WO2001010450A1 - Targeted thrombolytic agents - Google Patents

Abstract

Biocompatible, non-immunoreactive polymers are used in combination with both tissue or cellular receptor targeting ligands and thrombolytic agents to affect long acting yet localized lysis of thrombi.

Description

TARGETED THROMBOLYTIC AGENTS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Serial No. 09/218,660, filed on December 22, 1998, which is a continuation-in-part of U.S. application Serial No. 08/660,032, filed on June 6, 1996, which is a continuation-in-part of U.S. application Serial No. 08/640,464, filed May 1, 1996.

BACKGROUND OF THE INVENTION

Field of Invention

This invention relates in general to the field of cardiovascular therapeutics and in particular to thrombolytic agents which bear one or more targeting ligands selectively directed to thrombus.

Description of Related Art

Vascular thrombosis is an enormous clinical problem, particularly in developed Western countries. Indeed, vascular thrombosis accounts for about half of all deaths in these countries as a result of myocardial infarction, stroke, pulmonary emboli, and the like. Prompt detection and treatment of thrombosis with thrombolytic therapy is of paramount importance to improve patient survival and decrease morbidity. Therefore, a variety of thrombolytic agents such as streptokinase (SK), urokinase (UK), and tissue plasminogen activator (TPA) have been developed. These thrombolytic agents work by activating the protein plasminogen into plasmin. When this occurs, activated plasmin circulates throughout the vascular system, triggering the fibrinolytic cascade that dissolves the thrombi . Thrombolytic agents such as those described above have been used in humans for many years. However, more research directed at the refinement of thrombolytic agents has been reguired to address many problems involving their effectiveness and potentially harmful side effects.

One such problem relates to the relatively short half life of thrombolytic agent activity upon introduction into a patient's bloodstream. For example, SK protein, which comes from bacteria, is rapidly recognized as a foreign pathogen and then neutralized by immunoglobins. To address this problem, researchers have added certain chemical structures to thrombolytic agents such as SK in an attempt to "hide" them from immune system detection. To this end, Koide et al . disclosed a method of modifying SK through the addition of one or more activated polyethylene glycol (PEG) molecules of MW 5,000. Koide, Atsushi et al., FEBS Letters 143:1, 73-76 (1982). This modification resulted in a complete loss of SK antigenicity, but only 33% or less of SK anti-thrombic activity was retained. Similarly, Tomiya et al . chemically modified an acyl-plasmin-streptokinase complex with 5,000 MW PEG, rendering the resulting complexes completely resistant to neutralization with SK antibodies and 40% thrombolyticly active in human serum. Tomiya, Noboru et al . , FEBS Letters 193:1, 44-48 (1985).

Even more successfully, Rajagopalan et al . demonstrated that different sizes of PEG molecules can affect SK thrombolytic activity, antigenicity, and resistance to proteolytic degradation. Rajagopalan, Shrin et al . , J. Clin. Invest. 75: 413-419 (1985). By adding PEG of especially MW 2,000 to lysine residues of the SK protein, nearly complete thrombolytic activity was maintained with only minimal antigenicity (5% of unmodified SK) .

Furthermore, Rajagopalan discovered that PEG addition to SK protects newly activated plasmin complex from proteolytic degradation, thereby increasing therapeutic duration even more.

Unfortunately, these advances have also led to increased risk for patients due to the system-wide presence of long- lasting, active plasmin. In other words, because thrombolytics such as SK-PEG are so stable, they are slowly cleared from the bloodstream and remain active much longer than their unmodified versions. Thus, for example, stroke or hemorrhage can occur as a result of the inability to form blood clots anywhere within the vasculature during the time the thrombolytic agent is active .

To address this risk, and in contrast to prior advances, the present invention targets thrombolytic agents to the site of thrombosis so that plasmin is created at the site of the clot. Thus, in addition to more effective clot lysis, there is afforded a decreased chance for complications due to hemorrhage.

Blood platelet aggregation and secretion are known to play an essential role in the forming of thrombi during a variety of pathological events such as atherosclerosis or deep vein thrombosis. Hence, these cells have been targeted by another form of targeted clot therapy as taught by Degrado et al., U.S. Patent No. 5,635,477. This invention relates to novel cyclic compounds containing carbocyclic ring systems useful as antagonists of the platelet glycoprotein Ilb/IIIa complex. This integrin complex is found on the surface of platelet cells and is known to mediate their activation and aggregation during thrombogenesis. Accordingly, treating the GPIIb/IIIa complex with a chemical inhibitor or antagonist blocks platelet activation and aggregation. However, as with the other prior art, U.S. Patent No. 5,635,477 does not provide for a means of localizing thrombolytic effects to the thrombic site, but instead targets both activated and unactivated platelet sites throughout the vascular system. In contrast, the present invention can specifically target a thrombolytic drug to any one of multiple components of a thrombus , such as fibrin polymers, activated GPIIb/IIIa, or activated endothelial cells. Consequently, the targeted thrombolytics of the present invention are both more precisely localized and not solely dependent on the presence of high numbers of platelets. Furthermore, the thrombolytic drugs of the present invention can be targeted to receptors involved in different types of thrombogenic events, such as restenosis or other conditions associated with inflammation.

BRIEF SUMMARY OF THE INVENTION

It is an object of the invention to provide a therapeutically effective means of selectively localizing the effects of thrombolytic agents to the site or sites of thrombus .

A further object of the invention is to provide a therapeutically effective means of targeting a thrombolytic agent to a thrombus with minimal deactivation or destruction of the agent by thrombolytic inhibitors or cellular proteases.

Another object of the invention is to provide a therapeutically effective means of targeting a thrombolytic agent to a thrombus with minimal detection and destruction of the agent by the patient's immune system. A further object of the invention is to provide a less invasive and, thus, safer means of thrombolytic therapy through the administration of high affinity targeted thrombolytics intravenously rather than by catheterization.

Still another object of the invention is to provide for a more economical means of thrombolytic therapy through the administration of lesser quantities of more effective thrombolytic agents.

Yet another object of the invention is to provide a faster means of restoring blood flow in patients suffering from life-threatening or severely-damaging thrombic events.

In accordance with these objectives, the invention utilizes biocompatible, non-antigenic polymers in combination with both tissue or cellular receptor targeting ligands and thrombolytic agents to affect long acting yet localized lysis of thrombi.

The delivery of thrombolytic drugs that will not be detected and destroyed by the patient's immune system is accomplished by utilizing non-immunoreactive polymers such as the polyalkyleneoxides polyethylene glycol (PEG) and polypropylene glycol (PPG). Such polymers are known in the art to impart " stealth" through stearically hindering or otherwise blocking the binding of a patient's antibodies while still allowing for binding to plasminogen.

Yet even if thrombolytic drugs can evade immune system detection, cellular inhibitors and/or cellular proteases can reduce or eliminate thrombolytic activity. Thus, the presence of non-immunoreactive polymers further acts to stearically block or otherwise shield activated thrombolytics, such as plasmin, from inhibitory enzymes or proteases .

To minimize the possible side effects brought about by system-wide activation of thrombolysis, such as unchecked internal bleeding, targeting ligands are utilized to localize thrombolytic activity to the proximity of a thrombus. Targeting ligands may consist of any material or substance that promotes targeting of tissues and/or cellular receptors in vivo by the thrombolytic agents of the present invention. More specifically, a targeting ligand can be any synthetic, semi-synthetic, or naturally occurring material that displays binding affinity for one or more receptors on cell or tissue surfaces. Commonly used targeting ligands include, for example, proteins such as mono- and polyclonal antibodies, mono- or polysaccharides, bioactive agents, genetic materials, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a thrombolytic agent bound to one targeting ligand via a PEG spacer.

Figure 2A shows a thrombolytic agent connected to four molecules of targeting ligand via PEG spacers.

Figure 2B shows a thrombolytic agent connected to five molecules of targeting ligand via a star-PEG molecule.

Figure 3 shows a thrombolytic agent to which is attached a branched PEG. Each PEG branch bears one molecule of targeting ligand. DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the idea of selectively delivering a thrombolytic agent to the site of a thrombus. By localizing the activity of the agent to the actual thrombus or thrombi, thrombolysis is accomplished more effectively and with greatly reduced chances of a hemorrhage resulting from the system-wide activation of the thrombolytic pathway.

Accordingly, the invention comprises thrombolytic agents bearing targeting ligands to thrombi. Preferably each molecule of thrombolytic agent bears more than one targeting ligand and preferably each targeting ligand is covalently bound to the thrombolytic agent via a spacer molecule. In so doing, a "smart" thrombolytic agent is created which can then be targeted to create plasmin selectively at a site of vascular thrombosis. Thrombolytic agents useful in this invention are those which cause plasminogen or tissue plasminogen to be converted into plasmin. Such agents include SK, UK, and TPA. Other agents include but are not limited to scu-PA (single chain urokinase plasminogen activator), APSAC (acylated plasminogen/streptokinase activation complex), staphylokinase, prourokinase, anistreplase and alteplase. Of these, urokinase or scu-PA are preferred due to fewer immunological problems than streptokinase, a wider therapeutic window for administration than t-PA, and lower occurrences of cerebral hemorrhage than anistreplase and alteplase.

