EP1042003A1 - Apport local d'agents therapeutiques a action prolongee - Google Patents

Apport local d'agents therapeutiques a action prolongee

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Publication number
EP1042003A1
EP1042003A1 EP99916153A EP99916153A EP1042003A1 EP 1042003 A1 EP1042003 A1 EP 1042003A1 EP 99916153 A EP99916153 A EP 99916153A EP 99916153 A EP99916153 A EP 99916153A EP 1042003 A1 EP1042003 A1 EP 1042003A1
Authority
EP
European Patent Office
Prior art keywords
group
composition
ester
agents
hydroxysuccinimide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99916153A
Other languages
German (de)
English (en)
Inventor
Alan M. Ezrin
Peter G. Milner
Dominique P. Bridon
Darren L. Holmes
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ConjuChem Inc
Original Assignee
ConjuChem Inc
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Filing date
Publication date
Application filed by ConjuChem Inc filed Critical ConjuChem Inc
Publication of EP1042003A1 publication Critical patent/EP1042003A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K47/54—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 an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • This invention relates to the field of therapeutic agents in medicine.
  • this invention relates to the field of localized delivery of therapeutic agents wherein the agents are capable of covalently bonding to a site of interest in vivo, to provide increased tissue retention and pharmacodynamic duration of therapeutic benefit for the given drug.
  • therapeutic agents to localized sites such that the therapeutic agents have increased retention at the desired site and prolonged duration of action. These therapeutic agents are not as easily washed away from the site of administration so that reduced amounts of the agents can be supplied.
  • therapeutic agents capable of forming covalent bonds to localized sites such that the therapeutic agents have increased effective presence for therapeutic benefits.
  • the present invention is directed to therapeutic agents capable of forming covalent bonds to localized sites such that the therapeutic agents have increased tissue retention and half-lifes.
  • a first aspect of this invention relates to the modification of drugs at a site not involved in the pharmacophore receptor interaction.
  • a second aspect of this invention relates to novel chemistry involved in the non-specific formation of covalent bonds using homo and heterobifunctional cross-linking reagents.
  • the invention includes the non-specific labeling of fixed blood proteins and tissues with N-Hydroxy-Succinimide (NHS)- drugs and sulfo-NHS drugs, although other reactive groups, which are functional in an aqueous medium such as blood, may also be employed.
  • NHS N-Hydroxy-Succinimide
  • special reagents find use, such as azido, diazo, carbodiimide anhydride, hydrazine, dialdehydes, thiol groups, or amines to form amides, esters, imines, thioethers, disulfides, substituted amines, or the like.
  • the covalent bond which is formed should be able to be maintained during the lifetime of the blood component, unless it is intended to be a release site.
  • a major advantage of this technology is the small amount of drug necessary to provide an effective therapeutic window when compared to systemic administration. The reasons for this advantage are explained by the targeting of the delivery, the high yield of reaction between reactive entity and reactive functionality and the irreversible nature of the bond formed after reaction.
  • the therapeutic agent is localized in the vicinity of the therapeutic target and is not allowed to circulate in the blood stream like any other free drug.
  • Another advantage of the technology is its limited side effects that is a consequence of the advantage described above. Once bound to the membrane or tissue the therapeutic agent is not susceptible to liver metabolism, kidney filtration and excretion, and may even be protected from protease (inclusive of endopeptidase) activity which usually leads to loss of activity and accelerated elimination.
  • Another aspect of this invention relates to novel chemically reactive derivatives of radiolabeled molecules which can react with available functionalities on fixed blood proteins and on tissues to form covalent linkages, and in which the resulting covalently bound conjugates have radioactive properties.
  • the conjugated molecules As compared with free radiolabeled drugs the conjugated molecules have extended lifetimes in the bloodstream and are, therefore, capable of maintaining the radioactivity for extended periods of time as compared to the unconjugated parent drug, and provide such activity with reduced centrally mediated side effects.
  • the invention includes the non-specific labeling of fixed blood proteins and tissues with NHS-radiolabeled drugs and sulfo-NHS radiolabeled drugs although other reactive groups, which are functional in an aqueous medium such as blood, may also be employed.
  • special reagents find use, such as azido, diazo, carbodiimide, anhydride, hydrazine, dialdehydes, thiol groups, or amines to form amides, esters, imines, thioethers, disulfides, substituted amines, or the like.
  • the covalent bond which is formed should be able to be maintained during the lifetime of the blood or tissue component, unless it is intended to be a release site.
  • the invention further includes the conjugates of the radiolabeled derivatives with fixed blood components and tissues and methods for providing radioactivity in vivo comprising administering to a mammalian host the novel radiolabeled derivatives through the use of percutaneous catheter technology, endarterectomy or direct tissue incubation.
  • Another aspect of this invention relates to novel chemically reactive derivatives of RGD-containing peptides which can react with available functionalities on fixed blood proteins and on tissues to form covalent linkages, and in which the resulting covalently bound conjugates have RGD peptide activity.
  • the conjugated molecules As compared with RGD peptide drugs the conjugated molecules have extended lifetimes in the bloodstream or tissues and are, therefore, capable of maintaining RGD peptide activity for extended periods of time as compared to the unconjugated parent drug. Such activity is provided with reduced centrally mediated side effects due to the high efficiency of localized delivery, the irreversible covalent bond between therapeutic agent and membrane protein and the lower therapeutic doses used with this invention.
  • the invention includes the non-specific labeling of fixed blood proteins and tissues with NHS-RGD peptides and sulfo- NHS peptides although other reactive groups, which are functional in an aqueous medium such as blood, may also be employed.
  • special reagents find use, such as azido, diazo, carbodiimide anhydride, hydrazine, dialdehydes, thiol groups, or amines to form amides, esters, imines, thioethers, disulfides, substituted amines, or the like.
  • the covalent bond which is formed should be able to be maintained during the lifetime of the blood or tissue component, unless it is intended to be a release site.
  • the invention further includes the conjugates of the RGD peptide derivatives with fixed blood components and tissues and methods for providing RGD peptide activity in vivo comprising administering to a mammalian host the novel RGD peptide derivatives through the use of percutaneous catheter technology, endarterectomy, pericardial, advantitial, intracardiac or direct tissue incubation.
  • the invention further includes the method of use for conjugates of RGD derivatives with fixed blood components and tissues for providing RGD peptide to perform wound healing through platelet immobilization generated by the binding of the peptide onto glycoprotein llb/llla receptors.
  • the invention further includes the method of use for conjugates of RGD derivatives with fixed blood components and tissues for providing RGD peptide to produce anti-restenosis properties through direct interaction with known integrin receptors (alpha V beta III, llb/llla, alpha 5 beta III etc. ) to modulate the stimulation of platelets and inflammatory responses including induction of proliferation, migration, cell adhesion and tissue remodeling.
  • known integrin receptors alpha V beta III, llb/llla, alpha 5 beta III etc.
  • the invention further includes the method of use for conjugates of RGD derivatives with fixed blood components and tissues for providing RGD peptide to produce anti-angiogenic and antimetastatic properties through modification of adhesion and migration mediated by integrin receptor interaction with cell adhesion and cell to cell coupling as well as matrix degradation associated with cellular immigration and emigration.
  • the invention further includes a local delivery agent comprising a compound of the formula: X-Y-Z wherein X is selected from the group of wound healing agents, antibiotics, anti- inflammatories, antioxidants, anti-proliferatives, anti-restenosis, anti- angiogenic, immunosuppressants, anti-infectives and anti-cancer agents.
  • X is selected from the group of wound healing agents, antibiotics, anti- inflammatories, antioxidants, anti-proliferatives, anti-restenosis, anti- angiogenic, immunosuppressants, anti-infectives and anti-cancer agents.
  • Y is a linking group consisting of 0-30 atoms and Z is a chemically reactive entity capable of reaction with a reactive functionality on fixed blood components to form covalent bonds therewith.
  • Z may be selected from N-hydroxysuccinimide, N-hydroxy sulfosuccinimide, maleimide-benzoyl-succinimide, gamma- maleimido-butyryloxy succinimide ester, maleimidopropionic acid, isocyanate, thiolester, thionocarboxylic acid ester, imino ester, carbodiimide anhydride and carbonate ester.
  • X may be selected from peptides, organic molecules and radioactive molecules.
  • X may be an RGD containing peptide having wound healing properties.
  • the RGD peptide may have the sequence Ac-RIARGDFPDDRK(EGS)-NH 2 where EGS is ethylene glycol-bis(succinimidylsuccinate).
  • the invention further includes a method of increasing the retention time of a therapeutic agent locally administered to a site by delivering a compound of the formula: X-Y-Z wherein X is selected from the group of wound healing agents, antibiotics, anti- inflammatories, antioxidants, anti-proliferatives, anti-restenosis, anti- angiogenic, immunosuppressants, anti-infectives and anti-cancer agents.
