivery (or fluid extraction), may involve the use of a prepared catheter or other permanent or semi-permanent implanted medical device. Blood clots can form in, around, or attached to any device that is inserted into the vascular space, especially if the device remains in the vascular space for a long period of time, or if the device has one or more small openings. In addition, other fibrin-based occlusions may bind to any part of the prepared vascular access device, and may prevent or impair the proper functioning of the device.

It would be useful to have a quick and efficient treatment procedure, ie. destroying, dissolving, or lysing clots or other occlusions formed around, or attached to, a dissected vascular access device. In the absence of an effective procedure for treating clots formed in or around the vascular access device, this prepared device must be replaced by the physician, incurring additional cost and risk to the patient.

Therefore, it is an object of the present invention to provide a safe and effective method for the treatment of a blood clot or occlusion in the vicinity of a prepared vascular access device.

It is also an object of the present invention to provide a procedure for restoring the patency of a fully or partially occluded vascular access device.

The subject of this invention is also providing a procedure for restoring the function of a prepared device for vascular access, the function of which is to be changed by fibrin-based occlusion.

It is also an object of the present invention to provide a safe and biologically effective method for the use of topically administered fibrinolytic metalloproteinase for the lysis of blood clots in vivo.

In some embodiments, this invention is a method of therapeutic treatment of a blood clot in a prepared vascular access device by administering through said vascular access device an amount of fibrinolytic metalloproteinase in a pharmaceutically acceptable solution, where that amount does not exceed 1.7 mg of fibrinolytic metalloproteinase per kilogram of body weight of the treated human subject. In some embodiments, the vascular access device is implanted in an artery or vein of a human subject. In some embodiments, the prepared vascular access device is a catheter, shunt, access graft, needle, or cuff. In some embodiments, a vascular access device is used to introduce or remove fluid from the vascular domain. In some embodiments, the vascular access device of the present invention is used in connection with hemodialysis, blood transfusion, removal or replacement, chemotherapy, or any other procedure that requires the administration or withdrawal of fluid from a vein or artery.

In some embodiments, this invention is a procedure for the lysis of a blood clot in a prepared device for vascular access, through the local administration of a pharmaceutically acceptable solution into said clot, with an amount of fibrinolytic metalloproteinase that is complexed with a2-macroglobulin, an amount sufficient to facilitate the lysis of that clot, but whose content does not significantly exceed the saturation level of a2-macroglobulin in said human subject.

In some embodiments, the present invention provides a method for restoring the patency of a fully or partially occluded prepared vascular access device. In some embodiments, the present invention provides a method for restoring the function of a fabricated vascular access device.

Short description of the pictures

Figures 1A-1C. Figure 1A is a basic, baseline angiogram of the carotid artery of an adult pig, before the introduction of the balloon-catheter into the lesion and the formation of an occlusive thrombus; Figure 1B is an angiogram taken from the same animal after 4 days, prior to administration of NAT in accordance with the present invention. Figure 1C is an angiogram 2 h after administration of 30 mg of NAT through the "PRO" catheter (see Figure 3 for an illustration of this device).

Figure 2 illustrates a type of catheter device designed for localized delivery of a thrombolytic agent into a blood vessel.

Figure 3 is a cross-sectional view of an alternative type of catheter device for localized release of a thrombolytic agent in a blood vessel.

Figure 4 is a histogram of the estimated maximum dose of NAT in a patient with peripheral vascular occlusion.

Figure 5 illustrates the use of NAT on the Vitno catheter occlusion model.

Figure 6 illustrates the aggregated results of numerous experiments, and the averaged data obtained show that NAT dissolves occlusions in vitro up to 40% faster than Abbokinase<®>, Open Cath<®>, a commercial catheter cleaning urokinase product.

Detailed description of the invention

In some embodiments, this method is a method for treating blood clots in vivo, in human subjects, using fibrinolytic metalloproteinase, which consists of local administration of a safe, biologically effective amount of fibrinolytic metalloproteinase into the blood clot, such as using a catheter or other vascular access device. Typically, stationary fibrin clots are located in a blood vessel (arterial or venous, native or artificial (eg, graft)) of a human subject. In some embodiments, the present invention provides a method for treating a blood clot located in, or around, or attached to a prepared catheter, shunt, needle, or other vascular access device. In some embodiments, the present invention provides a method for restoring the patency of a fully or partially occluded prepared vascular access device. In some embodiments, the present invention provides a method for restoring the function of a vascular access device, where that function has been altered or partially affected by a fibrin-based occlusion.

A "safe, biologically effective" amount means an amount sufficient to break down fibrin and facilitate clot lysis (i.e., a thrombus), but not to significantly exceed the content of the saturation level of a2-macroglobulin in the bloodstream of the treated patient (i.e., contents that can damage the walls of blood vessels). Typically, this amount is within the range of between 0.025 and 1.7 mg per kg of body weight of the human subject being treated, as determined by testing blood samples from human subjects tested for α2-macroglobulinain content in vitro and binding capacity. From the in vitro results in this study, it was possible to define the level of saturation of <x2-macroglobulin in vivo, practically for all purposes, which made it possible to obtain a picture of the biologically effective amount, which gives not only the minimum level of fibrinolytic metalloproteinase necessary for biological efficiency, but also the maximum

level for tolerant prescribing. This assay is described in detail below and in the Examples.

The terms "local" or "localized" are applied to the way fibrinolytic metalloproteinase is released, and refer to intra-arterial or intra-venous administration, either into the blood clot itself (i.e. intra-thrombus) or in the immediate vicinity of the clot (either in front or behind, relative to blood flow), or close enough that most of the fibrinolytic metalloproteinase is absorbed by the clot. For the treatment of clots in, around, or attached to a vascular access device, or to restore function or a blocked vascular access device, the release of fibrinolytic metalloproteinase is usually accomplished within the vascular access device itself, and thus is undoubtedly local. In some embodiments, a secondary vascular access device, such as a catheter, can be used to deliver the fibrinolytic metalloproteinase into an implanted medical device, such as a stent.