Targeting ligands are utilized in this invention to selectively bind specific receptors or proteins on cell or tissue surfaces. For example, targeting ligands may bind to fibrin monomers, fibrin polymers, targets on activated endothelial cells such as p-selectin or ICAM-1 or the activated GPIIb/IIIa receptor of platelets. Moreover, specific ligand/target pairs may include, for example: sialyl lewis X binding to E-selectin (ELAM-1) on polymorphonuclear leukocytes or monocytes, mucin binding to L-selectin on high venular endothelium, sialyl lewisX binding to P-selectin on platelets, polymorphonuclear leukocytes, or monocytes, LFA-1 (CDlla/CD18) binding to ICAM-1 on leukocytes, VLA-4 binding to VCAM-1 (CD31) on lymphocytes, and hyaluronic acid binding to HCAM (CD44) on endothelial or matrix cells. From the above examples, P- selectin may be an especially useful target because it localizes on the luminal side of the endothelium during inflammation. Otherwise, it is retained only within the cytoplasm. Thus, P-selectin may serve as a target specific for inflamed tissue.

Targeting areas of inflammation is beneficial because fibrinous precipitates may form at areas undergoing inflammation, such as sites of vascular damage or plaque formation. Inflammation- or thrombus-associated binding targets may include any available cellular receptor involved in the fibrinolytic or thrombogenic cascades , for example, fibrinogen or von Willebrand factor on platelets, fibronectin or vitronectin on endothelial or smooth muscle cells, fibronectin or VCAM-1 on leukocytes, VLA-4 or CD49d/CD29 on monocytes or lymphocytes, ICAMs on neutrophils or leukocytes, and fibrin.

Additional targets for the thrombolytic drugs of the present invention may include cells that harbor receptors for particular chemoattractants as well as for thrombogenic or inflammation events. For example, such targets may include receptors for: N-formyl peptides and C5a on monocytes, neutrophils, eosinophils, or basophils; leukotrine B4 on monocytes or neutrophils; platelet activating factor, CTAP-III, and β-thromboglobulin on monocytes, neutrophils, or eosinophils; IL-8/NAP-1 on neutrophils or basophils; gro/MGSA on neutrophils, basophils, or fibroblasts; ENA-78 on neutrophils; MCP-1 on monocytes or basophils; MAP-lα,β on monocytes, neutrophils, t-lymphocyte subpopulations , basophils, or eosinophils; RANTES on monocytes, t-lymphocyte subpopulations, or eosinophils; 1-309 on monocytes.

The receptors in some of the examples above are G-protein coupled, large, and transmembranous . Moreover, they can, among other functions, direct migration and activation of integrin adhesion, with the preferred integrin target being the activated GPIIb/IIIa receptor of platelets. Thus, such receptors may be a good general class of targets for thrombolytic therapy.

At least one molecule of targeting ligand is bound to each molecule of thrombolytic agent. Preferably, each molecule of thrombolytic agent binds more than one molecule of targeting ligand. Up to about 100 molecules of targeting ligand may be bound per molecule of thrombolytic agent, but more preferably somewhere between 2 and 10 molecules of targeting ligand are bound per molecule of thrombolytic agent. Most preferably, somewhere between about 5 and about 10 targeting ligand molecules are bound to each molecule of thrombolytic agent. As one skilled in the art would recognize, however, correspondingly more and less targeting ligands may be bound to thrombolytic agents of higher and lower molecular weight.

The targeting ligand may be an antibody, an antibody fragment, a protein, a glycoprotein, a peptide, a glycopeptide, a polysaccharide, and oligosaccharide or a sugar molecule. If an antibody or antibody fragment is used as the targeting ligand, it is preferably fully or partially humanized and preferably produced via recombinant techniques. The preferred targeting ligand, however, is a peptide. Preferred peptides contain between 3 and 25 amino acids in length and still more preferably between 5 and 15 amino acids in length. The most preferred peptides have 6 amino acids. Preferred peptides may contain the 3 amino acid sequence of Arginine-Glycine- Aspartic acid (RGD) . Another preferred series of peptides bears the 3 amino acid sequence Alanine-Glycine-Aspartic Acid (AGD) . The peptide may be composed of D or L amino acids or a mixture thereof , most preferred, however, are peptides composed of L amino acids. The peptides may be linear or cyclized. Cyclized peptides are preferred because of increased resistance to hydrolysis and increased binding affinity for their target or targets. However, we have discovered that linear peptides can be used with great success for this invention. A particularly preferred peptide is the linear hexapeptide derived from the gamma carboxyl terminus of fibrinogen, SEQ ID NO.l Lysine-Glutamine-Alanine-Glycine-Aspartate- Valine (KQAGDV) . Unless noted otherwise, the standard one letter code for amino acids will be used hereinafter. Furthermore, all amino acid residues are considered to be the L-isomer unless specifically designated as a D isomer, in which case the amino acid will be preceded by (D). However, all chiral , diasteromeric and racemic forms are included in the present invention.

Other variant peptides of the RGD/AGD family having affinity for endothelial receptors include SEQ ID NO.2 GG(D)I(D)W(D)T(D)W(D)V, SEQ ID NO.3 G(D) I (D)W(D)T(D)W(D)V, SEQ ID NO.4 GIWTWV, SEQ ID NO.5 GGIWTWV, SEQ ID NO.6 NKTWTWV(NH₂) , SEQ ID NO.7 KTWTWV(NH₂) , or SEQ ID NO.8 TWTWV(NH₂), where (NH₂) represents C-terminal amidation of the peptide. These peptides, including those containing D-analogs, comprise portions of the sequence of P-, L-, E-selectins and inhibit leukocyte adhesion to endothelium or platelets. Accordingly, the invention may be used in the treatment of, for example, restenosis, angiogenesis in tumors, diabetic retinopathy, or reperfusion injury. Further ligands having affinity to endothelial cell receptors include, for example, PI of the gamma subunit of fibrinogen, which is defined as SEQ ID NO.9 KYGWTVFQAKRLDGSV or SEQ ID NO.10 KYGQKRLDGS . Still other effective peptides include those of the group consisting of SEQ ID NO.11 RYTDLVAI (NH₂) , SEQ ID NO.12 YTDLVAI (NH₂) , SEQ ID NO.13 YTDLVAIQNKNE(NH₂) , SEQ ID NO.14 DLVAIQNKNE(NH₂) , SEQ ID NO.15 LVAIQNKNE(NH₂) , SEQ ID NO.16 TDLVAIQN(NH₂) , and SEQ ID NO.17 Nif-TDLVAIQN(NH₂) , where Nif- is nifedipine. Additionally, examples of short RGD/AGD variant peptide ligands which find use in the invention include SEQ ID NO.18 GRGD, SEQ ID NO.19 ARGD, SEQ ID NO.20 VRGD, SEQ ID NO.21 LRGD , SEQ ID NO.22 SRGD, and SEQ ID NO.23 FRGD.

Fibrin-binding peptides also find utility in the invention. Among those favored are those identified as sequences of Factor XIIIA. Exemplary of the peptides of this type are SEQ ID NO.24 NKLIVRRGQSFYVQIDFSRPYDPRRDLFRVEYVIGRYPQENKGTYIPVPIVSELQSGK WGAKIVMR, SEQ ID NO.25

EDRSVRLSIQSSPKCIVGKFRMYVAVWTPYGVLRTSRNPETDTYILFN PWCEDDAVYLDNEKERE , and SEQ ID NO.26

EYVLNDIGVIFYGEVNDIKTRSWSYGQF-R' , where R' is -C0NH₂ or - NH₂. Fragments of these peptides may also be used. Common features of this group of peptides are the accessible SEQ ID NO.27 GQCWVF motif or chemical equivalents. Ho, K.C. , et al., J. Biol. Chem. 267:12664-12667 (1992).

Cyclic peptides in which the otherwise free amino terminus of a chain condenses with the otherwise free carboxyl end constitute another major class of ligands applicable to this invention. Within this broad group, one or more amino acyl residues may be of D-type stereochemistry or may contain modified arginine groups. Exemplary of this class of peptide ligands are those disclosed in Degrado, et al., U.S. Patent No. 5,635,477, which is incorporated by reference herein. Protective groups, such as tert- butyloxycarbonyl (t-BOC) , are used to mask reactive side chains in stepwise syntheses of cyclic peptides as disclosed in the '477 patent.

As shown in, for example, Table 1 of the '477 patent, there are a large number of cyclic targeting ligand formulations which will find use in the thrombolytic conjugates of the present invention. Such ligand formulations may be described generically as cyclic butapeptides containing the amino acids GD in combination with any two other modified amino acid residues. Illustrative of the thousands of cyclic peptide ligands operable in the present invention is cyclo-(D)V-(alpha-N- methyl)-RGD-(3-aminomethylbenzoic acid) .

Other classes of RGD/AGD variant peptide ligands suitable for the compositions of the invention include those with internal cysteine residues available for S-S bond dimerization. For example, SEQ ID NO.28 XaaRGDXaaXaa, where Xaa at position one is hydrogen or any amino acid and Xaa at positions five and six is Thr or Cys; SEQ ID NO.29 SYGRGDVRGDFKCGC , with the N-terminus acetylated; SEQ ID NO.30 RGDXaa, where Xaa is Ser, Thr, or Cys; SEQ ID NO.31 GRGDVRGDFKCGC , where the C-terminus is an amide;

SEQ ID NO.32 CGCRRRRRRRRRGDV; and

SEQ ID NO.33 SYGRGDVRGDFKCTCC, where the C-terminus is an amide.

