WO1992000748A1 - Copolymeres d'aminoacides et de poly(oxydes d'alkylene), vehicules de medicament et copolymeres charges bases sur lesdits vehicules - Google Patents

Copolymeres d'aminoacides et de poly(oxydes d'alkylene), vehicules de medicament et copolymeres charges bases sur lesdits vehicules Download PDF

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WO1992000748A1
WO1992000748A1 PCT/US1991/004797 US9104797W WO9200748A1 WO 1992000748 A1 WO1992000748 A1 WO 1992000748A1 US 9104797 W US9104797 W US 9104797W WO 9200748 A1 WO9200748 A1 WO 9200748A1
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group
pharmaceutically active
active compound
polymer
poly
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PCT/US1991/004797
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English (en)
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Samuel Zalipsky
Durgadas Bolikal
Aruna Nathan
Joachim Benjamin Kohn
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Enzon, Inc.
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Priority to CA002086528A priority Critical patent/CA2086528A1/fr
Priority to AU82355/91A priority patent/AU8235591A/en
Priority to JP91512668A priority patent/JPH05508879A/ja
Publication of WO1992000748A1 publication Critical patent/WO1992000748A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
    • 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
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/26—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/68—Polyesters containing atoms other than carbon, hydrogen and oxygen
    • C08G63/685—Polyesters containing atoms other than carbon, hydrogen and oxygen containing nitrogen
    • C08G63/6854—Polyesters containing atoms other than carbon, hydrogen and oxygen containing nitrogen derived from polycarboxylic acids and polyhydroxy compounds

Definitions

  • the present invention relates to copolymers of poly(alkylene oxides) and amino acids or peptide sequences, and more particularly to copolymers of polyalkylene oxides such as polyethylene glycol (PEG) , with amino acids or peptide sequences.
  • the present invention also relates to conjugates of such polymers formed with pharmaceutically active compounds covalently bonded to the amino acid or peptide sequence of the copolymer.
  • the present invention further relates to ionically conductive materials, hydrogel membranes and semi-interpenetrating polymer networks prepared from the copolymers of the present invention.
  • the conjugation of PEG begins with functionalization of the terminal hydroxyl groups of the polymer prior to coupling with a ligand of biological relevance, although some ligands are capable of covalently bonding to the terminal hydroxyl groups without functionalization.
  • a ligand of biological relevance although some ligands are capable of covalently bonding to the terminal hydroxyl groups without functionalization.
  • Zalipsky et al. J. Macromol. Sci-Chem. , A21, 839-845 (1984); and Zalipsky et al., Eur. Polym. J. . . 19. 1177-1183 (1983) .
  • One of the limitations of PEG is that it has only two reactive end groups available for functionalization.
  • Block copolymers of PEG with poly(L-proline) are disclosed by Jeon et als., J. Polvm. Sci. Part A Polvm. Chem.. 27, 1721-30 (1989) .
  • Block copolymers of PEG with poly(gamma-benzyl L-gl tamate) are disclosed by Cho et al., Makromol. Chem.. 191, 981-91 (1990) . In these references, the use of the PEG block copolymers as biomaterials is suggested.
  • PEG copolymers having multiple pendant functional groups at regular predetermined intervals that can be utilized for drug attachment or cross-linking reactions would be highly desirable.
  • a polymer in which the poly(alkylene oxide) and amino acid or peptide sequence are copolymerized by way of hydrolytically stable urethane linkages.
  • the polymer contains one or more recurring structural units independently represented by Formula I:
  • R- ⁇ is a poly(alkylene oxide)
  • R 2 is an amino acid or peptide sequence containing two amino groups and at least one pendant carboxylic acid group.
  • the pendant carboxylic acid group is not involved in the polymerization process and is thus retained as a pendant group on the polymer.
  • This pendant functional group can be further derivatized (e.g., converted to a different functional group), used for crosslinking or for the attachment of ligands, e.g., drugs.
  • R 2 is represented by Formula II:
  • R 3 and R 4 are independently selected from saturated and unsaturated, straight-chained and branched alkyl groups containing up to 6 carbon atoms and alkyl phenyl groups, the alkyl portions of which are covalently bonded to an a ine and contain up to 6 carbon atoms.
  • the values for a and b are independently zero or one.
  • 5 is independently selected from -NH- or -NH-AA-, wherein -AA- is an amino acid or peptide sequence, with the proviso that -AA- has a free N-terminus.
  • D is a pendant functional group having a structure represented by O 0
  • Y is selected from -OH, -NH-NH 2 , -O-Rg-
  • Y is a derivative of a pharmaceutically active compound covalently bonded to the pendant functional group by means of X, wherein X is a linkage selected from -NH-NH- in the case when in the underivatized pharmaceutically active compound an aldehyde or ketone is present at the position linked to the pendant functional group by means of X; -NH-NH-, -NH-Rg-NH-, -0-Rg-NH-, -0-Rg-O- or -NH-Rg-0- in the case when in the underivatized pharmaceutically active compound a carboxylic acid is present at the position linked to the pendant functional group by means of X; and 0 0 il )i
  • R g is selected from alkyl groups containing from two to six carbon atoms, aromatic groups, alpha-, beta-, gamma- and omega amino acids, and peptide sequences.
  • a polymer in which a poly(alkylene oxide) having terminal hydroxyl or terminal amino groups and an amino acid or peptide -6- sequence are copolymerized by way of hydrolytically stable amide linkages in the case of the poly(alkylene oxide) having terminal amino groups, and by way of hydrolyzable ester linkages in the case of poly(alkylene oxides) having terminal hydroxyl groups.
  • the polymer contains one or more recurring structural units independently represented by
  • R-L is a poly(alkylene oxide) , L is -0- or -NH- and R 2 is an amino acid or peptide sequence containing two carboxylic acid groups and at least one pendant amino group.
  • the pendant amino group is not involved in the polymerization process and is thus retained as a pendant group on the polymer that can be further derivatized, used for crosslinking, or for the attachment of ligands.
  • R is represented by Formula IV:
  • R 3 , R 4 , a and b are the same as described above with respect to Formula II.
  • R 5 is independently
  • -AA- is an amino acid or peptide sequence, with the proviso that -AA- has a free C-terminus.
  • D is a pendant functional group representing either -NHZ or -NH-X- j ⁇ -Z.
  • D is -NHZ
  • Z is hydrogen
  • Z is a pharmaceutically active compound covalently bonded to the pendant function group by means of X* ⁇ .
  • X- ⁇ is a linkage selected
  • a polymer in which a poly(alkylene oxide) having terminal amino groups and an amino acid or peptide sequence having at least one hydroxyl group are copolymerized by way of hydrolytically stable urethane linkages.
  • the polymer contains one or more recurring structural units independently represented by Formula III, in which L is -NH- and R 2 is an amino acid or peptide sequence having at least one activated hydroxyl group, one carboxylic acid group when only one activated hydroxyl group is present, and at least one pendant amino group that can be further derivatized, used for crosslinking or for the attachment of ligands, like the pendant amino group of Formula IV.
  • R 2 is preferably represented by Formula V: 0
  • R 3 , R 4 , a, b and D are the same as described above with respect to Formula IV.
  • R 5 is selected from: 0 0 0
  • the third embodiment does not require the amino acid or peptide sequence to have either two free amino groups or two free carboxylic acid groups.
  • natural amino acids such as hydroxylysine, serine, threonine, thyroxine and tyrosine, which can be polymerized through their hydroxyl and carboxylic acid groups, with the amino group remaining free as a pendant functional group.
  • polymerization processes are provided for the preparation of the copolymers of the present invention.
  • an interfacial polymerization process is provided for the preparation of the polymers of Formula I in which the poly(alkylene oxide) and amino acid or peptide sequence are copolymerized by means of stable urethane linkages.