  • X is selected from the group of wound healing agents, antibiotics, anti- inflammatories, antioxidants, anti-proliferatives, anti-restenosis, anti- angiogenic, immunosuppressants, anti-infectives and anti-cancer agents.
  • Y is a linking group consisting of 0-30 atoms and Z is a chemically reactive entity capable of reaction with a reactive functionality on fixed blood components to form covalent bonds therewith.
  • the invention further includes a method of promoting wound healing at a wound site, comprising administering a compound of the formula: X-Y-Z wherein X is a wound healing agent, Y is a linking group of 0-30 atoms and Z is a chemically reactive entity capable of reaction with a reactive functionality on fixed blood components to form covalent bonds therewith.
  • the invention further includes a method of treating a tumor comprising a compound of the formula X-Y-Z wherein X is an anti- cancer agent, Y is a linking group of 0-30 atoms and Z is a chemically reactive entity capable of reaction with a reactive functionality on fixed blood components to form covalent bonds therewith.
  • Figure 1 represents the association efficiency of [ 3 H J-NHS- propionate with the damaged rabbit carotid arteries following local incubation.
  • Figure 2 represents the retention efficiency of [ 3 H ]-NHS- propionate following a 3 minutes incubation period in damaged rabbit carotid arteries.
  • Figure 3 represents the 3 days retention efficiency of [ 3 H ]- NHS-propionate and [ 3 H ]-propionate following a 3 minutes incubation period with damaged rabbit carotid arteries.
  • Local delivery agents are agents that may be delivered to a local site of interest. Such agents include therapeutic agents. Local delivery includes topical application to both internal and external sites requiring therapeutic treatment.
  • Therapeutic agents are agents that have a therapeutic effect.
  • Therapeutic agents include wound healing agents, antibiotics, anti-infectives, anti-oxidants, chemotherapeutic agents, anti-cancer agents, anti-inflammatory agents, and antiproliferative drugs.
  • Fixed blood components are non- mobile blood components and include tissues, membrane receptors, interstitial proteins, fibrin proteins, collagens, platelets, endothelial cells, epithelial cells and their associated membrane and membraneous receptors, somatic body cells, skeletal and smooth muscle cells, neuronal components, osteocytes and osteoclasts and all body tissues especially those associated with the circulatory and lymphatic systems.
  • Mobile blood components are blood components that do not have a fixed situs for any extended period of time, generally not exceeding 5, more usually one minute.
  • Mobile blood components include soluble blood proteins such as immunoglobulins, serum albumin, ferritin, transferrin and the like.
  • Wound Healing Agents are agents that promote wound healing. Wound healing agents include integrins, cell adhesion molecules such as ICAM, ECAM, ELAM and the like, antibiotics, growth factors such as EGF, PDGF, IGF, bFGF, aFGF and
  • KGF fibrin, thrombin, RGD peptides and the like.
  • Antiproliferatives include antimetabolites, topoisomerase inhibitors, folic acid antagonists like methotrexate, purine antagonists like mercaptopurine, azathioprine, and pyrimidine antagonists like fluorouracil, cytarabine and the like.
  • Antioxidants are agents that prevents oxidative damage to tissue and include aspartate, orotate, tacophenol derivative (vitamin E), and free radical scavengers such as SOD, glutathione and the like.
  • Mammalian cells are continuously exposed to activated oxygen species such as superoxide, hydrogen peroxide, hydroxyl radical, and singlet oxygen. These reactive oxygen intermediates are generated in vivo by cells in response to aerobic metabolism, catabolism of drugs and other xenobiotics, ultraviolet and x-ray radiation, and the respiratory burst of phagocytic cells (such as white blood cells) to kill invading bacteria such as those introduced through wounds.
  • Hydrogen peroxide for example, is produced during respiration of most living organisms especially by stressed and injured cells.
  • lipid peroxidation which involves the oxidative degradation of unsaturated lipids.
  • Lipid peroxidation is highly detrimental to membrane structure and function and can cause numerous cytopathological effects.
  • Cells defend against lipid peroxidation by producing radical scavengers such as superoxide dismutase, catalase, and peroxidase. Injured cells have a decreased ability to produce radical scavengers.
  • Excess hydrogen peroxide can react with DNA to cause backbone breakage, produce mutations, and alter and liberate bases.
  • Hydrogen peroxide can also react with pyrimidines to open the 5,6-double bond, which reaction inhibits the ability of pyrimidines to hydrogen bond to complementary bases, Hallaender et al. ( 1 971 ).
  • oxidative biochemical injury can result in the loss of cellular membrane integrity, reduced enzyme activity, changes in transport kinetics, changes in membrane lipid content, and leakage of potassium ions, amino acids, and other cellular material.
  • Antioxidants have been shown to inhibit damage associated with active oxygen species. For example, pyruvate and other alpha- ketoacids have been reported to react rapidly and stoichiometrically with hydrogen peroxide to protect cells from cytolytic effects, O'Donnell-Tormey et al., J. Exp. Med., 1 65, pp. 500-514 (1 987).
  • Anti-infective agents are agents that inhibit infection and include anti-viral agents, anti-fungal agents and antibiotics.
  • Anti-viral agents are agents that inhibit virus and include vidarabine, acyclovir and trifluorothymidine.
  • Anti-fungal agents are agents that inhibit fungal growth.
  • Anti-fungal agents include anphoterecin B, myconazole, terconazole, econazole, isoconazole, thioconazole, biphonazole, clotrimazole, ketoconazole, butaconazole, itraconazole, oxiconazole, phenticonazole, nystatin, naphthyphene, zinoconazole, cyclopyroxolamine and fluconazole.
  • Antibiotics are natural chemical substances of relatively low molecular weight produced by various species of microorganisms, such as bacteria (including Bacillus species), actinomycetes (including Streptomyces) and fungi, that inhibit growth of or destroy other microorganisms. Substances of similar structure and mode of action may be synthesized chemically, or natural compounds may be modified to produce semi-synthetic antibiotics. These biosynthetic and semi-synthetic derivatives are also effective as antibiotics.
  • the major classes of antibiotics are (1 ) the beta-lactams, including the penicillins, cephalosporins and monobactams; (2) the aminoglycosides, e.g.
  • gentamicin tobramycin, netilmycin, and amikacin
  • tetracyclines e.g. ciprofloxacin
  • norfloxacin e.g. ciprofloxacin
  • vancomycin e.g. erythromycin, azithromycin, and clarithromycin
  • other antibiotics e.g., the polymyxins, chloramphenicol and the lincosamides.
  • Antibiotics accomplish their anti-bacterial effect through several mechanisms of action which can be generally grouped as follows: (1 ) agents acting on the bacterial cell wall such as bacitracin, the cephalosporins, cycloserine, fosfomycin, the penicillins, ristocetin, and vancomycin; (2) agents affecting the cell membrane or exerting a detergent effect, such as colistin, novobiocin and polymyxins; (3) agents affecting cellular mechanisms of replication, information transfer, and protein synthesis by their effects on ribosomes, e.g., the aminoglycosides, the tetracyclines, chloramphenicol, clindamycin, cycloheximide, fucidin, lincomycin, puromycin, rifampicin, other streptomycins, and the macrolide antibiotics such as erythromycin and oleandomycin; (4) agents affecting nucleic acid metabolism, e.g., the fluoroquinolones
  • Anti-cancer agents are natural or synthetic molecules which are effective against one or more forms of cancer. This definition includes molecules which by their mechanism of action are cytotoxic (anti-cancer chemotherapeutic agents), those which stimulate the immune system (immune stimulators) and modulators of angiogenesis. The outcome in either case is the slowing of the growth of cancer cells.
  • Anti-cancer therapy include radioactive isotopes such as 32 P used in the treatment of polycythemia vera and in chronic leukemia.
  • Radioactive phosphorus has a biological half-life of about 8 days in humans. It emits beta rays that exert a destructive effect on the rapidly multiplying cells. 32 P is usually administered in doses of about 1 mc daily for 5 days. Either the oral or intravenous route may be used and the doses are not greatly different. Radioactive iodine
  • radioactive gold 198 Au, and other isotopes are not as useful as 32 P. Nevertheless, 131 l has some limited applications in metastatic thyroid carcinoma.
  • Other radioactive isotopes can be used with our technology either as complexes of radioactive metal such as 51 Cr, 52 Mn , 52 Mg, 57 Ni, 55 Co and 56 P, 55 Fe , 103 Pd, 192 lr, 64 Cu and 67 Cu or as chelates of these metals using bifunctional chelating agents like (BFCs), 6-[p-(bromoacetamido)benzyl]-1 ,4,8,1 1 - tetraazacyclotetradecane-1 ,4,8,1 1 -tetraacetic acid (BAT), 6-[p-(isothiocyanato)benzyl]-1 , 4,8,1 1 - tetraazacyclotetradecane-1 ,4,8,1 1 -tetraacetic acid (SCN-TETA),
  • BFCs
  • agents useful in the treatment of neoplastic disease that are amenable to the embodiment of this application for local drug delivery and retention of the modified drug substance at the tumor site.