The term "catheter as a delivery device" or "vascular access device" is used herein in a conventional sense, and refers to a tubular medical device for insertion into channels, blood vessels, passages or body cavities, for the purpose of injecting or withdrawing fluids, or to keep such passages open. Typically, these means typically consist of an elongated, flexible catheter body, containing one or more internal passages (or "lumens"); the front portion, which allows the material (ie, clot lysis agent) to be introduced into the body of the catheter and to flow through the lumen; the rear part, which optionally has a notched spin; and one or more openings near the rear end, which allow material to exit the catheter in response to applied pressure.

The terms "proteolytic degradation," "dissolution," "lysis," and "therapeutic treatment" are all used herein to refer to the degradation, disintegration, breakdown, disruption, or other removal of a blood clot or other fibrin-based occlusion. A blood clot can partially or totally occlude the lumen of a blood vessel or medical device. Similarly, "restoration of patency" refers to any measurable increase in blood flow (eg, volume or velocity) through the lumen of a blood vessel, or medical device, or outlet, due to dissolution of a blood clot therein.

The term "blood clot" or "occlusion" is used herein to refer to any fibrin-based mass, cluster, obstruction, or growth. Typically, blood clots occur when blood coagulates in an artery or vein and slows, prevents, reduces, or blocks, completely or partially, the flow of blood through them. Blood clots can also form in or around foreign objects introduced into the vascular space. Blood clots can be stationary, such as a thrombus (a blood clot that forms in a blood vessel and stays there) or they can be mobile, such as an embolism (blood clots that travel from where they formed to another location in the body). Blood clots can also vary greatly in size, shape, and composition, and can be spherical masses as well as wings or other planar structures. Sometimes, a piece of atherosclerotic plaque, a small piece of tumor, fat globules, air, amniotic fluid, or other materials found in the blood can act in the same way as a blood clot. In a sense, these masses contain fibrin or other substances susceptible to degradation by fibrinolytic metalloproteinase, one of the methods of the present invention that would be useful for their treatment.

A further method of the present invention is illustrated below, and relates to peripheral arterial occlusion (PAO). PAO originates from peripheral vasculoma disease due to atherosclerosis. Symptoms develop slowly, over many years of progression of atherosclerosis, with the critical level of ischemia being reached in about 15 to 20% of patients with lower extremity disease. Medical therapy is limited and primarily aimed at prevention or risk reduction, using medications such as lipid-lowering or anti-platelet agents, smoking cessation programs, and physical exercise. Jackson and Clagett, Chest, 1998, 114, 666S-682S.

Clinical manifestations of peripheral vasculoma disease may include acute limb-threatening ischemic events, or the presence of chronic evidence of vasculoma disease (ie, occasional limping). In addition to the above-mentioned preventive ones

measure, chronic PAO is typically not treated until severe life-limiting or limb-threatening ischemia occurs. Depending on the affected vessel segment and the amount of occlusion, available medical interventions include percutaneous transluminal angioplasty, surgical revascularization, and thrombolysis. Studies have shown that intra-arterial infusion of clot lysis agents, especially in the early stages of occlusion, can avoid the need for surgical intervention. As shown in Rochetser's trial, which compares urokinase plasminogen activator thrombolysis with surgery, in the treatment of acute PAO (Ouriel et al., Journal of Vascular Surgery, 1994, 19, 1021-1030), 33% of patients in the thrombolysis group of this study were successfully treated with medication-only intervention, thus avoiding a more invasive procedure. In contrast, 98% of patients in the work group underwent an endovascular or surgical procedure.

Other medical disorders involving occlusive blood clots can be similarly effectively treated by this procedure, including, but not limited to, acute myocardial infarction, ischemic stroke, deep vein thrombosis, and pulmonary embolism. In some embodiments, the method of the present invention may also be applied to the dissolution of clots that occur chronically in implanted medical devices, such as prepared catheters and grafts for hemodialysis access. Other examples of implanted medical devices are shunts, access pieces, needles, cuffs, and other vascular access devices.

In some embodiments, the present invention is applicable to the therapeutic release of any fibrinolytic metalloproteinase that is capable of complexing with α2-macroglobulin. These fibrinolytic metalloproteinases, if found in nature, can be purified from their natural resources, e.g. fibrolase from snake venom. Alternatively, polypeptide fibrinolytic metalloproteinase agents, the nucleic acid and amino acid segments of which are known, can be produced using conventional recombinant isolation and purification methods.

Typically, recombinant methods employ a DNA molecule, encoding the fibrinolytic metalloproteinase of interest, which is inserted into an appropriate vector for secretion into a suitable host cell. This vector is chosen to be functional in the host cell used, ie. that it is compatible with the host cell system so that DNA secretion can occur. These vectors may also contain a 5'- flanking sequence (also referred to as a "promoter") and other secretion regulatory elements, as well as other elements, such as an initiation of replication element, a transcription termination element, a complete intronic sequence containing a donor and acceptor splice site, a signal peptide sequence, a ribosome element binding site, a polyadenylation sequence, a polylinker region for insertion of the encoded nucleic acid, and a selectable marker element. This vector may optionally also contain a "label" sequence, ie. an oligonucleotide sequence located at the 5' or 3' end of a sequence encoding a polypeptide, encoding a polyHis, or other small immunogenic sequence. This tag will be co-secreted with the desired protein and can serve as an affinity tag to purify this polypeptide from the host cell. If desired, this label can subsequently be removed from the purified polypeptide by various methods, for example using a selective peptidase.

In those cases where it is desirable for the polypeptide to be secreted from the host cell, a signal sequence can be used to direct the polypeptide out of the host cell in which it was synthesized. Typically, the signal sequence is located in the coding region of the nucleic acid sequence, or directly at the 5' end of the coding region. Many signal sequences have been identified, and any can be used if it is functional in the selected host cell.

After constructing the vector and inserting the nucleic acid into the appropriate site of the vector, the vector thus completed can be inserted into a suitable host cell for amplification and/or secretion of the polypeptide. Host cells can be prokaryotic (such as E. coli) or eukaryotic (such as yeast cells, insect cells, or vertebrate cells).