  • the process includes the steps of intimately admixing a solution of an activated poly(alkylene oxide) in a water-immiscible organic solvent with an amino acid or peptide sequence in an aqueous solution having a pH of at least 8.0, which amino acid or peptide sequence has protected C-terminals and at least two free amino groups; and recovering from the organic solvent the resulting copolymer of the poly(alkylene oxide) and the amino acid or peptide sequence.
  • a solution polymerization process is provided for the preparation of the polymers of Formula III in which the poly(alkylene oxide) and amino acid or peptide sequence are copolymerized by way of hydrolytically stable amide or hydrolyzable ester linkages.
  • the process includes the steps of contacting a hydroxyl-terminated or amino-terminated poly(alkylene oxide) with an amino acid or a peptide sequence in an organic solvent in the presence of coupling reagent and an acylation catalyst, which amino acid or peptide sequence has at least two free carboxylic acid groups, with the proviso that when the poly(alkylene oxide) is hydroxyl-terminated, the amino acid or peptide sequence has protected N-terminals.
  • the resulting copolymer of the poly(alkylene oxide) with the amino acid or peptide sequence is then recovered.
  • a solution polymerization process is provided for the preparation of polymers according to Formula III in which L is -NH-.
  • a poly(alkylene oxide) having terminal amino groups is copolymerized with an amino acid or peptide sequence by way of urethane linkages formed with activated hydroxyl groups.
  • the process includes the step of providing an amino acid or peptide sequence having at least one hydroxyl group and protected C-terminals and activating the hydroxyl group in an organic solvent with an activating reagent in the presence of an acylation catalyst.
  • the activated hydroxyl groups are then reacted with an amino-terminated poly(alkylene oxide) in the organic solvent and the resulting copolymer of the poly(alkylene oxide) with the amino acid or peptide sequence is then recovered. If the amino acid of peptide sequence has one hydroxyl group, the copolymer will be polymerized by way of alternating urethane and amide linkages.
  • polymerization can be performed exclusively through these groups by way of urethane linkages and the carboxylic acid groups of the amino acid or peptide sequence can also be protected and remain free as pendant f nctional groups.
  • methods are provided for preparing polymer conjugates of the copolymers of the present invention and pharmaceutically active compounds. Hydrolytically stable conjugates are utilized when the pharmaceutical compound is active in conjugated form. Hydrolyzable conjugates are utilized when the pharmaceutical compound is inactive in conjugated form. The properties of the poly(alkylene oxide) dominate the copolymer and conjugate thereof.
  • the pharmaceutically active compound can be directly conjugated to the pendant functional group of the copolymer, or it may be conjugated by means of a bifunctional linker.
  • the linker should contain a functional group capable of coralently bonding with the pendant functional group or a functionalized derivative thereof, and a functional group capable of covalently bonding with the pharmaceutically active compound or a functionalized derivative thereof.
  • the linker should also contain a spacer moiety such as an aliphatic or aromatic moiety, amino acid or peptide sequence. Examples of linkers include alkanol amines, diamines, hydrazines, and the like.
  • a linker compound When a linker compound is employed the order of reaction is not important.
  • the linker may first be attached to the pendant functional group of the copolymer and then attached to the pharmaceutically active compound.
  • the linker may first be attached to the pharmaceutically active compound and then attached to the copolymer.
  • a method is provided for preparing a polymer conjugate of a pharmaceutically active compound which compound prior to conjugation has an amino or hydroxyl group, and a copolymer of a poly(alkylene oxide) and an amino acid or peptide sequence, which amino acid or peptide sequence has, prior to conjugation, a pendant carboxylic acid group, by directly attaching the pharmaceutically active compound to the pendant functional groups of the copolymer.
  • the method includes the steps of contacting, in an organic solvent, in the presence of an coupling reagent and an acylation catalyst, the pharmaceutically active compound and the copolymer.
  • the resulting conjugate of the copolymer and the pharmaceutically active compound is then recovered.
  • a hydrolytically stable amide bond is formed when the pharmaceutically active compound has an amino group prior to conjugation, linking the pharmaceutically active compound to the copolymer.
  • a hydrolytically unstable ester bond is formed linking the pharmaceutically active compound to the copolymer.
  • the copolymer can optionally have activated pendant carboxylic acid groups.
  • a method for preparing a polymer conjugate of a pharmaceutically active compound, which compound has a carboxylic acid group prior to conjugation, and a copolymer of a poly(alkylene oxide) and an amino acid or peptide sequence, which amino acid or peptide sequence has, prior to conjugation, a pendant carboxylic acid group or active ester thereof, using an alkanol amine linker.
  • the method includes the steps of reacting, in an aqueous solution, in the presence of a water-soluble coupling reagent, the pendant carboxylic acid group of the copolymer with a alkanol amine, so that an alkanol amide of the carboxylic acid group is formed.
  • the pharmaceutically active compound and the copolymer are then contacted in a suitable solvent so that an ester linkage is formed between the alkanol amide of the copolymer and the carboxylic acid group of the pharmaceutically active compound, and the resulting conjugate of the copolymer and the pharmaceutically active compound is then recovered.
  • the order of reaction may be reversed, so that the alkanol amine is first reacted with the carboxylic acid group of the pharmaceutically active compound to form an alkanol amide . of the carboxylic acid group.
  • the pharmaceutically active compound and the copolymer are then contacted in the organic solvent so that an ester linkage is formed between the alkanol amide of the pharmaceutically active compound and the pendant carboxylic acid group of the copolymer.
  • a method for preparing a polymer conjugate of a pharmaceutically active compound, which compound has a carboxylic acid group prior to conjugation, and a copolymer of a poly(alkylene oxide) and an amino acid or peptide sequence, which amino acid or peptide sequence has, prior to conjugation, a pendant carboxylic acid group or an active ester thereof, using a diamine linker.
  • the method includes the steps of reacting, in an organic solvent, in the presence of an activating reagent and an acylation catalyst, the copolymer and a diamine, so that an amino amide of the pendant functional group is formed, and then contacting, in the organic solvent, the copolymer with the pharmaceutically active compound.
  • the resulting conjugate of the copolymer and the pharmaceutically compound is then recovered.
  • the order of reaction may be reversed so that an amino amide is first formed with the carboxylic acid group of the pharmaceutically active compound, which amino amide is then reacted with the pendant carboxylic acid group of the copolymer.
  • the pharmaceutically active compound may be reacted with an excess of copolymer, together with an additional quantity of the diamine, thereby conjugating the pharmaceutically active compound with the pendant carboxylic acid groups by way of amido amide linkages and forming available amino amide linkages with unconjugated pendant carboxylic acid groups.
  • the available amino amide linkages of the copolymer conjugate are then further reacted, in the organic solvent, in the presence of sodium borohydride or sodium cyanoborohydride, with a monoclonal antibody having oxidized carbohydrate moieties, so that the carbohydrate moieties covalently attach to the available amino amide linkages.
  • the resulting conjugate of the copolymer, the pharmaceutically active compound and the monoclonal antibody is then recovered.
  • a method for preparing a polymer conjugate of a pharmaceutically active compound, which compound has an aldehyde, ketone or carboxylic acid group prior to conjugation, and a copolymer of a poly(alkylene oxide) and an amino acid or peptide sequence, which amino acid or peptide sequence, prior to conjugation, has a pendant carboxylic acid group, using a hydrazine linker.
  • the method includes the steps of reacting, in an organic solvent, in the presence of a coupling reagent and an acylation catalyst, the copolymer with an alkyl carbazate, so that an alkyl carbazate of the pendant functional group is formed, and then converting the alkyl carbazate to an acyl hydrazine.
  • the pharmaceutically active compound is then contacted in the organic solvent with the copolymer, and the resulting conjugate of the copolymer and the pharmaceutically active compound is then recovered.
  • the pharmaceutically active compound may be reacted with an excess of copolymer, so that fee acyl hydrazine groups remain as pendant functional groups.