  • agents derivitized with this technology can include anti- metabolites such as metotrexate (folic acid derivatives), fluoroaucil, cytarabine, mercaptopurine, thioguanine, petostatin (pyrimidine and purine analogs or inhibitors), a variety of natural products such as vincristine and vinblastine (vinca alkaloid), etoposide and teniposide, various antibiotics such as miotomycin, plicamycin, bleomycin, doxorubicin, danorubicin, dactomycin; a variety of biological response modifiers including interferon-alpha; a variety of miscellaneous agents and hormonal modulators including cisplatin, hydroxyurea, mitoxantorne, procarbo
  • anti- metabolites such as meto
  • taxoids various modifications and attachments to the basic ring structure (taxoid nucleus) as may be shown to be efficacious for reducing cancer cell growth and which can be constructed by organic chemical techniques known to those skilled in the art.
  • Chemotherapeutics include podophyllotoxins and their derivatives and analogues. Another important class of chemotherapeutics useful in this invention are camptothecins.
  • chemotherapeutics useful in this invention are the anthracyclines (adriamycin and daunorubicin).
  • chemotherapeutics are compounds which are drawn from the following list: Taxotere, Amonafide, llludin
  • Other compounds useful in the invention include: 1 ,3-bis(2- chloroethyD-1 -nitrosurea ("carmustine” or “BCNU”), 5-fluorouracil, doxorubicin ("adriamycin”), epirubicin, aclarubicin, Bisantrene (bis(2- imidazolen-2-ylhydrazone)-9, 1 0-anthracenedicarboxaldehyde, mitoxantrone, methotrexate, edatrexate, muramyl tripeptide, muramyl dipeptide, lipopolysaccharides, vidarabine and its 2-fluoro derivative, resveratrol, retinoic acid and retinol, carotenoids, and tamoxifen.
  • chemotherapeutic agents useful in the application of this invention include: Decarbazine, Lonidamine, Piroxantrone, Anthrapyrazoles, Etoposide, Camptothecin, 9-aminocamptothecin, 9- nitrocamptothecin, camptothecin-1 1 ("Irinotecan'), Topotecan, Bleomycin, the Vinca alkaloids and their analogs [Vincristine, Vinorelbine, Vindesine, Vintripol, Vinxaltine, Ancitabine], 6- aminochrysene, and Navelbine.
  • RGD Peptides The RGD peptide for conjugation to tissues or fixed endogenous proteins in accordance with the present invention includes a sequence of amino acids, preferably naturally occurring L-amino acids and glycine, having the following formula:
  • R r Arg-Gly-Asp-R 2 In this formula, R, and R 2 represent an amino acid or a sequence of more than one amino acid or a derivatized or chemically modified amino acid or more than one derivatized or chemically modified amino acids.
  • Delivery devices are devices useful for local delivery of therapeutic agents. Delivery devices include catheters, syringes, trocars and endoscopes.
  • Reactive entities are entities capable of forming a covalent bond. Such reactive agents are coupled or bonded to a therapeutic agent of interest. Reactive entities will generally be stable in an aqueous environment and will usually be carboxy, phosphoryl, or convenient acyl group, either as an ester or an anhydride, or an imidate, thereby capable of forming a covalent bond with an amino group at the target site to form an amide or amide derivative. For the most part, the esters will involve phenolic compounds, or be thiol esters, alkyl esters, phosphate esters, or the like. While the reactive entity is usually chosen to react with an amino group at the target site, other reactive functionalities at the target site may be exploited.
  • the reactive functionality may comprise various phosphinyl or phosphonyl derivatives for the bonding to available hydroxyl functions at the target site or may comprise an imine, thioimine or disulfide for bonding to thiol residues.
  • the reactive functionalities available on vascular proteins for covalent bond formation with the reactive group are primarily amino, carboxyi and thiol groups. While any of these may be used as the target for the reactive entity, for the most part, bonds to amino groups will be employed, particularly with the formation of amide bonds.
  • bonds to amino groups will be employed, particularly with the formation of amide bonds.
  • special reagents find use such as diazo, azido, carbodiimide, anhydride, hydrazine, or thiol groups, depending on whether the reaction is in vivo or in vitro, the target, the specificity of the reactive entity, and the like.
  • IC50 Concentration of an enzyme inhibitor at which 50% of the enzymatic activity is inhibited.
  • Protective groups are chemical moieties utilized to protect reactive entities from reacting with themselves. Various protective groups are disclosed in U.S. 5,493,007 which is hereby incorporated by reference. Such protective groups include acetyl, fluorenylmethyloxycarbonyl (FMOC), t-butyloxy carbonyl (BOC), benzyloxycarbonyl (CBZ), and the like.
  • Linking groups are chemical moieties that link or connect reactive entities to therapeutic agents.
  • Linking groups may comprise one or more alkylene, alkyleneoxy, alkenylene, alkynylene or amino group substituted by alkyl groups; cycloalkylene groups, polycyclic groups, aryl groups, polyaryl groups, substituted aryl groups, heterocyclic groups, and substituted heterocyclic groups.
  • Linking group will have from 2-100, more usually from 2- 18, preferably from 6-12 atoms in the chain, particularly carbon, oxygen, phosphorous and nitrogen, more particularly carbon and oxygen.
  • the invention is directed to the local delivery of therapeutic agents which have been modified with reactive entities so that they will covalently react and bond in vivo with reactive functionalities onto a fixed blood component and provide increased half lifes for the therapeutic agents.
  • the derivatized therapeutic agent of the present invention will, for the most part, have the following formula: X-Y-Z wherein:
  • X is selected from the group consisting of wound healing agents, antibiotics, anti-inflammatories, antioxidants, antiproliferatives, immunosuppressants, anti-infective and chemotherapeutic agents;
  • Y is a linking group of from 0-30, more usually of from 2-1 2, preferably of from 4-1 2 atoms, particularly carbon, oxygen, phosphorous and nitrogen, more particularly carbon and oxygen, where the oxygen is preferably present as oxy ether, where Y may be alkylene, oxyalkylene, or polyoxyalkylene, where the oxyalkylene group has from 2-3 carbon atoms, and the like.
  • a linking group of 0 atoms is preferred when it is desired to place X as close to Z as possible;
  • Z is a reactive entity, such as carboxy, carboxy ester, where the ester group is of 1 -8, more usually 1 -6 carbon atoms, particularly a physiologically acceptable leaving group which activates the carboxy carbonyl for reaction with amino groups in an aqueous system, e.g. N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide,
  • the reactive functionalities which are available on proteins for covalently bonding to the chemically reactive entity of the derivatized therapeutic agent are primarily amino groups, carboxyi groups and thiol groups. While any of these may be used as the target of the chemically reactive entity on the therapeutic agent, for the most part, bonds to amino groups will be employed, particularly with formation of amide bonds. To form amide bonds, one may use as a chemically reactive group a wide-variety of active carboxyi groups, particularly esters, where the hydroxyl moiety is physiologically acceptable at the levels required.
  • N-hydroxysuccinimide (NHS) and N-hydroxy sulfosuccinimide, (sulfo-NHS), maleimide-benzoyl- succinimide (MBS), gamma-maleimido-butyryloxy succinimide ester (GMBS) and maleimidopropionic acid (MPA), although other alcohols, which are functional in an aqueous medium such as blood, may also be employed.
  • reagents find use, such as azido, diazo, carbodiimide, anhydride, hydrazine, dialdehydes, thiol groups, or amines to form amides, esters, imines, thioethers, disulfides, substituted amines, or the like.
  • the covalent bond which is formed should be able to be maintained during the lifetime of the blood or tissue component, unless it is intended to be a release site.
  • the functional group on this protein will be a thiol group and the chemically reactive group will be a maleimido-containing group such as GMBA or MPA.
  • GMBA stands for gamma-maleimide-butyrylamide.
  • the manner of producing the derivatized therapeutic agents of the present invention will vary widely, depending upon the nature of the various elements comprising the molecule.
  • the synthetic procedures will be selected so as to be simple, provide for high yields, and allow for a highly purified product.
  • the chemically reactive group will be created as the last stage, for example, with a carboxyi group, esterification to form an active ester will be the last step of the synthesis.
  • Methods for the production of derivatized therapeutic agents of the present invention are described in examples 1 -6.
  • Each therapeutic agent selected to undergo the derivatization with a linker and a reactive agent will be modified according to the following criteria: if a carboxylic group, not critical for the retention of pharmacological activity is available on the original molecule and no other reactive functionality is present on the molecule, then the carboxylic acid will be chosen as attachment point for the linker-reactive entity modification. If no carboxylic acids are available, then any other functionalities not critical for the retention of pharmacological activity will be selected as attachment point for the linker-reactive entity modification. If several functionalities are available on a therapeutic agent, a combination of protecting groups will be used in such a way that after addition of the linker/reactive entity and deprotection of all the protected functional groups, retention of pharmacological activity is still obtained.