Suitable host cells or cell strains may be mammalian cells, such as Chinese Hamster Ovary (CHO) cells or 3T3 cells. The selection of suitable host cells and procedures and methods of transformation, cultivation, amplification, testing and product production and purification are known in the art. Other suitable mammalian cell lines are the monkey cell lines COS-1 and COS-7 and the CV-1 cell line. Other mammalian host cell lines include, for example, primate cell lines and rodent cell lines, including transformed cell lines. Also suitable are normal diploid cells, cell strains derived from primary tissue culture in vitro, as well as primary explants. Candidate cells may be genotypically deficient in gene selection, or may contain a dominantly acting selection gene. Other suitable mammalian cell lines include, but are not limited to, HeLa, murine L-929 cells, 3T3 strains, derived from Swiss, Balb-C or NIH mice, BHK or HaK guinea pig cell lines.

Beneficial host cells are also bacterial cells, for example, various strains of E. colii various strains of yeast cells.

Introduction (also known as insertion, "transformation" or "transfection") of a vector into a selected host cell can be done using methods such as calcium phosphate, electroporation, microinjection, lipofection, or the DEAE-dextran method. The method chosen will be a function, in part, of the type of host cells used. These procedures and other suitable methods are well known to those skilled in the art.

Host cells, when cultured under appropriate conditions, can synthesize the desired fibrinolytic metalloproteinase. The host cells can be cultured using standard media, well known as versed

to an expert. These media usually contain all the nutrients required for cell growth and survival. A suitable medium for culturing cellsE. coli, for example, Luria Broth (LB) and/or Terrific Broth (TB). Suitable media for culturing eukaryotic cells are RPMI 1640, MEM, DMEM, all supplemented with serum and/or growth factors, as required for each particular cell strain being cultured.

Typically, an antibiotic or other compound useful for the selective growth of transformed cells is added as the sole supplement to the medium. The compound to be used is dictated by the selected marker element present in the plasmid with which the host cells are transformed. For example, if the selectable marker element is resistant to kanamycin, the compound added to the culture medium will be kanamycin.

The amount of protein produced in host cells is assessed using standard methods known in the art, including Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, and/or activity assays, such as DNA gel binding shift assays.

If the protein is secreted from host cells other than Gram-negative bacteria, it is likely that the bulk will be present in the culture medium. If it is not secreted, it will be present in the cytoplasm. For an intracellular protein, typically the host cells are first mechanically disrupted. For a protein that has a periplasmic location, either mechanical disruption or osmotic treatment can be used to release the periplasmic contents into a buffered solution, and the polypeptide is then isolated from that solution. After that, purification from the solution can be done using various techniques.

If a protein is synthesized to contain a tag, such as a hexahistidine or other small peptide, either at the carboxyl or amino terminus, it can essentially be purified in a one-step process, by passing that solution through an affinity column, where the matrix of that column has a high affinity for that tag, or directly, to the polypeptide (ie, a monoclonal antibody). If that polypeptide is unlabeled and antibodies are not available, other purification procedures can be used, for example, ion exchange chromatography, molecular sieve chromatography, reverse phase chromatography, HPLC, native gel electrophoresis (nucleotides 1-783), in addition to the "mature" polypeptide (nucleotides 784-1392). NAT secretion in such a system typically comprises a DNA molecule with the sequence SEQ ID NO: 6, which encodes a "prepro" sequence (nucleotides 1-783), in addition to a "mature" polypeptide (nucleotides 784-1386).

The fibronolytic metalloproteinase used in accordance with the present invention, whether it is NAT, fibrolase or some other fibrinolytic metalloproteinase, is administered in the form of a pharmaceutically acceptable solution, alone or containing additional pharmaceutically acceptable ingredients. If desired, these solutions may contain, in addition to the fibrinolytic metalloproteinase and the solvent (ie, distilled water or physiological saline), standard ingredients, such as stabilizers (to prevent protein aggregation or physical or chemical degradation in the aqueous medium), bulking agents (to provide bulk), diluents, antibacterial agents, viscosity adjusting agents, antioxidants, etc., in conventional amounts. Known additives that can be included in this formulation are polyols (including mannitol, sorbitol and glycerin); sugars (including glucose and sucrose); and amino acids (including alanine, glycine, and glutamic acid). See, for example, "Remington's Pharmaceutical Sciences", Mack Publishing Company, Easton, Pennsylvania. See also, WO 01/24817 A2 (incorporated herein in its entirety by reference), which describes various preparations of fibrinolytic agents useful in the present invention.

Preferably, the pharmaceutical preparation is buffered (with a biocompatible buffering agent, e.g., citric acid or a salt of citric acid) to a pH that is at or near neutral (pH 7.0) prior to administration, and the pH is typically between 6.5 and about 8.0 (±0.5).

If zinc is a metal ion of a fibrinolytic metalloproteinase, such as fibriolase or NAT, it may be desirable to add zinc compounds in water as a stabilizer (for example, zinc-sulfate or zinc-acetate). To enhance the long-term stability and storage time of this preparation, it may also be convenient to freeze the solution or convert it to a lyophilized product (freeze drying), which is revealed or reconstituted before use, as appropriate.

By way of illustration, a liquid medical preparation that can be frozen, and can be used in the process of the present invention, contains fibrolase or NAT, a zinc salt dissolved in water, citric acid as a buffer, optionally with an additional stabilizer, which is selected from the group consisting of calcium salts dissolved in water and optionally a bulking agent (eg, mannitol). A surfactant, such as Tween<®>80 (BASF, Gummee, Illinoius), may also be added to increase freeze/bleed stability. Tris buffer (Sigma, St. Louis, Missouri) or other buffer may be added to stabilize the pH at or around 7.4. Most of these ingredients will be present in low concentrations, ranging from 0.001 to 2.0 mM, or less than 10%{m/V).The buffering agent is added in an amount sufficient to achieve the desired pH, and this amount may vary depending on the specifics of the formulation.