  • the method can then further include the step of reacting, in the organic solvent, in the presence of sodium borohydride, the pendant acyl hydrazine groups with a monoclonal antibody having oxidized carbohydrate moieties, so that the oxidized carbohydrate moieties form diacyl hydrazides with the pendant functional group.
  • the resulting conjugate of the copolymer, the pharmaceutically active compound and the monoclonal antibody is then recovered.
  • a method for preparing a polymer conjugate of a pharmaceutically active compound, which compound has a carboxylic acid group prior to conjugation, and a copolymer of a poly(alkylene oxide) and an amino acid or peptide sequence, which amino acid or peptide sequence has a pendant amino group prior to conjugation, by directly attaching the pharmaceutically active compound to the pendant functional group of the copolymer.
  • the method includes the steps of reacting, in an organic solvent, in the presence of an activating reagent and an acylation catalyst, the pharmaceutically active compound and the copolymer, and then recovering the resulting conjugate of the copolymer and the pharmaceutically active compound.
  • the pharmaceutically active compound may be reacted with an excess of the copolymer, so that pendant amino groups remain.
  • the method then further includes the step of reacting, in the organic solvent, in the presence of sodium borohydride, the remaining pendant amino groups with a monoclonal antibody having oxidized carbohydrate moieties, so that the oxidized carbohydrate moieties covalently attach to the pendant amino groups.
  • the resulting conjugate of the copolymer with the pharmaceutically active compound and the monoclonal antibody is then recovered.
  • a conductive composition is provided of an alkali metal electrolyte salt combined with a copolymer of a poly(alkylene oxide) and an amino acid or peptide sequence, which amino acid or peptide sequence has pendant carboxylic acid groups protected by C-terminus protecting groups.
  • the alkali metal electrolyte salt is a lithium salt selected from LiAsFg, LiPFg, Lil, LiBr, LiBFg, LiAlCl 4 , LiCF 3 C0 2 and LiCF 3 S0 3 .
  • the conductive composition of the present invention is utilized as a solid electrolyte in an electrochemical cell.
  • the electrochemical cell includes a cathode, an anode and the conductive material of the present invention.
  • the cathode includes a cathode- active material capable of intercalating lithium and the anode is preferably a counter-electrode capable of intercalating lithium.
  • More preferred embodiments utilize a lithiated transition metal chalcogenide as the cathode-active material and a graphitic carbon as the counter-electrode.
  • Still yet another aspect of the present invention provides hydrogel membranes and semi-interpenetrating polymer networks prepared from the polymers of the present invention.
  • the hydrogel membranes have high equilibrium water content and good mechanical strength, and, as such, are suitable for many biomedical applications such as wound dressings and implants.
  • the urethane linkages are non-degradable under physiological conditions.
  • the cross link density of the membrane can be controlled by varying the length of the poly(alkylene oxide) chain used in the cross linking reaction.
  • a second embodiment of this aspect of the present invention provides hydrogel membranes of polymer matrices formed from copolymers of poly(alkylene oxides) and amino acids or peptide sequences, which amino acids or peptide sequences have pendant acyl hydrazine groups.
  • the copolymers are cross linked by way of hydrolytically labile acyl semicarbazide linkages between a diisocyanate and the pendant acyl hydrazine groups of the polymer.
  • Hydrogel membranes of this aspect of the present invention when incorporated with water, demonstrate high water content and high mechanical strength.
  • a third embodiment of this aspect of the present invention provides semi-interpenetrating polymer networks (IPN) of a linear, preformed second polymer entrapped within the polymer matrices of the present invention.
  • the second polymer is chosen to be biocompatible and to improve a physical characteristic, such as tensile strength, of the polymer matrix. Polymers that are ordinarily immiscible may be combined to form the semi-IPN's of the present invention.
  • the semi-IPN's of the present invention can be formed from polymers that would not be physically blendable by any other means.
  • the second polymer is poly(BPA carbonate) or poly(desaminotyrosyl tyrosine hexyl ester carbonate) .
  • Still yet another aspect of the present invention provides methods by which the hydrogel membranes and semi-IPN's of the present invention may be prepared.
  • a method is provided for preparing a cross linked polymer matrix of a copolymer of a poly(alkylene oxide) and an amino acid or peptide sequence, wherein at least one terminus of the copolymer is the poly(alkylene oxide) .
  • the method includes the steps of providing a first solution of the copolymer dissolved in an organic solvent in which the polymer matrix is soluble, protecting the pendant C-terminals or N-terminals of the amino acid or peptide sequence of the copolymer and then forming in the first solution an active ester of the poly(alkylene oxide) terminus of the copolymer.
  • the first solution is then mixed with a second solution of an equivalent quantity of a trifunctional amine in a solvent in which the polymer matrix is soluble so that urethane linkages form between the active ester and the tris(amino) amine.
  • the resulting cross-linked copolymer polymer matrix is then recovered.
  • a method for preparing a semi-IPN by first dissolving a linear, pre-formed second polymer in the first solution before mixing the first solution with the second solution so that the second polymer is entrapped within the cross-linked polymer matrix, as the polymer matrix is formed.
  • a method for preparing a cross-linked polymer matrix of a copolymer of a poly(alkylene oxide) and an amino acid or peptide sequence, which amino acid or peptide sequence has a pendant acyl hydrazine group.
  • the method includes the steps of providing a solution of the copolymer in an organic solvent in which the polymer matrix is soluble and adding an equivalent quantity of diisocyanate to the solution so that acyl semicarbazide linkages form between the pendant acyl hydrazines and the diisocyanate.
  • the resulting cross-linked copolymer polymer matrix is then recovered.
  • a method for preparing a semi-IPN by dissolving a linear, pre-formed second polymer in the first solution before mixing the first solution with the second solution so that the second polymer is trapped within the cross-linked copolymer matrix as the polymer matrix is formed.
  • the present invention provides a versatile family of poly(alkylene oxide) copolymers having multiple pendant functional groups at regular predetermined intervals. By being capable of forming linkages through the pendant functional groups, which linkages have varying degrees of hydrolytic stability or instability, the copolymers are useful for a variety of biomedical end-use applications.
  • FIG. 1 depicts the weight loss with time of a hydrogel membrane of the present invention in phosphate buffer (pH 7.4) at 60°C.
  • FIG. 2 depicts an Arrhenius plot of conductivity versus temperature for an ionically conductive material of the present invention.
  • the polymers of the present invention are copolymers of poly(alkylene oxides) and amino acids or peptide sequences.
  • the polymers thus include one or more recurring structural units in which the poly(alkylene oxide) and the amino acid or peptide sequence are copolymerized by means of urethane linkages, which structural units are independently represented by Formula I disclosed above.
  • R- ⁇ is a poly(alkylene oxide) and R 2 is an amino acid or peptide sequence containing two amino groups and at least one pendant carboxylic acid group.
  • poly(alkylene oxides) suitable for use in the polymers of the present invention include polyethylene glycol (PEG), polypropylene glycol, poly(isopropylene glycol), polybutylene glycol, poly(isobutylene glycol) and copolymers thereof.
  • Preferred poly(alkylene oxides) for use with the present invention have the structure:
  • R 8 , R 9 and R 10 are independently selected from straight-chained and branched alkyl groups containing up to 4 carbon atoms, c is an integer between about 1 and about 100, inclusive, and d and e are independently integers between 0 and about 100, inclusive, with the proviso that the sum of c, d and e is between about 10 and about 100, inclusive.
  • the most preferred poly(alkylene oxide) is PEG.
  • the molecular weight of the poly(alkylene oxide) is not critical, and would depend mainly upon the end use of a particular copolymer. Those of ordinary skill in the art are capable of determining molecular weight ranges suitable for their end-use applications. In general, the useful range of molecular weight is a number average molecular weight between about 600 and about 200,000 daltons, and preferably between about 2,000 and about 50,000 daltons. Because the copolymers are hydrolytically stable, lower molecular weight polyalkylene oxides are preferred to insure that the resulting polymer is not too large to be eliminated by the kidney. Preferably, the molecular weight of the resulting polymer should not exceed 50,000 daltons.
