EP1176985A2 - Conjugues de medicaments polymeres actives par voie enzymatique - Google Patents

Conjugues de medicaments polymeres actives par voie enzymatique

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Publication number
EP1176985A2
EP1176985A2 EP00928630A EP00928630A EP1176985A2 EP 1176985 A2 EP1176985 A2 EP 1176985A2 EP 00928630 A EP00928630 A EP 00928630A EP 00928630 A EP00928630 A EP 00928630A EP 1176985 A2 EP1176985 A2 EP 1176985A2
Authority
EP
European Patent Office
Prior art keywords
conjugate
gly
pro
linker
biologically active
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Withdrawn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
EP00928630A
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German (de)
English (en)
Inventor
James M. Pachence
Benjamin A. Belinka
Thulasi Ramani
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.)
Vectramed Inc
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Vectramed Inc
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Publication date
Application filed by Vectramed Inc filed Critical Vectramed Inc
Publication of EP1176985A2 publication Critical patent/EP1176985A2/fr
Withdrawn legal-status Critical Current

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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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine 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

Definitions

  • the present invention relates generally to polymeric drug conjugates composed of biologically active agents attached to regular repeating linear co-polymers or branched copolymers by means of an enzymatically cleavable linker.
  • the linker may contain one or more chemical bonds that are cleaved by enzymes, and, in some cases, may be further cleaved by changes in pH, ionic, or redox conditions.
  • the polymeric drug conjugates of the present invention can be specifically designed to provide for optimal enzymatic approach and cleavage of the linker by modifying the co-polymer.
  • the invented polymeric drug conjugates may be used to modify drug solubility, drug bioavailability, drug residence time, drug absorption characteristics, and/or drug bioactivity.
  • Novel formulation technologies have been developed to improve the delivery of many pharmaceutical agents, primarily to overcome issues of aqueous solubility, drug toxicity, bioavailability, and patient compliance (e.g., increase the time period between drug administrations).
  • Drug delivery techniques can increase the therapeutic range of a compound by decreasing toxicity, thereby broadening the indications and clinical use of important drugs.
  • clinical indications of important pharmaceutical agents, particularly anti-cancer drugs are often dose-limited because of systemic toxicity.
  • the toxicity of potentially important compounds can be significantly decreased by direct delivery of the active agent to specific tissues using implantable, bioresorbable polymers Langer, 1990).
  • implantable polymer technologies for drug delivery have inherent limitations, such as, for example, the fact that drug release is a function of hydrolytic degradation of the associated polymer or of simple diffusion of the drug from the polymer matrix, and therefore, the release characteristics are not a function of the disease being treated but are actually independent of the disease.
  • Polymer or lipid encapsulation systems do not have inherent properties which provide for site-specific (targeted) release of the encapsulated agent.
  • polymer encapsulation systems are normally only suitable for water-soluble drugs, while liposomal formulations are restricted to those lipid soluble drugs which will partition in the liposomal bilayers without disruption of the bilayer integrity.
  • Macromolecules in the form of synthetic, natural, or semi-synthetic (chemically modified natural macromolecules) polymers have been utilized as carriers for a variety of pharmaceutical agents (see for example, Pachence and Kohn, 1998).
  • Various chemical spacer groups have been previously used to covalently couple active agents to the polymer to create a conjugate capable of controlled or sustained release of a drug within the body. These spacer groups provide biodegradable bonds that permit controlled drug release.
  • the size and nature of the spacer groups, and the charge and structure of the polymer are important characteristics to consider in the design of a drug with controlled or sustained release characteristics. As a result, the release of a drug often depends on hydrolytic cleavage of the bond between the polymer and active agent. However, while such a method does provide sustained release, it does not inherently target the active agent to a specific tissue.
  • polymeric drug conjugates can in general provide a method for controlled drug release or directed drug distribution in the body (and thereby improve the drug therapeutic index), the successful application of polymeric drug delivery systems depends to a great extent on: (1) the ability to reproducibly prepare well-defined polymer/drug conjugates;
  • MMPs matrix metalloproteinases
  • ECM ECM matrix metalloproteinases
  • Thrombin-like alanine aminopeptidase and elastase-like enzymatic activity are known to be common in bacterial infections (Finlay and Cossart, 1997), and the amino acid cleavage sequences of such enzymes are well-documented.
  • Kopecek, et al. (U.S. Patents No. 5,037,883 and 5,258,453) describe the use of polymeric carriers attached to drugs with a linking chain that is cleaved by intracellular enzymes. This method, however, is limited by the necessity of the polymeric carrier-linking chain-drug conjugate to be taken into the cell before enzymatic cleavage can occur. Particularly, these patents describe a polymer with a targeting moiety wherein degradation of the drug-carrier linkage occurs via intracellular lysosomal hydrolysis. The technology of Kopecek further relies on chemically linking drugs to preformed polymers.
  • sulfated polyanionic polysaccharides such as heparin
  • synthetic organic sulfated polymers such as polystyrene sulfonate, sulfated polyvinyl alcohol, or polyethylene sulfonate
  • the tissue- targeting component of the Thorpe invention is the endothelial cell-binding binding portion of heparin and similar polymers.
  • the active agents are randomly conjugated to pre-formed polymers. Furthermore, these polymers are not inherently water soluble, nor are they taught to extend drug residence time.
  • the biologically releasable bonds linking the active agent to the polymer in Thorpe are generally hydrolyzable, and are not disease specific. Thorpe does not describe drug releasing conditions which would lead to tissue-localized high concentrations of the active agent.
  • Other groups have described alternating co-polymers of PEG that result in water- soluble polymers for drug delivery.
  • Zalipsky, et al. U.S. Patents No. 5,219,564 and 5,455,027) describe a linear preformed polymer of PEG and the amino acid lysine to yield functional pendant groups (such as the terminal carboxyl group of lysine) at regular intervals. However, these methods do not describe methods of providing drug attachment along the polymer backbone at regular intervals.
  • the concept of enzymatically cleavable linking groups which would provide site-directed drug delivery is not disclosed.
  • the present invention relates generally to polymeric drug conjugates composed of biologically active agents attached to regular repeating linear co-polymers or branched copolymers by means of an enzymatically cleavable linker More specifically, the present invention relates to a polymeric drug conjugate comprising one or more biologically active agents conjugated via an enzymatically cleavable linker to either a regular repeating linear unit comprising a water soluble polymer segment and a multifunctional chemical moiety, or a branched polymer comprising two or more water soluble polymer segments each bound to a common multifunctional chemical moiety In a preferred embodiment, the one or more biologically active agents are conjugated via the linker to the multifunctional chemical moiety of the regular repeating linear unit In another preferred embodiment, the one or more biologically active agents are conjugated via the linker to at least one of the two or more water soluble polymer segments
  • the linker may contain one or more chemical bonds that may be cleaved by enzymes and, in some cases, additionally by changes in pH, ionic, or redox conditions, which are present in high concentration near, in, and /or on the surface of diseased tissues More specifically, the linker may be further cleaved by hydrolysis, reduction reactions, oxidative reactions, pH shifts, photolysis, or combinations thereof The linker may also be further cleaved by a non-specific enzyme reaction In a preferred embodiment, the linker may WO 00/64486 PCT/USOO/l 1670
  • the linker may be cleaved by a membrane-bound enzyme.
  • the linker is cleaved by an enzyme that is available at a target site, wherein the enzyme may also be up-regulated at the target site.
  • the target site may be diseased tissue or biological fluid, wherein the diseased tissue may be present in skin, bone, cartilage, muscle, connective tissue, neural tissue, reproductive organs, endocrine tissue, lymphatic tissue, vasculature, or visceral organs and the biological fluid may be blood, pleural fluid, peritoneal fluid, joint fluid, pancreatic fluid, bile, or cerebral-spinal fluid.
  • the linker may be cleaved by any of an enzyme resulting from a microbial infection, a skin surface enzyme, or an enzyme secreted by a cell, by an enzyme secreted by a cancer cell, by an enzyme located on the surface of a cancer cell, by an secreted by a cell associated with a chronic inflammatory disease, by an enzyme secreted by a cell associated with rheumatoid arthritis, or by an enzyme secreted by a cell associated with osteoarthritis.
  • the conjugates of the present invention can be designed to provide for optimal enzymatic approach to and cleavage of the linker by modifying the water soluble polymer segments of the linear co-polymer and/or the multifunctional chemical moieties of the branched co-polymer.
  • the polymeric drug conjugate of the present invention may be used to modify drug solubility, drug bioavailability, drug residence time, drug absorption characteristics, and/or drug bioactivity.
  • General structures of the polymeric drug conjugates are shown below.
  • the copolymer backbones of the conjugate of the present invention are composed of water soluble polymer segments attached to multifunctional chemical moieties to form either regular linear repeating or branched scaffolds. These scaffolds are designed to provide a series of evenly spaced chemical functionalities for the attachment of biologically active moieties through an enzymatically cleavable linking group.
  • the composition of the co-polymer can be modified to allow for optimal enzymatic approach to the enzymatically cleavable linkers by the modification of the size or chemical environment of the individual polymer segments and/or the multifunctional chemical moieties.
  • the polymer construct poly[D-L-M-P] consists of a multifunctional chemical moiety, M, that is used to join water soluble polymer segments, P, to form a regular repeating linear co- polymer backbone and additionally to provide the chemical substituents for the attachment of a biologically active agent, D, via an enzymatically cleavable linker, L.
  • M multifunctional chemical moiety
  • the water soluble polymeric drug conjugate can be designed to increase the water solubility of D and be formulated to be administered through injection, oral, topical, inhalation delivery, subcutaneous deposition (implant), deposition using minimally invasive surgical procedures such as laproscopy, or other previously described physical delivery methods.
  • the pharmaceutical agent released from the co-polymer/linking agent conjugate by enzymatic activity provides reconstituted pharmaceutical activity in a high local tissue concentration.
  • the metabolically sensitive linking group, L is designed to be cleaved by enzymes that are present at high concentration (higher than non-pathological levels) at or near the targeted disease site.
  • the branched polymeric drug conjugate of the formula Q(-P-L-D) k consists of a common multifunctional chemical moiety, Q, that is used to attach k number of water soluble polymer segments, P, attached to a biologically active agent, D, via an enzymatically cleavable linker, L.