  • the chemically reactive entity is at a site, so that when the therapeutic agent is bonded to the fixed blood component, the therapeutic agent retains a substantial proportion of the parent compound's inhibitor activity.
  • the derivatized therapeutic agent of the present invention will generally have substantially lower IC 50 's generally in the range of about 0.5-0.01 of the IC 50 of the parent molecule. Desirably, the IC 50 should be not less than 0.05, preferably not less than about 0.1 .
  • the amount of the derivatized therapeutic agent administered will also vary.
  • the determination of the nature and length of the linker will be performed through an empirical optimization phase and will be measured by the retention or the loss of biological activity. For instance, with a given inhibitor enzyme interactions, an iteration of the modification of the nature and the length of the linker and a measure of the biological enzymatic activity may be necessary to determine the most favored linker length and nature. Preferably a short hydrophiiic 4-1 2 atom linker easily synthesized will be favored to start the iteration process.
  • a minimum distance from the target has to be respected based on the nature of the isotope and its penetration.
  • the length and nature of the linkers are not as important as they are for an enzyme inhibitor combination. For instance an isotope that emits a beta rays like 32 P should be positioned within 5 mm from the target to have maximum efficiency (99%) with limited or no effect coming from a small change on the nature and length of the linker.
  • the reactive entities have to be chosen in such a way that most of the reactive entity reacts with the reactive functionality in the shortest amount of time for an in vivo application.
  • Some surgical applications require a maximum incubation time of three minutes, due to biological constrains (interruption of arterial blood flow for instance).
  • Some other surgical intervention like vascular grafting which is performed ex vivo may take several minutes or hours if the graft tissue is kept under appropriate conditions of conservation.
  • the reactive entities may thus be different for the type of application based on their rate of reaction with the reactive functionalities.
  • 1 -30 % of the molecule added will bond to the target reactive functionality in vivo within a 3 minutes incubation window. More preferably 10-90% of the reactive entity will bond to the reactive functionality in vivo within a 3 minute window.
  • the reaction will be over within 5 minutes.
  • Modified therapeutic agents are infused either via standard balloon catheters, using pressure or iontophoresis to produce an efficient distribution of the infused drug substance, or introduced via standard techniques of infusion, lavage or topical application so that the drug is locally delivered to the site of interest.
  • Drugs are modified with appropriate connectors to retain activity when bound to exposed proteins in the vessel wall.
  • covalent attachment is achieved through the use of either non-specific NHS or other crosslinking agents or through the use of site specific covalent affinity probes to specific proteins. Once bound, the modified drugs retain biological activity.
  • the covalent attachment of the therapeutic agents provides a long lasting effect superior in time to existing methods or local drug delivery.
  • RGD peptides for Improved Wound Healing RGD (Arg-Gly-Asp)-containing peptides and proteins such as
  • Osteopontin have been recognized to accelerate wound healing in animal models.
  • the mechanism by which these peptides are thought to work is through stimulation of fibroblast and epithelial cell migration and adhesion into the wound.
  • the RGD containing peptides bind to a defined receptor, the alpha(v)beta3 integrin receptor on these cells which stimulates attachment and migration.
  • RGD containing peptides have failed to improve wound healing, i.e. acceleration or increased strength, in human clinical trials. These peptides most likely failed to work because the residence time in the dermal ulcer/wound was too brief and/or because of lack of attachment to fixed structures in the wound substratum.
  • RGD containing peptide useful in the present invention is the following:
  • RGD containing peptide such as those that are the subject of the current application, could be covalently coupled to the wound substratum using reactive chemistry and, in combination with the appropriate spacer/linker, be available for a more prolonged but correct presentation to wound healing elements than are conventionally applied RGD containing peptides. Binding and attachment to cellular wound healing elements such as fibroblasts and epithelial cells through the alpha(v)beta3 receptor is not altered by the chemistry employed in the current application.
  • the drug product described here could be applied topically to skin wounds and incisions including burns, dermal ulcers, trophic ulcers, diabetic ulcers, surgical incisions, skin graft donor and acceptor sites, surgical flap repairs, excision biopsy sites, fistulae, fissures, as well as sites of gastrointestinal ulceration in the stomach, duodenum, colon or rectum.
  • the agent could also be applied to sites of intestinal or other visceral anastamosis, or peritoneal, pleural, or mucosal surfaces to stimulated repair and healing.
  • the agent would be applied to the raw surface after adequate debridment either physically be irrigation or cleaning, or following enzymatic preparation and cleaning with agents such as hyaluronidase, papain, etc.
  • the drug could be applied to a cleaned and slightly moist wound surface either in an aqueous solution, in DMSO, as a powder, an aerosolized mist, ointment, lotion, emulsion, or as a film or as a component of a bandage or wrap. Unreacted drug may be removed after a period of time such as 5-20 mins of application.
  • the wound should be infection free and kept clean and most likely bandaged with wet to dry dressings post application.
  • Tumor metastasis is characterized by a series of steps involving interaction of various host cells (endothelial cells, platelets, lymphocytes) and an extracellular matrix such as collagens I and IV, fibronectin, laminin and sulfated glycosaminoglycans.
  • host cells endothelial cells, platelets, lymphocytes
  • extracellular matrix such as collagens I and IV, fibronectin, laminin and sulfated glycosaminoglycans.
  • integrins cell surface receptors.
  • Some synthetic peptides derived from adhesion molecules that are present in the extracellular matrix have been shown to modulate the mechanism involved in the metastasizing function of tumor cells. For instance several integrins recognize the amino acid sequence RGD which mediates the adhesion of normal and tumor cells to components of the extracellular matrix.
  • Angiogenesis is a multistep process involving matrix degradation, cellular proliferation and migration and recolonization in which capillary endothelial cells sever their normal cell cell attachment, migrate through the extracellular matrix and reform cell cell attachment to create new capillary.
  • the RGD peptide for conjugation to tissues or fixed endogenous proteins in accordance with the present invention includes a sequence of amino acids, preferably naturally occurring L-amino acids and glycine, having the following formula: R Arg-Gly-Asp-R 2 ln this formula, R and R 2 represent an amino acid or a sequence of more than one amino acid or a derivatized or chemically modified amino acid or more than one derivatized or chemically modified amino acids.
  • R ⁇ represents XY(Z) friendship, in which X, Y and Z independently represent an amino acid; and n represents 0 or 1 ; R 2 represents OH or NH 2 ; or any amino acid; or a sequence of more than one amino acid or a derivatized or chemically modified amino acid.
  • R 2 represents an amino acid other than serine, threonine or cysteine or the amide thereof wherein the amino acid is rendered a carboxyamide.
  • R 2 is more than one amino acid, the first amino acid in the sequence, which is attached to aspartic acid, being other than serine, threonine or cysteine, or the amide of any free carboxyi groups wherein R 2 includes a derivatized or chemically modified amino acid.
  • R 2 includes a linking group having a chemically reactive group which covalently bonds to reactive functionalities or proteins and R 1 includes a protective group to prevent the chemically reactive group of R 2 from reacting with R
  • R includes a linking group having a chemically reactive group which covalently bonds to reactive functionalities on proteins and R 2 includes a protective group to prevent the chemically reactive group of R ⁇ from reacting with R 2 .
  • both R, and R 2 include a linking group having a chemically reactive entity which covalently bonds to functionalities on fixed proteins.
  • the linking groups may be similar or different.
  • I, R and R 2 may include any amino acid or sequence thereof.
  • the amino acids are preferably naturally occurring. The most common naturally-occurring amino acids are shown in Table I:
  • R and R 2 in the RGD peptide of this invention are not limited to the 20 natural-amino acids.
  • R and R 2 can be D-amino acids, non-classical amino acids or cyclic peptides or peptidomimetics (chemical peptide analogs).
  • Non- classical amino acids include but are not limited to the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids, and amino acid analogs in general.
  • the Arg and/or Asp in the RGD sequence can be the D (dextrarotary) or L (levorotary) amino acid.
  • the polypeptide for conjugation to fixed blood proteins can be any size, and encompasses what might otherwise be called an oligopeptide, a protein, an organic molecule or a polymer such as polyethylene glycol.
  • the polypeptide will have no more than about 1 ,000 amino acids.
  • the polypeptide may be prepared by methods that are known in the art. For example, in brief, solid phase peptide synthesis consists of coupling the carboxyi group of the C-terminal amino acid to a resin and successively adding N-alpha protected amino acids.
  • the protecting groups may be any known in the art.
  • Integrins, growth factors and cell adhesion molecules may be modified using the NHS/linker technology disclosed here so that they will covalently bond to the base of an esophageal, gastric or duodenal ulcer when applied topically to the lesion via an endoscope.