  • the amino acid or peptide sequence represented by R 2 in Formula I preferably has a structure according to Formula II wherein R 3 and R 4 are independently selected from saturated and unsaturated, straight- chained and branched alkyl groups containing up to 6 carbon atoms and alkylphenyl groups, the alkyl portions of which are covalently bonded to an amine and contain up to 6 carbon atoms. Included within the definition of the alkyl or phenyl portions of the alkyl phenyl groups are alkyl or phenyl groups substituted by one or more substituents selected from hydroxyl, halogens, amino, and the like. The values for a and b are independently 0 or 1.
  • R 5 is -NH- or -NH-AA-, wherein -AA- is an amino acid or peptide sequence, with the proviso that -AA- contains a free N-terminus so that, when present, R 2 represents a peptide sequence of two or more amino acids.
  • the polymers of Formula I possess pendant functional groups at regular intervals within the polymer having the structure:
  • the pendant functional groups are carboxylic acid groups.
  • the pendant carboxylic acid groups may be further functionalized, in which case Y is selected from
  • Y can also be a C-terminus protecting group or a derivative of a pharmaceutically active compound covalently bonded to the recurring structural unit by the pendant functional group.
  • R 6 is selected from alkyl groups containing from 2 to 6 carbon atoms, aromatic groups alpha-, beta-, gamma- and omega amino acids, and peptide sequences.
  • R 3 and R 4 are preferably alkyl groups containing from 1 to 4 carbon atoms, inclusive.
  • R 2 is an amino acid
  • R 5 is -NH-.
  • R 5 is a peptide sequence
  • R 5 is -NH-AA-, wherein the -AA- of R 5 is bonded to R 3 or R 4 by way of the -NH- group of R 5 .
  • the single amino acids and the two or more amino acids making up the peptide sequences are preferably alpha amino acids, in which case either a or b, or both, is zero, and -AA- represents one or more alpha amino acids.
  • the amino acids and the two or more amino acids making up the peptide sequences are natural amino acids, in which instance, R 3 (when b is zero) or R 4 (when a is zero) is -CH 2 -CH 2 -CH 2 - in the case of ornitine -CH 2 -CH 2 -CH 2 -CH 2 - in the case of lysine, -CH-CH 2 -S-CH 2 -
  • the peptide sequences of 2 are preferably sequences containing from 2 to about 10 amino acid residues, in which case -AA- would preferably contain from 1 to about 9 amino acid residues.
  • the peptide sequences of R 2 even more preferably contain from 3 to 7 amino acid residues, inclusive, in which case, -AA- would contain from 2 to 6 amino acid residues, inclusive.
  • Y of the pendant functional group can be a C-terminus protecting group.
  • C-terminus protecting groups are well-known to those of ordinary skill in the art and include those disclosed in Bodanszky, The Practice of Peptide Synthesis (Springer- Verlag, New York, 1984) , the disclosure of which is herein incorporated by reference thereto.
  • Preferred C-terminus protecting groups are alkyl, aryl and silicon protecting groups.
  • the pendant carboxylic acid groups may be further functionalized.
  • Y is preferably -NH-NH 2 .
  • R 6 when present, is preferably an ethyl group, a natural alpha-amino acid or a peptide sequence containing from 2 to 10 natural amino acid residues.
  • Y can be a derivative of a pharmaceutically active compound covalently bonded to the recurring structural unit by means of the pendant functional group.
  • Y is covalently bonded to the recurring structural unit by means of an amide bond in the case when in the underivatized pharmaceutically active compound a primary or secondary amine is present at the position of the amide bond in the derivative.
  • underivatized pharmaceutically active compounds containing a primary or secondary amine examples include acyclovir, cephradine, melphalan, procaine, ephedrine, adriamycin, daunomycin, and the like.
  • Y is covalently bonded to the recurring structural unit by means of an ester bond in the case when in the underivatized pharmaceutically active compound a primary hydroxyl is present at the position of the ester bond in the derivative.
  • underivatized pharmaceutically active compounds containing a primary hydroxyl group include acyclovir, plumbagin, atropine, quinine, digoxin, quinidine and the like, as well as biologically active peptides.
  • Y can also be a derivative of a pharmaceutically active compound covalently bonded to the recurring structural unit by means of -X-, so that the pendant functional group has the structure:
  • -C-X-Y X is a linkage derived from the above-described further functionalized pendant carboxylic acid groups.
  • X is -NH-NH- in the case when in the underivatized pharmaceutically active compound an aldehyde or ketone is present at the position linked to the pendant functional group of the recurring structural unit by means of X.
  • underivatized pharmaceutically active compounds containing an aldehyde or ketone include adriamycin, daunomycin, testosterone, and the like. Steroids such as ketones and aldehydes are also easily generated by conventional methods.
  • X is -NH-NH-, -NH-R 6 -NH-, -O-Rg-NH-, -0-R g -0- or -NH-R g -O- in the case when in the underivatized pharmaceutically active compound a carboxylic acid is present at the position linked to the pendant functional group of the recurring structural unit by means of X.
  • underivatized pharmaceutically active compounds containing a carboxylic acid examples include chlorin e 6 , cephradine, cephalothin, melphlan, penicillin V, aspirin, nicotinic acid, chemodeoxycholic acid, chlorambucil, and the like, as well as biologically active peptides.
  • underivatized pharmaceutically active compounds include those compounds listed above with respect to amide and ester linkages.
  • Y can also be a derivative of a monoclonal antibody having oxidized carbohydrate moieties in the case when in the underivatized oxidized monoclonal antibody a ketone or aldehyde is present at the position linked to the recurring structural unit by means of X.
  • the polymer preferably contains a recurring structural units having an oxidized monoclonal antibodies covalently bonded thereto at the pendant functional group and recurring structural units having a derivative of a pharmaceutically active compound covalently bonded thereto at the pendant functional group.
  • the monoclonal antibody and the pharmaceutically active compound are preselected so that the monoclonal antibody targets cells for which it is specific for treatment by the pharmaceutically active compound it is co-conjugated with.
  • chlorin e g is a photosensitizer that can be co-conjugated with an anti-T cell monoclonal antibody to target the photosensitizer to T-cell leukemia cells.
  • Only one monoclonal antibody is required to be bound to a polymer to bind the polymer to a cell for which the monoclonal antibody is specific.
  • the ratio of pharmaceutically active compound to monoclonal antibody should be between about 4 and about 100. Preferably, the ratio is between about 6 and about 20.
  • the polymers of the present invention can have one or more recurring structural units in which the poly(alkylene oxide) and the amino acid or peptide sequence are copolymerized by means of amide or ester linkages, which structural units are independently represented by Formula III disclosed above.
  • R- ⁇ is a poly(alkylene oxide)
  • L is -0- or -NH-
  • R 2 is an amino acid or peptide sequence containing two carboxylic acid groups and at least one pendant amino group.
  • the poly(alkylene oxides) of R* ⁇ , and the preferred species of same, are the same as described above with respect to Formula I.
  • the amide and ester linkages are hydroslytically labile, there is no preference for limiting the molecular weight of the poly(alkylene oxide) below 50,000 daltons.
  • R 2 is preferably an amino acid or peptide sequence having a structure according to Formula IV disclosed above, wherein R 3 , R 4 , a and b are the same as described above with respect to Formula II.
  • R 5 is selected from:
  • -AA- is an amino acid or peptide sequences, with the proviso that -AA- contains a free C-terminus, so that when present, R 2 represents a peptide sequence of two or more amino acids.
  • the polymers of Formula III also possess pendant functional groups at regular intervals within the polymer, having the structure -NHZ or -NH-X- ⁇ -Z.