Any of the above peptide ligands can serve as targeting moieties to the appropriate receptors. Synthetic methods for a few of the more preferred conjugates are illustrated in the Examples to follow. Those skilled in the biosynthetic art will appreciate that basic, thiol, hydroxyl and/or acidic functional groups in the amino acyl residues have individual chemistries such that protection and deprotection involves different blocking groups, such as t-BOC, FMOC, and others.

The targeting ligands of the present invention preferably are covalently bound to the thrombolytic agent via a spacer molecule or tether. The spacer molecule or tether preferably comprises a blood soluble or hydrophilic polymer, the preferred polymer being PEG. However, a variety of different hydrophilic polymers or tethers may be used to bind the targeting ligand(s) to the thrombolytic agent. Such polymers include but are not limited to copolymers of polyethylene oxide and polyvinyl alcohol , polyhydroxyporopylene glycol , polypropylene glycol , polymethylpropyleneglycol and polyhydroxypropyleneoxide, heteropolymers of small alkoxy monomers, such as a polyethylene/polypropylene-glycol, polyalkylether, such as the methoxy- or ethoxy- capped analogs, dextran, or starch. All of these can be obtained commercially in a variety of polymer sizes, for example, from 120-20,000 daltons. Alternatively, a homo- or heteropolymer can be formed by known polymer synthesis methods to achieve a desired monomeric composition and size.

The hydrophilic polymer used as the tether to bind the targeting ligand to the thrombolytic agent may vary in molecular weight from about 100 to 150,000. More preferably, the molecular weight of the polymer varies from about 1,000 to 50,000 and even more preferably from about 3,000 to about 30,000 daltons. The preferred polymer is a di- or bifunctional PEG derivative. Such PEG derivatives include PEG diol , star-PEGs , multi-arm branched PEGs, di-amino-PEGs, amino acid esters of PEG, hydrazine hydrochloride derivatives of PEG, nucleophilic PEG derivatives such a PEG-thiol, carboxylate PEGs such as PEG succinate, carboxymethylated PEG and PEG-propionic acid, PEG amino acids and electrophilically activated PEGs such as PEG succinimidyl succinate, the succinamide derivative of PEG propionic acid, the succinimide derivative of carboxymethylated PEG, PEG-2-succinimide, the benzotriazole carbonate derivative of PEG, active esters of amino acid PEGs, pendant modified PEG-NHS esters, the glycidyl ether or epoxide derivatives of PEG, oxycarbonylimidazole derivatives of PEG, p- nitrophenylcarbonate derivatives of PEG, PEG tresylate, PEG aldehyde, PEG isocyanate and maleic anhydride copolymers of PEG, sulfhydryl selective PEGs such as PEG vinylsulfone, PEG iodoacetamide, PEG maleimide and PEG- orthopyridyl-disulfide .

In the foregoing series of PEG derivatives, most preferably both termini of the PEG molecule are functionalized, i.e. neither end comprises the monomethoxy derivative. Both ends of the PEG molecule are functionalized so that one end is free to react with a group on the thrombolytic agent and one end is free to react with the targeting ligand, e.g. a peptide. Although both ends of the PEG molecule may be the same, more preferably the PEG is heterofunctional , i.e., both ends comprise a different reactive group. Additional heterofunctional PEGs include H0-PEG-NH₂, HO-PEG-COOH, and NH₂-PEG-C00H. Also the heterofunctional PEGs include NHS- PEG-vinylsulfone and NHS-PEG-maleimide.

Biotinylated PEGs may also be employed such as biotin-PEG- biotin, biotin-PEG-NHS and biotin-PEG-maleimide. When a biotinylated PEG is used, it is bound to avidin. The avidin molecule is bound to either the targeting ligand (e.g. antibody or peptide) or more preferably the avidin is bound to the thrombolytic agent. Because the avidin/biotin binding interaction is so strong, the targeted thrombolytic can be administered in tandem if desired; in other words, the targeting ligand-PEG conjugate containing a free terminal biotin is administered first, and then, after some time for the conjugate to bind to the thrombus (perhaps 5 minutes), the avidin labeled thrombolytic is administered so as to bind the biotin on the ligand-PEG conjugate.

More preferably, however, the targeted thrombolytic is administered in one step and not in succession. Vinyl derivatives of PEG can also be used such as PEG-0CH₂CH=CH₂ (allyl-PEG), PEG-0₂CCH=CH₂ (acryl-PEG), and PEG- methacrylate. Additional PEG derivatives which may be useful in this invention include PEG-S-propionic Acid and PEG-trichlorophenylcarbonate.

In a preferred embodiment of this invention a branched PEG is used to couple more than one targeting ligand to one molecule of thrombolytic agent. It is believed that this configuration increases the affinity of the targeted thrombolytic for the substrate thrombus. The creation of a branched PEG may be achieved by attaching 2 molecules of a di-functional PEG to lysine to produce a branched acid. For example, t-BOC-amino-PEG-COOH is activated with N- hydroxysuccinimide to yield the active ester of t-BOC- (PEG)₂NHS. The branched structure of the PEG has a relatively large molecular volume which helps to protect the thrombolytic agent from hydrolysis and helps to prolong the circulation half-life of the thrombolytic agent, and is particularly useful for low molecular weight thrombolytic agents (below 30,000 MW) .

A particularly preferred branched PEG is the star-PEG, which is a multi-armed PEG made by polymerization of ethylene oxide from a cross-linked divinyl benzene core (Gnanou, Y., et al. (1988) Makromol . Chem. 189, 2885; Rein, D., et al . (1993) Acta Polymer., 44, 225.). Both the number and length of PEG branches or "arms" can be controlled, allowing for the accommodation of more molecules of ligand or thrombolytic agent as desired. Moreover, having more "arms" can impart more rotational degrees of freedom to the conjugate in its approach to the receptor binding sites .

We discovered that the polydentate nature produced by attaching binding ligands to branched PEGs increases the affinity of the targeted thrombolytic agent for the clot. When a branched PEG is used in this manner, preferably both free arms of the PEG molecule are bound to targeting ligands and the central portion of the PEG is bound to the thrombolytic agent.

Depending upon the affinity of the targeting ligand for thrombus, the particular thrombolytic agent, and other factors, it is possible to have variations on this theme. For example, one end of the di-PEG molecule can be the monomethoxy derivative and the targeting ligand affixed to only one terminus. On the other hand, the thrombolytic agent may be bound to both free ends of the di-PEG molecule and the targeting ligand attached to the center of the di-PEG. This latter conformation works well when bulky targeting ligands such as antibodies are used to target multiple thrombolytic agents per molecule of targeting ligand. It is even possible in this regard to produce agents which comprise two different thrombolytic molecules tethered together by a di-PEG directed to the thrombus by a single targeting ligand. Most preferably, however, when a di-PEG is used, both free arms of the PEG are bound to a peptide, and the central portion of the di- PEG is bound to a single molecule of thrombolytic agent. Furthermore, it is possible to use PEGs of higher orders of branching so that a single molecule of PEG may bind three or more targeting ligands and 1 or more thrombolytic agent .

The targeting ligand(s) may first be bound to the PEG spacer, and this in turn is bound to the thrombolytic agent. The functionalized PEG-peptide conjugate is bound to the thrombolytic agent using reactive groups and conditions which will not inactivate the enzymatic properties of the thrombolytic agent. The thrombolytic agents contain a variety of different groups which can be used to bind the PEG-peptide conjugate. Generally, these groups are found within the amino acids which form the backbone of the thrombolytic agent. Such groups include primary and secondary amines, primary and secondary carboxyl groups, hydroxyl groups, and sulfhydryl moieties. Alternatively, artificially-added linkers, such as thiol groups, may be used to couple a thrombolytic agent to the PEG-peptide conjugate (Unger, PCT publication WO96/40285, published 12/19/96).

Some of the thrombolytic agents contain saccharide or other sugar molecules. Hydroxyl groups on these sugar molecules can be activated into aldehydes, and these can be use to bind the reactive groups on the ends of the PEG- peptide conjugates. The particular groups on the thrombolytic agent selected to attach to the targeting ligand will vary depending upon the particular thrombolytic agent. The selection for sites of attachment, for example, lysine versus cysteine, is generally chosen so as to not adversely affect the enzymatic activity of the thrombolytic agent. The most frequently used derivatives for attachment to a lysine reside on the thrombolytic agent are the N-NHS active esters of PEG such as PEG succinimidyl succinate (SS-PEG- peptide) and succinimidyl propionate (SPA-PEG). Usually, several PEG-peptides can be attached to a single protein molecule of thrombolytic agent at room temperature at pH 8-9.5 within 30 minutes. Increasing the pH will increase the rate of reaction; conversely, lowering the pH will decrease the rate of reaction.

The general scheme of a reaction using lysine active PEG'S and a protein based thrombolytic agent is shown below: 1-10 parts SPA-PEG-peptide-peptide-PEG-0CH₂CH₂-C0₂-NHS + 1 part Protein-NH₂ — > Peptide-PEG-0-CH₂CH₂-C0NH- Protein (when reacted at pH 7-9 for 30min) .