By way of illustration, a pharmaceutical preparation that can be lyophilized, or that has been lyophilized, and that can be used in the process of the present invention, contains fibrolase or NAT, a zinc stabilizer (eg, water-soluble zinc, as above), and citric acid as a buffer, with or without additives (eg, a bulking agent, such as mannitol, glycine, and the like). The lyophilized preparation may also contain a disaccharide sugar, such as sucrose or trehalose, as well as a lyoprotectant. A surfactant, such as Tween<®>80, may be added to protect the fibrinolytic metalloproteinase (eg, fibrolase, or NAT) from lyophilization stress. Ideally, the pH should be maintained at 8.0 ± 0.5, using a suitable buffer with a pKau in this range (for example, Tris). The amounts of the ingredients are the same as those given above.

As mentioned, in some embodiments, the method of the present invention is used to locally administer a biologically effective amount of fibrinolytic metalloproteinase, which is within a dose range between 0.025 and 1.7 mg/kg. Preferably, this amount is in the range of about 0.1 to about 0.5 mg/kg. The strength of the solution is formulated accordingly, and dilution is carried out, if necessary, during administration.

In contrast to the treatment of thrombosis in a native artery or vein, such as PAO, the treatment of a blood clot that has occurred in an implanted or prepared vascular access device presents an additional challenge. For example, in the case of an occluded central venous vessel, the "dead volume" of the catheter (ie, the internal diameter of the catheter multiplied by its length) defines the maximum volume that can be contained within that catheter. For example, a catheter with an internal diameter of 1.0 mm and a length of 40.0 cm has a dead volume of -0.3 mL. Because this volume is so limited, it is important to define the appropriate strength of the solution. Accordingly, increasing the solution's strength, or concentration, is an effective way to increase the amount of drug available to dissolve a targeted blood clot.

In some embodiments, the method of the present invention relates to the use of a fibrinolytic metalloproteinase in the treatment of occlusions associated with a vascular access device. These clots can occur in, around, or attached to vascular access devices. Typically, clots form in the lumen or outlet of a device, such as a catheter, and can interfere with the catheter's function. Clots can also form on the outside of a vascular access device, such as a flap that can cover the exit port, preventing drug delivery or blood withdrawal. Clots can also become attached to a vascular access device, interfering with the proper functioning of that device. This interference can be the result of partial occlusion or blockage, ie. when some fluid can pass, or complete occlusion, when fluid does not pass.

To demonstrate one method of the present invention, an in vitro catheter occlusion model was used to stimulate a clot in a prepared catheter type device. In this model, collagen-coated Pasteur pipettes are used to simulate the dissected portion of the catheter. Human blood is drawn in by capillary action in a length of 3.0 mm and allowed to clot. The tip of the pipette is inserted into the vial containing the saline solution, and the other into the solution with 4 mg of NAT (100 µL of a 40 mg/mL solution). Figure 5 illustrates the results of this catheter occlusion model in vitro, showing that the clot in saline is intact, while the clot in 40 mg/mL solution is active in terms of clot dissolution.

In the following experiment, a separate group of pipettes (each containing a blood clot at its tip) is inserted and incubated in a vial containing: 100 uL of saline solution, 100 uL of urokinase solution (5000 international units/mL) and 100 uL of NAT solution, where the amount of drug varies from 0.5 to 4 mg, with increasing solution strength from 5 mg/mL to 40 mg/mL. Figure 6 illustrates the aggregated results of a number of experiments, leading to averaged data showing that NAT dissolves occlusions in vitro up to 40% faster than Abbokinase<®>OpenCath<®>, a commercial catheter cleaning urokinase product (second column, UK 500 Int. Units). Figure 6 also shows that NAT is dose-dependent in terms of the rate at which NAT acts on the proteolytic degradation of the blood clot.

For the treatment of relatively small occlusions in a prepared catheter, such as those simulated in an in vitro catheter occlusion model, the preferred treatment range would be approximately 5 to 40 mg/mL of fibrolase, NAT, or other fibrinolytic metalloproteinase. However, depending on the type and size of the vascular access device or other medical device, and the location, size, and extent of the occluded clot, either much lower or higher doses may be required. For example, a clot in a relatively large shunt is likely to require higher concentrations of fibrinolytic metalloproteinase than a clot blocking the exit port of a 1.0 mL diameter catheter. Accordingly, concentrations for this indication may range from approximately 0.1 mg/mL or less, to over 80 mg/mL.

For blood clots located in, around, or attached to a prepared vascular access device, the effective dose can be delivered in a number of ways, including intermittent infusion, continuous infusion, bolus administration, or a combination of all three. Considering the difficulties indicated by "dead volume" limitations, bolus administration is usually preferred.

In a typical case, the procedure of the present invention is performed in conjunction with a catheter-directed thrombolysis procedure. That procedure involves the use of a presterilized catheter-type drug delivery device, the sidewalls of which are made of a thin semi-rigid or flexible biocompatible material (eg, polyolefin, fluoropolymer, or other inert polymer). Typically, a suitable catheter contains at least one internal cavity (or lumen) that extends the entire length of the device. The material from which the catheter is constructed is flexible enough to be moved through the interior of the vasculature, without causing injury to the walls of the blood vessel, yet rigid enough to be stretched along the entire distance to the treatment site, while the internal cavity of the device remains completely open. Typically, such a catheter device ranges from 2 to 20 cm, on the French scale for catheter diameters (1/3 mm is equal to 1 French division), and in length from about 60 cm to about 2 m.

Exemplary catheter devices for intravascular delivery of thrombolytic medication in accordance with the present invention are illustrated in Figures 2 and 3, and their practical application is described in detail in the following examples. However, any conventional catheter or vascular access device suitable for this procedure may be used, including, but not limited to, the specific devices referred to herein.

For example, Figure 2 illustrates a type of catheter device designed to locally deliver a thrombolytic agent into a blood vessel. The device, which is shown in cross-section, contains "side holes" at the release end, through which the infusate (thrombolytic agent) is released, under the pressure of the applied fluid. The diameters of the catheter tubes in this image and the next image, relative to their overall size, have been exaggerated to better show the details. See also Figure 3, which illustrates a cross-section of an alternative type of catheter device for localized release of a thrombolytic agent into a blood vessel. This device contains thin slits, called "pressure vents" (PROs), cut into the catheter wall at regular intervals so that the infusate is released when the fluid pressure inside the catheter reaches a critical point, causing these slits to open. This device can be used in conjunction with an automated, piston-driven pulsatile infusion device (not shown, but exemplified below), which is capable of delivering pulsed drug infusions.