  • topically applied growth factors such as, but not limited to, GF,PDGF, IGF, bFGF, aFGF and KGF, and integrins and cell adhesion molecules such as those mentioned above will stimulate reepithelialization and ulcer healing.
  • NSAIDS, steroids and other antiinflammatory drugs could also be modified by our technology to produce ulcer healing.
  • antibacterials or bacteriostatic agents such as bismuth sulphate designed to kill or inactivate Heliobacter pylorii could be applied to the base of peptic ulcers in order to achieve persistent and high local concentrations of these therapies in the region of the ulcer to aid healing.
  • H. pylorii is recognized as an etiological factor in peptic ulcers and its continued presence in the ulcer is recognized to interfere with healing.
  • Intraabdominal and intrathoracic surgery is often complicated by adhesions which develop within weeks to months postoperatively. These adhesions may remain undetected and of no consequence; however, often months to years postoperatively they may cause problem with the function of an organ or viscus, such as the intestines, or cause intractable pain requiring reoperation.
  • antiinflammatory peptides and drugs such as inhibitors of cell and matrix adhesion molecules coupled via spacers to NHS, topically applied during surgery to key sites that are prone to adhesions such as small or large intestinal anastomoses, adhesions can be prevented.
  • Intraoperative Administration to Prevent Bleeding Fibrin, thrombin or tissue factor peptides, and fragments of these and other procoagulant proteins and drugs can be attached to sites of bleeding in the body using NHS/linker technology to effect hemostasis to prevent bleeding.
  • These agents can be topically administered to sites of bleeding during open or laparascopic or other minimally invasive surgery, including but not limited to arthroscopy, thorascopic, or culdoscopic and endoscopic surgery.
  • agents could also be used to prevent bleeding post arteriotomy, or locally at sites following arterial puncture with catheters such as angiographic, angioplasty or hemodialysis or hemoperfusion catheters to reduce the risk of post puncture hemorrhage or intrauterine for post partum hemmorhage or dysfunctional uterine bleed.
  • catheters such as angiographic, angioplasty or hemodialysis or hemoperfusion catheters to reduce the risk of post puncture hemorrhage or intrauterine for post partum hemmorhage or dysfunctional uterine bleed.
  • These agents could also be of great value to prevent potentially serious hemorrhage intrathoracically post cardiac surgery, intracranially following neurosurgery, or after repair of an aneurysm either intracranially, intrathoracically, or intraabdominally.
  • the technology of this invention may be used to affect retention and the local effect of antiproliferatives to prevent and/or minimize adhesions in opthalmic surgery.
  • it could be used during anterior chamber surgery for glaucoma to prevent postoperative growth of a proliferative tissue in the region of the trabecular meshwork or anterior chamber resorptive apparatus.
  • topical use of protease inhibitors derivatized with NHS esters could be utilized to inhibit bacterial damage to the sclera as well as directly injected into the retinal to inhibit angiogenesis and macula degeneration.
  • Urology In uroiogical procedures, postoperative hemorrhage is common and the procoagulants mentioned above when coupled, via linker technology, to reactive NHS esters can be applied topically via a cystoscope or in an open operative field to sites of potential hemorrhage.
  • the technology of the invention may be applied postoperatively for the prevention of hemorrhage following prostate surgery including but not limited to TURP or open prostatectomy, polyp removal, bladder cancer removal or partial bladder resection.
  • the technology can be used to apply bacteriostatic or bactericidal agents perioperatively via a cystoscope or postoperatively via a Mey catheter and bladder irrigation to prevent or treat infection.
  • Anti- inflammatory agents can be similarly applied to prevent postoperative urethral stricture formation.
  • Endoscopy The same technology could be used in the prevention and treatment of esophageal stricture formation following esophageal surgery or due to benign causes such as Barrett's esophagus or severe reflux esophagitis. Endoscopy application of phosphodiesterase inhibitors such as methylxanthines (pentoxifyline, aminophylline, theophylline and related derivatives) as well as the local covalent linkage of selected anti-inflammatory agents may be useful in managing patients in acute asthmatic attack or cystic fibrosis. 8. Biliary Surgery
  • NHS/linker coupled anti-inflammatory agents or bacteriostatic/bactericidal agents such as those described above can be applied topically to sites of anastomosis/resection during open or laparoscopic biliary surgery to prevent adhesions, strictures and infection.
  • the technology may be utilized colonoscopically in the management of inflammatory bowel disease (IBD) to apply and sustain high local concentrations of select small molecule anti- inflammatory drugs such as steroids, NSAIDS and aspirin.
  • select small molecule anti- inflammatory drugs such as steroids, NSAIDS and aspirin.
  • Selectins and inhibitors of cell adhesion and antiproliferatives can be applied topically to the inflamed section/sections of the colon affected by Crohn's disease or Ulcerative Colitis in order to accelerate healing, reduce inflammation, and prevent stricture formation.
  • NHS during open surgical repair of either a transected or injured spinal cord, or damaged or transected peripheral nerve can be undertaken using this technology to improve nerve regeneration.
  • the technology can be used to apply anti-inflammatory and antibacterial agents to sites of biopsy or stricture in the airway when identified and visualized endobronchially.
  • hemostasis can be achieved or aided by the topical application post-biopsy of procoagulant factors/peptides attached via linkers to NHS esters.
  • Topical application of selected thrombin inhibitors attached via linkers to NHS esters may aid in the inhibition of small cell lung carcinoma and act as a an adjunctive therapy to surgical resection procedures.
  • Procoagulant and antibacterial factors can be applied to tooth sockets postextraction to prevent bleeding and infection.
  • Local application of selected antibiotics such as tetracyclin can result in the inhibition of proteases that are associated with gingivitis and gum deterioration by permanently adhering the drug for prolonged periods of time.
  • procoagulant and antibacterial drugs coupled to NHS via linkers can be applied topically to head and neck sites during surgery such as tumor resection to prevent bleeding and infection.
  • the procoagulant coupled NHS agents can be applied topically to nosebleeds, especially severe posterior septal bleeds which are prone to recurrence and are difficult to access and control.
  • 192 lr and 32 P are known to reduce coronary restenosis in patients with previous restenosis.
  • the intracoronary radiotherapy has been shown to reduce the intimal hyperplasia that is part of restenosis.
  • 137 Cs, 89 Sr and 90 Sr, 32 P, Iodine 125 l and 131 l, 192 lr, 90 Y, 90 Y/Sr, bismuth, radium are used internal therapy of various forms of cancers including those in the mouth, lip, breast, anus, vagina, thyroid, bone marrow, lungs and prostate.
  • the radiotherapy has been shown to shrink the tumor or reduce the risk of spreading before and during surgery. Internal radiotherapy is also given to kill off any tiny amounts of the tumor that may have been left after surgery. All these isotopes could be covalently attached to the tumor or in the vicinity of the tumor itself for either palliative, curative and radical treatments or as an adjuvant to other therapies.
  • 32 P can be covalently attached to tissue or membrane proteins through the use of an NHS phosphodiester or triester.
  • radioisotopes can be used with our technology either as complexes of radioactive metal such as 51 Cr, 52 Mn , 52 Mg, 57 Ni, 55 Co and 56 P, 5 ⁇ Fe , or as chelates of these metals.
  • Preferred radioactive isotopes are beta ray and gamma ray emitters.
  • immuno-suppressant agents such as cyclosporin and derivatives, corticosteroids, sulfasalazine, thalidomide, methotrexate, OKT3, peptide-T, or agents that inhibit T-cell activation or adhesion would be useful to locally apply to organs prior to transplantation to mask immune responsiveness and organ rejection. Such agents could be applied locally at the time of tissue harvest (e.g. heart, lung, liver harvest) or immediately prior to restitution of blood flow in the recipient. Such immuno-suppressant agents would prevent the recognition of foreign antigen from the donor tissue that would facilitate short term acceptance and facilitate longer term ability for the host to accommodate the transplanted organ.
  • Such drugs delivered in this matter could include NHS derivatives of anti-arrhythmic agents such as disopyramide, quinidine, amiodarone to control cardiac rate and rhythm disturbances; antibiotics or anti inflammatory agents such as corticosteroids into the pericardial space to control pericardititis; growth factors such as VEGF or FGF to induced topical revascularization, or inotropic agents such as methylxanthines, digitalis or phosphodiesterase inhibitors to improve contractility and ejection fraction in the failing heart.
  • anti-arrhythmic agents such as disopyramide, quinidine, amiodarone to control cardiac rate and rhythm disturbances
  • antibiotics or anti inflammatory agents such as corticosteroids into the pericardial space to control pericardititis
  • growth factors such as VEGF or FGF to induced topical revascularization, or inotropic agents such as methylxanthines, digitalis or phosphodiesterase inhibitors to improve contractility and ejection fraction in the failing heart.