Sulfhydryl-selective PEGs such as vinylsulfone, iodoaceta ide, maleimide, and dithioorthopyridine derivatives may be used to react with sulfhydryl groups (e.g. cysteine residues) on the thrombolytic protein. Typically, the reaction times for these derivatives will be 0.5 to 2 hours at pH 7-8. For attachment to stearically hindered sulfhydryl groups on thrombolytic agents, however, the reaction times may be significantly longer.

After the product is prepared, unbound PEG-peptide may be removed by chromatographic separation, differential solubilization, or dialysis. The resultant product may be stored as an aqueous solution of peptide-PEG-thrombolytic agent or stored as a dried powder. If necessary, the product can be dried through lyophylization to prolong shelf life. To increase the stability during lyophylization, the product may be mixed with human serum albumin or human albumin derived from recombinant sources. Amounts approaching 95% by weight and higher of human serum can be mixed with the peptide-PEG-thrombolytic material to help protect it during lyophylization. Alternatively, or in addition, the peptide-PEG- thrombolytic material may be mixed with one or more cryoprotectant materials, such as trehalose, sucrose, maltose, dextran, or any of a variety of sugar-based material as is well known in the art. Most preferably, however, the peptide-PEG-thrombolytic agent is stored as an aqueous solution, ready to use.

In general, the targeted thrombolytic agent is administered intravenously. Indeed, the high specificity of these new agents allows thrombolysis to be attained in a less invasive manner than was hitherto possible. For example, acute myocardial infarction due to thrombosis in the coronary artery is commonly treated at present by injecting a thrombolytic agent directly into the coronary artery via catheter. Because the targeted thrombolytics bind at the site of the clot, they can often be administered intravenously yet still achieve the same effect as intra-arterial administration, thus avoiding complications, such as further vascular injury, caused by using a catheter.

Nonetheless, targeted thrombolytics can be administered intra-arterially, whereupon a longer duration effect is achieved. Targeted thrombolytics are also useful following angioplasty, stent, and graft placement to prevent clot- or inflammation-related restenosis. In this application, the embodiments of this invention with targeting ligands directed to activated endothelial cells can be used to prevent thrombosis following vascular procedures. Targeted thrombolytics can also be used in concert with ultrasound enhanced sonothrombolysis, sonothrombolysis enhanced by microbubbles , and mechanical clot fragmentation/lysis procedures.

The following examples are provided to further describe and clarify the invention. However, such description discloses only some of the various ways in which the invention may be practiced. Examples numbers 1-4,6,7, and 9 are actual examples, while numbers 5,8, and 10 are prophetic in nature.

Example 1

This example is directed to the synthesis of hexalinear

SEQ ID NO. 1 KQAGDV peptide.

Initially, 1. Og of N-( 9-fluorenylmethyloxycarbonyl ) -L- valine p-alkoxybenzyl alcohol resin ester (containing 0.56 mmole of amino acid) is shaken with 20ml of 20% (v/v) piperdine in methylene chloride for 1 hour to remove the FMOC (9-fluorenylmethyloxycarbonyl) group. The mixture is filtered and the resin washed with methylene chloride. The deprotected resin is then treated with 0.92g of N- FMOC-L-aspartic acid-beta-t-butyl ester in 15ml of dimethylformamide in the presence of 0.43g l-(3- dimethylaminopropyl)-3-ethylcarbondiimide hydrochloride (EDC), 0.31ml triethylamine, and 0.30g 1- hydroxybenzotriazole (HOBT) for 1.5 hrs. The resin is again filtered out, washed with methylene chloride, and treated with 20% piperdine in methylene chloride as above to remove the FMOC group. The resulting resin derivative is then treated as above with 1.36g N-alpha-FMOC-N-omega- ( 4-methoxy-2 , 3 , 6-trimethylbenzenesulfonyl ) -L-glycine in the presence of triethylamine, EDC, and HOBT. The FMOC group is removed again as above.

The peptide then is removed from the resin by treating with 20ml of 95% trifluoroacetic acid for two hours. Subsequent treatment with N-FMOC adducts of L-alanine and L-glutamine follow analogously. Finally, the resulting resin derivative is treated as above with 1.36g N-alpha- FMOC-N-omega-( 4-methoxy-2 , 3 , 6-trimethylbenzenesulfonyl )-L- lysine in the presence of triethylamine, EDC, and HOBT. The lysine epsilon amino group is deprotected by overnight treatment with concentrated trifluoroacetic acid. The resulting solution is diluted with 0.5% (v/v) acetic acid, washed with 3 portions of ethyl acetate, than lyophilized to give L-lysyl-L-glutaninyl-L-alanyl-L-glycyl-L-aspartyl- L-valine as the ditrifluoroacetate salt, the melting point of which is 90-95° C.

Example 2

This example is directed to the preparation of a conjugate form by SEQ ID NO. 1 KQAGDV and PEG.

A. Preparation of PEG-succinimide

To a cooled (0 to 5 C) 250 ml round bottom flask containing a solution of 200 mg of PEG (MW 3,500 daltons; Shearwater Polymers, Inc., Huntsville, AL) , 6 mg of N- hydroxysuccinimide, 2 mg DMAP (4-dimethylaminopyridine) , and 40 ml of acetonitrile, a solution of 12mg DCC (dicyclohexylcarbodiimide) in 10 ml of acetonitrile was added in a dropwise fashion. The mixture was stirred for 5 hours and the resulting white solid (dicyclohexylurea) was removed by filtration. The filtrate was concentrated in vacuo to produce 200 mg of PEG-succinimide as a white solid.

B. Preparation of SEQ ID N0.1 and PEG conjugate To a cooled (0 to 5°C), stirred solution containing 20 mg of SEQ ID NO.l (Shearwater Polymers or synthesized as in Example 1) in 20 ml aqueous buffer at a pH of 8.5, a 4 mg solution of PEG-succinimide in 10 ml acetonitrile from Step A was added dropwise. The temperature of the resulting mixture was equilibrated to room temperature and the reaction mixture was stirred for about 48 hours. The mixture was concentrated in vacuo and the residual salts were washed away using a dialysis bag having a molecular weight cutoff of about 3500 and equilibrated against water. The resulting dialyzed solution was frozen and lyophilized to yield 12 mg of PEG-SEQ ID NO.l as a white solid.

Example 3

This example is directed to the synthesis of cyclized RGD bearing peptides.

The chemical t-Butyloxycarbonyl-3-aminomethylbenzoic acid (Boc-Mamb) is coupled to oxime resin by modification of the method described by Degrade and Kaiser [(1980) J. Org. Chem. 45:11295-11300] using one equivalent of the 3- aminomethylbenzoic acid, one equivalent of HBTU [2-(lH- Benzotriazol-1-yl)-1 , 1 , 3 , 3-tetramethyluronium hexafluorophosphate] , and three equivalents of NMM (N- methyl orpholine) . Alternatively, 1 equivalent of Boc- Mamb may be coupled to oxime resin using one equivalent each of DCC and DMAP in methylene chloride. Coupling times range from 15 to 96 hours. The substitution level is then determined using either the picric acid test [Gisin, (1972) Anal. Chim. Acta, 58:248-249] or the quantitative ninhydrin assay [Jones et al., (1975) In Vitro, 11:41-45]. Unreacted oxime groups are blocked using 0.5 M trimethylacetylchloride/0.5 M diisopropylethylamine in DMF (N,N-dimethylformamide) for 2 hours. Deprotection of the Boc protecting group is accomplished using 25% TFA

(trifluoroacetic acid) in DCM (dichloromethane) (v/v) for 30 minutes.

The remaining amino acids or derivatives are coupled using a two to tenfold excess (based on the loading of the first amino acid or amino acid derivative) of the appropriate amino acid or derivative and HBTU in approximately 8 mis of DMF. The resin is then neutralized in situ using three equivalents of NMM, and the coupling times range from one hour to several days. The completeness of coupling is monitored by qualitative ninhydrin assay in cases where the amino acid was coupled to a secondary amine. Amino acids are recoupled if necessary based on these results.

After the linear peptide has been assembled, the N- terminal Boc group is removed by treatment with 25% TFA in DCM for 30 minutes. The resin is then neutralized by treatment with 10% DIEA (diisopropylethylamine) in DCM. Cyclization with the concomitant cleavage of the peptide is accomplished using the method of Osapay and Taylor, J.Am.Chem. Soc (1990), 112:6046-6051. Briefly, the resin is suspended in approximately 10 mls/g of DMF, adding one equivalent of acetic acid and stirring at 50-60 °C for 50- 72 hours. Following filtration through a sintered glass funnel, the DMF filtrate is evaporated, redissolved in acetic acid or 1 : 1 acetonitrile:water, and lyophilized to obtain protected cyclized material. Alternatively, the material may be dissolved in methanol and precipitated with ether to obtain protected cyclized material. This is then treated using standard procedures with anhydrous hydrogen fluoride (Lebl and Hruby, Tetrahedron Lett. (1984) 25:2067-2068. containing lml/g m- cresol or anisole as scavenger at 0° C for 20 to 60 minutes to remove side chain protecting groups. The crude product may be purified by reverse phase HPLC using a 2.5 cm preparative Vydac C18 column with a linear acetonitrile gradient containing 0.1% TFA to produce pure cyclized material .

