WO2001010450A1 - Agents thrombolytiques cibles - Google Patents

Agents thrombolytiques cibles Download PDF

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Publication number
WO2001010450A1
WO2001010450A1 PCT/US2000/021418 US0021418W WO0110450A1 WO 2001010450 A1 WO2001010450 A1 WO 2001010450A1 US 0021418 W US0021418 W US 0021418W WO 0110450 A1 WO0110450 A1 WO 0110450A1
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seq
peg
peptides
pegs
group
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PCT/US2000/021418
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Evan C. Unger
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Imarx Therapeutics, Inc.
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Priority to AU65224/00A priority Critical patent/AU6522400A/en
Publication of WO2001010450A1 publication Critical patent/WO2001010450A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid

Definitions

  • 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.
  • 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.
  • SK streptokinase
  • UK urokinase
  • TPA tissue plasminogen activator
  • 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.
  • SK protein which comes from bacteria, is rapidly recognized as a foreign pathogen and then neutralized by immunoglobins.
  • researchers have added certain chemical structures to thrombolytic agents such as SK in an attempt to "hide” them from immune system detection.
  • 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.
  • PEG polyethylene glycol
  • thrombolytics such as SK-PEG are so stable, they are slowly cleared from the bloodstream and remain active much longer than their unmodified versions.
  • 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 .
  • the present invention targets thrombolytic agents to the site of thrombosis so that plasmin is created at the site of the clot.
  • thrombolytic agents to the site of thrombosis so that plasmin is created at the site of the clot.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • non-immunoreactive polymers such as the polyalkyleneoxides polyethylene glycol (PEG) and polypropylene glycol (PPG).
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • non-immunoreactive polymers further acts to stearically block or otherwise shield activated thrombolytics, such as plasmin, from inhibitory enzymes or proteases .
  • 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.
  • 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.
  • FIG. 3 shows a thrombolytic agent to which is attached a branched PEG.
  • Each PEG branch bears one molecule of targeting ligand.
  • 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.
  • the invention comprises thrombolytic agents bearing targeting ligands to thrombi.
  • 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.
  • 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.
  • scu-PA single chain urokinase plasminogen activator
  • APSAC acylated plasminogen/streptokinase activation complex
  • staphylokinase prourokinase
  • anistreplase anistreplase and alteplase.
  • 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.
  • 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.
  • 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.
  • ELAM-1 E-selectin
  • monocytes mucin binding to L-selectin on high venular endothelium
  • sialyl lewisX binding to P-selectin on platelets polymorphonuclear leukocytes, or monocytes
  • 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.
  • P-selectin may serve as a target specific for inflamed tissue.
  • 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.
  • 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 ⁇ -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-l ⁇ , ⁇ on monocytes, neutrophils, t-lymphocyte subpopulations , basophils, or eosinophils; RANTES on monocytes, t-lymphocyte subpopulation
  • 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.
  • 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.
  • 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 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) .
  • ALD Alanine-Glycine-Aspartic Acid
  • 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) .
  • KQAGDV SEQ ID NO.l Lysine-Glutamine-Alanine-Glycine-Aspartate- Valine
  • 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(NH 2 ) , SEQ ID NO.7 KTWTWV(NH 2 ) , or SEQ ID NO.8 TWTWV(NH 2 ), where (NH 2 ) represents C-terminal amidation of the peptide.
  • peptides comprising 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 (NH 2 ) , SEQ ID NO.12 YTDLVAI (NH 2 ) , SEQ ID NO.13 YTDLVAIQNKNE(NH 2 ) , SEQ ID NO.14 DLVAIQNKNE(NH 2 ) , SEQ ID NO.15 LVAIQNKNE(NH 2 ) , SEQ ID NO.16 TDLVAIQN(NH 2 ) , and SEQ ID NO.17 Nif-TDLVAIQN(NH 2 ) , where Nif- is nifedipine.
  • 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
  • EYVLNDIGVIFYGEVNDIKTRSWSYGQF-R' where R' is -C0NH 2 or - NH 2 . 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.
  • 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.
  • 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) .
  • RGD/AGD variant peptide ligands suitable for the compositions of the invention include those with internal cysteine residues available for S-S bond dimerization.