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte/monocyte colony stimulating factor
  • erythropoetin thrombopoetin
  • interleukin-3 erythropoetin-3
  • Agents used for such purposes could include antithrombotics, antiproliferative agents (methotrexate, colchicine, angiopeptin, or heparin derivatives etc) to limit restenosis, protease inhibitors to limit vascular breakdown associated with aneurism and restenosis, radioactive therapy or DNA delivery to limit proliferation, angiostatic agents such as NHS derivatives of endostatin or angiostatin to limit vascular development or oligonucleotides, cDNA or naked DNA, and growth factors VEGF, FGF or to encourage vascular regeneration.
  • antithrombotics antiproliferative agents (methotrexate, colchicine, angiopeptin, or heparin derivatives etc) to limit restenosis, protease inhibitors to limit vascular breakdown associated with aneurism and restenosis, radioactive therapy or DNA delivery to limit proliferation
  • angiostatic agents such as NHS derivatives of endostatin or angiostatin to limit vascular development or oligonucleotides, cDNA
  • nitroso donors such as nitroprusside or other nitric oxide donors to restore normal vascular function in injured tissue
  • agents such as nitroso donors such as nitroprusside or other nitric oxide donors to restore normal vascular function in injured tissue would prove to be beneficial.
  • Other agents such as oligosaccharides, cyclodextrans or mannose derivatives could be attached covalently to vasculature lumenal interface via
  • NHS derivatization for the purpose of inhibiting leukocyte cell adhesion and rolling to limit vascular damage and inflammation in response to injury such as reperfusion, angioplasty or stent placement.
  • Such agents would benefit from the derivatization with the reactive functionalities in order to facilitate placement and covalent adherence and enhance cellular loading and uptake to improve tissue retention and drug delivery.
  • the therapeutic compound of the invention will be packaged in vials as a sterile liquid (preferably for room temperature storage), or as a lyophilisate in a vial for reconstitution.
  • the solution (or diluent in the case of the lyophilisate) may contain a low concentration of some type of organic solvent in order to ensure solubility and stability.
  • the liquid or post-reconstitution solution will contain as high a concentration as possible of the therapeutic compound.
  • Each vial of therapeutic compound will contain an overage of therapeutic material required to treat each of the indications for the compound or to diagnose indications for the disease or condition.
  • a) Open surgical field lavaqe There are a number of indications for local therapeutic compounds which would entail administration of the therapeutic compound as an adjunct to open surgery. In these cases, the therapeutic compound would either be lavaged in the surgical site (or a portion of that site) prior to closure, or the therapeutic compound would be incubated for a short time in a confined space (e.g., the interior of a section of an artery following an endarterectomy procedure or a portion of GI tract during resection) and the excess fluid subsequently evacuated.
  • a confined space e.g., the interior of a section of an artery following an endarterectomy procedure or a portion of GI tract during resection
  • tissue grafts such as autologous and xenobiotic vein/artery and valve grafts as well as organ grafts can be pretreated with therapeutic compounds that have been modified to permit covalent bond formation by either incubating them in a therapeutic solution and/or perfusing them with such a solution.
  • Catheter delivery A catheter is used to deliver the therapeutic compound either as part of an endoscopic procedure into the interior of an organ (e.g., bladder, GI tract, vagina/uterus) or adjunctive to a cardiovascular catheter procedure such as a balloon angioplasty. Standard catheters as well as newer drug delivery and iontophoretic catheters can be utilized.
  • Direct injection For certain poorly vascularized spaces such as intra-articular joint spaces, a direct injection of a therapeutic compound may be able to bioconjugate to surface tissues and achieve a desirable duration of drug effect.
  • Other applications could include intra medullary, intratumor, intravaginal, intrauterine, intra intestinal, intra eustachian tube, intrathecal, subcutaneous, intrarticular, intraperitoneal or intraocular injections as weel as via bronchoscope, via nasogastiric tube and via nophrostomy.
  • the compounds of this invention may be administered to a mammal, preferably a human. Because the delivery of a local therapeutic compound is targeted to achieve a local instead of a systemic effect, dosing as a function of patient weight or body surface area is not appropriate. For some therapeutic compounds, there is no significant risk of a local area safety or toxicity effect of the compound as a result of an overdosage. In such cases the therapeutic compound concentration formulation is optimized to deliver the highest possible density of conjugative bonding to the cells and proteins lining the targeted local area in order to retain therapeutic levels of localized drug activity at the site for as long a period of time as possible. However, some therapeutic compounds may have an optimal dose level (usually calculated based on conjugation density per surface area). In such cases, the density of conjugation per surface area is controlled as a function of a) surface site preparation, b) reaction time and conditions (e.g., temperature), c) therapeutic compound concentration of solution, and d) solution pH and buffers.
  • the key factors in localized delivery of therapeutic agents are as follows: a) maintain the consistency of conjugation bonding density to the target tissues; b) minimize any local tissue irritation or other adverse effects of the therapeutic agent administration and c) minimize the amount of local therapeutic compound which goes systemic.
  • Optimizing the compound solution, pH and buffers results in maximizing the level of conjugation bonding to the site surface. Removal of any residual liquid solution from an open operating field minimizes the amount of the therapeutic compound that may go systemic.
  • an array of bifunctional linker can be used to convert the molecule into a reactive NHS derivative.
  • ethylene glycol- bis(succinimydylsuccinate) (EGS) and triethylamine dissolved in DMF and added to the free amino containing molecule (with a ratio of 10: 1 in favor of EGS) will produce the mono NHS derivative.
  • EGS ethylene glycol- bis(succinimydylsuccinate)
  • GMBS N-[ ⁇ -maleimidobutyryloxy]succinimide ester
  • the maleimido group will react with the free thiol and the NHS derivative will be purified from the reaction mixture by chromatography on silica or by HPLC.
  • this invention is applicable to any molecule with preferably one chemical step only to derivatize the molecule (as described in example 1 or 3) or two steps (as described in example 2 and involving prior protection of a sensitive group) or three steps (protection, activation and deprotection). Under exceptional circumstances only, would we require to use multiple steps (beyond three steps) synthesis to transform a molecule into an active NHS or maleimide derivative.
  • Example 5 Example 5
  • an maleimide derivative from a amino derivatized molecule, one can use N-[ ⁇ -maleimidobutyryloxy]succinimide ester (GMBS) and triethylamine in DMF.
  • GMBS N-[ ⁇ -maleimidobutyryloxy]succinimide ester
  • the succinimide ester group will react with the free amino and the maleimide derivative will be purified from the reaction mixture by crystallization or by chromatography on silica or by HPLC.
  • an array of bifunctional crosslinking reagentsl can be used to convert the molecule into a reactive NHS derivative.
  • maleimidopropionic acid (MPA) can be coupled to the free amine to produce a maleimide derivative through reaction of the free amine with the carboxylic group of MPA using HBTU/HOBt/DIEA activation in DMF.
  • Rhodamine GreenTM-X succinimidyl ester, hydrochloride mixed isomers is commercially available from Molecular Probes (Eugene Oregon) as illustrated below:
  • New Zealand rabbits (2 Kg), male or female, were intramuscularly anesthetized with Xylazine (20 mg/kg), Ketamine (50 mg/kg) and Acepromazine (0.75 mg/kg) prior to surgical exposure of left carotid artery. Both carotid arteries were isolated and blood flows were measured. A catheter (22G) was inserted in the arterial segment and rinsed with 0.9% sodium chloride via catheter until there was no more visible evidence of blood in the segment.
  • a 1 -cm incubation chamber was created by ligatures in the segment area.
  • the incubation chamber was flushed three times with 1 mL of 0.9% sodium chloride.
  • a solution of 1 00 ⁇ l of 500 ⁇ M NHS- Rhodamine was prepared and incubated in the incubation chamber for 3 minutes.
  • the excess of rhodamine was withdrawn with a 1 mL syringue.
  • the incubation chamber was washed once again with 3 times 1 00 mL of 0.9% sodium chloride.
  • the incubation chamber was then removed from the rabbit, cut in three pieces and dipped in 10% formalin for further evaluation.
  • the NHS-Rhodamine treated arteries exhibited dramatic levels of fluorescence whereas those arteries treated solely with Rhodamine exhibited little fluorescence over background.
  • New Zealand rabbits (2 kg), male or female, were intramuscularly anesthetized with Xylazine (20 mg/kg), Ketamine (50 mg/kg) et Acepromazine (0.75 mg/kg) prior to surgical exposure of left carotid artery. Segments of 10 mm of carotids, were transiently isolated by temporary ligatures and rinsed with C.9% sodium chloride via a cannula until there was no more visible evidence of blood components.
  • a catheter (1 8G) was inserted in the arterial segment and served to introduce the angioplasty balloon (2.5 mm of diameter, over the wire/Boston Scientific Inc.).
  • a vascular damage (angioplasty) was performed on the isolated segment in order to eliminate the layer of endothelial cells.
  • the angioplasty balloon was serially inflated at different atmospheres (4, 6, 8 and 10) during 1 minute, with 45 seconds of delay between inflations. At 4 atmospheres a balloon traction was performed 5 times and 1000 U/kg of heparin were infused in the blood circulation.