  • the water soluble polymeric drug conjugate can be designed to increase the water solubility of D and can be formulated to be administered through injection, oral, topical, inhalation delivery, subcutaneous deposition, deposition using minimally invasive surgical procedures (e.g. laproscopy), or other previously described physical delivery methods.
  • the pharmaceutical agent released from the polymeric conjugate by enzymatic activity provides reconstituted pharmaceutical activity in a high local tissue concentration.
  • the metabolically sensitive linking group, L is designed to be cleaved by enzymes that are present at high concentration (higher than non-pathological levels) at or near the site of disease.
  • the present invention also includes pharmaceutical compositions comprising the novel conjugate and a physiologically acceptable carrier, wherein the pharmaceutical composition is preferably suitable for injection, or oral, topical, inhalation, or implantation methods of administration.
  • the present invention also includes methods of alleviating a pathological condition comprising administering an effective amount of novel conjugates of the present invention, wherein the pathological condition may include neoplastic diseases, chronic inflammatory diseases acute inflammatory diseases, cardiac diseases, renal diseases, liver diseases, lung diseases, neurological diseases, musculoskeletal diseases, and immunological disorders.
  • the pathological condition may include neoplastic diseases, chronic inflammatory diseases acute inflammatory diseases, cardiac diseases, renal diseases, liver diseases, lung diseases, neurological diseases, musculoskeletal diseases, and immunological disorders.
  • the method may include regulating cardiac function, renal function, liver function, lung function, or neurological function, modulating immunological function, modulating hormonal function, treating microbial infections, or regulating scar tissue
  • the present invention provides novel compositions for creating tissue-targeting formulations consisting of pharmaceutical agents conjugated to water soluble polymers
  • the present invention provides a method of targeting drug release by using chemical linking groups between the polymer and the pharmaceutical agent that can be specifically cleaved at the site of disease
  • the pharmaceutical agents according to the present invention are spaced along the water soluble polymer backbone at regular intervals, wherein the interval between each active agent is controlled by synthetic methods
  • a unique aspect of the conjugate of the present invention is a construct which consists of a drug and linker which repeats on a water soluble polymer backbone
  • the spacing between attachment of the drug/linker group complex can be controlled by the synthetic methods presented herein
  • One preferred method of forming the conjugate of the present invention is that the drug, linker, and monomer be conjugated first, and then the resulting product is coupled to a water soluble polymer which then forms the polymer conjugate This provides for a high degree of drug-linker substitution on the polymer construct (typically greater than 90%), providing
  • the present invention also requires that the linking group be cleavable by enzymatic activity, such as by enzymes which may be present in high concentrations near, in and/or on the surface of diseased tissue
  • enzymatic activity such as by enzymes which may be present in high concentrations near, in and/or on the surface of diseased tissue
  • This invention describes polymeric drug conjugates formed by covalently attaching a biologically active agent to a co-polymeric backbone via an enzymatically cleavable linker.
  • the conjugates poly[D-L-M-P] and Q(-P-L-D) k are administered to a patient, wherein the biologically active agent is released from the polymer backbone by a physiological process, and the agent's activity is reconstituted.
  • the linker consists of chemical chains with one or more bonds that are susceptible to physiological cleavage, preferably enzymatic cleavage.
  • the structures of the invented constructs are shown in General Polymeric Drug Conjugate Formulas.
  • the invented polymer construct of formula poly[D-L-M-Pl consists of a multifunctional chemical moiety , M, that is composed of organic compounds, amino acids, or a combination of both which contains chemical functionalities that can be used to form covalent bonds with the polymer segments, P, and the linking group, L;
  • L is a "linker” that can consist of, either independently or in combination, amino acids, sugars, nucleic acids, or other organic compounds which possess one or more chemical bonds that are enzymatically cleavable;
  • D is a biologically active agent which possesses a chemical substituent that can form a covalent bond to the linking group, L;
  • P is a water soluble polymer or copolymer that contains at least two functionalities that can form covalent chemical bonds to substituents on the monomer, M; and
  • m is the number of polymer repeats, typically ranging from 5 to 12 but can extend over the range of 2-25.
  • the water-soluble polymer segment comprises poly(ethylene glycol) with a molecular weight of about 2,000
  • the multifunctional chemical moiety comprises N-(2-hydroxyacetyl)serine
  • the linker comprises (H-Leu-Gly-Pro-Ala-NH-
  • the biologically active agent comprises doxorubicin- 14-O-hemiglutarate.
  • the water-soluble polymer segment comprises poly(ethylene glycol) with a molecular weight of about 1,000
  • the multifunctional chemical moiety comprises tris(2-aminoethyl)amine
  • the linker comprises (H-Val-Pro-Arg-OH)
  • the biologically active agent comprises N*-(aminoiminomethyl)-N 2 -(3 -mercapto- 1-oxopropyl-L- lysylglycyl-L- ⁇ -aspartyl-L-tryptophyl-L-prolyl-L-cysteinamide, cyclic (1 - 6)-disulfide.
  • the water-soluble polymer segment comprises poly(ethylene glycol) with a molecular weight of about 2,000
  • the multifunctional chemical moiety comprises N ⁇ -(- ?-aminophenylacetyl)- ?-aminophenylalanyl hydrazide
  • the linker comprises (OCH-CO-Gly-Pro-Leu-Gly-Pro-OH)
  • the biologically active agent comprises a pharmaceutical analog of Leu-Gly- ⁇ - 5 -fluorouracil.
  • the water-soluble polymer segment comprises poly(ethylene glycol) with a molecular weight of about 1,000
  • the multifunctional chemical moiety comprises 3,5-dihydroxyphenylacetic acid
  • the linker comprises (H-Cys(S-CH 2 -CH 2 - CO-NH-CH 2 -CH 2 -NH_)-Glu-Glu-Glu-OH)
  • the biologically active substance comprises a pharmaceutical analog of Leu-Gly- ⁇ -5 -fluorouracil.
  • the water-soluble polymer segment comprises poly(ethylene glycol) with a molecular weight of about 2,000
  • the multifunctional chemical moiety comprises lysine
  • the linker comprises (H-Gly-Pro-Tyr-Ala-Tyr-T ⁇ -Lys-NH 2 )
  • the biologically active agent comprises methotrexate.
  • the water-soluble polymer segment comprises poly(ethylene glycol) with a molecular weight of about 2,000
  • the multifunctional chemical moiety comprises lysine
  • the linker comprises (H-Gly-Pro-Lys-Pro-Val-Gly-Nva-Trp-Lys- OH)
  • the biologically active agent comprises methotrexate.
  • the water-soluble polymer segment comprises poly(ethylene glycol) with a molecular weight of about 2,000
  • the multifunctional chemical moiety comprises lysine
  • the linker comprises (H-Gly-Pro-Leu-Gly-Pro-Lys-NH )
  • the biologically active agent comprises methotrexate.
  • the invented polymer construct of formula Q(-P-L-D) k consists of a common multifunctional chemical moiety, Q, that is used to attach k number of water soluble polymer segments, P, and a n enzymatically cleavable linker, L, that connects P to D, a biologically active agen, wherein k is preferably an integer from 1 to about 100, more preferably an integer from 2 to about 100.
  • the water soluble polymer conjugate prodrug system can be designed to increase the water solubility of D and can be formulated to be administered through injection, oral, topical, inhalation delivery, subcutaneous deposition, deposition using minimally invasive surgical procedures such as laproscopy, or other previously described physical delivery methods.
  • polymer construct Q(-P-L-D) allows for multiple equally spaced drug-linker substituents on each common multifunctional chemical moiety, Q.
  • the structure, chemical composition, or size of the polymer segment, P can be easily changed to allow for a facile approach of the enzyme to the enzymatically cleavable linker, L, and optimize the biological utility of the product construct.
  • the pharmaceutical agent released from the polymer/linking agent conjugate by metabolic activity would provide reconstituted pharmaceutical activity in a high tissue local concentration.
  • the water-soluble polymer segment comprises poly(ethylene glycol) with a molecular weight of about 4,000
  • the common multifunctional chemical moiety comprises pentaerythritol or a pentaerythritol analog
  • the linker comprises
  • the biologically active agent comprises methotrexate, and said integer is 4.
  • the water-soluble polymer segment comprises poly(ethylene glycol) with a molecular weight of about 4,000
  • the common multifunctional chemical moiety comprises an 8 arm dendrimer
  • the linker comprises (H-Gly-Pro-Leu-Gly-
  • the biologically active agent comprises methotrexate, and the integer is 8.
  • the water-soluble polymer segment comprises poly(ethylene glycol) with a molecular weight of 4,000
  • the common multifunctional chemical moiety comprises pentaerythritol or a pentaerythritol analog
  • the linker comprises (H-Glu-Glu-
  • the biologically active agent comprises methotrexate, and the integer is 4.
  • the water-soluble polymer segment comprises poly(ethylene glycol) with a molecular weight of 4,000
  • the common multifunctional chemical moiety comprises an 8 arm dendrimer
  • the linker comprises (H-Glu-Glu-Glu-Lys( ⁇ -CO-CH 2 - CH -OH)-NH 2
  • the biologically active agent comprises methotrexate
  • the integer is 8.
  • the water-soluble polymer segment comprises poly(ethylene glycol) with a molecular weight of 4,000
  • the common multifunctional chemical moiety comprises pentaerythritol or a pentaerythritol analog
  • the linker comprises (H-Gly-Pro- Lys-Pro-Val-Gly-Nva-T ⁇ -Lys( ⁇ -CO-CH 2 -CH 2 -OH)-NH 2
  • the biologically active substance comprises methotrexate
  • the integer is 4.
  • the water-soluble polymer segment comprises poly( ethylene glycol) with a molecular weight of 4,000
  • the common multifunctional chemical moiety comprises an 8 arm dendrimer
  • the linker comprises (H-Gly-Pro-Lys-Pro-Val-Gly-Nva- T ⁇ -Lys( ⁇ -CO-CH 2 -CH 2 -OH)-NH 2
  • the biologically active agent comprises methotrexate
  • the integer is 8.
  • the water-soluble polymer segment comprises poly(ethylene glycol) with a molecular weight of 4,000
  • the common multifunctional chemical moiety comprises pentaerythritol or a pentaerythritol analog
  • the linker comprises (H-Gly-Pro- Tyr-Ala-Tyr-T ⁇ -Lys( ⁇ -CO-CH 2 -CH 2 -OH)-NH 2
  • the biologically active agent comprises methotrexate
  • the integer is 4.