  • the pendant functional groups are amino groups.
  • the pendant amino groups may be further functionalized, in which case Z is selected from:
  • Z can also be an N-terminus protecting group or a derivative of a pharmaceutically active compound covalently bonded to the recurring structural unit by the pendant functional group.
  • R 6 and the preferred species thereof are the same as described above with respect to Formula II.
  • R 3 and R 4 are again preferably alkyl groups containing from 1 to 4 carbon atoms, inclusive.
  • R 5 is a carboxyl group.
  • R 5 is:
  • the single amino acids and the two or more amino acids making up the peptide sequences are preferably alpha-amino acids, in which case a or b, or both, is zero, and -AA- represents one or more alpha-amino acids. More preferably, the single amino acids and the two or more amino acids making up the peptide sequences are natural amino acids, in which instance R 3 (when b is zero) or R 4 (when a is zero) is ⁇ CH 2 - in the case of aspartic acid, -CH 2 -CH 2 - in the case of glutamic acid, and
  • Z of the pendant amino group of the recurring structural unit can represent a N-terminus protecting group.
  • N-terminus protecting groups are well-known to those of ordinary skill in the art and include those disclosed in the above-cited Bodanszky, The Practice of Peptide Synthesis, the disclosure of which is herein incorporated by reference thereto.
  • the preferred N-terminus protecting groups are benzyloxycarbonyl and tert-butoxycarbonyl groups.
  • Z could also be a derivative of a pharmaceutically active compound covalently bonded to the recurring structural unit by the pendant functional group.
  • Z is covalently bonded to the recurring structural unit by means of an amide bond in the case when in the underivatized pharmaceutically active compound a carboxylic acid group is present in the position of the amide bond in the derivative.
  • underivatized pharmaceutically active compounds containing carboxylic acid groups include those described above for Y with respect to Formula II.
  • Z can also be a derivative of a pharmaceutically active compound covalently bonded to the recurring structural unit by means of -X-_ ⁇ , so that the pendant functional group has the structure -NH-X- ⁇ -Z.
  • - ⁇ is a linkage derived from the above-described further functionalized pendant amino groups.
  • X ⁇ ⁇ is a linkage selected from: 0 0
  • underivatized pharmaceutically active compound a carboxylic acid is present at the position linked to the pendant functional group of the recurring structural unit by means of x ⁇ .
  • examples of underivatized pharmaceutically active compounds containing carboxylic acid groups have been previously listed.
  • X* ⁇ is:
  • underivatized pharmaceutically active compounds containing a primary or secondary amine or primary hydroxyl are the same as those listed above for Y with respect to Formula II.
  • Z can also be a derivative of a monoclonal antibody having oxidized carbohydrate moieties covalently bonded to the pendant amino group of the recurring structural unit by means of an amide bond in the case when in the underivatized oxidized monoclonal antibody a ketone or aldehyde is present at the position of the amide bond in the derivative.
  • the polymer having pendant amino groups preferably contains both recurring structural units having oxidized monoclonal antibodies covalently bonded thereto at the pendant functional group and recurring structural units having a derivative of a pharmaceutically active compound covalently bonded thereto at the pendant functional group, with the monoclonal antibody and the pharmaceutically active compound preselected so that the monoclonal antibody targets cells for which it is specific for treatment by the pharmaceutically active compound it is co-conjugated with.
  • R 2 can also be an amino acid or peptide sequence having at least one activated hydroxyl group, one carboxylic acid group when only one activated hydroxyl group is present, and at least one pendant amino group.
  • R 3 , R 4 , a, b, Z and AA and the preferred species thereof are the same as disclosed above for Formula IV and R 5 is selected from:
  • R 3 when b is zero or R 4 (when a is zero) is -CH 2 - in the case of serine, and:
  • polymers of the present invention can also have both the amide and ester recurring structural units of Formula III, so that, with respect to Formula III, L is -O- for some recurring structural units and -NH- for other recurring structural units.
  • L is -O- for some recurring structural units
  • -NH- for other recurring structural units.
  • the polymers of Formulas I and III have an absolute weight average molecular weight in the range of from about 10,000 to about 200,000 daltons, with about 20,000 to about 50,000 daltons being preferred for drug conjugate end-use applications. Molecular weights are determined by gel permeation chromatography relative to polyethylene glycol. Stated another way, the polymers of the present invention have from about 10 to about 100 repeating units represented by one of the structures of Formulas I and III, depending upon the molecular weight of the poly(alkylene oxide) used. As noted above, the molecular weight of the polymer should preferably not exceed 50,000 daltons, when the backbone of the polymer is not hydrolytically labile. Interfacial Polymerization
  • the polymers of Formula I are prepared by an interfacial polymerization process in which the poly(alkylene oxide) and amino acid or peptide sequence are copolymerized by means of stable urethane linkages.
  • the interfacial polymerization utilizes a water-immiscible organic solution containing one or more activated poly(alkylene oxides) .
  • the poly(alkylene oxides) are described above and include compounds specifically enumerated as preferred. Activated poly(alkylene oxides) and the preparation of same, are well-known to those of ordinary skill in the art.
  • poly(alkylene oxides) can be activated by reaction with cyanuric chloride, or by succinylation of terminal hydroxyl groups followed by dicyclohexylcarbodiimide-mediated condensation with N-hydroxy succinimide, or by the formation of imidazolyl formate derivatives using carbonyl diimideazole, or by reaction with chloroformates of 4-nitrophenol and 2,4,5-trichlorphenol.
  • the preferred activated form of the poly(alkylene oxide) is the succinimidyl carbonate prepared by reacting the terminal hydroxyl groups of the poly(alkylene oxide) with phosgene to form the chloroformate, which is then reacted with N-hydroxy succinimide to form the succinimidyl carbonate.
  • the preparation of poly(alkylene oxide) succinimidyl carbonates is described in co-pending U.S. Patent
  • the solution of the active carbonate of the poly(alkylene oxide) in the organic solvent is added to an aqueous solution containing one or more of the amino acids or peptide sequences described above, including compounds specifically enumerated as preferred, having protected C-terminals and at least two free amino groups.
  • the aqueous solution is buffered to a pH of at least 8.0. Suitable buffers include NaHC0 3 and Na 2 C0 3 .
  • the organic solution is added to the aqueous solution with vigorous stirring, which stirring is continued for several hours between about 4°C and about 40°C and preferably at ambient temperature. Slightly higher or lower temperatures are also suitable, depending upon the requirements of the reactants, which can be readily determined by those of ordinary skill in the art without undue experimentation.
  • the activated poly(alkylene oxide) reacts with the amino acid or peptide sequence to produce the copolymer of Formula I.
  • the mixture is then acidified to a pH of about 2.0 or lower.
  • the two phases separate, with the organic phase containing the polymer.
  • the reaction rate is a function of the concentration of the two phases, with the reaction rate increasing as phase concentration increases.
  • phase concentration is the solubility of the reactants in each phase.
  • suitable water-immiscible organic solvents include methylene chloride, chloroform, dichloroethane and the like. Equimolar ratios of activated poly(alkylene oxide) to amino acid or peptide sequence starting materials are employed to maximize polymer length.
  • the polymer can then be purified by conventional purification techniques, such as by dialysis against distilled water with a molecular weight sizing membrane or by elution with a molecular weight sizing chromatography column.
  • Solution Polymerization - First Mode The polymers of Formula III are prepared by a solution polymerization process in which the poly(alkylene oxide) and the amino acid or peptide sequence are copolymerized by means of hydrolytically stable amide or hydrolyzable ester linkages.
  • the poly(alkylene oxide) should first be dried by the azeotropic removal of water by distillation in toluene, followed by drying under vacuo.
  • the solution polymerization is carried out in an organic solvent such as methylene chloride, chloroform, dichloroethane and the like.
  • the poly(alkylene oxides) utilized in the reaction can have either hydroxyl terminals or amino terminals and are otherwise as described above and include compounds specifically enumerated as preferred.