  • the angioplasty balloon was then retrieved from the artery and the catheter was reintroduced.
  • the arterial segment was rinsed 3 times with saline, and 100 ⁇ M of [ 3 H ]-NHS-propionate was incubated within the isolated segment of the artery for either 30 seconds, 3 minutes or 30 minutes.
  • the excess of incubation liquid was withdrawn from the artery, and the segment was rinsed 5 times with saline.
  • the treated artery was immediately harvested, and incorporation of [ 3 H] -labeled compounds within the artery was evaluated by scintillation counting (see figure 1 ).
  • After 30 seconds of incubation we recorded an association efficiency of 2.55%.
  • At 3 min and 30 min we recorded an association efficiency of 5.5 and 6.5%, respectively.
  • the yield of the reaction can usually be improved by using EDC as the coupling reagent, as exemplified below.
  • EDC the coupling reagent
  • New Zealand rabbits (2 kg), male or female, were anesthetized with xylazine (20 mg/kg), ketamine (50 mg/kg) and acepromazine (0.75 mg/kg) intramuscularly prior to surgical exposure of left carotid artery.
  • Carotid arteries were surgically dissected and segments of approximately 10 mm length were isolated. The vessels were cannulated and rinsed with 0.9% sodium chloride until there was no more visible evidence of blood components.
  • a catheter (18G) was inserted in the arterial segment and served to introduce the angioplasty balloon (2.5 mm of diameter, over the wire/Boston Scientific Inc.).
  • Vascular damage (angioplasty) was performed on the isolated segment in order to eliminate the layer of endothelial cells.
  • the angioplasty balloon was serially inflated at different atmospheres (4, 6, 8 and 10) for 1 minute, with 45 seconds of delay between inflations. At 4 atmospheres a balloon traction was performed 5 times and 1 000 U/kg of heparin were infused in the blood circulation.
  • the angioplasty balloon was then retrieved from the artery and the catheter was reintroduced.
  • the arterial segment was rinsed 3 times with saline, and 100 /M of [ 32 P]- NHS-[linker] was incubated within the isolated segment of the artery for 3 minutes. At the end, the excess of incubation liquid was withdrawn from the artery, and the segment was rinsed 5 times with saline.
  • the vessel was sutured closed, blood flow restored and surgical wounds repaired. Animals were returned to the vivarium for periods up to four weeks. Tissue retention of [ 32 P]-NHS-[linker] was evaluating using whole animal radiography at selected periods of time after injury.
  • Tissue response to this therapy can be evaluated using standard histomophometric analysis quantifying if the extent of tissue proliferation and neoitimal formation in the treated versus control animals to determine if this form of brachotherapy can limit the response to vascular injury and hyperproliferative overgrowth classical observed under these conditions.
  • the yield of the reaction can usually be improved by using EDC as the coupling reagent, as exemplified below.
  • EDC the coupling reagent
  • New Zealand rabbits (2 Kg), male or female, were anesthetized with xylazine (20 mg/kg), ketamine (50 mg/kg) and acepromazine (0.75 mg/kg) intramuscularly prior to surgical exposure of left carotid artery.
  • Carotid arteries were surgically dissected and segments of approximately 10 mm length were isolated. The vessels were cannulated and rinsed with 0.9% sodium chloride until there was no more visible evidence of blood components.
  • a catheter ( 1 8G) was inserted in the arterial segment and served to introduce the angioplasty balloon (2.5 mm of diameter, over the wire/Boston Scientific Inc.).
  • Vascular damage (angioplasty) was performed on the isolated segment in order to eliminate the layer of endothelial cells.
  • the angioplasty balloon was serially inflated at different atmospheres (4, 6, 8 and 10) for 1 minute, with 45 seconds of delay between inflations. At 4 atmospheres a balloon traction was performed 5 times and 1000 U/kg of heparin were infused in the blood circulation.
  • the angioplasty balloon was then retrieved from the artery and the catheter was reintroduced.
  • the arterial segment was rinsed 3 times with saline, and 100 ⁇ M of [ 131 l]-NHS-[linker] was incubated within the isolated segment of the artery for 3 minutes. At the end, the excess of incubation liquid was withdrawn from the artery, and the segment was rinsed 5 times with saline.
  • the vessel was sutured closed, blood flow restored and surgical wounds repaired. Animals were returned to the vivarium for periods up to four weeks. Tissue retention of [ 131 l]-NHS-[linker] was evaluated using whole animal radiography at selected periods of time after injury.
  • Tissue response to this therapy can be evaluated using standard histomophometric analysis quantifying if the extent of tissue proliferation and neoitimal formation in the treated versus control animals to determine if this form of brachotherapy can limit the response to vascular injury and hyperproliferative overgrowth classical observed under these conditions.
  • Analytical HPLC were performed using a Varian (Rainin) binary HPLC system: gradient elution of 5-60% B (0.045% TFA in H 2 O (A) and 0.045% TFA in CH 3 CN (B)) at 0.5 mL/min using a Dynamax C 18 , 60A, 8 ⁇ m, 4.6 mm x 25 cm column equipped with a Dynamax C 18 , 6 ⁇ , 8 ⁇ m guard module and an UV detector (Varian Dynamax UVD II) detecting at ⁇ 21 4 and 254 nm. Mass spectrometry was performed on a PE Sciex API III electro-spray Biomolecular Mass Analyzer.
  • the peptide was removed from the resin by shaking 609 mg of Ac-RIARGDFPDDRK-MBHA-Resin with two-5 mL portions of a cleavage cocktail (comprised of: 1 0 mL of trifluoroacetic acid (TFA); 0.75 g of phenol; 0.25 g of thioanisole; 0.5 mL of ethanedithiol
  • a cleavage cocktail (comprised of: 1 0 mL of trifluoroacetic acid (TFA); 0.75 g of phenol; 0.25 g of thioanisole; 0.5 mL of ethanedithiol
  • Human umbilical vein endothelial cells from ATCC are grown to confluence in Medium 1 99 containing 2.2 mg/mL of sodium bicarbonate supplemented with 20% heat-inactivated FBS, ECGS 1 50 ⁇ g/mL, penicillin 100 Ui/mL, streptomycin 100 ⁇ g/mL. Cells are grown in 75 cm 2 flask at 37° C under 5% CO2 and the medium is replaced on the first day of seeding and every two days there after.
  • the cells are used between second and fourth passages.
  • Selected RGD containing agents are evaluated for their ability to inhibit the growth and proliferation of these cells under normal conditions as well as in confluent cultures that are injured mechanically by scraping a lesion into the confluence and measuring the rate of wound healing or trough repopulation.
  • fibronectin 100 ⁇ L fibronectin (5 and 10 ⁇ g/mL), vitronectin (5-10 ⁇ g/mL) or HSA 1 in PBS overnight and air dried. Endothelial and somatic cells are harvested by treating with trypsin (0.25%, w/v)/EDTA (1 mM) (5 mL/25cm 2 of surface area), washed twice in PBS and suspended at 5x10 5 cells/mL in PBS containing 10 ⁇ g/mL of fluorescein isothiocyanate (FITC) for 30 minutes at 37° C.
  • trypsin 0.5%, w/v
  • EDTA 1 mM
  • Cells are harvested by treating with trypsin/EDTA (5mL/25 cm 2 of surface area), washed twice in PBS and suspended at 5x1 0 5 cells/mL in PBS containing different concentrations of the HSA-RGD peptide.
  • the isolated cells are Incubate for 30 minutes at 37° C and then added to the upper chamber of the Transwell (Costar 8.0 ⁇ m) chamber that had been precoated with a type I collagen as a chemotractant. After 6-1 8 hours of incubation at 37° C, 5% CO 2 the membrane insert was recovered and cells remove with Q-tip cotton swab and placed into 1 % crystal violet in 20 % methanol 80% water. The cells were stained for 20 minutes and extracted with 10% acetic acid and absorbance measured at 600 nm. The number of migrated cells are calculated from a standard curve.
  • Endothelial cells spontaneously form capillary tube when seeded on natural ECM.
  • Basement membrane matrix (Sigma) is diluted at 4 mg/mL with cold PBS and added to 24-well plates (Costar) in a total volume of 200 ⁇ L in each well. Plates are left at 37° C for 30 minutes to form a gel layer into which HUVEC cells 2x10 5 in a medium with 20% FBS supplemented with concentrations of peptides are applied to each well and incubated at 37° C for 24 hours with 5% CO 2 . After incubation, cells are washed, fixed in 2% glutaldehyde for 10 minutes and subjected to inverted contrast-phase microscopy. The effect of the RGD peptides on tube forming sprouts are visually measured to determine the effects on capillary formation.
  • the CAM in the chick embryo is a classical in ovo assay to quantitate drug effect on vascular angiogenesis.
  • Ten days old chicken embryos are incubated at 37° C with 60% humidity.