  • the water-soluble polymer segment comprises poly( ethylene glycol) with a molecular weight of 4,000
  • the common multifunctional chemical moiety comprises 8 arm dendrimer
  • the linker comprises (H-Gly-Pro-Tyr-Ala-Tyr-Trp-Lys( ⁇ - CO-CH 2 -CH 2 -OH)-NH 2
  • the biologically active agent comprises methotrexate
  • the integer is 8.
  • Polymeric drug conjugate poly[D-L-M-P] of this invention can be assembled using four separate units; the multifunctional chemical moiety, M, an enzymatically cleavable linker, L, a biologically active agent, D, and a water soluble polymer segment, P. These individual units are initially substituted with one or more reactive functional groups that are used to form stable chemical bonds with the other units of the construct.
  • 1 to 100% of the multifunctional chemical moieties, M, inco ⁇ orated into the copolymer backbone may be substituted with the biologically active agent-linker conjugate, D-L.
  • the chemical linkage connecting the water soluble polymer segment, P, to the multifunctional chemical moiety, M may include a carbamate, thiocarbamate, ether, urea, thiourea, carbonate, thiocarbonate, or ester function
  • Construct Q(-P-L-D) of this invention can also be assembled using four units; the common multifunctional chemical moiety, Q, an enzymatically cleavable linker, L, a biologically active agent, D, and a water soluble polymer segment , P. These individual units are initially substituted with one or more reactive functional groups that are used to form stable bonds with the other units of the construct.
  • 1 to 100% of the water soluble polymer segments, P, inco ⁇ orated onto the common multifunctional chemical moiety, Q may be substituted with the biologically active agent-linker conjugate, D-L.
  • the chemical linkage connecting the water soluble polymer segment, P, to the common multifunctional chemical moiety, Q, or the linker, L may include a carbamate, thiocarbamate, ether, urea, thiourea, carbonate, thiocarbonate, or ester function.
  • the multifunctional chemical moiety, M is designed to covalently join the water soluble polymer segments, P, and also bind the linker, L. It is designed and synthesized to provide the structure and chemical functionalities illustrated in General Formula 1.
  • Xi, X 2 , and X 3 are chemical substituents that can be used to form covalent bond with the polymer segments, P, or the enzymatically cleavable linker, L.
  • Xj, X 2 , and X 3 can independently be or be derived from, but are not restricted to, hydroxyl, amino, thiol, alkyl or aryl disulfide, isothiocyanate, thiocarbonylimidazole, thiocarbonylchloride, aldehyde, ketone, carboxylic acid, carboxylic acid ester, sulfonic acid, sulfonic acid ester, sulfonyl chloride, phosphoric acid, alkyl or aryl succinimidyl carbonate, alkyl or aryl chlorocarbonate, alkyl or aryl succinimidylthiocarbonate, alkyl or aryl chlorothiocarbonate, halide, or thioester
  • Ri, R 2 , or R 3 act as spacers that initially separate the reactive functional groups to provide an optimal chemical and steric environment for the assembly of the final polymeric drug conjugate, and ultimately separate the polymer segments, P, and linker , L, to allow for optimal biological activity of the invented construct.
  • the multifunctional chemical moiety is derived from N-(2- hydroxyacetyl)serine, lysine, tris(2-aminoethyl)amine, N-(p -nitrophenylacetyl)- ?- nitrophenylalanine acid hydrazide, 3,5-dihydroxyphenylacetic acid, 3,5-diaminobenzoic acid, or 6-amino-4-(2-aminoethyl)hexanoic acid
  • X4 and X 5 are chemical substituents that can be used to form covalent bonds with the multifunctional chemical moiety, M, and the biologically active agent, D.
  • X 4 and X 5 can independently be or be derived from, but are not restricted to, hydroxyl, amino, thiol, alkyl or aryl disulfide, isothiocyanate, thiocarbonylimidazole, thiocarbonylchloride, aldehyde, ketone, carboxylic acid, carboxylic acid ester, sulfonic acid, sulfonic acid ester, sulfonyl chloride, phosphoric acid, alkyl or aryl succinimidyl carbonate, alkyl or aryl chlorocarbonate, alkyl or aryl succinimidylthiocarbonate, alkyl or aryl chlorothiocarbonate, halide, or thioest
  • the linker may include an amino acid, a sugar, a nucleic acid, or other organic compounds, or combinations thereof. In another preferred embodiment, the linker may include a peptide sequence.
  • the peptide sequence may be cleaved by a serine protease such as thrombin, chymotrypsin, trypsin, elastase, kallikrein, or substilisin.
  • a serine protease such as thrombin, chymotrypsin, trypsin, elastase, kallikrein, or substilisin.
  • the thrombin-cleavable peptide sequence may include -Gly-Arg-Gly-Asp-, -Gly-Gly-Arg-, -Gly- Arg-Gly-Asp-Asn-Pro-, -Gly-Arg-Gly-Asp-Ser-, -Gly-Arg-Gly-Asp-Ser-Pro-Lys-, -Gly-Pro- Arg-, -Val-Pro-Arg-, or -Phe- Val -Arg-.
  • the elastase-cleavable peptide sequence may include -Ala-Ala-Ala-, -Ala-Ala-Pro- Val-, -Ala-Ala-Pro-Leu-, -Ala-Ala-Pro-Phe-, -Ala-Ala-Pro-Ala-, or -Ala-Tyr-Leu-Val-.
  • the linker may include a peptide sequence which may be cleaved by a cysteine proteinase such as papain, actinidin, bromelain, lysosomal cathepsins, cytosolic calpain, and parasitic protease.
  • a cysteine proteinase such as papain, actinidin, bromelain, lysosomal cathepsins, cytosolic calpain, and parasitic protease.
  • the parasitic protease may be derived from Trypanosoma or Schistosoma.
  • the linker may include a eptide sequence which may be cleaved by an aspartic proteinase such as pepsin, chymosin, lysosomal cathepsins D, a processing enzyme, a fungal protease, and a viral proteinase.
  • an aspartic proteinase such as pepsin, chymosin, lysosomal cathepsins D
  • a processing enzyme may include renin
  • the fungal protease may include penicillopepsin, rhizopuspepsin, or endothiapepsin
  • the viral protease may include the protease from the AIDS virus.
  • the linker may include a peptide sequence that can be cleaved by a matrix metalloproteinase such as collagenase, stromelysin, and gelatinase.
  • a matrix metalloproteinase such as collagenase, stromelysin, and gelatinase.
  • the matrix metalloproteinase may include -Gly-Pro-Y-Gly-Pro-Z-, -Gly-Pro-
  • the matrix metalloproteinase includes -Leu-Gly-, or Ile-Gly.
  • collagenase-cleavable peptide sequence may include -Pro-Leu-Gly-Pro-D-Arg-Z-, -Pro- Leu-Gly-Leu-Leu-Gly-Z-, -Pro-Gln-Gly-Ile-Ala-Gly-Trp-, -Pro-Leu-Gly-Cys(Me)-His-, -Pro-Pro-
  • the stromelysin-cleavable peptide sequence may include -Pro-Tyr-
  • gelatinase-cleavable peptide sequence may include -Pro-Leu-
  • the linker may include a peptide sequence that can be cleaved by an angiotensin converting enzyme, wherein the angiotensin converting enzyme may include -Asp-Lys-Pro-, -Gly-Asp-Lys-Pro-, or -Gly-Ser-Asp-Lys-Pro-.
  • the linker may include a peptide sequence that can be cleaved by a prostate specific antigen or a prostate specific membrane antigen, wherein the linker may include -(Glu) n - , where n is an integer from 1 to 10.
  • the biologically active agent, D consists of any biologically useful agent, analog, or metabolite, or a mixture of several systems, which possess (or can be modified to possess) at least one chemical functionality (for example, including, but not limited to, hydroxyl, amino, thiol, alkyl or aryl disulfide, isothiocyanate, thiocarbonylimidazole, thiocarbonylchloride, aldehyde, ketone, carboxylic acid, carboxylic acid ester, sulfonic acid, sulfonic acid ester, sulfonyl chloride, phosphoric acid; alkyl, aryl succinimidyl carbonate, alkyl or aryl chlorocarbonate, alkyl or aryl succinimidylthiocarbonate, alkyl or aryl chlorothiocarbonate, halide, or thioester) for covalent attachment to the linker, L.
  • chemical functionality for example, including, but not limited
  • Biologically active agents that may be delivered by the system of the present invention include, but are not restricted to, analgesics, anesthetics, antifungals, antibiotics, antiinflammatories, anthelmintics, antidotes, antiemetics, antihistamines, antihypertensives, antimalarials, antimicrobials, antipsychotics, antipyretics, antiseptics, antiarthritics, antituberculotics, antitussives, antivirals, cardioactive drugs, cathartics, chemotherapeutic agents, corticoids (steroids), antidepressants, depressants, diagnostic aids, diuretics, enzymes, expectorants, hormones, hypnotics, minerals, nutritional supplements, parasympathomimetics, potassium supplements, radiation sensitizers, sedatives, sulfonamides, stimulants, sympathomimetics, tranquilizers, urinary antiinfectives, vasoconstrictors, vasodilators, vitamins,
  • the chemotherapeutic agent comprises a nitrogen mustard, an ethylenimine, a methylmelamine, a nitrosourea, an alkyl sulfonate, a triazene, a folic acid analog, a pyrimidine analog, a purine analog, a vinca alkaloid, an epipodophyllotoxin, an antibiotic, an enzyme, a biological response modifier, a platinum complex, a methylhydrazine derivative, an adrenocorticol suppressant, a somatostatin, a somatostatin analog, a hormone, a hormone antagonist, or combinations thereof.
  • the chemotherapeutic agent comprises methotrexate, taxol, aminopterin, doxorubicin, bleomycin, camptothecin, etoposide, estramustine, prednimustine, melphalan, hydroxyurea, or 5-fluorouracil.
  • the peptide based pharmaceutical agent comprises a cytokine, a growth factor, a cell receptor antagonist, or a cell receptor agonist.
  • the biologically active agent comprises an eptifibatide and other platelet binding proteins, a granulocyte colony stimulating factor, a human growth factor, a vascular endothelial growth factor, a bone mo ⁇ hogenic protein, an interferon, or an interleukin.