Example 4 This example is directed to the synthesis of PEG- streptokinase.

PEG-streptokinase coupling is achieved as described in Rajagopalan et al., J. Clin. Invest. _r 75:413-419 (1985). Briefly, PEG was dissolved in dioxane at 37 °C at a concentration of 50mM. l,l'-carbonyldiimidazole was added to a final concentration of 0.5 M and the solution was incubated at 37 °C for 2hrs with stirring. The solutions then were dialyzed extensively against H₂0 using Spectrapor membranes with M-. inclusion limits of 1,000 for PEG-2, 2,000 for PEG-4, and 3,500 for PEG-5. Activated PEG preparations were lyophilized and stored desiccated at 4°C.

Activated PEG then was reacted with streptokinase (luM) in lOmM sodium borate buffer, pH 8.5, at 4°C for 72 hrs with 40mM of activated PEG. Up to 80mM of activated PEG may be used in the reaction above with no appreciable loss in SK activity.

Example 5

This example is directed to the synthesis of SEQ ID NO.l- PEG-Streptokinase .

First, the compound ω, ω'-dimethylenecarboxy- polyethyleneglycol anhydride is made by mixing 0.34g of cold (0-5°C) ω, ω'-dimethylenecarboxy-polyethyleneglycol with 20ml of methanol in a 100ml round-bottomed flask together with 0.02g of dicyclohexylcarbodiimide (DCC) in 5ml of methanol. This solution should be stirred overnight, followed by removal of the resulting white precipitate (dicyclohexylurea) by filtration. The filtrate is then concentrated by evaporation to yield a 0.3g white crystal product.

Second, the cooled product from the first step is reacted with a mixture containing 3mg of DCC in 2ml of acetonitrile, 1.8mg of N-hydroxy-succinimide, and 0.2mg of DMAP in 6.0ml of acetonitrile. After 3 hours of stirring at 5°C, the mixture is equilibrated at room temperature and stirring is continued overnight. The white precipitate (dicyclohexylurea) is removed by filtration as above and the filtrate is concentrated by evaporation to yield 60mg of succinymidyl-PEG-succinimide.

Next, the product from the second step is dissolved in 8ml of acetonitrile, which then is added dropwise to a 10°C solution of 30ml of pH 8.5 phosphate buffer containing 20mg each of streptokinase and KQAGDV. The mixture is stirred at room temperature for 48 hours and then concentrated under vacuum and dialyzed against lOmM phosphate buffer using dialysis tubing with a cutoff of 3500 daltons. The resulting dialysate is then lyophilized to yield 15mg of a mixture containing three conjugates, i.e., SEQ ID N0.1-PEG-SEQ ID NO.l, SEQ ID NO.l-PEG- streptokinase, and streptokinase-PEG-streptokinase. The three products are resolved by gel filtration chromatography, with the desired SEQ ID N0.1-PEG- streptokinase eluting as a band between the other two conjugates. Example 6

This example is directed to the synthesis of cyclic RGD- PEG-streptokinase , where cyclic RGD-PEG is prepared as described in Example 3.

Depending upon the structure of the cyclic RGD peptide, a number of different chemical groups can be used to attach the peptide to the PEG molecule. For example, the gamma- COOH group of the aspartic acid residue can be reacted with PEG-amine as generally described by Zalipsky et al . , (1983) Eur. Polym. J 19:1177. Alternatively, a R-group carboxyl distally located from the RGD area could be reacted with PEG-amine. In any case, standard protection techniques involving t-BOC can be utilized to block the groups on the peptide that one does not wish to react as described in Kelly and McNeil, Tetrahedron Lett. (1994), 35:

9003-6. The cyclized RGD-PEG can then be chemically attached to streptokinase by reacting a free -OH group on PEG with lysyl-NH₂ groups on streptokinase. Alternatively, a Merrifield or modified Merrifield peptide synthesis protocol may be used as described in U.S. Patent No. 3,784,523.

Example 7

This example is directed to the synthesis of SEQ ID NO.l- PEG-Streptokinase , where the peptide-PEG conjugate is coupled with a cysteine residue of streptokinase.

A bifunctional PEG-thiol, such as PEG-(SH)₂ (Shearwater Polymers, Huntsville, AL) , is reacted with a hexalinear SEQ ID NO.l peptide to produce the intermediate SEQ ID NO.l-PEG-SH according to the general method of Zalipsky et al., J.Macro ol.Sci. (1984) A21 6-7:839-45. Since thiol groups readily undergo disulfide formation, this intermediate subsequently is reacted with cysteine residues on streptokinase to produce SEQ ID N0.1-PEG- streptokinase (Musu, et al., Appl .Biochem.Biotechnol. (1996) 56:243-263).

Example 8

This example is directed to the synthesis of urokinase- PEG-SEQ ID NO.l, where the PEG-peptide is reacted with sugar moieties of urokinase.

Using PEG-(amine)₂ (Shearwater Polymers), amine-PEG- peptide is made according to the method of Zalipsky et al., (1983) Eur.Polym. J, 19:1177. Since urokinase contains sugar rings harboring ether linkages, these rings are "opened-up, " i.e. the ether linkage is converted to an aldehyde, by exposing the urokinase to a mild oxidizing agent. The amine-PEG-peptide is then reacted with the newly-formed sugar aldehydes to yield peptide-PEG- urokinase. Both of these steps are described in detail in Ohya, Y., et al . , (1991) J. Macro ol . Sci . Chem. , A28:743- 60.

Example 9

This example is directed to the synthesis of peptide-PEG- t-PA.

PEG-peptide is synthesized as described in Example 4. The PEG-peptide intermediate is then reacted with t-PA in the following manner. To a cooled (5-10° C) , stirred solution of t-PA (20mg in 20ml of aqueous buffer) at a pH of 8.5 was added dropwise to a solution of PEG-peptide and acetonitrile (10ml). The temperature of the resulting mixture was equilibrated to room temperature and the reaction mixture was stirred for about 48 hours. The mixture was concentrated in vacuo and the residual salts were dialyzed away using a dialysis bag having a molecular weight cutoff of about 3500, equilibrated against water. The resulting dialyzed solution was frozen and lyophilized to yield 12mg of peptide-PEG-t-PA as a white solid.

Example 10

This example is directed at the synthesis to streptokinase-starPEG-SEQ ID N0.1KQAGDV, where a branched PEG is used to generate a conjugate with multiple targeting ligands tethered to a single thrombolytic- starPEG.

Multi-armed or "star" PEG-peptide (Shearwater Polymers, Huntsville, Alabama) was synthesized in the same manner described in Example 2, except that a 10 molar excess of SEQ ID NO.l was reacted per mole of star PEG. Subsequently, starPEG-peptide molecules are chemically attached to streptokinase by reacting a free -OH group on PEG with lysyl-NH2 groups on streptokinase. Alternatively, a free hydroxyl group on PEG can be oxidized to -COOH so that the starPEG-peptide and streptokinase can be joined by a quasi peptide type condensation reaction utilizing the same conditions as in Example 4 except that 8 equivalents of peptide are used per equivalent of star-PEG.

As would be understood by those skilled in the art, any number of functional equivalents may exist in lieu of the preferred embodiments described above. Thus, as will be apparent to those skilled in the art, changes in the details, steps and materials that have been described may be within the principles and scope of the invention illustrated herein and defined in the appended claims. Therefore, while the present invention has been shown and described in what is believed to be the most practical and preferred embodiment, it is recognized that departures can be made therefrom within the scope of the invention, which is therefore not to be limited to the details disclosed herein but is to be accorded the full scope of the claims so as to embrace any and all equivalent products and methods.

Claims

We claim:

1. A targeted molecular composition comprising a thrombolytic agent, a non-immunoreactive polymer, and a targeting ligand.

2. The targeted molecular composition of Claim 1 , wherein said thrombolytic agent is selected from the group consisting of streptokinase, urokinase, tissue plasminogen activator, single-chain urokinase plasminogen activator, acylated plasminogen/streptokinase activation complex, staphylokinase, prourokinase , anistreplase, and alteplase, or mixtures thereof.

3. The targeted molecular composition of Claim 1, wherein said non-immunoreactive polymer is selected from the group consisting of polyethylene glycol, copolymers of polyethylene oxide and polyvinyl alcohol, polyhydroxypropylene glycol, polypropylene glycol, polymethylpropyleneglycol , polyhydroxypropyleneoxide, heteropolymers of alkoxy monomers, polyethylene/polypropyleneglycol, polyalkylether , PEG diol, star PEGs, multi-arm branched PEGs, di-amino-PEGs, amino acid esters of PEG, hydrazine hydrochloride derivatives of PEG, nucleophilic PEG derivatives, carboxylate PEGs, PEG amino acids, electrophilically activated PEGs, PEG succinimidyl succinamide, a succinamide derivative of PEG propionic acid, a succinimide derivative of carboxymethylated PEG, PEG 2 succinimide, a benzotriazole carbonate derivative of PEG, active esters of amino acid PEGs, pendant modified PEG NHS esters, the glycidyl ether or epoxide derivatives of PEG, oxycarbonylimidazole derivatives of PEG, p- nitrophenylcarbonate derivatives of PEG, PEG tresylate, PEG aldehyde, PEG isocyanate and maleic anhydride copolymers of PEG, sulfhydryl selective PEGs, PEG vinylsulfone, PEG iodoacetamide, PEG malei ide, and PEG- orthopyridyl-disulfide, or mixtures thereof.