  • a 1 cm 2 window is made to access the underlying CAM.
  • Angiogenesis is induced by injecting 200 ⁇ L of TNF ⁇ , 5ng/mL on the CAM.
  • test RGD peptides are applied in a volume of 50 ⁇ L.
  • the window is covered with sterile cellophane tape and the embryos are incubated for a further 48 hours at 37° C with 60% humidity.
  • CAM tissue is resected and angiogenesis is visualized on microscope.
  • the inhibition of vascular formation is semi quantitively scored to assess the effects of the RGD peptides in inhibiting angiogenesis.
  • Rats are anesthetized and small pocket is made on the cornea to inserted material to induce blood vessel formation.
  • Pellets of a slow releasing polymer (Hydron) containing angiogenic factor (VEGF) and different concentrations of RGD peptides are implanted into the pocket. (See preparation mode of mixture in Hydron in D'Amato technique. Angiogenesis 1 996). After 3 to 5 days, the animals will be killed and corneal vessel is photographed. The density of blood vessel growth is scored and activity of test RGD peptides defined as reflected by the extent of inhibiting blood vessel formation.
  • Hydron slow releasing polymer
  • VEGF angiogenic factor
  • Nude mice are anesthetized with sodium pentobarbital (25 mg/kg ip) and skin lesions produced with 0.5 mm silver nitrate cautery device. The lesion produced heals in 3-5 days. The rate of cellular infiltrate and the limitation of scarring is assessed in animals treated with RGD peptides to facilitate wound healing. In advanced animal models RGD peptides for surgical wound healing will be evaluated in models of gastrointestinal surgery. Under surgical anesthesia rats with undergo a laparotomy and a complete transection of the duodenum. AT the time of surgery the cut and adjacent ends are treated with RGD peptides and resutured.
  • the time to healing is assessed in comparison to controls based upon the rate of histopathological healing as well as restitutioning GI function as assessed by charcoal transit times evaluated in these animals 24 - 72 hours after surgical injury.
  • the ability of the RGD peptides to heal the cut ends of the GI tract will be characterized over a several week period of time to assess the enhancement of the healing process.
  • Example 25 In vivo angiogenesic and anti-metastatic activity
  • the anti-metastatic activity of such RGD peptides is evaluated in nude mice inoculated with different human cancer cell lines.
  • the tumor are established to a defined mass greater than 2 cm using defined growth and mass curves.
  • Animals are injected with the NHS RGD peptides directly into the established human tumor and the effects of the local application of the RGD peptide on the progression of tumor size and mass is determined in comparison to vehicle treated animals.
  • the anti-proliferative activity and effects on angiogenesis is determined by direct quantification of capillary formation, cell number, tumor density and blood flow defined into the tumor after treatment.
  • New Zealand rabbits (2 Kg), male or female, were intramuscularly anesthetized with Xylazine (20 mg/kg), Ketamine (50 mg/kg) et Acepromazine (0.75 mg/kg) prior to surgical exposure of left carotid artery. Segments of 1 0 mm of carotids, were transiently isolated by temporary ligatures and rinsed with C.9% sodium chloride via a cannula until there was no more visible evidence of blood components.
  • the carotid artery is injured by standard balloon angioplasty and RGD applied at the time of injury. The surgical incisions are repaired and the animals returned to the vivarium for periods of time up to one month after injury.
  • the injured vessel is isolated and inspected with perfusion fixation and harvesting.
  • the response to injury is assessed histomorphologically using computer imaging of the cross sectional areas and calculating the intimal to medial ratios as well as evaluating using BrDU the extent of cellular proliferation in this in vivo assay.
  • the effects of local RGD peptides on preventing the hyperproliferative response in this model reveals drug interactions in stabilizing the injured vessel.
  • New Zealand rabbits (2 kg), male or female, were intramuscularly anesthetized with Xylazine (20 mg/kg), Ketamine (50 mg/kg) et Acepromazine (0.75 mg/kg) prior to surgical exposure of left jugular vein.
  • Segments of 20 mm of carotids were transiently isolated by temporary ligatures and rinsed with 0.9% sodium chloride via a cannula until there was no more visible evidence of blood components.
  • the vessels are removed and surgical transplanted into the descending aorta. Twenty four hours after surgery the animals are reanesthetized and blood flow measured using Transonic flow probes to determine if thrombosis has occurred.
  • the application of NHS RGD to the vascular graft can act as a procoagulant or an anticoagulant.
  • the local application of the RGD will be measured in different uses to prevent bleeding at the suture sites as well as to define the effects on platelet deposition and arterialization at various time points in this model.

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Abstract

L'invention concerne des méthodes et des compositions permettant un apport localisé d'agents thérapeutiques, lesquels sont capables de former des liaisons covalentes avec un site recherché. Les agents thérapeutiques pouvant être utilisés dans cette invention sont notamment les cicatrisants, les antibiotiques, les anti-inflammatoires, les antioxydants, les antiprolifératifs, les immunosuppresseurs, les anti-infectieux, et les anticancéreux.
EP99916153A 1998-03-23 1999-03-23 Apport local d'agents therapeutiques a action prolongee Withdrawn EP1042003A1 (fr)

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
US7897498P 1998-03-23 1998-03-23
US78974P 1998-03-23
US8635298P 1998-05-20 1998-05-20
US86352P 1998-05-20
US8620598P 1998-05-21 1998-05-21
US86205P 1998-05-21
US10739198P 1998-11-06 1998-11-06
US107391P 1998-11-06
PCT/US1999/006344 WO1999048536A2 (fr) 1998-03-23 1999-03-23 Apport local d'agents therapeutiques a action prolongee

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CA (1) CA2291066A1 (fr)
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WO (1) WO1999048536A2 (fr)

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US20030203976A1 (en) 1993-07-19 2003-10-30 William L. Hunter Anti-angiogenic compositions and methods of use
NZ511762A (en) 1993-07-19 2003-09-26 Univ British Columbia Anti-angiogenic compositions and methods of use
EP1591453A1 (fr) * 1999-05-17 2005-11-02 ConjuChem Inc. Péptides-yy modifiées et ses conjugués
US7144854B1 (en) 1999-09-10 2006-12-05 Conjuchem, Inc. Long lasting anti-angiogenic peptides
ES2209885T3 (es) 1999-05-17 2004-07-01 Conjuchem, Inc. Peptidos insulinotropicos de larga duracion.
US6887470B1 (en) 1999-09-10 2005-05-03 Conjuchem, Inc. Protection of endogenous therapeutic peptides from peptidase activity through conjugation to blood components
US7601691B2 (en) 1999-05-17 2009-10-13 Conjuchem Biotechnologies Inc. Anti-obesity agents
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US6514500B1 (en) 1999-10-15 2003-02-04 Conjuchem, Inc. Long lasting synthetic glucagon like peptide {GLP-!}
JP4216480B2 (ja) 1999-05-17 2009-01-28 コンジュケム バイオテクノロジーズ インコーポレイテッド ウイルス感染の長期持続性融合ペプチドインヒビター
CN1698881A (zh) * 1999-05-17 2005-11-23 康久化学公司 病毒感染的长效融合肽抑制剂
DE19926154A1 (de) * 1999-06-09 2000-12-14 Ktb Tumorforschungs Gmbh Verfahren zur Herstellung einer injizierbaren Arzneimittelzubereitung
WO2001017568A2 (fr) * 1999-09-07 2001-03-15 Conjuchem, Inc. Diffusion pulmonaire permettant la bioconjugaison
US6706892B1 (en) 1999-09-07 2004-03-16 Conjuchem, Inc. Pulmonary delivery for bioconjugation
EP1889639A3 (fr) * 1999-09-07 2008-04-09 ConjuChem Biotechnologies Inc. Procédés et compositions pour produire des agents antinéoplastiques de très longue durée
US7090851B1 (en) 1999-09-10 2006-08-15 Conjuchem Inc. Long lasting fusion peptide inhibitors of viral infection
US6713454B1 (en) 1999-09-13 2004-03-30 Nobex Corporation Prodrugs of etoposide and etoposide analogs
US6380405B1 (en) * 1999-09-13 2002-04-30 Nobex Corporation Taxane prodrugs
US6541508B2 (en) 1999-09-13 2003-04-01 Nobex Corporation Taxane prodrugs
JP2004520395A (ja) 2001-02-02 2004-07-08 コンジュケム，インコーポレーテッド 長期持続性成長ホルモン放出因子誘導体
JP4280070B2 (ja) 2001-02-16 2009-06-17 コンジュケム バイオテクノロジーズ インコーポレイテッド 胃腸疾患および胃腸障害の処置のための長期持続性グルカゴン様ペプチド２（ｇｌｐ−２）
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IL133053A0 (en) 2001-03-19
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AU3452799A (en) 1999-10-18
WO1999048536A3 (fr) 2001-05-17
CA2291066A1 (fr) 1999-09-30

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