  • the water soluble polymer segment, P consists of is a relatively short, water soluble polymeric system (for example, average M.W. 400-25,000) which contains at least two chemical functionalities (for example, including but not limited to, hydroxyl, amino, thiol, alkyl or aryl disulfide, isothiocyanate, thiocarbonylimidazole, thiocarbonylchloride, aldehyde, ketone, carboxylic acid, carboxylic acid ester, sulfonic acid, sulfonic acid ester, sulfonyl chloride, phosphoric acid, alkyl or aryl succinimidyl carbonate, alkyl or aryl chlorocarbonate, alkyl or aryl succinimidylthiocarbonate, alkyl or aryl chlorothiocarbonate, halide, or thioester) that can be used for covalent attachment to the multifunctional chemical moiety, M.
  • chemical functionalities for example,
  • P can be, but is not restricted to, poly(ethylene glycol), poly(vinyl alcohol), poly(2-hydroxyethyl methacrylate), poly(acrylic acid), poly(methacrylic acid), poly(maleic acid), poly(lysine), and the like or a copolymer consisting of a mixture of these or other polymeric entities possibly substituted with organic functional groups.
  • the common multifunctional chemical moiety, Q is designed to couple to k number of short water soluble polymer segments, P. It is designed and synthesized to provide the structure and chemical functionalities illustrated in General Formula 3.
  • X ⁇ is a chemical substituent that can be used to form covalent bonds with the polymer se ments
  • P. X « can independently be or be derived from, but are not restricted to, hydroxyl, amino, thiol, alkyl or aryl disulfide, isothiocyanate, thiocarbonylimidazole, thiocarbonylchloride, aldehyde, ketone, carboxylic acid, carboxylic acid ester, sulfonic acid, sulfonic acid ester, sulfonyl chloride, phosphoric acid, alkyl or aryl succinimidyl carbonate, alkyl or aryl chlorocarbonate, alkyl or aryl succinimidylthiocarbonate, alkyl or aryl chlorothiocarbonate, halide, or thioester functions (possibly substituted with appropriate protecting groups that can be removed before further chemical reaction)
  • J acts as a spacer that initially separate the reactive functional
  • the common multifunctional chemical moiety includes pentaerythritol, dendrimers, or branched lysine trees
  • Assembly Method I The method of assembly, illustrated in Reaction Scheme 1, requires an initial covalent coupling of the linker, L, to the biologically active agent, D, producing a conjugate, D-L.
  • the D-L construct is then reacted with multifunctional chemical moiety, M, producing the system D-L-M
  • the D-L-M is then covalently coupled to the appropriate water soluble polymeric segment, P, to give the desired regular repeating linear polymeric drug conjugate poly[D-L- M-P]
  • linker L
  • M M
  • the second reaction of this pathway requires the chemical coupling of L-M to agent, D, yielding the D-L-M which is then attached to the appropriate polymeric system, P, to form invented construct poly[D-L-M-P].
  • M can be initially coupled to the linker, L, (see Reaction Scheme 3) to prepare the conjugate L-M.
  • L-M is then reacted with P and then D to prepare the invented construct.
  • M can be covalently attached to the polymer, P, to yield the construct poly[M-P] (see Reaction Scheme 4).
  • the co-polymer conjugate is then reacted with the linker, L, to yield poIy[L-M-P] and is then coupled to the biologically active agent, D, to yield product poly[D-L-M-P].
  • the product constructs may or may not have each linker, L, substituted with a biologically active agent, D.
  • the D-L can then be synthesized independently and attached to poly[M-P], to yield the invented construct, poly[D-L-M-P]
  • the product constructs may or may not have each multifunctional moiety, M, substituted with a D-L.
  • the product constructs may or may not have each polymer segment, P, substituted with D-L.
  • Q can first be coupled to two or more polymer segments, P, to form Q(- P) k .
  • P polymer segment
  • the second reaction of this pathway requires the chemical coupling of Q-(P)k to the linker L, yielding the Q-(P-L) k macromer which is then attached to the appropriate biologically active agent, D, to form invented construct Q(-P-L-D) k .
  • the product constructs may or may not have each linker, L, substituted with a biologically active agent, D
  • the biologically active agent, D can be initially coupled to the linker, L, (see Reaction Scheme 8) to prepare conjugate L-D.
  • L-D is then reacted with P to produce the macromer P-L-D, which is in turn attached to the common multifunctional moiety, Q, to prepare the invented construct
  • the invented constructs are assembled by the covalent coupling of structural portions, D, L, P and M or with D, L, and with Q These units are attached by means of the substituents Xi-X ⁇ which are chosen to allow for the formation of stable covalent bonds between the various units of the polymeric drug conjugates Attachment of the Pharmaceutically Active Agent, D, to the Linker, L
  • substituents can independently be or be derived from, but are not restricted to, hydroxyl, amino, thiol, alkyl or aryl disulfide, isothiocyanate, thiocarbonylimidazole, thiocarbonylchloride, aldehyde, ketone, carboxylic acid, carboxylic acid ester, sulfonic acid, sulfonic acid ester, sulfonyl chloride, phosphoric acid, alkyl or aryl succinimidyl carbonate, alkyl or aryl chlorocarbonate, alkyl or aryl succinimidylthiocarbonate, alkyl or aryl chlorothiocarbonate, halide, or thioester functions
  • additional reagents are added during the coupling reactions to begin or enhance the covalent attachments.
  • the reagents and synthetic techniques needed for the coupling of D to L are commonly known to those of ordinary skill in the art of organic, peptide, or oligonucleotide synthesis.
  • t is chemically protected to prevent reaction with D.
  • functional group, X 5 on L is the N-hydroxysuccinimidyl ester on tetrapeptide, Fmoc-Cys(Dnp)-Glu-Glu-Glu-OSu (Compound 40) and the reactive substituent on D is the amino group on cancer drug analog H-Leu-Gly( ⁇ -5-fluorouracil)-OH (Compound 34).
  • the covalently coupled amide product D-L (Compound 41) is shown in Reaction Scheme 9 below.
  • X5 is the amino group on tetrapeptide conjugate, H-Leu-Gly- Pro-Ala-M-P (Compound 12) and the reactive functionality on D is the carboxylic acid substituent of N-Fmoc-doxorubicin-14-O-hemiglutarate (Compound 13).
  • Activating agent 1,3-dicyclohexylcarbodiimide is added to the reaction mixture enabling the coupling reaction to take place.
  • the covalently coupled amide product, regular repeating linear polymer po!y[D- L-M-P], (Compound 14) is shown in Reaction Scheme 10 below.
  • X 5 is the carboxylic acid function of an Integrilin analog (Compound 19) and the reactive function on D is the terminal amino group on tripeptide, H- Val-Pro-Arg(Boc) 2 -OMe (Compound 20).
  • Activating agent 1,3-dicyclohexylcarbodiimide is added to the reaction mixture enabling the coupling reaction to take place.
  • the covalently coupled amide product D-L (Compound 21) is shown in Reaction Scheme 11 below.
  • functional group, X 5 on L is the N-hydroxysuccinimidyl ester of pentapeptide, Boc-Gly-Pro-Leu-Gly-Pro-OSu (Compound 33) and the reactive substituent on D is the amino group on cancer drug analog H-Leu-Gly( ⁇ -5-fluorouracil)-OH (Compound 33).
  • the water soluble polymer segment, P consists of a short, water soluble polymer or co-polymer system which contains at least two chemical functionalities for covalent attachment to the monomer, M.
  • the reagents and synthetic techniques needed for the coupling of M to P are commonly known to those of ordinary skill in the art of organic, peptide, or oligonucleotide synthesis.
  • P can be, but is not restricted to, polyethylene glycol, poly(vinyl alcohol), poly(2-hydroxyethyl methacrylate), poly(acrylic acid), poly(methacrylic acid), poly(maleic acid) or various copolymers or analogs of each.
  • the preparation of the various polymers and analogs are accomplished using standard techniques known to those of average knowledge in the field of polymer synthesis.
  • the attachment of common multifunctional moiety, Q, to polymer segment, P is accomplished using synthetic reactions similar to those for the conjugation of M to P.
  • groups, Xi and X 2 on , M are both amino functions (Compound 17) and the polymer, P, is an N-hydroxysuccinimidyl carbonate substituted analog of polyethylene glycol-1000 (Compound 16).
  • the resulting biscarbamate conjugate, regular repeating linear co-polymer, P-M, (Compound 18) is shown in Reaction Scheme 13 below.
  • Xi and X 2 are both hydroxy group (Compound 7) and the reactive functionality on P (Compound 8) is a toluene sulfonate ester.
  • a base sodium hydride
  • a base sodium hydride
  • Substituents X 3 and X can independently be or be derived from, but are not restricted to, hydroxyl, amino, thiol, alkyl or aryl disulfide, isothiocyanate, thiocarbonylimidazole, thiocarbonylchloride, aldehyde, ketone, carboxylic acid, carboxylic acid ester, sulfonic acid, sulfonic acid ester, sulfonyl chloride, phosphoric acid, alkyl or aryl succinimidyl carbonate, alkyl or aryl chlorocarbonate, alkyl or aryl succinimidylthiocarbonate, alkyl or aryl chlorothiocarbonate, halide, or thioester functions (possibly substituted with appropriate protecting groups that can be removed before further chemical reaction).
  • group X 3 is an amine on M, already attached to the polymer system, P (Compound 10), and X» is a carboxylic acid on the linker, L (Compound 11), that is activated with 1,3-dicyclohexylcarbodiimide in situ to form the poly[L-M-P] portion of the invented construct.
  • the structure of the resultant product (Compound 12) is shown in
  • X 3 is an acid hydrazide on Compound 32 and X_t is an aldehyde on Compound 38 (see Reaction Scheme 16).
  • X 3 is a pyridyl disulfide on Compound 50 and is a thiol group on Compound 42 (see Reaction Scheme 17).
  • the linker, L is assembled using standard synthesis methodologies and is designed to connect individual biologically active agent, D, to the polymeric scaffolds of the invented polymeric drug conjugates. It is also engineered to possess one or more enzymatically cleavable bonds, the breaking of which allows for the release of the biologically active agent or its analog from the polymeric constructs.