  • 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.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.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.
  • 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.
  • 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
  • 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.
  • 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-NH 2 , HO-PEG-COOH, and NH 2 -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.
  • a biotinylated PEG 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.
  • 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.
  • the targeted thrombolytic is administered in one step and not in succession.
  • Additional PEG derivatives which may be useful in this invention include PEG-S-propionic Acid and PEG-trichlorophenylcarbonate.
  • 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) 2 NHS.
  • 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.
  • having more "arms” can impart more rotational degrees of freedom to the conjugate in its approach to the receptor binding sites .
  • one end of the di-PEG molecule can be the monomethoxy derivative and the targeting ligand affixed to only one terminus.
  • 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.
  • agents which comprise two different thrombolytic molecules tethered together by a di-PEG directed to the thrombus by a single targeting ligand.
  • a di-PEG 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.
  • 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.
  • 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).
  • 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).
  • PEG succinimidyl succinate SS-PEG- peptide
  • SPA-PEG succinimidyl propionate
  • 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.
  • 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.
  • sulfhydryl groups e.g. cysteine residues
  • the reaction times for these derivatives will be 0.5 to 2 hours at pH 7-8.
  • the reaction times may be significantly longer.
  • 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.
  • 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.
  • 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.
  • cryoprotectant materials such as trehalose, sucrose, maltose, dextran, or any of a variety of sugar-based material as is well known in the art.
  • the targeted thrombolytic agent is administered intravenously.
  • the high specificity of these new agents allows thrombolysis to be attained in a less invasive manner than was hitherto possible.
  • 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.
  • 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.
  • 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.
  • 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.
  • This example is directed to the synthesis of hexalinear
  • 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.
  • EDC dimethylaminopropyl-3-ethylcarbondiimide hydrochloride
  • HOBT 1- hydroxybenzotriazole
  • 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.
  • This example is directed to the preparation of a conjugate form by SEQ ID NO. 1 KQAGDV and PEG.
  • This example is directed to the synthesis of cyclized RGD bearing peptides.
  • Boc-Mamb 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) .
  • 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
  • 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.
  • 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.
  • the DMF filtrate is evaporated, redissolved in acetic acid or 1 : 1 acetonitrile:water, and lyophilized to obtain protected cyclized material.
  • 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 H 2 0 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.
  • This example is directed to the synthesis of SEQ ID NO.l- PEG-Streptokinase .
  • the compound ⁇ , ⁇ '-dimethylenecarboxy- polyethyleneglycol anhydride is made by mixing 0.34g of cold (0-5°C) ⁇ , ⁇ '-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.
  • DCC dicyclohexylcarbodiimide
  • 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.
  • 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.
  • This example is directed to the synthesis of cyclic RGD- PEG-streptokinase , where cyclic RGD-PEG is prepared as described in Example 3.
  • a number of different chemical groups can be used to attach the peptide to the PEG molecule.
  • 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.
  • a R-group carboxyl distally located from the RGD area could be reacted with PEG-amine.
  • 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:
  • the cyclized RGD-PEG can then be chemically attached to streptokinase by reacting a free -OH group on PEG with lysyl-NH 2 groups on streptokinase.
  • a Merrifield or modified Merrifield peptide synthesis protocol may be used as described in U.S. Patent No. 3,784,523.
  • 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).
  • 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.
  • 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.
  • 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.
  • t-PA 20mg in 20ml of aqueous buffer
  • 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.
  • 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.
  • 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.