For blood clots that form in a vein or artery and are not associated with the introduction of a vascular or other medical device, an effective dose of fibrinolytic metalloproteinase can be delivered through a catheter to the local treatment site by pulsatile infusion, continuous infusion, bolus administration, or a combination of all three. The solution strength (ie, concentration) of fibrinolytic metalloproteinase in the treatment solution is also an important parameter for these types of clots. More specifically, a range should be chosen between the minimum dilution of fibrinolytic metalloproteinase to achieve efficiency, from the lower end (which is particularly important for bolus administration) and the maximum solubility of fibrinolytic metalloproteinase, from the upper end. Typically, solution strengths in the range of about 0.1 to about 80 mg/mL are used. The volume of the bolus (or the total volume of multiple boluses, in the case of "pulse" release) is then selected according to the release of an effective amount of fibrinolytic metalloproteinase within the range prescribed above.

This invention is further illustrated by the following examples, which are understood to be illustrative only and not to limit the invention to the embodiments described. In these examples and throughout the description of the present invention, "kg" refers to the subject's body mass in kilograms, "mg" refers to milligrams, "mL" refers to milliliters, and "min" refers to minutes. The illustrated fibrinolytic metalloproteinase, namely Novel Acting Thrombolytic (NAT), was recombinantly derived and made according to the methods referred to above.

Example 1

Thrombolysis of subacute thrombosis of the common part of the carotid artery

adult pigs

NAT was tested in a subacute carotid artery thrombosis model in adult pigs, average weight 75 kg, in a service laboratory (Charles River Laboratories, Southbridge, Massachusetts). The purpose of this study is to determine the fibrinolytic activity of NAT in a thrombosis model relevant to peripheral artery occlusion in humans.

In this animal model, thrombosis of the carotid artery was caused along its entire length (approximately 20 cm from the beginning of the aorta to the carotid branch) by a combination of balloon, thrombin and path injury. The size of the thrombus approximates the size of the thrombus seen clinically in humans with peripheral artery occlusion. After successful thrombosis, the animal is allowed to recover over a period of several days. A four-day period was chosen to allow extensive fibrin cross-linking, thrombus remodeling and cell infiltration. Incidentally, both the size and age of the thrombus in this model reasonably represent the magnitude and duration of ischemic symptoms reported in the recently published TOPAS Thrombolysis Trial with Plasminogen Activators in Peripheral Artery Occlusion in Humans. For literature, see Ouriel et al., New England Journal of Medicine, 1998, 338, 1105-1111.

Briefly, in the common part of the carotid artery, thrombosis can be obtained along its entire length using a fluoroscopically guided balloon injury. The balloons used are oversized and inappropriate. The balloons are inflated with a pressure of up to 12 bar, which causes the injury of tearing the intimate layer of the blood vessel. As the balloon inflates, it moves back and forth to scrape the endothelium of the blood vessel.

This balloon insertion procedure creates extensive trauma and a vessel surface that is highly susceptible to thrombus formation, and is repeated along the entire length of the common carotid artery. After complete injury of this entire artery, the balloon is pulled out in an anterior position, close to the aorta, and inflated to occlude blood flow through this blood vessel. While occluded, 50 units of bovine thrombin are injected through the distal opening of the balloon catheter to stimulate coagulation. The balloon is kept inflated for 30 min, which leads to thrombotic occlusion of the blood vessel. After 30 minutes, the balloon is deflated and an angiogram is taken to confirm that the blood vessel is occluded. With this procedure, occlusion of the blood vessel is achieved in more than 90% of cases.

Figure 1A represents a baseline, baseline angiogram of the carotid artery of an adult pig, before balloon catheter injury and occlusive thrombus formation. The arrow indicates the position and presence of contrast medium in the common part of the left carotid artery, indicating that blood is flowing through the artery without obstruction (ie, the vessel is "patent" or has ""). Figure 1B is an angiogram taken on day four of the same animal, prior to administration of NAT, according to the method of the present invention. The arrow shows the position of the common part of the left carotid artery, but the contrast medium does not flow through the artery due to the presence of an occlusive thrombus. Figure 1C is an angiogram taken 2 h after administration of 30 mg of NAT, through the "PRO" catheter.

(see Figure 3 for an illustration of this device). The arrow shows the presence of contrast medium in the blood vessel, which shows that patency in the left carotid artery has been restored. Minimal residual thrombus is visible in the lumen of the artery.

Once a thrombus has formed, the balloon catheter, guide catheter, and access cuff are removed, and the animal is allowed to recover over a 4-day period. On day 4, the animals are re-anesthetized, occlusion reconfirmed, and a multiple side-hole drug delivery catheter (see Figures 2 and 3) is inserted, under fluoroscopic guidance, and positioned so that the side holes are located within the thrombus. Thrombolysis with NAT was observed angiographically, using a fixed dosage of NAT, which ranged from 10 to 30 mg (or approximately 0.1 to 0.4 mg/kg, on a weight-adjusted basis), as shown in Figures 1A-1C.

As illustrated in Figure 1B, NAT is effective in restoring previous flow as assessed by angiography. To quantify this, the flow through the target blood vessel is quantitatively assessed according to a 4-point scale (ranging from 0 to 3), where:

0 = no flow

1 = flow estimated to be less than 30% than in the contralateral carotid (no thrombus) 2 = flow estimated to be less than 30-80% than in the contralateral carotid 3 = flow indistinguishable from the same in the contralateral carotid.

What is shown in Figure 1B is scored on the upper scale as 3, which is a common score in thrombus vessels treated with 30 mg of NAT (approximately 0.4 mg/kg in a 75 kg pig). Tables 1-3 in the following examples illustrate treatment regimens and mean flow values obtained from serial angiograms. In all these trials, respiration, body temperature, heart rate and arterial blood pressure were continuously monitored and remained within physiological limits, with no changes observed after the administration of NAT.