  • the linker, L may also include spacer groups, F and R5, that contain one or more hydrolytically, oxidatively, or photolytically cleavable chemical bonds. Since L contains two active functional group, X 4 and X 5 , one or more of these functions can be chemically protected during the assembly of the construct and then deprotected when required.
  • the enzymatically cleavable bond of the linker may be spaced from the co-polymeric backbone and biologically active agent via the spacer groups to allow for enhanced exposure of the linking group to enzymes or to provide an optimal chemical environment for cleavage.
  • L which consists of independently or in combination, amino acids, sugars, nucleic acids, or other organic compounds joined by saturated and unsaturated, straight and branched chain alkyl, aryl, or alkylaryl, heteroalkyl, heteroaryl, or heteroalkyaryl groups which may contain up to 20 carbon atoms
  • alkyl, aryl, or alkylaryl, heteroalkyl, heteroaryl, or heteroalkyaryl groups which may contain up to 20 carbon atoms
  • reagents and techniques known to those of average skill in the art of organic, peptide and oligonucleotide chemistry.
  • the enzymatically cleavable linker is derived from the tripeptide, FMOC-Cys(DNP)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-OSu which can be assembled using standard amino acid coupling techniques (see Lloyd-Williams, et al. "Chemical Approaches to the Synthesis of Peptides and Proteins," CRC Press, New York, 1997).
  • the cleavable linker is derived from the tripeptide, H-Val-Pro- Arg(Boc) 2 -OMe. In another embodiment, the cleavable linker is derived from the tetrapeptide, H-Leu-
  • the cleavable linker is derived from the pentapeptide, H-Gly- Pro-Leu-Gly-Pro-OSu.
  • M The multifunctional chemical moiety, M, is prepared using reagents and techniques known to one of average knowledge in the art. Since M contains three active functional group, Xi, X 2 , and X 3 , one or more of these functions can be chemically protected during synthesis and then deprotected when required.
  • M contains three active functional group, Xi, X 2 , and X 3 , one or more of these functions can be chemically protected during synthesis and then deprotected when required.
  • the multifunctional chemical moieties, Q, used to prepare the branched polymeric drug conjugates of the invention are prepared using reagents and techniques known to one of average knowledge in the art. Since Q contains multiple reactive functions X 6 that are designed to react with polymer segment P alone, no protecting groups are usually required. In some cases however, when more than one type of biologically active agent, D, enzymatically cleavable linker, L, or water soluble polymer segment, P, are desired on a single invented polymeric drug conjugate, some of the reactive functional groups may be protected during certain synthetic steps and then deprotected when required. For a description of the many reactions which can be used to attach one portion of the construct to another see March, J.;
  • the biologically active agent, D is covalently coupled to the polymeric drug conjugates poly[D-L-M-P] and Q(-P-L-D) k via a enzymatically cleavable bond present in the linker, L.
  • the rate of release of D from either conjugate will depend on the mechanism of cleavage in vivo.
  • D can be cleaved from the constructs by biological or physiological processes, or by chemical reactions D will be released either directly from the polymeric drug conjugate or in the form of a complex D-L' (a compound containing D coupled to all or part of L).
  • the release of D may involve a combination of both enzymatic and non-enzymatic processes Cleavage (either by single or multiple steps) resulting in the release of the active agent
  • D may be brought about by non-enzymatic processes
  • chemical hydrolysis for example, at an ester bond
  • Hydrolytic cleavage may result in the release of the complex L- D or the free active compound D.
  • Cleavage can also be initiated by pH changes
  • the prodrug conjugate poly[D-L-M-P] may be dissolved in a minimally buffered acidic or basic pH solution before delivery, the bond between L and D or M and L of the prodrug conjugate poly[D-L-M-P] would be characterized by a high degree of chemical lability at physiological pH of 7 4, and would thus be cleaved when the conjugate is delivered to the tissue or circulatory system of the organism, releasing either the active agent D or the complex L-D If necessary, a second reaction, either chemical or enzymatic, would result in the cleavage of D from the complex L-D It is known to those in the art that N-Mannich base linkages exhibit this type of activity
  • Cleavage can also occur due to an oxidative/reductive reaction
  • a disulfide linkage can be created between L and D or M and L of the prodrug conjugate poly[D-L-M-P]
  • Such prodrug complexes would be stable at physiological pH, the bond between L and D or M and L of the prodrug conjugate poly[D-L-M-P] would be characterized by a high degree of chemical lability in reducing environments, such as in the presence of glutathione
  • a second reaction either chemical or enzymatic, would result in the cleavage of D from the complex L-D
  • Proteolytic enzymes are produced in or near diseased tissues and organs as a result of biological signals from infectious agents, blood-bome cytokines, diseased tissues itself, or fluids near diseased tissue
  • the linking group L is designed to be cleaved between L and D or M and L of the conjugate poly[D-L-M-P],
  • Such proteolytic enzymes
  • thrombin a serine protease that is activated during the clotting cascade cleaves the Arg-Gly bond in the following sequences
  • Matrix metalloproteinases and other extracellular matrix proteases are prevalent in healing and metabolism
  • this enzyme family is also implicated in various pathological processes, including chronic inflammation, arthritis, and cancer
  • MMPs are active during tumor growth and are necessary for metastasis
  • One major extracellular protein is collagen, which has a characteristic repeat amino acid sequence -Gly- Pro-Y-Gly-Pro-Z (where Y and Z are any amino acids, except for Pro or Hypro)
  • Matrix metalloproteinases and other extracellular matrix proteases cleave primarily at Leu-Gly or Ile- Gly bonds
  • Amino acid sequences which are cleaved by this family of enzymes include, but are not restricted to
  • sequences which are cleaved by this family of enzymes include, but are not restricted to:
  • angiotensin converting enzyme Another example of an enzyme that is up-regulated due to disease, and therefore can be exploited in the current invention, is the angiotensin converting enzyme. This enzyme cleaves at amino acid sequences which include, but not restricted to:
  • Cells at the site of diseased tissue will produce numerous enzymes, growth factors, and cytokines that are present elsewhere in the organism at much lower concentrations.
  • cells that are involved with inflammation that produce secreted and cell-surface enzymes include: granulocytes (neutrophils, eosinophils, basophils), monocytes/ macrophages, and lymphocytes
  • Activated macrophages are known to secrete elastase, collagenase and other MMPs, plasminogen activator, and other proteolytic enzymes
  • Activated peritoneal macrophages are known to produce hydrogen peroxide, which can be used to cleave D from the invented prodrug conjugates Eosinophils, activated at the site of inflammation, produce lysosomal enzymes, peroxidase, histaminase, and other enzymes
  • various cancer cells (example, from prostate tumors) produce secreted or cell-surface enzymes that
  • the present example (see Synthetic Pathway 1) describes the preparation of a regular repeating linear polymeric drug conjugate of the invention in which the biologically active agent, D, is derived from Fmoc-doxorubicin-14-O-hemiglutarate, the enzymatically cleaved region of the linker, (L ⁇ -L n ), is the tetrapeptide, Val-Gly-Pro-Ala, the multifunctional chemical moiety, M, is compound 7, and the water soluble polymer segment, P, is poly(ethylene glycol) with an average M W of about 2000 (PEG-2000)
  • the resultant mixture is filtered, the solvents are removed by rotary evaporation, and lOOmL of a solution of acetic acid:tetrahydro-furan:water/4:2:l is added.
  • the reaction mixture is heated to 45 ° C for 3.5h, the solvents are removed by rotary evaporation, and the distillation residue is crystallized using an appropriate solvent(s) to afford purified product amide, 7.
  • a 500mL round bottom flask equipped with a thermometer, addition funnel, magnetic stir bar, and dry N 2 inlet-outlet is charged with 0.86g g (2.2mmol) of a 60% dispersion of sodium hydride in mineral oil.
  • the dispersion is washed three times with 25mL of hexane under a blanket of dry nitrogen and then 250mL of dry tetrahydrofuran (dried by refluxing over sodium benzophenone ketal) is added to the reaction flask.
  • HC1 is slowly added to acidify the reaction mixture.
  • the solvents are removed by rotary evaporation and the distillation residue is dissolved in 30mL of water.
  • the aqueous solution is dialyzed against distilled water (Spectrapor membrane with molecular weight cutoff of 12,000-14,000) and the resultant solution is lyophilized to give polymer conjugate, 9 Average molecular weight of the product is determined by GPC relative to PEG standards.
  • a 200mL hydrogenation flask is charged with l.Og (0.5mmol) of conjugate 9, lOOmL of acetic acid, and O. lg of 5% palladium-carbon catalyst.
  • the reaction mixture is shaken and hydrogenated at 40psi of hydrogen for 16h at room temperature.
  • the reaction solution is filtered through a pad of Celite and the solvents are removed by rotary evaporation to leave a viscous oil.
  • the distillation residue is dissolved in 30mL of water and the aqueous solution is dialyzed against distilled water (Spectrapor membrane with molecular weight cutoff of 12,000-14,000). The resultant aqueous solution is lyophilized to give polymer conjugate, 10.
  • a 500mL round bottom flask equipped with a magnetic stir bar, thermometer, Drierite filled drying tube and addition funnel is charged with l.Og (0.5mmol) of polymer conjugate, 10, 200mL of dichloromethane, 0.23g (0.5mmol) of Boc-Leu-Gly-Pro-Ala-OH, ll,(Bachem)and 0.1 lg (5mmol)of 1,3-dicyclohexylcarbodiimide (Aldrich).
  • the reaction mixture is allowed to stir magnetically for 4h at room temperature, 50mL of water is then added, the organic layer is separated from the aqueous layer, and the aqueous layer is extracted with 3xl00mL portions of dichloromethane.
  • the present example describes the preparation (see Synthetic Pathway 2) of a regular repeating linear polymeric drug conjugate in which the pharmaceutical agent, D, is derived from Integrilin (N 6 -(aminoiminomethyl)-N 2 -(3 -mercapto- 1 -oxopropyl-L-lysylglycyl-L- aspartyl-L-tryptophyl-L-prolyl-L-cysteinamide, cyclic (l->6)-disulfide), the enzymatically cleaved region of the linker, (L ⁇ -L strict), is the tripeptide, Val-Pro-Arg, the multifunctional chemical moiety, M, is compound 17, the water soluble polymer segment, P, is poly(ethylene glycol) with an average M.W. of about 1000 (PEG-1000).