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Abstract

Selon cette invention, on utilise des polymères biocompatibles non immunoréactifs en combinaison avec des agents thrombolytiques et des ligands ciblant des récepteurs de cellules et de tissus pour agir sur une lyse durable mais localisée de thrombus.
PCT/US2000/021418 1999-08-10 2000-08-04 Agents thrombolytiques cibles WO2001010450A1 (fr)

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WO2007095659A1 (fr) * 2006-02-23 2007-08-30 Fibrex Medical Research & Development Gmbh Peptides et dérivés peptidiques, leur préparation et leur utilisation pour produire un médicament ayant une activité thérapeutique et/ou prophylactique
WO2007095661A1 (fr) * 2006-02-23 2007-08-30 Fibrex Medical Research & Development Gmbh Peptides et derives peptidiques, leur production et leur utilisation dans la preparation d'une composition pharmaceutique active therapeutiquement et/ou a titre preventif
WO2010092114A1 (fr) 2009-02-13 2010-08-19 Guerbet Utilisation de tampons pour la complexation de radionucléides
WO2012084981A1 (fr) 2010-12-20 2012-06-28 Guerbet Nanoemulsion de chelate pour irm
WO2013045333A1 (fr) 2011-09-26 2013-04-04 Guerbet Nanoemulsions et leur utilisation comme agents de contraste
WO2014114724A1 (fr) 2013-01-23 2014-07-31 Guerbet Magneto-emulsion vectorisee
US8926945B2 (en) 2005-10-07 2015-01-06 Guerbet Compounds comprising a biological target recognizing part, coupled to a signal part capable of complexing gallium
US8986650B2 (en) 2005-10-07 2015-03-24 Guerbet Complex folate-NOTA-Ga68
CN109897179A (zh) * 2019-04-02 2019-06-18 中国科学院长春应用化学研究所 一种多臂聚乙二醇-聚（l-谷氨酸酯）嵌段共聚物及其制备方法和应用
CN110499349A (zh) * 2019-08-05 2019-11-26 中南民族大学 具有携氧潜能的第一小分子肽、可产生活性氧的第二小分子肽及其制备方法
WO2020007822A1 (fr) 2018-07-02 2020-01-09 Conservatoire National Des Arts Et Metiers (Cnam) Nanoparticules de bismuth métallique (0), procédé de fabrication et utilisations de celles-ci
EP4000597A1 (fr) * 2020-11-17 2022-05-25 The Boots Company plc Tétrapeptide et compositions comprenant des tétrapeptides

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006102395A3 (fr) * 2005-03-22 2007-05-10 Medstar Health Inc Systemes et methodes d'administration pour le diagnostic et le traitement de maladies cardio-vasculaires
WO2006102395A2 (fr) * 2005-03-22 2006-09-28 Medstar Health Inc Systemes et methodes d'administration pour le diagnostic et le traitement de maladies cardio-vasculaires
US8926945B2 (en) 2005-10-07 2015-01-06 Guerbet Compounds comprising a biological target recognizing part, coupled to a signal part capable of complexing gallium
US8986650B2 (en) 2005-10-07 2015-03-24 Guerbet Complex folate-NOTA-Ga68
WO2007095659A1 (fr) * 2006-02-23 2007-08-30 Fibrex Medical Research & Development Gmbh Peptides et dérivés peptidiques, leur préparation et leur utilisation pour produire un médicament ayant une activité thérapeutique et/ou prophylactique
WO2007095661A1 (fr) * 2006-02-23 2007-08-30 Fibrex Medical Research & Development Gmbh Peptides et derives peptidiques, leur production et leur utilisation dans la preparation d'une composition pharmaceutique active therapeutiquement et/ou a titre preventif
US8067533B2 (en) 2006-02-23 2011-11-29 Fibrex Medical Research & Development Gmbh Peptides and peptide derivatives, the production thereof as well as their use for preparing a therapeutically and/or preventively active pharmaceutical composition
WO2010092114A1 (fr) 2009-02-13 2010-08-19 Guerbet Utilisation de tampons pour la complexation de radionucléides
US9770520B2 (en) 2010-12-20 2017-09-26 Guerbet Chelate nanoemulsion for MRI
WO2012084981A1 (fr) 2010-12-20 2012-06-28 Guerbet Nanoemulsion de chelate pour irm
WO2013045333A1 (fr) 2011-09-26 2013-04-04 Guerbet Nanoemulsions et leur utilisation comme agents de contraste
WO2014114724A1 (fr) 2013-01-23 2014-07-31 Guerbet Magneto-emulsion vectorisee
WO2020007822A1 (fr) 2018-07-02 2020-01-09 Conservatoire National Des Arts Et Metiers (Cnam) Nanoparticules de bismuth métallique (0), procédé de fabrication et utilisations de celles-ci
CN109897179A (zh) * 2019-04-02 2019-06-18 中国科学院长春应用化学研究所 一种多臂聚乙二醇-聚（l-谷氨酸酯）嵌段共聚物及其制备方法和应用
CN109897179B (zh) * 2019-04-02 2021-08-17 中国科学院长春应用化学研究所 一种多臂聚乙二醇-聚(l-谷氨酸酯)嵌段共聚物及其制备方法和应用
CN110499349A (zh) * 2019-08-05 2019-11-26 中南民族大学 具有携氧潜能的第一小分子肽、可产生活性氧的第二小分子肽及其制备方法
EP4000597A1 (fr) * 2020-11-17 2022-05-25 The Boots Company plc Tétrapeptide et compositions comprenant des tétrapeptides
WO2022106056A1 (fr) * 2020-11-17 2022-05-27 The Boots Company Plc Tétrapeptide et compositions comprenant des tétrapeptides

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