Example 2

Catheter selection and pulsed spray release

In the clinical treatment of peripheral artery occlusion, thrombolytic agents are delivered through catheters placed near or immersed in the thrombus. As shown in Figures 2 and 3, there are typically two types of catheters that are used.

One variant, the "side-hole" catheter (Figure 2) has laterally small, round holes (2) cut into the catheter (4) near the closed distal end 6, and an inlet port 8 (for fibrinolytic metalloproteinase solution), in an attached ring 10, fixed at the front end 12. The catheter is constructed as a tube of flexible, long, biocompatible polymer material, which is hollow, thin-walled, and has a uniform diameter of 2 to 20 French divisions, preferably from 3 to 5 French divisions. This catheter, on the outer surface, near the distal end 6, contains two radiologically opaque markers 14; which limit that part of the catheter that contains the side holes 2. In practice, this catheter is inserted through a surgically made opening in the occluded artery or vein, while being observed through fluoroscopy, in accordance with the standard accepted procedure, and is carefully moved through - the blood vessel, so that the distal end 6 can be placed in or near the thrombus. The markers 14, which are clearly visible in the fluoroscopic field of view, can serve to guide and position that part of the catheter, so that the infusate coming out of the side holes 2 comes directly into contact with the thrombus. Then, a pharmaceutical solution of fibrinolytic metalloproteinase is injected, under slight pressure, from the syringe-like reservoir 16, through the inlet part 8, then flows towards the distal part 6, exiting through the holes 2 into the thrombus, which causes the breakdown of the fibrinous material.

When infusions of fibrinolytic metalloproteinase are given, using this type of catheter, most of the solution containing the fibrinolytic agent tends to exit through the nearest side holes (ie those closest to the inlet port 8), which has a negative effect on the uniformity of drug release at the treatment site. There is also the possibility of backflow of blood into the catheter through the side openings 2, due to negative pressure.

Another variant of the catheter, also made of a round, biocompatible, thin-walled polymeric material, shown in Figure 3, has extremely thin slits 2, laser-cut in the flexible catheter 4, at regular intervals near the distal end 6. These slits, which will be referred to as pressure vent exit ports ("PRO"), are tight enough that the infusate does not escape unless the fluid pressure inside the catheter reaches a critical value that causes the slits to simultaneously rupture. inflate and thus temporarily open. This catheter may also contain external radiopaque markers 8, which assist in positioning the device at the site of the thrombus.

Ideally, these "PRO" infusion catheters are used in conjunction with an automated, reciprocating pulsatile infusion device (not shown), which is capable of releasing regulated pulses of a small volume of infused medication into the inlet port 10, via an attached ring 12 secured to the front end 14 of the catheter 4. When such a pulse is released, the pressure within the catheter instantly increases. The answer is to momentarily open the pressure outlet vents (slits 2) and allow the infusate (eg, a pharmaceutical solution of fibrinolytic metalloproteinase) to escape. The theoretical advantage of the pulsed release of the infusate and the PRO type catheter is that the infusate is released uniformly through the slits, along the entire length of the catheter, while the infusate released through the "side holes" of the catheter (Figure 2) follows the scheme of least resistance and flows through the holes closer to the front, non-uniformly, as already mentioned.

A four-day-old porcine carotid thrombosis model is used to test the performance of the two catheter types mentioned above, using fixed doses of 30 mg NAT (approximately 0.4 mg/kg in a 75 kg pig). The results are summarized in Table 1.

As shown in Table 1, the results of angiographic flow in the group treated with a variant of the pulse desolvation showed slightly higher initial flow results on the 30-min angiogram, which did not change until the time of 4 h. Although no statistical differences were obtained, the angiographic results were evaluated as better. Therefore, a PRO-type catheter, combined with pulsed spray release, is the preferred method of releasing fibrinolytic metalloproteinase, in accordance with the present invention.

Example 3

Drug release time testing

Acute peripheral arterial occlusion is typically treated with plasminogen activators, such as urokinase, delivered by infusion, often for 24 h and occasionally for 48 h. The duration of the infusion is assumed to maintain a low level of plasmin generation for an extended period of time, with the goal of effectively dissolving the occlusive thrombus. Since both NAT and fibrolase are fibrinolytic metalloproteinases, such prolonged infusions may not be necessary. To assess whether release rate affects clot lysis angiographically, a fixed dose of 30 mg NAT (roughly 0.4 mg/kg 75 kg pig) is delivered using a PRO catheter and a pulsatile spray device. Using pulse volumes of 0.1 mL, a 5 mg/mL solution of NAT is released over 6 min (ten pulses per minute), or over 60 min (1 pulse per minute). The results are shown in Table 2.

As shown in Table 2, the release of 30 mg of NAT over 6 min gives a mean score of 2.7 in 3 animals, on the 30 min angiogram, which remains unchanged until the 4 h time. In contrast, the release of 30 mg NAT by pulsatile infusion over 60 min is far less impressive. Although these data were not statistically compared, these results support the release of NAT by the faster pulsing regimen.

Example 4

Pulse volume optimization

The pulsation nebulizer infusion device is programmable in terms of volume delivered per pulse, from 0.1 to 0.5 mL per pulse. To determine whether pulse volume has any effect on angiographic outcome in a porcine model, 0.2 mL pulse volumes were compared with 0.4 mL pulse volumes. NAT was released at a fixed dose of 10 mg (equivalent to 0.15 mg/kg pig). The results are shown in Table 3.

As shown in Table 3, at 30 min the mean agiographic score was slightly higher in the 0.4 mL pulse volume group. However, at 4 h, the group mean is slightly higher for a pulse volume of 0.2 mL. Therefore, no conclusion can be drawn from these studies that one pulse volume is better than another.

The results in the foregoing text and tables show that virtually all of these NAT treatment regimens are effective in treating peripheral artery occlusions with the release method of the present invention, and that these results are at least comparable to treatment with plasminogen activators, such as urokinase, which is today's treatment of choice with thrombolytic agents.

These results indicate that the PRO catheter with pulsatile nebulization produces better angiographic results. For a given animal weight (70-100 kg), a fixed dose of 30 mg is roughly equivalent to 0.3-0.4 mg/kg adjusted for weight.