  • Integrilin N 6 -(aminoiminomethyl)-N 2 -(3 -mercapto- 1 -oxopropyl-L-lysyl
  • a 500mL round bottom flask equipped with a magnetic stir bar, thermometer, Drierite filled drying tube, and addition funnel is charged with 7.30g (50mmol) of tris(2- aminoethyl)amine, 15 (Aldrich) and 200mL of 1,4-dioxane.
  • the reaction mixture is cooled to 5°C and then 2.60g (lOmmol) of 9-fluorenylmethyl chlorocarbonate, FMOC-C1 (Aldrich), is slowly added.
  • the reaction mixture is stirred for 12h at room temperature and then poured onto ice-water.
  • the pH of the reaction mixture is adjusted to 8 using aqueous sodium carbonate solution and the aqueous solution is extracted with 3xl00mL of dichloromethane.
  • a clean dry 500mL round bottom flask equipped with a magnetic stir bar, thermometer, Drierite filled drying tube, and addition funnel is charged with 0.37g (lmmol) of N-fluorenylmethoxy-carbonyl-tris(2-aminoethyl)amine, 17, 5.0mL of 0.1M borate buffer at pH 9.3, and 1.14g (lmmol) of the bis-succinimidylcarbonate of poly(ethylene glycol) avg. M.W.
  • the reaction mixture is cooled to 10°C and 1.44g of di-tert-butyl dicarbonate (6.6 mmol; Aldrich) is added.
  • the resultant solution is stirred at room temperature for lh and the solvents are removed by rotary evaporation.
  • the distillation residue is cooled in an ice-water bath and stirred in 50mL of ethyl acetate.
  • a dilute solution of KHSO is added slowly until a pH of 3 is attained and then the aqueous layer is extracted with 3xl5mL of ethyl acetate.
  • a 500mL round bottom flask equipped with a magnetic stir bar and Drierite filled drying tube is charged with 200mL of dichloromethane, 1.12g (1 mmol) of Integrilin analog, 19, 0.58g (1 mmol; Bachem) of H-Val-Pro-Arg(Boc)2-OMe, 20, and 0.21g (lmmol) of 1,3- dicyclohexylcarbodiimide (Aldrich).
  • the resultant solution is magnetically stirred at room temperature for 5h and then 50mL of water is added.
  • the reaction mixture is filtered, the aqueous layer is separated from the organic layer, and the organic layer is dried over anhydrous sodium sulfate for 4h.
  • the resultant slurry is filtered and the solvents removed by rotary evaporation to leave a syrup.
  • the syrup is stirred with 1.1 mL of IN lithium hydroxide in 3mL of methanol at 5°C for 15h and then crystallized from an appropriate solvent to give peptide conjugate, 21.
  • a 500mL round bottom flask equipped with a magnetic stir bar, thermometer, Drierite filled drying tube and addition funnel is charged with 0.5g (0.5mmol) of polymer conjugate, 18, 200mL of dichloromethane, 0.84g (0.5mmol) of 21, and O. lOg (0.3mmol) of 1,3- dicyclohexylcarbodiimide (Aldrich).
  • the reaction mixture is allowed to stir magnetically for 4h at room temperature, 50mL of water is added, the organic layer is separated from the aqueous layer, and the aqueous layer is extracted with 3xl00mL portions of dichloromethane.
  • the organic layers are combined, washed with lxlOOmL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate for 4h, filtered, and the solvents are removed by rotary evaporation. 25mL of 3N HCl and 75mL of ethyl acetate are added to the distillation residue and the resulting slurry is stirred at room temperature for 30min. The solvents are removed by rotary evaporation, the residue is dissolved in 25mL of water, and the aqueous solution is dialyzed (Spectrapor membrane with molecular weight cutoff of 12,000-14,000) against distilled water. The solution is lyophilized to give the invented drug linked polymer construct, 22. The average molecular weight of 22 is determined by GPC analysis, the presence of the carbamate linkages is confirmed by IR spectroscopy, and the degree of substitution of 21 onto the product construct is calculated using UV and NMR spectroscopy.
  • the present example describes the preparation (see Synthetic Pathway 3) of a regular repeating linear polymeric drug conjugate of the invention in which the biologically active agent, D, is derived from H-Leu-Gly- ⁇ (5-fluorouracil)-OH, the enzymatically cleaved region of the linker, (L ⁇ -L n ), is the heptapeptide, Gly-Pro-Leu-Gly-Pro-Leu-Gly, the multifunctional chemical moiety, M, is compound 29, and the water soluble polymer segment, P, is poly(ethylene glycol) with an average M.W. of about2000 (PEG-2000).
  • a clean dry 250mL round bottom flask equipped with a magnetic stir bar, thermometer, and Drierite filled drying tube is charged with 1.8 lg (lOmmol) of p- nitrophenylacetic acid (Aldrich), 23, 200mL of 1,2-dimethoxyethane, 1.15g (lOmmol) of N- hydroxysuccinimide (Aldrich), and 2.06g (lOmmol) of 1,3-dicyclohexylcarbodiimide (Aldrich).
  • the reaction mixture is stirred magnetically at room temperature for 12h, filtered, and the solvent is removed by rotary evaporation.
  • the distillation residue is crystallized using an appropriate solvent(s) to give p-nitrophenylacetic acid, N-hydroxysuccinimidyl ester, 25.
  • a clean dry 250mL round bottom flask equipped with a magnetic stir bar, thermometer, and Drierite filled drying tube is charged with 2.78g (lOmmol) of p- nitrophenylacetic acid, N-hydroxysuccinimidyl ester, 25, lOOmL of dry 1,2-dimethoxyethane, 1.40mL (lOmmol) of triethylamine (Aldrich), and 2.10g (lOmmol) of p-nitrophenylalanine (Schweizerhall, South Plainfield, NJ), 24.
  • the reaction mixture is stirred for 14 hour at room temperature and then poured onto a mixture lOOmL of ice-water in lOOmL of dichloromethane.
  • the reaction mixture is filtered, the organic layer is separated from the aqueous layer, and the aqueous layer is extracted with 2xl00mL of dichloromethane.
  • the organic layers are combined, washed with lx200mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate for 4h, filtered, and the solvents are removed by rotary evaporation.
  • the distillation residue is crystallized using an appropriate solvent(s) to give amide, 26.
  • the reaction mixture is stirred for 14 hour at room temperature, filtered, and the solvents are removed by rotary evaporation.
  • the distillation residue is dissolved in a minimal amount of a mixture of acetonitrile in water and then purified using C-18 reverse phase HPLC (eluent: acetonitrile:water:0.1% trifluoroacetic acid).
  • the collected eluent fractions are combined, and the solvents removed by rotary evaporation to give an oil that is dissolved in a minimal amount of distilled water.
  • the aqueous solution is lyophilized to give the amine product, 35.
  • the distillation residue is mixed with 25mL of water, filtered and the filtrate is dialyzed (Spectrapor membrane with molecular weight cutoff of 12,000-14,000) against distilled water.
  • the aqueous solution is lyophilized to afford the polymer conjugate product, 31. GPC analysis against PEG standards is used to determine the molecular weight of the conjugate.
  • a clean dry 250mL round bottom flask equipped with a magnetic stir bar, thermometer, and Drierite filled drying tube is charged with 3.44g (lOmmol) of disuccinimidyl tartarate, 36 (Pierce), lOOmL of dry 1,2-dimethoxyethane, 1.40mL (lOmmol) of triethylamine (Aldrich), and 7.37g (lOmmol) of 35
  • the reaction mixture is stirred for 14 hour at room temperature, and then poured onto lOOmL of ice-water in lOOmL of dichloromethane.
  • the reaction mixture is filtered, the organic layer is separated from the aqueous layer, and the aqueous layer is extracted with 2xl00mL of dichloromethane.
  • the organic layers are combined, washed with lx200mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate for 4h, filtered, and the solvents are removed by rotary evaporation.
  • the distillation residue is crystallized using an appropriate solvent(s) to afford peptide adduct, 37.
  • a clean 250mL round bottom flask equipped with a thermometer and magnetic stirrer is charged with 1.59g (2mmol) of peptide adduct, 37, and lOOmL of 0.015M aqueous sodium periodate.
  • the pH of the reaction mixture is adjusted to 7.0 using sodium phosphate buffer and the reaction solution is allowed to stir for 2h at room temperature.
  • the solvents are removed by rotary evaporation and the distillation residue is dissolved in a minimal amount of a 1 : 1 mixture of water and acetonitrile.
  • the resultant solution is injected onto a semi- preparative C-18 HPLC column and the product is eluted using a water: acetonitrile: 0.1%TFA gradient.
  • the collected fractions are combined and lyophilized to give peptidoaldehyde, 38.
  • a clean 250mL round bottom flask is charged with l.Og of polymer conjugate, 31, 25mL of water and 5mL of trifluoroacetic acid.
  • the reaction mixture is shaken and the solvents are removed by rotary evaporation.
  • the distillation residue is dissolved in a minimal amount of water, filtered, and dialyzed (Spectrapor membrane with molecular weight cutoff of 12,000-14,000) against distilled water.
  • the aqueous solution is lyophilized to afford acid hydrazide substituted polymer conjugate, 32.
  • a clean 250mL round bottom flask equipped with a thermometer and magnetic stir bar is charged with 50mL of phosphate buffered saline solution at pH 5.0, l.Og (0.5mmol) of acid hydrazide substituted polymer conjugate, 32, and 0.40g (0.5mmol) of peptidoaldehyde, 38.
  • the reaction mixture is stirred for 20h at room temperature and then dialyzed against distilled water.
  • the aqueous solution is lyophilized to give the invented drug linked polymer conjugate, 39.
  • the average molecular weight of 39 is determined by GPC analysis, the presence of the carbamate linkages is confirmed by IR spectroscopy, and the degree of substitution of 38 onto the product construct is calculated using UV and NMR spectroscopy.
  • the present example describes the preparation (see Synthetic Pathway 4) of a regular repeating linear polymeric drug conjugate of the invention in which the pharmaceutical agent, D, is derived from H-Leu-Gly- ⁇ (5-fluorouracil)-OH; the enzymatically cleaved region of the linker, (L ⁇ -Lfinity), is hexapeptide, Cys-Glu-Glu-Glu-Leu-Gly; the multifunctional chemical moiety, M, is compound 51: and the water soluble polymer segment, P, is poly(ethylene glycol) (PEG-1000) with an average M.W. of about 1000.