  • poly(alkylene oxide) is dissolved in the solvent and stirred under argon. An equimolar quantity is then added of one or more of the amino acids or peptide sequences described above, including compounds specifically enumerated as preferred, having protected
  • the reaction mixture may be heated slightly to dissolve the amino acid or peptide.
  • the solution concentration of either compound is not critical.
  • An excess quantity of a coupling reagent is also added to the reaction mixture, together with an excess quantity of an acylation catalyst.
  • Suitable coupling reagents and the quantities to employ are well-known and disclosed by the above-cited Bodanszky, Principles of Peptide Synthesis, the disclosure of which is hereby incorporated herein by reference thereto.
  • Examples of such coupling reagents include, but are not limited to, carbodiimides such as ethyl dimethylaminopropyl carbodiimide (EDC) , diisopropyl carbodiimide and 3-[2-morpholinyl-(4)-ethyl] carbodiimide, p-toluene sulfonate, 5-substituted isoxazolium salts, such as Woodward's Reagent K, and the like.
  • EDC ethyl dimethylaminopropyl carbodiimide
  • diisopropyl carbodiimide diisopropyl carbodiimide and 3-[2-morpholinyl-(4)-ethyl] carbodiimide, p-toluene sulfonate, 5-substituted isoxazolium salts, such as Woodward's Reagent K, and the like.
  • Suitable acylation catalysts and the quantities to employ are also well-known, and include, but are not limited to, dimethylaminopyridiniu toluene sulfonate, hydroxybenzotriazole, imidazoles, triazole, dimethyl amino pyridene, and the like.
  • reaction mixture is then stirred between about 4°C and about 40°C and preferably at room temperature until completion of the reaction, typically within 24 hours, usually overnight.
  • the poly(alkylene oxide) reacts with the amino acid or peptide sequence to produce the copolymer of Formula III.
  • a urea precipitate is removed by filtration, and the polymer is then precipitated with cold ether, filtered and dried under vacuum.
  • the polymer can then be further purified by conventional methods, typically by reprecipitation from isopropanol.
  • the polymers of Formula III can also be prepared by a solution polymerization process in which a poly(alkylene oxide) having amino terminals and an amino acid or peptide sequence having at least one hydroxyl group are copolymerized in an organic solvent by means of hydrolytically stable urethane linkages.
  • the one or more hydroxyl groups of the amino acid or peptide sequence should first be activated in the organic solvent.
  • the activation step is well-known and essentially conventional.
  • the hydroxyl group can be activated by reacting it with an alkyl chloroformate, or with p-nitrophenyl chloroformate.
  • the hydroxyl group can be activated as described above with respect to the activation of poly(alkylene oxides) for the interfacial polymerization process of the present invention, preferably utilizing the process disclosed by U.S. Patent Application Serial No. 340,928 by Zalipsky, incorporated herein by reference thereto.
  • the activation is carried out in the presence of one or more of the activating reagents and acylation catalysts described above with respect to the first mode of solution polymerization.
  • the same ratio of activating reagent and acylation catalyst to amino acid or peptide sequence should also be utilized.
  • the same solvents are utilized as described above with respect to the first mode solution polymerization.
  • the poly(alkylene oxide) should first be dried as described above with respect to the first mode solution polymerization. After the activation of the one or more hydroxyl groups of the amino acid or peptide sequence is complete, the poly(alkylene oxide) is then added to the reaction mixture with stirring. As with the first mode solution polymerization equimolar quantities of reactants are preferred. The reaction mixture is then stirred at room temperature until completion of the reaction, typically within 24 hours, usually overnight.
  • the poly(alkylene oxides) utilized in the reaction have amino terminals and are otherwise as described above and include compounds specifically enumerated as preferred. The poly(alkylene oxide) reacts with the activated hydroxyl groups of the amino acid or peptide sequence to form urethane linkages.
  • the resulting polymer is then precipitated, separated and purified as described above with respect to the first mode solution polymerization.
  • the polymers of the present invention can be used in the preparation of drug carriers by conjugating the pendant functional groups either directly with reactive functional groups on a drug molecule, or by first further functionalizing the pendant functional group to improve its reactivity with or selectivity for a functional group on a candidate drug molecule. Accordingly, the copolymers of the present invention can be conjugated with candidate drug molecules by one of the modes of conjugation set forth below.
  • the polymers of Formula I having pendant carboxylic acid groups, can be directly conjugated with pharmaceutically active compounds that, prior to conjugation, have an amino or hydroxyl group.
  • the polymers of Formula I are described above, and include polymers specifically enumerated as preferred.
  • Pharmaceutically active compounds having amino or hydroxyl groups are also described above.
  • the conjugation reaction utilizes an organic solvent in which the reactants are soluble.
  • suitable organic solvents include DMF, CH 3 CN, CH 2 C1 2 , and the like.
  • the appropriate quantities of the polymer and the pharmaceutically active compound are dissolved in the solvent.
  • the solvent may be heated slightly to dissolve the reactants. An excess of the pharmaceutically active compound is preferred to insure substantial conjugation of the pendant functional groups of the polymers.
  • the total solution concentration include DMF, CH 3 CN, CH 2 C1 2 , and the like.
  • a urea product precipitates, which is removed by filtration.
  • the polymer conjugate is then precipitated with a solvent in which the polymer has poor solubility, e.g., ether, hexane and the like, filtered and purified by further reprecipitation crystalization, from such solvent as ethanol, ethyl acetate, iso-propanol and the like.
  • a solvent in which the polymer has poor solubility e.g., ether, hexane and the like
  • filtered and purified by further reprecipitation crystalization from such solvent as ethanol, ethyl acetate, iso-propanol and the like.
  • the product then is dried in vacuo.
  • a hydrolytically unstable ester bond is formed linking the pharmaceutically active compound to the copolymer by means of the pendant functional group.
  • a hydrolytically stable amide bond is formed linking the pharmaceutically active compound to the copolymer by means of the pendant functional group. If the pharmaceutical compound is active in conjugated form, then a hydrolytically stable bond is desirable. However, if the pharmaceutical compound is inactive in conjugated form, then a hydrolytically unstable bond is desirable.
  • the pharmaceutically active compound has both an amino and a hydroxyl group, the question of which group to conjugate to will thus depend upon the activity of the pharmaceutical compound in conjugated form. Once a decision is made to conjugate to either the amino group or the hydroxyl group, the group through which conjugation is not to occur should be protected to prevent the formation of undesirable conjugates. The attachment of such protective groups is well-known to those of ordinary skill in the art.
  • the pendant carboxylic acid group at the copolymer is preferably an activated pendant carboxylic acid group.
  • the activation of such carboxylic acid groups is well-known and essentially conventional.
  • the pendant carboxylic acid group can be reacted with N-hydroxy succinimide in the presence of a coupling agent such as dicyclohexyl carbodiimide in a solvent such as DMF, CHC1 3 , pyridine and the like.
  • the polymers of Formula I having pendant carboxylic acid groups can also be conjugated with pharmaceutically active compounds that, prior to conjugation, have a carboxylic acid group, by first reacting the pendant carboxylic acid group of the copolymer with a alkanol amine, so that an amide of the pendant carboxylic acid group is formed.
  • the polymers of Formula I are disclosed above and include polymers specifically enumerated as preferred. Pharmaceutically active compounds having a carboxylic acid group are also described above.
  • Pharmaceutically active compounds having a carboxylic acid group can also be formed from pharmaceutically active compounds having hydroxyl groups by forming an acid ester of the hydroxyl group with a dicarboxylic acid anhydride, such as succinic anhydride, or an N-dicarboximide, such as N-hydroxy succinimide.
  • a dicarboxylic acid anhydride such as succinic anhydride
  • an N-dicarboximide such as N-hydroxy succinimide.