Reducing the fixed dose of NAT to 10 mg resulted in a decrease in the mean angiographic results of the group, and patency was not achieved in some animals. This indicates that a dose of 10 mg is considered the threshold dose of biological activity in this model. For a given animal weight in these studies (70-100 kg), a fixed dose of 10 mg is roughly equivalent to 0.1-0.15 mg/kg on a weight-adjusted basis. Varying the pulse volume from 0.2 mL to 0.4 mL was not shown to profoundly affect angiographic patency results.

Example 5

Determination of safe, well-tolerated, biologically effective

dose range in humans

There is no satisfactory literature regarding the serum concentration or biochemical activity of a2-macroglobulin in elderly patients with peripheral vascular disease (PVD). Since <x2-macroglobulin concentrations are a key determinant of safety, and are likely to be related to tolerance of fibrinolytic metalloproteinases in vivo, a cross-over epidemiologic study was performed in PVD patients to assess a2-macroglobulin concentration and fibrinolytic metalloproteinase binding capacity (using NAT as a test agent).

At two centers, 216 patients were enrolled (Cleveland Clinic Foundation, Cleveland, OH, and Rochester General Hospital, Rochester, NY). Demographic information and other patient characteristics were collected, and serum was obtained by measuring a2-macroglobulin, NAT binding capacity (by titration of serum samples from individual patients, using an HPLC assay that detects unbound NAT) and other serum hernia parameters. The primary task is to determine the relationship between the concentration of a.2-macroglobulin in serum and the amount of NAT (in ug/mL of serum) that can be neutralized in vitro (binding capacity

NAT).

Comparison of patient characteristics in this trial with those of the two largest published studies of PAO thrombolysis (ie, the STILE and TOPAS studies) shows that the patient population in this trial is also representative of previous studies of PAO thrombolysis; for other details in previous studies, see Annals of Surgery, 1994, 220, 251-266 and Ouriel et al., New England Journal of Medicine, 1998, 338, 1105-1111, respectively.

For each patient, the estimated maximum dose (EMD) of NAT was calculated, using the binding capacity of NAT, and the plasma volume estimate for each patient. The results of the study predict that the average patient can receive a dose of 1.7 mg/kg (delivered or topically or systemically) without exceeding the binding capacity of α2-macroglobulin to bind and neutralize NAT. The results of this test are summarized in Figure 4.

In Figure 4, the estimated maximum dose of NAT was calculated for each of the 216 patients in this trial. The results of this test are shown as a histogram, in which a bell-shaped distribution can be seen visually. The average patient in this trial is predicted to be able to tolerate 1.7 mg/kg NAT (bell-shaped distribution peak). Doses prescribed in animal studies are shown as reference (right) and can be seen to be higher than the estimated maximum doses of NAT in 99% of the study population.

Therefore, the prescribed range of 0.025 to 1.7 mg/kg in the present invention represents a rational estimate of the dose of patients who, for example, can receive it safely (based on plasma volume and NAT binding capacity for <x2-macroglobulin) without the appearance of free NAT in the bloodstream.

In conclusion, the results of the pharmacological studies exemplified in Examples 1-4 above demonstrate the biological efficacy of fibrinolytic metalloproteinase as a clot lysis agent in an animal model of thrombosis, where the thrombus is comparable in size and age to those frequently encountered in peripheral arterial occlusions in humans. Doses identified in animal models were obtained without considering or testing potential toxicity in the animal. Dose efficacy in rabbits and dogs (3.7 and 4.0 mg/kg, respectively), as described by Ahmed et al. and Markland et al. (above), may enable the use of fibrinolytic metalloproteinases in veterinary medicine. However, when viewed in the presence of human data, prescribing doses of 3.7 and 4.0 mg/kg could result in overdose in 99% of patients, according to a population-based study. Therefore, the published animal studies do not allow for the therapeutic use of fubrinolytic metalloproteinases in humans in a manner that would be safe and biologically effective. On the other hand, the data given in Example 5 allow such use in human beings.

Claims (54)