  • reaction mixture is stirred for one hour at room temperature and then poured into a 400mL of ice-cold water. A sufficient amount of 0.1N HCl is slowly added to bring the pH of the solution to pH 5, the precipitates are filtered, dried, and dissolved in a minimal amount of a mixture of acetonitrile and water.
  • the resultant solution is chromatographed using C-18 reverse phase HPLC (eluent: acetonitrile:water:0.1% trifluoroacetic acid), the collected eluent fractions are combined, and the solvents removed by rotary evaporation to give Fmoc-L-Cys(DNP)-L-Glu(OtBu)-L-Glu(OtBu)-L-Glu(OtBu)-L- Leu-2-(5-fluorouracil-l-yl)-L,D-gly-OH, 41.
  • the organic layer is separated from the aqueous layer, the organic layer is dried over anhydrous sodium sulfate for 4h, filtered and the solvents are removed by rotary evaporation to afford 3,5-(2- ditetrahydrofi ⁇ ranyloxyxy)-phenylacetic acid .
  • a clean, dry, 250mL round bottom flask equipped with a magnetic stir bar, thermometer, and Drierite filled drying tube is charged with 3.08g (lOmmol) of 3,5-(2- ditetrahydrofuranyloxy)-phenylacetic acid, 44, 200mL of 1,2-dimethoxyethane, 1.15g (lOmmol) of N-hydroxysuccinimide (Aldrich), and 2.06g of 1,3-dicyclohexylcarbodiimide (Aldrich).
  • the reaction mixture is stirred magnetically at room temperature for 12h, filtered, and the solvents are removed by rotary evaporation.
  • the distillation residue is crystallized using an appropriate solvent(s) to give 3,5-(2-ditetrahydrofuranyloxy)-phenylacetic acid, N- hydroxy succinimidyl ester, 45.
  • a clean dry 250mL round bottom flask equipped with a magnetic stir bar, thermometer, and Drierite filled drying tube is charged with 4.05g (lOmmol) of 3,5-(2- ditetrahydrofuranyloxy)-phenylacetic acid, N-hydroxy succinimidyl ester, 45, lOOmL of dry 1,2-dimethoxyethane, 1.40mL (lOmmol) of triethylamine (Aldrich), and 2.82g (lOmmol) of N- fluorenylmethoxycarbonylethanediamine trifluoroacetate, 46 (prepared according to the procedure of Adamczyk, et al., Organic Preparations and Procedures International, 1995, 27, 239).
  • the reaction mixture is stirred for 14 hours at room temperature and then 50mL of water and lOOmL of dichloromethane is added.
  • the organic layer is separated from the aqueous solution and the aqueous layer is washed with 2xl00mL of dichloromethane.
  • the organic layers are combined, dried over anhydrous sodium sulfate for 4h, filtered, and the solvents are removed by rotary evaporation.
  • the distillation residue is crystallized using an appropriate solvent(s) to give 3,5-(2-ditetrahydrofuranyloxy)-phenylacetic acid, N-(N'- fluorenylmethoxy-carbonyl) ethanediamine amide, 47.
  • a clean dry, 250mL round bottom flask equipped with a magnetic stir bar, thermometer, and Drierite filled drying tube is charged with 5.72g (lOmmol) of 3,5-(2- ditetrahydro-fi ⁇ ranyloxy)-phenylacetic acid, N-(N'-fluorenylmethoxycarbonyl) ethanediamine amide, 47, lOOmL ofN,N-dimethylformamide, and l lmL of piperidine (Aldrich).
  • the reaction mixture is stirred magnetically at room temperature for 1.5h and the solvents are removed by rotary evaporation.
  • a clean dry 250mL round bottom flask equipped with a magnetic stir bar, thermometer, and Drierite filled drying tube is charged with 3.50g (lOmmol) of 3,5-(2- ditetrahydrofuranyloxy)-phenylacetic acid, N-ethanediamine amide, 48, lOOmL of dry 1,2- dimethoxyethane, 1.40mL (lOmmol) of triethylamine, and 3.12g (lOmmol) of N-succinimidyl- 3-(2-pyridyldithio)-propionate, 49 (Pierce; Rockford, IL).
  • the reaction mixture is stirred for
  • a 500mL round bottom flask equipped with a thermometer, addition funnel, condenser, magnetic stir bar, and dry N 2 inlet-outlet is charged with lOOmL of acetonitrile, 0 28g (2mmol) of finely powdered potassium carbonate, 0 25g of 18-crown-6 (Aldrich), lOmL of water, and 1 49g (lmmol) of dihydroxyphenyl-disulfide adduct, 51
  • the reaction mixture is heated to reflux and 2 Og of DTS-PEG1000, 52, (see synthesis of ditosylate, 8, above) is then added.
  • the reaction mixture is heated to reflux, allowed to stir for 18h at room temperature, and then 0 IN HCl is slowly added to acidify the reaction mixture
  • the solvents are removed by rotary evaporation and 5mL of piperidine dissolved in 25mL of N,N-dimethylformamide (DMF) is added
  • the reaction mixture is stirred at room temperature for lh and the solvents are removed by rotary evaporation
  • the distillation residue is dissolved in 5mL of trifluoroacetic acid (TFA) and 30mL of water and then the solvents are removed by rotary evaporation
  • TFA trifluoroacetic acid
  • the distillation residue is dissolved in 25mL of water and dialyzed against distilled water (Spectrapor membrane with molecular weight cutoff of 12,000-14,000)
  • the resultant solution is lyophilized to give invented drug polymer conjugate, 53
  • the average molecular weight of 53 is determined by GPC analysis and the degree of substitution of 51 into the product construct is
  • the present example describes the preparation (see Synthetic Pathway 5) of a branched polymeric drug conjugate of the invention in which the pharmaceutical agent, D, is derived from methotrexate; the enzymatically cleavable region of the linker, (L L View), is hexapeptide, Gly-Pro-Leu-Gly-Pro-Lys-NH 2 , the common multifunctional chemical moiety, Q, is pentaerythritol, and the water soluble polymer segment, P, is poly(ethylene glycol) with an average M.W. of about 4000.
  • a clean dry 250mL round bottom flask equipped with a magnetic stir bar, thermometer, and Drierite filled drying tube is charged with lOmmol of 4 arm branched PEG- propionic acid system, 54 (Shearwater Polymers, Huntsville, AL), lOOmL of dry N,N- dimethylformamide, 7.0 mL (50mmol) of triethylamine (Aldrich), and 40mmol of 55 (Anaspec,
  • the reaction mixture is stirred for ten hours at room temperature and then poured into a 400mL of ice-cold water. A sufficient amount of 0.1N HCl is slowly added to bring the pH to 4 and the solvents are removed under reduced pressure. The resultant precipitates are crystallized from an appropriate solvent to give invented drug polymer construct, 56.
  • the average molecular weight of 56 is determined by GPC analysis and the degree of substitution of 55 onto the product construct is calculated using UV and NMR spectroscopy.
  • the present example (see Synthetic Pathway 6 below) describes the preparation of a regular repeating linear polymeric drug conjugate of the invention in which the biologically active agent, D, is methotrexate, the enzymatically cleaved region of the linker, (L ⁇ -Lfinity), is the heptapeptide, Gly-Pro-Tyr-Ala-Tyr-Trp-Lys, the multifunctional chemical moiety, M, is lysine, and the water soluble polymer segment, P, is poly(ethylene glycol) with an average MW of about 2000 (PEG-2000).
  • a clean dry 250mL round bottom flask equipped with a magnetic stir bar, heating mantle, Dean-Stark water removal trap, condenser, and Drierite filled drying tube ischarged with 120mL of toluene and lO.Og (5mmol) of PEG-2000 (Aldrich, Milwaukee, WI).
  • the resultant solution is refluxed for 2 hours, during which time approximately lOO ⁇ L of water is collected in the Dean-Stark trap, and the heating mantle is then removed.
  • the reaction mixture was allowed to cool to ambient temperature and 6.0g (33.7mmol) of 1,1'- thiocarbonyldiimidazole, tech., 90% (Aldrich) is added in one portion.
  • the reaction mixture is stirred at RT for 70h and approximately 3 ⁇ of the solvent is then removed by rotary evaporation to give an orange oil.
  • the oil is slowly added to 300mL of rapidly stirring ethyl ether to give a two phase mixture.
  • the ethereal (upper) layer is decanted and the viscous oil in the lower layer is slowly added to 300mL of fresh ethyl ether.
  • the ethereal layer is again decanted and the oily lower layer is dissolved in 70mL of ethyl acetate.
  • the ethyl acetate solution is slowly dropped into 300mL of rapidly stirring cold ethyl ether to give off-white precipitates.
  • the distillation residue is dissolved in 3mL of deionized water and dialyzed (Spectrapor membrane with molecular weight cutoff of 3,500) against distilled water.
  • the aqueous solution remaining in the dialysis bag is lyophilized to afford 64mg of linear copolymer backbone, 59, as a white solid.
  • GPC Polymer Lab PL-GEL 10 3 and 10 5 column, 0.1% LiBr in DMF eluent shows the product copolymer to have a M consult of 19,022 with a polydispersity of 1.42.
  • a 50mL round bottom flask equipped with a thermometer, magnetic stir bar and Drierite filled drying tube is charged with 20mL of acetonitrile, 4.74g (2.0mmol) of linear copolymer backbone, 59, l.OmL of pyridine, and 1.54g ( ⁇ .Ommol) of N, N'-disuccinimidyl carbonate, tech. (Aldrich).
  • the resultant solution is magnetically stirred at room temperature for 17h and then added dropwise to 300mL of rapidly stirring ethyl ether.
  • the ethereal mixture is stirred for 0.5h, filtered, and the collected precipitates are washed with 2x300mL of fresh ether.
  • the isolated solids are dried under vacuum to give activated copolymer backbone,
  • a 5mL reaction vial equipped with a magnetic spin vane is charged with 500 ⁇ L of N,N-dimethylformamide, 1 17mg (50 ⁇ mol) of activated copolymer backbone, 61, 87 ⁇ L
  • the ethereal supernatant is decanted from the yellow precipitates and the remaining solids are washed with 7mL of fresh ethyl ether
  • the collected solids are dissolved in 7.5mL of deionized water, the pH is adjusted to 2.5 by adding concentrated hydrochloric acid, and the resultant solution is dialyzed (Spectrapor membrane with molecular weight cutoff of 3,500) against distilled water.