4. The targeted molecular composition of Claim 1, wherein said targeting ligand is selected from the group consisting of antibodies, antibody fragments, proteins, glycoproteins, peptides, glycopeptides , polysaccharides, oligosaccharides, and monosaccharides, or mixtures thereof .

5. The targeted molecular composition of Claim 4, wherein said peptides are between 3 and 66 amino acids in length.

6. The targeted molecular composition of Claim 5, wherein said peptides are selected from the group consisting of SEQ ID NO.2 GG(D)I(D)W(D)T(D)W(D)V, SEQ ID NO.3 G(D)I(D)W(D)T(D)W(D)V, SEQ ID NO.4 GIWTWV, SEQ ID NO.5 GGIWTWV, SEQ ID NO.6 NKTWTWV(NH₂) , SEQ ID NO .7 KTWTWV(NH₂) , SEQ ID NO.8 TWTWV(NH₂), SEQ ID NO.11 RYTDLVAI (NH₂) , SEQ ID NO.12 YTDLVAI(NH₂) , SEQ ID NO.13 YTDLVAIQNKNE(NH₂) , SEQ ID NO.14 DLVAIQNKNE(NH₂) , SEQ ID NO.15 LVAIQNKNE (NH₂) , SEQ ID NO.16 TDLVAIQN(NH₂) , and SEQ ID NO.17 Nif-TDLVAIQN(NH₂) , SEQ ID NO.18 GRGD, SEQ ID NO.19 ARGD, SEQ ID NO.20 VRGD, SEQ ID NO.21 LRGD, SEQ ID NO.22 SRGD, and SEQ ID NO.23 FRGD, or mixtures thereof.

7. The targeted molecular composition of Claim 5, wherein said peptides are selected from the group consisting of

SEQ ID NO.24

NKLIVRRGQSFYVQIDFSRPYDPRRDLFRVEYVIGRYPQENKGTYIPVPIVSELQSGK WGAKIVMR, SEQ ID NO.25

EDRSVRLSIQSSPKCIVGKFRMYVAVWTPYGVLRTSRNPETDTYILFN PWCEDDAVYLDNEKERE , and SEQ ID NO.26

EYVLNDIGVIFYGEVNDIKTRSWSYGQF-R', where R' is -C0NH₂ or - NH₂ , or fragments thereof .

8. The targeted molecular composition of Claim 5, wherein said peptides are selected from the group consisting of

SEQ ID NO.28 XaaRGDXaaXaa , where Xaa at position one is hydrogen or any amino acid and Xaa at position five or six is Thr or Cys; SEQ ID NO.29 SYGRGDVRGDFKCGC , with the N- terminus acetylated; SEQ ID NO.30 RGDXaa, where Xaa is Ser, Thr, or Cys; SEQ ID NO.31 GRGDVRGDFKCGC , where the C- terminus is an amide; SEQ ID NO.32 CGCRRRRRRRRRGDV; and SEQ ID NO.33 SYGRGDVRGDFKCTCC , where the C-terminus is an amide , or mixtures thereof .

9. The targeted molecular composition of Claim 5 , wherein said peptides are selected from a group consisting of SEQ ID NO.9 KYGWTVFQAKRLDGSV and SEQ ID NO.10 KYGQKRLDGS, or mixtures thereof.

10. The targeted molecular composition of Claim 5, wherein said peptides are cyclic peptides.

11. The targeted molecular composition of Claim 10, wherein said cyclic peptides comprise a butapeptide containing the amino acids G and D in combination with any two other amino acids.

12. The targeted molecular composition of Claim 11, wherein said butapeptide comprise the sequence cyclo-(D)V- (alpha-N-methyl)-RGD-(3-aminomethylbenzoic acid) .

13. The targeted molecular composition of Claim 5, wherein said peptides consist of linear hexapeptides containing the amino acids RGD or AGD.

14. The targeted molecular composition of Claim 13, wherein said peptides comprise the amino acids SEQ ID NO.l KQAGDV.

15. A molecular composition comprising a thrombolytic agent, a non-immunoreative polymer, and a targeting ligand, wherein said targeting ligand targets a fibrinolytic or thrombogenic cascade protein or receptor on a cell selected from the group consisting of platelets, endothelial cells, monocytes, smooth muscle cells, leukocytes, lymphocytes, neutrophils, fibroblasts, basophils, eosinophils, matrix cells, and mixtures thereof .

16. The composition of Claim 15, wherein said thrombolytic agent is selected from the group consisting of streptokinase, urokinase, tissue plasminogen activator, single chain urokinase plasminogen activator, acylated plasminogen/streptokinase activation complex, staphylokinase, prourokinase, anistreplase, and alteplase, or mixtures thereof.

17. The composition of Claim 15, wherein said non- immunoreactive polymer is selected from the group consisting of polyethylene glycol, copolymers of polyethylene oxide and polyvinyl alcohol , polyhydroxypropylene glycol , polypropylene glycol , polymethylpropyleneglycol , polyhydroxypropyleneoxide , heteropolymers of alkoxy monomers, polyethylene/polypropyleneglycol, polyalkylether, PEG diol, star PEGs, multi-arm branched PEGs, di-amino-PEGs, amino acid esters of PEG, hydrazine hydrochloride derivatives of PEG, nucleophilic PEG derivatives, carboxylate PEGs, PEG amino acids, electrophilically activated PEGs, PEG succinimidyl succinamide, the succinamide derivative of PEG propionic acid, the succinimide derivative of carboxymethylated PEG, PEG 2 succinimide, the benzotriazole carbonate derivative of PEG, active esters of amino acid PEGs, pendant modified PEG NHS esters, the glycidyl ether or epoxide derivatives of PEG, oxycarbonylimidazole derivatives of PEG, p- nitrophenylcarbonate derivatives of PEG, PEG tresylate, PEG aldehyde, PEG isocyanate and maleic anhydride copolymers of PEG, sulfhydryl selective PEGs, PEG vinylsulfone, PEG iodoacetamide, PEG maleimide, and PEG- orthopyridyl-disulfide, or mixtures thereof.

18. The composition of Claim 15, wherein said targeting ligand is selected from the group consisting of antibodies, antibody fragments, proteins, glycoproteins, peptides, glycopeptides, polysaccharides, oligosaccharides, and monosaccharides , or mixtures 5 thereof.

19. The composition of Claim 18, wherein said peptides are between 3 and 66 amino acids in length.

10 20. The composition of Claim 19, wherein said peptides are selected from the group consisting of SEQ ID NO.2 GG(D)I(D)W(D)T(D)W(D)V, SEQ ID NO.3 G(D) I (D)W(D)T(D)W(D)V, SEQ ID NO.4 GIWTWV, SEQ ID NO.5 GGIWTWV, SEQ ID NO.6 NKTWTWV(NH₂) , SEQ ID NO.7 KTWTWV(NH₂) , SEQ ID NO.8

15 TWTWV(NH₂), SEQ ID NO.11 RYTDLVAI (NH₂) , SEQ ID NO.12

YTDLVAI(NH₂) , SEQ ID NO.13 YTDLVAIQNKNE(NH₂) , SEQ ID NO.14 DLVAIQNKNE(NH₂) , SEQ ID NO.15 LVAIQNKNE(NH₂) , SEQ ID NO.16 TDLVAIQN(NH₂) , and SEQ ID NO.17 Nif-TDLVAIQN(NH₂) , SEQ ID NO.18 GRGD, SEQ ID NO.19 ARGD, SEQ ID NO.20 VRGD, SEQ ID

20 NO.21 LRGD, SEQ ID NO.22 SRGD, and SEQ ID NO.23 FRGD, or mixtures thereof.

21. The composition of Claim 19, wherein said peptides are selected from the group consisting of SEQ ID NO.24 5 NKLIVRRGQSFYVQIDFSRPYDPRRDLFRVEYVIGRYPQENKGTYIPVPIVSELQSGK

WGAKIVMR, SEQ ID NO.25

EDRSVRLSIQSSPKCIVGKFRMYVAVWTPYGVLRTSRNPETDTYILFN

PWCEDDAVYLDNEKERE , and SEQ ID NO.26

EYVLNDIGVIFYGEVNDIKTRSWSYGQF-R' , where R' is -C0NH₂ or - 0 NH₂, or fragments thereof.

22. The composition of Claim 19, wherein said peptides are selected from the group consisting of SEQ ID NO.28 XaaRGDXaaXaa, where Xaa at position one is hydrogen or any 5 amino acid and Xaa at position five or six is Thr or Cys; SEQ ID NO.29 SYGRGDVRGDFKCGC , with the N-terminus acetylated; SEQ ID NO.30 RGDXaa, where Xaa is Ser, Thr, or Cys; SEQ ID NO.31 GRGDVRGDFKCGC , where the C-terminus is an amide; SEQ ID NO.32 CGCRRRRRRRRRGDV; and SEQ ID NO.33 SYGRGDVRGDFKCTCC , where the C-terminus is an amide, or mixtures thereof.