  • the hydroxyl group of the pharmaceutically active compound can be reacted with succinic anhydride in the presence of a base such as triethylamine in a suitable solvent such as DMF.
  • a base such as triethylamine
  • suitable solvent such as DMF
  • Alkanol amines are defined as including, in addition to compounds such as ethanol amine or 3-propanol amine, amino acids and peptide sequences having free hydroxyl and amino groups, so that alkanol amines suitable for use in the present invention have the structure HO-Rg- NH 2 , wherein Rg and the preferred species thereof are the same as described above with respect to Formula II.
  • the reaction between the copolymer and the alkanol can be performed in aqueous solution.
  • the polymer is dissolved in the solution with an excess, perferably at least a ten-fold excess of a alkanol amine.
  • the pH of the solution is then adjusted to between about 4.5 and about 6 by the addition of 0.1 N HCl.
  • At least a ten-fold excess of a water-soluble coupling reagent is then added with maintenance of the pH within the above range by the addition of l N HCl.
  • the reaction mixture should be stirred, for about 5 to about 48 hours, acidified, and extracted into an organic solvent such as methylene chloride, CHC1 3 dichloroethane, and the like.
  • the solvent extract is then washed with 1 N HCl followed by washing with saturated NaCl.
  • the extract is then dried over anhydrous MgS0 4 , filtered and concentrated to a viscous syrup.
  • the polymer product is then precipitated using cold ether.
  • the polymer product can then be purified by reprecipitation from isopropanol, followed by washings with hexane and complete drying in vacuo.
  • Suitable water-soluble coupling reagents are well-known and disclosed by the above-cited Bodanszky, Principles of Peptides Synthesis, the disclosure of which is hereby incorporated herein by reference thereto.
  • the examples of such coupling reagents include, but are not limited to, water-soluble carbodiimides such as EDC, and 3-[morpholinyl-(4)-ethyl] carbodiimide, p-toluene sulfonate, 5-substituted isoxazoliu salts, such as Woodward's Reagent K, and the like.
  • the alkanol amide of the copolymer is then reacted with the carboxylic acid group of the pharmaceutically active compound in a solvent such as DMF, CH 2 C1 2 , pyridine, and the like.
  • a solvent such as DMF, CH 2 C1 2 , pyridine, and the like.
  • the appropriate quantities of the hydroxyl amide the polymer and the pharmaceutically active compound are combined in the solvent, which may be heated slightly to dissolve the reactants Again, excess quantities of the pharmaceutically active compound are preferbly employed to insure substantial conjugation of the pendant hydroxyl amides of the polymer.
  • the reaction is carried out in the presence of one or more of the coupling reagents and acylation catalysts described above with respect to the first mode of drug conjugation.
  • the amount of coupling reagent and acylation catalyst should be equivalent to or in excess of the amount of pharmaceutically active compund.
  • the carboxylic acid group of the pharmaceutically active compound is preferably an activated carboxylic acid group.
  • the carboxylic acid group of the pharmaceutically active compound can be activated by the method described above for activation of the pendant carboxylic acid group of the polymer. Other activating methods are well known and essential conventional.
  • reaction mixture is then stirred at between about 4 and about 40°C, and preferably about room temperature, until completion of the reaction, typically within 24 hours, usually overnight.
  • the hydroxyl group of the pendant alkanol amide then reacts with the carboxylic acid group of the pharmaceutically active compound to form an ester linkage.
  • a urea product precipitates that is removed by filtration.
  • the product is then precipitated, filtered, dried and purified according to the procedure described above with respect to the first mode of drug conjugation.
  • the above order of reaction may be reversed, so that the alkanol amine is first reacted with a pharmaceutically active compound having a carboxylic acid group, which carboxylic acid group may be optionally activated, to form a alkanol amide thereof.
  • Suitable optional activating steps are well-known and essentially conventional.
  • the carboxylic acid group of the pharmaceutically active compound can be reacted with an alkyl or p-nitrophenyl chloroformate in the presence of a base such as triethyl amine in a suitable solvent such as DMF.
  • the pharmaceutically active compound with the activated carboxylic acid group is then precipitated, dried and purified by conventional means and reacted with the alkanol amine by the process described above for the copolymer.
  • the resulting alkanol amide of the pharmaceutically active compound is then reacted with the carboxylic acid group of the copolymer, following the procedure described above so that an ester linkage forms between the alkanol amide of the pharmaceutically active compound and the pendant carboxylic acid group of the copolymer, following the procedure described above for the formation of the ester linkage between the alkanol amide of the copolymer and the carboxylic acid group of the pharmaceutically active compound.
  • the pendant carboxylic acid group of the copolymer is preferably activated in accordance with the optional procedures set forth for this mode when the alkanol amide of the pendant carboxylic acid group of the copolymer is first formed.
  • the pendant carboxylic acid groups of the copolymer are preferably activated pendant carboxylic acid groups, which carboxylic acid groups can be activated in the manner described above with respect to the first mode of drug conjugation.
  • the polymers of Formula I can also be directly conjugated with pharmaceutically active compounds having, prior to conjugation, carboxylic acid groups, by first reacting the pendant carboxylic acid groups of the polymer with a diamine, so that an amino amide of the pendant carboxylic acid group is formed. The amino amide is then reacted with the carboxylic acid group of the pharmaceutically active compound to form an amido amide linkage between the pendant carboxylic acid group of the copolymer and the carboxylic acid group of the pharmaceutically active compound.
  • the polymers of Formula I are described above and include polymers specifically enumerated as preferred. Pharmaceutically active compounds having a carboxylic acid group are also described above.
  • Pharmaceutically active compounds having a carboxylic acid group can also be formed from pharmaceutically active compounds having hydroxyl groups by forming an acid ester of the hydroxyl group as described above with respect to the second mode of drug conjugation.
  • Pharmaceutically active compounds having a hydroxyl group are also described above.
  • Diamines are defined as including, in addition to compounds such as ethylene diamine, amino acids and peptide sequences having two free amino groups, so that diamines suitable for use with the present invention have the structure H 2 N-Rg-NH 2 , wherein R g and the preferred species thereof are the same as described above with respect to Formula II.
  • the reaction between the copolymer and the diamine utilizes an aqueous solution.
  • the polymer is dissolved in the solution with an excess, preferably at least a ten-fold excess of the diamine, which excess is utilized in order to minimize undesirable cross linking reactions.
  • the pendant carboxylic acid groups of the copolymer are preferably activated pendant carboxylic acid groups, which pendant carboxylic acid groups are activated as described above with respect to the second mode of drug conjugation.
  • the diamine is reacted with the pendant carboxylic acid group of the copolymer by the same method described above with respect to the second mode reaction between the alkanol amine and the pendant carboxylic acid group of the copolymer.
  • the reaction mixture is made basic and extracted with an organic solvent such as methylene chloride. The solvent extract is washed, dried, filtered, concentrated, precipitated and purified by the procedure described above with respect to the alkanol amide of the copolymer prepared pursuant to the second mode of drug conjugation.
  • the pendant amino amide of the copolymer is then reacted with the carboxylic acid group of the pharmaceutically active compound as described above with respect to the second mode of drug conjugation.
  • the carboxylic acid group of the pharmaceutically active compound is preferably an activated carboxylic acid group.
  • the carboxylic acid group can be activated by the conventional means mentioned above with respect to the second mode of drug conjugation for the reaction of the amine portion of the alkanol amine with the carboxylic acid group of the pharmaceutically active compound.
  • reaction mixture is then stirred under the conditions described above with respect to the first mode of drug conjugation.
  • the pendant amino amide then reacts with the carboxylic acid group of the pharmaceutically active compound to form an amide linkage.
  • the work up and isolation of the polymer product is the same as described above with respect to the first mode of drug conjugation.
  • the above order of reaction may be reversed so that the diamine is first reacted with a pharmaceutically active compound having a carboxylic acid group following the procedure described above for the reaction of the amino amide of the copolymer with the carboxylic acid group of the pharmaceutically active compound.