  • the aqueous solution is lyophilized to give the drug linked polymer constuct, 63.
  • the average molecular weight of 63 is determined by GPC analysis, the presence of the thiocarbamate linkages is confirmed by IR spectroscopy, and the degree of substitution of 62 onto the product construct is calculated using UV and NMR spectroscopy.
  • the present example describes the preparation (see Synthetic Pathway 7) of a regular repeating linear polymeric drug conjugate in which the pharmaceutical agent, D, is methotrexate, the enzymatically cleaved region of the linker, (L ⁇ -L n ), is the peptide, Gly-Pro- Lys-Pro-Val-Gly-Nva-T ⁇ -Lys, the multifunctional chemical moiety, M, is lysine, and the water soluble polymer segment, P, is ⁇ oly( ethylene glycol) with an average MW of about 2000 (PEG-2000).
  • a 5mL reaction vial equipped with a magnetic spin vane is charged with lmL of N,N- dimethylformamide, 74.8mg (32 ⁇ mol) of activated copolymer backbone, 64 (prepared by the method of Kohn, et al, Macromolecules, 1992, 25, 4476), 55 ⁇ L (320 ⁇ mol) of N,N- diisopropylethylamine (Chemlmpex, Wood Dale, IL), and 50mg (32 ⁇ mol) of peptide conjugate, 65 (AnaSpec, Inc., San Jose, CA). The reaction mixture is stirred for 60h at room temperature and then 5mL of water is added.
  • aqueous solution is stirred for 24h at room temperature, 200 ⁇ L of hydrazine (Aldrich) is added, and the resultant aqueous solution is dialyzed (Spectrapor membrane with molecular weight cutoff of 3,500) against distilled water.
  • the homogeneous solution in the dialysis membrane is lyophilized to give the drug linked polymer constuct, 66.
  • the average molecular weight of 66 is determined by GPC analysis, the presence of the carbamate linkages is confirmed by IR spectroscopy, and the degree of substitution of 65 onto the product is calculated using UV and NMR spectroscopy.
  • the present example describes the preparation (see Synthetic Pathway 8 below) of a regular repeating linear polymeric drug conjugate of the invention in which the biologically active agent, D, is methotrexate, the enzymatically cleaved region of the linker, (L ⁇ -Lfinity), is the heptapeptide, Gly-Pro-Tyr-Ala-Tyr-Trp-Lys, the multifunctional chemical moiety, M, is lysine, and the water soluble polymer segment, P, is poly(ethylene glycol) with an average MW of about 2000 (PEG-2000).
  • a clean dry 250mL round bottom flask equipped with a condenser, magnetic stir bar, addition funnel, drying tube, heating mantle, Dean-Stark water removal trap, and HCl trap is charged with lO.Og (5mmol) of PEG-2000 (Aldrich) and 120mL of toluene.
  • the polymer solution is azeotropically dried for 2h under reflux, cooled to room temperature, and then 2.2mL (29mmol) of thiophosgene (Aldrich) is added in one portion.
  • the reaction mixture is allowed to stir for 18h at room temperature and then the solvents are removed at reduced pressure.
  • the pH is lowered to 2 by the careful addition of concentrated hydrochloric acid and the organic layer is separated from the aqueous layer.
  • the organic layer is washed with 2xl00mL of saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, filtered, and the solvents are removed by rotary evaporation.
  • the crude polymer is dissolved in 50mL of water and dialyzed against distilled water using a SPECTRAPOR membrane with a molecular weight cut-off of 12,000-14,000 daltons.
  • the aqueous solution remaining in the dialysis membrane is lyophilized to give linear repeating copolymer, 59, as a white solid.
  • GPC is used to determine the average molecular weight of the product
  • IR spectroscopy confirms the presence of thiocarbamate linkages
  • 1H NMR spectroscopy is used to confirm the presence of lysine protons.
  • the present example describes the preparation (see Synthetic Pathway 9) of a regular repeating linear polymeric drug conjugate of the invention in which the pharmaceutical agent, D, is methotrexate, the enzymatically cleaved region of the linker, (L L Starting), is hexapeptide, Gly-Pro-Leu-Gly-Pro-Lys, the multifunctional chemical moiety, M, is lysine, and the water soluble polymer segment, P, is poly(ethylene glycol) (PEG-2000) with an average MW of about 2000.
  • a clean dry 50mL reaction vial equipped with a magnetic stir bar and drying tube is charged with 25mL of N,N-d.methylformamide, l .OOg (lmmol) of methotrexate-peptide conjugate, 68, (AnaSpec, Inc.), 1.74mL (lOmmol) of N,N-diisopropylethylamine (Aldrich), and 0.44g (lmmol) of N ⁇ ,N ⁇ -di-t-Boc-L-lysine, N-hydroxysuccinimide ester (Sigma, St.
  • the reaction mixture is allowed to stir at room temperature for 18h and then the solvents are removed by rotary evaporation.
  • the distillation residue is dissolved in water and the desired product is isolated by reverse phase C-18 chromatography using acetonitrile: water (0.1%TFA) elution.
  • the product containing eluent fractions are combined and lyophilized to give methotrexate-peptide conjugate, 70, as a yellow solid.
  • the identity of the product is determined by 1H NMR and mass spectrometry.
  • a clean dry 50mL reaction vial equipped with a magnetic stir bar and drying tube is charged with 25mL of N,N-dimethylformamide, 1.13g (lmmol) of methotrexate-peptide conjugate, 70, 1.74mL (lOmmol) of N,N-diisopropylethylamine (Aldrich), and 2.22g (lmmol) of the bis(imidazolyl) thiocarbamate of poly(ethylene glycol), 58.
  • the reaction mixture is allowed to stir at room temperature for 18h and then the solvents are removed by rotary evaporation.
  • the distillation residue is dissolved in water and the resultant solution is dialyzed (Spectrapor membrane with molecular weight cutoff of 10,000) against distilled water to give the invented drug linked polymer construct 71 as a yellow solid.
  • the average molecular weight of the construct is determined by GPC, the presence of the thiocarbamate linkages is confirmed by ER spectroscopy, and the degree of substitution of 70 into the product construct is calculated using UV and NMR spectroscopy.
  • the present example describes the preparation (see Synthetic Pathway 10) of a regular repeating linear polymeric drug conjugate of the invention in which the pharmaceutical agent, D, is methotrexate, the enzymatically cleaved region of the linker, (L ⁇ -L n ), is hexapeptide, Gly-Pro-Leu-Gly-Pro-Lys, the multifunctional chemical moiety, M, is lysine, and the water soluble polymer segment, P, is poly( ethylene glycol) (PEG-2000) with an average MW of about 2000.
  • the reaction mixture is allowed to stir at room temperature for 18h and then the solvents are removed by rotary evaporation.
  • the distillation residue is dissolved in water, the pH is adjusted to 2.5 using cone. HCl, and the resultant solution is dialyzed (Spectrapor membrane with molecular weight cutoff of 10,000) against distilled water to give the invented drug linked polymer construct 72 as a yellow solid.
  • the average molecular weight of the construct is determined by GPC, the presence of the thiocarbamate linkages is confirmed by IR spectroscopy, and the degree of substitution of 70 into the product is calculated using UV and NMR spectroscopy.
  • the present example describes the preparation (see Synthetic Pathway 11) of a regular repeating linear polymeric drug conjugate of the invention in which the pharmaceutical agent, D, is methotrexate, the enzymatically cleaved region of the linker, (L ⁇ -Lfinity), is heptapeptide, Gly-Pro-Tyr-Ala-Tyr-T ⁇ -Lys, the multifunctional chemical moiety, M, is lysine, and the water soluble polymer segment, P, is poly(ethylene glycol) (PEG-2000) with an average MW of about 2000.
  • a 5mL reaction vial equipped with a magnetic spin vane is charged with 0.5mL of N,N-dimethylformamide, 117mg (50 ⁇ mol) of activated copolymer backbone, 64, (prepared by the method of Kohn, et al, Macromolecules, 1992, 25, 4476), 87 ⁇ L (lOequiv.) of N,N- diisopropylethylamine (Chemlmpex), and 50mg (50 ⁇ mol) of peptide conjugate, 62 (AnaSpec).
  • the reaction mixture is stirred for 24h at room temperature and added dropwise to 7mL of ethyl ether to give yellow precipitates.
  • the liquid is decanted from the precipitates and the solids are washed with 7mL of fresh ethyl ether.
  • the supernatant is again decanted, the solid residue is dried at reduced pressure, and 7.5mL of deionized water is added.
  • the resultant aqueous solution is dialyzed (Spectrapor membrane with molecular weight cutoff at 3,500 daltons) against deionized water and lyophilized to give 95mg (28.5mmol, 57%yield) of invented drug linked construct, 73, as a yellow solid.
  • GPC Polymer Lab PL-GEL 10,000 and 100,000 column with DMF, 0.1% LiBr as eluent
  • GPC shows the average molecular weight of the polymer-drug construct to be 73,072 with a polydispersity of 4.17.
  • the extent of inco ⁇ oration of 62 onto the PEG-lysine backbone is determined to be 39% by UV spectroscopy.
  • MMP3 enzyme (Chemicon International, Temecula, CA) results in the formation of methotrexate-peptide fragment 74.
  • the rate of cleavage of 74 from 73 is determined by RP-18 HPLC to be 33% over 2h at 37 °C.

Abstract

La présente invention concerne un conjugué de médicament polymère contenant au moins un agent actif d'un point de vue biologique conjugué via un bras clivable par voie enzymatique soit à un motif linéaire régulier répétitif contenant un segment polymère hydrosoluble et un fragment chimique multifonctionnel, soit à un polymère ramifié comprenant au moins deux segments polymères hydrosolubles liés chacun à un fragment chimique multifonctionnel commun. L'invention concerne également des procédés d'obtention de ces conjugués, ainsi que des compositions pharmaceutiques les contenant et l'utilisation desdits conjugués dans le traitement d'états pathologiques.
EP00928630A 1999-04-28 2000-04-28 Conjugues de medicaments polymeres actives par voie enzymatique Withdrawn EP1176985A2 (fr)

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WO2000064486A2 (fr) 2000-11-02
CA2369999A1 (fr) 2000-11-02

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