5 23. The composition of Claim 19, wherein said peptides are selected from a group consisting of SEQ ID NO.9 KYGWTVFQAKRLDGSV and SEQ ID NO.10 KYGQKRLDGS, or mixtures thereof.

10 24. The targeted molecular composition of Claim 19, wherein said peptides are cyclic peptides.

25. The targeted molecular composition of Claim 24, wherein said cyclic peptides comprise a butapeptide

15 containing the amino acids G and D in combination with any two other amino acids.

26. The targeted molecular composition of Claim 25, wherein said butapeptide comprise the sequence cyclo-(D)V-

20 (alpha-N-methyl)-RGD-(3-aminomethylbenzoic acid).

27. The composition of Claim 19, wherein said peptides consist of linear hexapeptides containing the amino acids RGD or AGD. 5

28. The composition of Claim 27, wherein said peptides comprise the amino acids SEQ ID NO.l KQAGDV.

29. A targeted molecule for creating plasmin selectively 0 at a site of vascular thrombosis comprising a thrombolytic agent, a non-immunoreactive polymer, and a targeting ligand.

30. The targeted molecule of Claim 29, wherein said 5 thrombolytic agent is selected from the group consisting of streptokinase, urokinase, tissue plasminogen activator, single chain urokinase plasminogen activator, acylated plasminogen/streptokinase activation complex, staphylokinase, prourokinase, anistreplase, and alteplase, or mixtures thereof.

31. The targeted molecule of Claim 29, wherein said non- immunoreactive polymer is selected from the group consisting of polyethylene glycol, copolymers of polyethylene oxide and polyvinyl alcohol , polyhydroxypropylene glycol , polypropylene glycol , polymethylpropyleneglycol , polyhydroxypropyleneoxide, heteropolymers of alkoxy monomers, polyethetylene/polypropyleneglycol, polyalkylether, PEG diol, star PEGs, multi-arm branched PEGs, di-a ino-PEGs , amino acid esters of PEG, hydrazine hydrochloride derivatives of PEG, nucleophilic PEG derivatives, carboxylate PEGs, PEG amino acids, electrophilically activated PEGs, PEG succinimidyl succinamide, the succinamide derivative of PEG propionic acid, the succinimide derivative of carboxymethylated PEG, PEG 2 succinimide, the benzotriazole carbonate derivative of PEG, active esters of amino acid PEGs, pendant modified PEG NHS esters, the glycidyl ether or epoxide derivatives of PEG, oxycarbonylimidazole derivatives of PEG, p- nitrophenylcarbonate derivatives of PEG, PEG tresylate, PEG aldehyde, PEG isocyanate and maleic anhydride copolymers of PEG, sulfhydryl selective PEGs, PEG vinylsulfone, PEG iodoacetamide , PEG maleimide, and PEG- orthopyridyl-disulfide, or mixtures thereof.

32. The targeted molecule of Claim 29, wherein said targeting ligand is selected from the group consisting of antibodies, antibody fragments, proteins, glycoproteins , peptides, glycopeptides, polysaccharides, oligosaccharides, or a sugar molecule, or mixtures thereof.

33. The targeted molecule of Claim 32, wherein said peptides are between 3 and 66 amino acids in length.

34. The targeted molecules of Claim 33, wherein the peptides are selected from the group consisting of SEQ ID NO.2 GG(D)I(D)W(D)T(D)W(D)V, SEQ ID NO.3 G(D)I(D)W(D)T(D)W(D)V, SEQ ID NO.4 GIWTWV, SEQ ID NO.5 GGIWTWV, SEQ ID NO.6 NKTWTWV(NH₂) , SEQ ID NO.7 KTWTWV(NH₂) , SEQ ID NO.8 TWTWV(NH₂), SEQ ID NO.11 RYTDLVAI (NH₂) , SEQ ID NO.12 YTDLVAI(NH₂) , SEQ ID NO.13 YTDLVAIQNKNE(NH₂) , SEQ ID NO.14 DLVAIQNKNE(NH₂) , SEQ ID NO.15 LVAIQNKNE(NH₂) , SEQ ID NO.16 TDLVAIQN(NH₂) , and SEQ ID NO.17 Nif-TDLVAIQN(NH₂) , SEQ ID NO.18 GRGD, SEQ ID NO.19 ARGD, SEQ ID NO.20 VRGD, SEQ ID NO.21 LRGD, SEQ ID NO.22 SRGD, and SEQ ID NO.23 FRGD, or mixtures thereof.

35. The targeted molecules of Claim 33, wherein said peptides are selected from the group consisting of SEQ ID

NO.24

NKLIVRRGQSFYVQIDFSRPYDPRRDLFRVEYVIGRYPQENKGTYIPVPIVSELQSGK

WGAKIVMR, SEQ ID NO.25

EDRSVRLSIQSSPKCIVGKFRMYVAVWTPYGVLRTSRNPETDTYILFN PWCEDDAVYLDNEKERE, and SEQ ID NO.26

EYVLNDIGVIFYGEVNDIKTRSWSYGQF-R' , where R' is -C0NH₂ or -

NH₂, or fragments thereof.

36. The targeted molecules of Claim 33, wherein said peptides are selected from the group consisting of SEQ ID NO.28 XaaRGDXaaXaa, where Xaa at position one is hydrogen or any amino acid and Xaa at position five or six is Thr or Cys; SEQ ID NO.29 SYGRGDVRGDFKCGC , with the N-terminus acetylated; SEQ ID NO.30 RGDXaa, where Xaa is Ser, Thr, or Cys; SEQ ID NO.31 GRGDVRGDFKCGC , where the C-terminus is an amide; SEQ ID NO.32 CGCRRRRRRRRRGDV; and SEQ ID NO.33 SYGRGDVRGDFKCTCC , where the C-terminus is an amide, or mixtures thereof .

37. The targeted molecules of Claim 33, wherein said peptides are selected from a group consisting of SEQ ID NO.9 KYGWTVFQAKRLDGSV and SEQ ID NO.10 KYGQKRLDGS, or mixtures thereof.

38. The targeted molecular composition of Claim 33, wherein said peptides are cyclic peptides.

39. The targeted molecular composition of Claim 38, 5 wherein said cyclic peptides comprise a butapeptide containing the amino acids G and D in combination with any two other amino acids.

40. The targeted molecular composition of Claim 39,

10 wherein said butapeptide comprise the sequence cyclo-(D)V- (alpha-N-methyl)-RGD-(3-aminomethylbenzoic acid) .

41. The targeted molecules of Claim 33, wherein said peptides consist of linear hexapeptides containing the

15 amino acids RGD or AGD.

42. The targeted molecules of Claim 41, wherein said peptides comprise the amino acids SEQ ID NO.l KQAGDV.

20 43. A method of treating vascular thrombosis comprising the step of administering to a patient a therapeutically effective amount of a composition which includes a thrombolytic agent, a non-immunoreactive polymer, and a targeting ligand.

25

44. The method of Claim 43, wherein said composition is administered by intravenous injection.

45. The method of Claim 43, wherein said composition is 30 administered in situ through a catheter.

46. The method of Claim 43, wherein said thrombolytic agent is selected from the group consisting of streptokinase, urokinase, tissue plasminogen activator,

35 single chain urokinase plasminogen activator, acylated plasminogen/streptokinase activation complex, staphylokinase, prourokinase, anistreplase, and alteplase, or mixtures thereof.

47. The method of Claim 43, wherein said non- immunoreactive polymer is selected from the group consisting of polyethylene glycol , copolymers of polyethylene oxide and polyvinyl alcohol ,

5 polyhydroxyporopylene glycol, polypropylene glycol, polymethylpropyleneglycol , polyhydroxypropyleneoxide , heteropolymers of alkoxy monomers, polyethetylene/polypropyleneglycol, polyalkylether, PEG diol, star PEGs, multi-arm branched PEGs, di-amino-PEGs ,

10 amino acid esters of PEG, hydrazine hydrochloride derivatives of PEG, nucleophilic PEG derivatives, carboxylate PEGs, PEG amino acids, electrophilically activated PEGs, PEG succinimidyl succinamide, the succinamide derivative of PEG propionic acid, the

15 succinimide derivative of carboxymethylated PEG, PEG 2 succinimide, the benzotriazole carbonate derivative of PEG, active esters of amino acid PEGs, pendant modified PEG NHS esters, the glycidyl ether or epoxide derivatives of PEG, oxycarbonylimidazole derivatives of PEG, p-

20 nitrophenylcarbonate derivatives of PEG, PEG-tresylate, PEG aldehyde, PEG isocyanate and maleic anhydride copolymers of PEG, sulfhydryl selective PEGs, PEG vinylsulfone, PEG iodoacetamide, PEG-maleimide , and PEG- orthopyridyl-disulfide, or mixtures thereof.

25

48. The method of Claim 43, wherein said targeting ligand is selected from the group consisting of antibodies, antibody fragments, proteins, glycoproteins, peptides, glycopeptides , polysaccharides , oligosaccharides, or a

30 sugar molecule, or mixtures thereof.