1. Procedure for the lysis of a blood clot in, around, or attached to a prepared device for vascular access to a human being, indicated by the fact that it consists of prescribing through the mentioned device for vascular access an amount of fibrinolytic metalloproteinase that does not exceed 1.7 mg of fibrinolytic metalloproteinase per kg of body weight of a human being. 2. Procedure for lysis of a blood clot in, around, or attached to a prepared device for vasculami access to a human being, indicated by the fact that it consists of administering through the mentioned device for vasculami access an amount of fibrinolytic metalloproteinase that complexes with a2-macroglobulin, in an amount sufficient to facilitate the lysis of that clot, but not significantly exceeding the saturation level of a2-macroglobulin in the said human being. 3. The method according to Claim 1 or 2, characterized in that it contains 0.1 to 80 mg/mL of fibrinolytic metalloproteinase. 4. The method according to Claim 1 or 2, characterized in that it contains 0.1 to 50 mg/mL of fibrinolytic metalloproteinase. 5. The method according to Claim 1 or 2, characterized in that said vascular access device is a catheter. 6. The method according to Claim 1 or 2, characterized in that said vascular access device is a shunt. 7. The method according to Claim 1 or 2, characterized in that the mentioned vascular access device is an access graft. 8. The method according to Claim 1 or 2, characterized in that the said vascular access device is a needle. 9. Method according to Claim 1 or 2, characterized in that the vascular access device is used to introduce a liquid preparation into a human artery or vein. 10. The method according to Claim 1 or 2, characterized in that the vascular access device is used for taking blood from a human artery or vein. 11. The method according to Claim 1 or 2, characterized in that the vascular access device is used in connection with a hemodialysis procedure. 12. The method according to Claim 1 or 2, characterized in that the vascular access device is used in connection with a blood transfusion procedure. 13. The method according to Claim 1 or 2, characterized in that the vascular access device is used in connection with chemotherapy. 14. The method according to Claim 1 or 2, characterized in that it is used to treat peripheral arterial occlusions. 15. The method according to Claim 1 or 2, characterized in that the vascular access device is used in connection with blood collection. 16. The method according to Claim 1 or 2, characterized in that said blood clot is located on the inner surface of the prepared device for vascular access. 17. The method according to Claim 1 or 2, characterized in that said "blood clot" is located on the outer surface of the prepared vascular access device. 18. Method according to Claim -1 or 2, characterized in that said blood clot is attached to a prepared device for vascular access. 19. The method according to Claim 1 or 2, characterized in that said vascular access device is located in an artery. 20. The method according to Claim 1 or 2, characterized in that said vascular access device is located in a vein. 21. The method according to Claim 1 or 2, characterized in that said vascular access device consists of a catheter device with "side holes". 22. The method of Claim 1 or 2, wherein said vascular access device comprises a "pressure vent" (PRO) catheter release device. 23. Method according to Claim 1 or 2, characterized in that the fibrinolytic metalloproteinase solution is administered as a bolus. 24. The method according to Claim 1 or 2, characterized in that the fibrinolytic metalloproteinase is a Novel Acting Thrombolytic (NAT). 25. The method according to Claim 1 or 2, characterized in that the fibrinolytic metalloproteinase is fibrolase. 26. A method for restoring the patency of a prepared vascular access device, which is used in human beings with a fibrin-based occlusion, indicated by the fact that the amount of fibrinolytic metalloproteinase that is complexed with a2-macroglobulin is administered through said vascular access device, in an amount sufficient to facilitate clot lysis, but whose content does not significantly exceed the saturation level of a2-macroglobulin in said human being, where said amount does not exceeds 1.7 mg of fibrinolytic metalloproteinase per kg of human body weight. 27. Method for restoring the function of a prepared vascular access device, which is used in human beings with a fibrin-based occlusion, indicated by the fact that the amount of fibrinolytic metalloproteinase that is complexed with a2-macroglobulin is prescribed through said vascular access device, in an amount sufficient to facilitate clot lysis, but whose content does not significantly exceed the saturation level of a2-macroglobulin in said human being, where said amount does not exceed 1.7 mg of fibrinolytic metalloproteinase per kg of human body weight. 28. Use of fibrinolytic metalloproteinase in an amount that does not exceed 1.7 mg of fibrinolytic metalloproteinase per kg of body weight of a human subject, in a pharmaceutically acceptable solution, for the production of medicanet for the therapeutic treatment of a blood clot in, around, or attached to a prepared device for vascular access. 29. Use of a fibrinolytic metalloproteinase complexed with a2-macroglobulin, in an amount sufficient to facilitate clot lysis, but not significantly exceeding the saturation level of ct2-macroglobulin in, around, or attached to a prepared vascular access device, in a pharmaceutically acceptable solution, for the production of a medicament for proteolytic decomposition of a blood clot in, around, or attached to said prepared vascular access device. 30. Use according to Claim 28 or 29, wherein the fibrinolytic metalloproteinase has a concentration of 0.1 to 80 mg/mL. 31. Use according to Claim 28 or 29, wherein the fibrinolytic metalloproteinase has a concentration of 0.1 to 50 mg/mL. 32. Use according to Claim 28 or 29, wherein said vascular access device is a catheter. 33. Use according to Claim 28 or 29, wherein said vascular access device is a shunt. 34. Use according to Claim 28 or 29, wherein said vascular access device is an access graft. 35. Use according to Claim 28 or 29, wherein said vascular access device is a needle. 36. Use according to Claim 28 or 29, wherein the vascular access device is used to introduce a liquid preparation into a human artery or vein. 37. Use according to Claim 28 or 29, wherein the vascular access device is used to draw blood from a human artery or vein. 38. Use according to Claim 28 or 29, wherein the vascular access device is used in connection with a hemodialysis procedure. 39. Use according to Claim 28 or 29, wherein the vascular access device is used in connection with a blood transfusion procedure. 40. Use according to Claim 28 or 29, wherein the vascular access device is used in conjunction with chemotherapy. 41. Use according to Claim 28 or 29, wherein the vascular access device is used to treat peripheral arterial occlusions. 42. Use according to Claim 28 or 29, wherein the vascular access device is used in connection with blood donation. 43. Use according to Claim 28 or 29, wherein said blood clot is located on the inner surface of a prepared vascular access device. 44. Use according to Claim 28 or 29, wherein said blood clot is located on the outer surface of a prepared vascular access device. 45. Use according to Claim 28 or 29, wherein said blood clot is attached to a prepared vascular access device. 46. Use according to Claim 28 or 29, wherein said vascular access device is located in an artery. 47. Use according to Claim 28 or 29, wherein said vascular access device is located in a vein. 48. Use according to Claim 28 or 29, wherein said vascular access device comprises a "side hole" catheter device. 49. Use according to Claim 28 or 29, wherein said vascular access device comprises a catheter release device having a "pressure vent" (PRO). 50. Use according to Claim 28 or 29, wherein said medicament is prepared for bolus administration. 51. Use according to Claim 28 or 29, where the fibrinolytic metalloproteinase is a Novel Acting Thrombolytic (NAT). 52. Use according to Claim 28 or 29, wherein the fibrinolytic metalloproteinase is fibrolase. 53. Use of fibrinolytic metalloproteinase in an amount that is complexed with a2-macroglobulin, and in an amount sufficient to facilitate clot lysis, but not significantly exceeding the saturation level of a2-macroglobulin in a human subject, in a pharmaceutically acceptable solution, where said amount does not exceed 1.7 mg of fibrinolytic metalloproteinase per kg of body weight of a human subject, for the production of a medicament for restoring the patency of a prepared device for vascular access which has a fibrin-based occlusion. 54. Use of fibrinolytic metalloproteinase in an amount that is complexed with a2-macroglobulin, and in an amount sufficient to facilitate clot lysis, but not significantly exceeding the level of saturation content of a2-macroglobulin in a human subject, in a pharmaceutically acceptable solution, where said amount does not exceed 1.7 mg of fibrinolytic metalloproteinase per kg of body weight of a human subject, for the production of a medicament for restoring the function of a prepared device for vascular access that has fibrin-based occlusion.