49. The method of Claim 48, wherein said peptides are between 3 and 66 amino acids in length.

35 50. The method of Claim 49, wherein the peptides are selected from the group consisting of SEQ ID NO.2 GG(D)I(D)W(D)T(D)W(D)V, SEQ ID NO.3 G(D) I (D)W(D)T(D)W(D)V, SEQ ID NO.4 GIWTWV, SEQ ID NO.5 GGIWTWV, SEQ ID NO.6 NKTWTWV(NH₂) , SEQ ID NO.7 KTWTWV(NH₂) , SEQ ID NO.8 TWTWV(NH₂), SEQ ID NO.11 RYTDLVAI (NH₂) , SEQ ID NO.12 YTDLVAI(NH₂) , SEQ ID NO.13 YTDLVAIQNKNE(NH₂) , SEQ ID NO.14 DLVAIQNKNE(NH₂) , SEQ ID NO.15 LVAIQNKNE(NH₂) , SEQ ID NO.16 5 TDLVAIQN(NH₂), and SEQ ID NO.17 Nif-TDLVAIQN(NH₂) , SEQ ID NO.18 GRGD, SEQ ID NO.19 ARGD, SEQ ID NO.20 VRGD, SEQ ID NO.21 LRGD, SEQ ID NO.22 SRGD, and SEQ ID NO.23 FRGD, or mixtures thereof.

10 51. The method of Claim 49, wherein said peptides are selected from the group consisting of SEQ ID NO.24 NKLIVRRGQSFYVQIDFSRPYDPRRDLFRVEYVIGRYPQENKGTYIPVPIVSELQSGK WGAKIVMR, SEQ ID NO.25 EDRSVRLSIQSSPKCIVGKFRMYVAVWTPYGVLRTSRNPETDTYILFN

15 PWCEDDAVYLDNEKERE , and SEQ ID NO.26

EYVLNDIGVIFYGEVNDIKTRSWSYGQF-R' , where R' is -C0NH₂ or - NH₂ , or fragments thereof .

52. The method of Claim 49, wherein said peptides are 20 selected from the group consisting of SEQ ID NO.28

XaaRGDXaaXaa, where Xaa at position one is hydrogen or any amino acid and Xaa at position five or six is Thr or Cys; SEQ ID NO.29 SYGRGDVRGDFKCGC , with the N-terminus acetylated; SEQ ID NO.30 RGDXaa, where Xaa is Ser, Thr, or 25 Cys; SEQ ID NO.31 GRGDVRGDFKCGC, where the C-terminus is an amide; SEQ ID NO.32 CGCRRRRRRRRRGDV; and SEQ ID NO.33 SYGRGDVRGDFKCTCC , where the C-terminus is an amide, or mixtures thereof .

30 53. The method of Claim 49, wherein said peptides are selected from a group consisting of SEQ ID NO.9 KYGWTVFQAKRLDGSV and SEQ ID NO.10 KYGQKRLDGS, or mixtures thereof .

35 54. The method of Claim 49, wherein said peptides are cyclic peptides.

55. The method of Claim 54, wherein said cyclic peptides comprise a butapeptide containing the amino acids G and D in combination with any two other amino acids.

56. The method of Claim 55, wherein said butapeptide

5 comprise the sequence cyclo-(D)V-(alpha-N-methyl)-RGD-(3- aminomethylbenzoic acid) .

57. The method of Claim 49, wherein said peptides consist of linear hexapeptides containing the amino acids RGD or

10 AGD.

58. The method of Claim 57, wherein said peptides comprise the amino acids SEQ ID NO.l KQAGDV.

15 59. A method of preparing a targeted molecular composition comprising the steps of combining together a thrombolytic agent, a non-antigenic polymer, and a targeting ligand through synthetic chemistry.

20 60. The method of Claim 59, wherein said thrombolytic agent is selected from the group consisting of streptokinase, urokinase, tissue plasminogen activator, single chain urokinase plasminogen activator, acylated plasminogen/streptokinase activation complex,

25 staphylokinase, prourokinase, anistreplase, and alteplase, or mixtures thereof .

61. The method of Claim 59, wherein said non- immunoreactive polymer is selected from the group

30 consisting of polyethylene glycol, copolymers of polyethylene oxide and polyvinyl alcohol , polyhydroxyporopylene glycol , polypropylene glycol , polymethylprpyleneglycol , polyhydroxypropyleneoxide , heteropolymers of alkoxy monomers,

35 polyethetylene/polypropyleneglycol, polyalkylether, PEG diol, star PEGs, multi-arm branched PEGs, di-amino-PEGs, amino acid esters of PEG, hydrazine hydrochloride derivatives of PEG, nucleophilic PEG derivatives, carboxylate PEGs, PEG amino acids, electrophilically activated PEGs, PEG succinimidyl succinamide, the succinamide derivative of PEG propionic acid, the succinimide derivative of carboxymethylated PEG, PEG 2 succinimide, the benzotriazole carbonate derivative of PEG, active esters of amino acid PEGs, pendant modified PEG NHS esters, the glycidyl ether or epoxide derivatives of PEG, oxycarbonylimidazole derivatives of PEG, p- nitrophenylcarbonate derivatives of PEG, PEG tresylate, PEG aldehyde, PEG isocyanate and maleic anhydride copolymers of PEG, sulfhydryl selective PEGs, PEG vinylsulfone, PEG iodoacetamide , PEG maleimide, and PEG- orthopyridyl-disulfide, or mixtures thereof.

62. The method of Claim 59, wherein said targeting ligand is selected from the group consisting of antibodies, antibody fragments, proteins, glycoproteins, peptides, glycopeptides, polysaccharides, oligosaccharides, or a sugar molecule, or mixtures thereof.

63. The method of Claim 62, wherein said peptides are between 3 and 66 amino acids in length.

64. The method of Claim 63, wherein the peptides are selected from the group consisting of SEQ ID NO.2

GG(D)I(D)W(D)T(D)W(D)V, SEQ ID NO.3 G(D) I (D)W(D)T(D)W(D)V, SEQ ID NO.4 GIWTWV, SEQ ID NO.5 GGIWTWV, SEQ ID NO.6 NKTWTWV(NH₂) , SEQ ID NO.7 KTWTWV(NH₂) , SEQ ID NO.8 TWTWV(NH₂), SEQ ID NO.11 RYTDLVAI (NH₂) , SEQ ID NO.12 YTDLVAI(NH₂) , SEQ ID NO.13 YTDLVAIQNKNE(NH₂) , SEQ ID NO.14 DLVAIQNKNE(NH₂) , SEQ ID NO.15 LVAIQNKNE(NH₂) , SEQ ID NO.16 TDLVAIQN(NH₂) , and SEQ ID NO.17 Nif-TDLVAIQN(NH₂) , SEQ ID NO.18 GRGD, SEQ ID NO.19 ARGD , SEQ ID NO.20 VRGD , SEQ ID NO.21 LRGD, SEQ ID NO.22 SRGD, and SEQ ID NO.23 FRGD, or mixtures thereof.

65. The method of Claim 63, wherein said peptides are selected from the group consisting of SEQ ID NO.24 NKLIVRRGQSFYVQIDFSRPYDPRRDLFRVEYVIGRYPQENKGTYIPVPIVSELQSGK WGAKIVMR, SEQ ID NO.25

EDRSVRLSIQSSPKCIVGKFRMYVAVWTPYGVLRTSRNPETDTYILFN PWCEDDAVYLDNEKERE , and SEQ ID NO.26 5 EYVLNDIGVIFYGEVNDIKTRSWSYGQF-R' , where R' is -C0NH₂ or - NH₂, or fragments thereof.

66. The method of Claim 63, wherein said peptides are selected from the group consisting of SEQ ID NO.28

10 XaaRGDXaaXaa, where Xaa at position one is hydrogen or any amino acid and Xaa at position five or six is Thr or Cys; SEQ ID NO.29 SYGRGDVRGDFKCGC , with the N-terminus acetylated; SEQ ID NO.30 RGDXaa, where Xaa is Ser, Thr, or Cys; SEQ ID NO.31 GRGDVRGDFKCGC, where the C-terminus is

15 an amide; SEQ ID NO.32 CGCRRRRRRRRRGDV; and SEQ ID NO.33 SYGRGDVRGDFKCTCC , where the C-terminus is an amide, or mixtures thereof.

67. The method of Claim 63, wherein said peptides are 20 selected from a group consisting of SEQ ID NO.9

KYGWTVFQAKRLDGSV and SEQ ID NO.10 KYGQKRLDGS, or mixtures thereof .

68. The method of Claim 63, wherein said peptides are 25 cyclic peptides.

69. The method of Claim 64, wherein said cyclic peptides comprise a butapeptide containing the amino acids G and D in combination with any two other amino acids.

30

70. The method of Claim 65, wherein said butapeptide comprise the sequence cyclo-(D)V-(alpha-N-methyl)-RGD-( 3- amino ethylbenzoic acid) .

35 71. The method of Claim 63, wherein said peptides consist of linear hexapeptides containing the amino acids RGD or AGD.

72. The method of Claim 71, wherein said peptides comprise the amino acids SEQ ID NO.l KQAGDV.

73. The method of Claims 50, 52, 53, 57, or 58, wherein use of said peptides further comprises the step of inhibiting leukocyte adhesion to endothelium at a site of inflammation.

74. The method of Claims 54, 55, or 56, wherein the use of said peptides further comprises the step of inhibiting leukocyte adhesion to platelets at a site of inflammation.