  • the reaction forms an amino amide of the carboxylic acid group of the pharmaceutically active compound.
  • the carboxylic group of the pharmaceutically active compound is preferably an activated carboxylic acid group, which may be activated by conventional means, such as by reaction with a carbodiimide.
  • the resulting amino amide of the carboxylic acid group of the pharmaceutically active compound is then reacted with the pendant carboxylic acid group of the copolymer following the procedure described above for the reaction of the diamide with the pendant carboxylic acid group of the copolymer.
  • An amide linkage is formed between the amino amide and the pendant carboxylic acid group.
  • the pendant carboxylic -42- acid group is preferably an activated carboxylic acid group, prepared as described above with respect to the first mode of drug conjugation.
  • Drug Conjugation - Fourth Mode The polymers of Formula I, having pendant carboxylic acid groups, can also be conjugated with pharmaceutically active compounds that, prior to conjugation, have an aldehyde, ketone or carboxylic acid group.
  • the fourth mode of drug conjugation first forms pendant acyl hydrazine groups from the pendant carboxylic acid groups of the polymer, which acyl hydrazine is then reacted with the aldehyde, ketone or carboxylic acid group of the pharmaceutically active compound to form a hydrazone or diacyl hydrazide linkage between the copolymer and the pharmaceutically active compound.
  • the polymers of Formula I are described above and include polymers specifically enumerated as preferred. Pharmaceutically active compounds having an aldehyde, ketone or carboxylic acid group are also described above.
  • Pharmaceutically active compounds having a carboxylic acid group can also be formed from pharmaceutically active compounds having hydroxyl groups by forming an acid ester of the hydroxyl group as described above with respect to the second mode of drug conjugation. Pharmaceutically active compounds having a hydroxyl group are also described above.
  • the fourth mode of drug conjugation first forms pendant acyl hydrazine groups on the polymer by reacting the pendant carboxylic acid groups of the polymer with an alkyl carbazate, so that the pendant carboxylic acid groups form pendant alkyl carbazate groups.
  • the alkyl portion is acting as a protecting group. It is removed in the subsequent step to yield acyl hydrazine.
  • the reaction utilizes an organic solvent such as methanol in which the polymer and an excess, preferably at least a ten-fold excess of an alkyl carbazate are reacted at between about 4°C and about 40°C in the presence of an excess quantity of a coupling reagent.
  • the most preferred alkyl carbazate is t-butyl carbazate.
  • suitable coupling reagents include those listed above with respect to the first mode of drug conjugation.
  • the work-up and isolation of the polymer product is the same as described above with respect to the first mode of drug conjugation.
  • the alkyl carbazate group is then removed to form pendant acyl hydrazine groups by mixing the polymer with a 4 M solution of HCl in dioxane. The mixture is stirred for between about 30 min. and about 2 hours at room temperature, with the polymer settling at the bottom as an oil. The hydrochloride salt of the hydrazine is then worked-up and isolated as described above.
  • the polymer having pendant acyl hydrazine groups is then conjugated with the pharmaceutically active compound.
  • the conjugation reaction utilizes an organic solvent in which the reactants are soluble.
  • suitable organic solvents include pyridine, DMF, CH 2 C1 2 , THF, and the like.
  • the polymer having pendant acyl hydrazine groups and the pharmaceutically active compound are dissolved in the solvent and reacted as disclosed above with respect to the first mode of drug conjugation.
  • the pendent acyl hydrazine groups of the polymer react with the aldehyde and ketone to form a hydrazaone or with the carboxylic acid group of the pharmaceutically active compound to form a diacyl hydrazide linkage.
  • Hydrazaones can be formed with aldehyde or ketone containing drugs (adriamycine, testosterone) or when aldehydes or ketones are introduced (e.g., by oxidation of carbohydrate residues of glycopeptides such as disclosed by the co-pending U.S. patent application Serial No. 673,696 by Zalipsky et als, filed March 15, 1991, the disclosure of which is hereby incorporated herein by reference thereto) .
  • the -44- work-up and isolation of the polymer product is the same as described above with respect to the first mode of drug conjugation.
  • the carboxylic acid group is preferably an activated carboxylic acid group, substituted with a suitable leaving group capable of being displaced by the pendant acyl hydrazine group of the polymer.
  • suitable leaving groups are disclosed by Bodanszky, Principals of Peptide Synthesis, cited above, the disclosure of which is hereby incorporated herein by reference thereto.
  • Such leaving groups include, but are not limited to, imidazolyl, triazolyl, N-hydroxy succinimidyl, N-hydroxy norbornene dicarboximidyl and phenolic leaving groups, and are substituted onto the carboxylic acid group of the pharmaceutically active compound by reacting the carboxylic acid group in the presence of an activating reagent with the corresponding imidazole, triazole, N-hydroxy succinimide, N-hydroxy norbornene dicarboximide and phenolic compounds.
  • Suitable activating reagents include those disclosed above with respect to the first mode of drug conjugation.
  • the polymers of Formula III having pendant amino groups can be directly conjugated with pharmaceutically active compounds that, prior to conjugation, have a carboxylic acid group.
  • the polymers of Formula III are described above, and include polymers specifically enumerated as preferred.
  • Pharmaceutically active compounds having carboxylic acid groups are also described above.
  • Pharmaceutically active compounds having a carboxylic acid group can also be formed from pharmaceutically active compounds having hydroxyl groups by reacting the hydroxyl group as described above with respect to the second mode of drug conjugation.
  • Pharmaceutically active compounds having a hydroxyl group are described above.
  • the polymer and the pharmaceutically active compound are reacted and recovered as described above in the fourth mode of drug conjugation for the reaction between the polymer having pendant acyl hydrazine groups and the pharmaceutically active compound having carboxylic acid groups.
  • the pendant amino group of the polymer reacts with the carboxylic acid group of the pharmaceutically active compound to form an amide linkage.
  • the carboxylic acid group of the pharmaceutically active compound is preferably an activated carboxylic acid group, substituted with a suitable leaving group capable of being displaced by the pendant amino group of the polymer.
  • the activation of such carboxylic acid groups is well-known and essentially conventional.
  • the carboxylic acid groups of the pharmaceutically active compounds can be activated as described above with respect to the fourth mode of drug conjugation.
  • polymers of Formulas I and III can also be conjugated with biologically active polypeptides and glycopolypeptides.
  • biologically active polypeptides and glycopolypeptides of interest include those listed in the above-incorporated copending U.S. Patent Application
  • the biologically active polypeptides and glycopolypeptides contain aldehyde, ketone and carboxylic acid groups that can be conjugated with the polymers of the present invention according to the third, fourth and fifth modes of drug conjugation, or according to the methods described in the above-incorporated U.S. Patent Application Serial
  • Monoclonal antibodies contain carbohydrate moieties capable of being oxidized to form aldehydes and ketones.
  • the groups can be generated on the carbohydrate moieties, for example, by oxidizing the vicinal diols of the carbohydrate moieties with excess periodate, or enzymatically, e.g. by use of galactose oxidase, using the methods described in the above-incorporated U.S. Patent Application Serial No. 673,696 by Zalipsky et als.
  • ketones and aldehydes of the oxidized carbohydrate moieties of monoclonal antibodies can be coupled with the polymers of Formula I by the fourth mode of drug conjugation disclosed above.
  • Sodium borohydride or sodium cyanoborohydrate is added to the reaction mixture to reduce the resulting hydrazone to a more stable alkyl hydrazide.
  • the oxidized carbohydrate moieties of monoclonal antibodies will also react with amino amides formed from pendant carboxylic acid groups of the polymers of Formula I, according to the third mode of drug conjugation, as well as with the pendant amino groups of the polymers of Formula III, according to the fifth mode of drug conjugation, in the presence of sodium borohydride.
  • the attachment of a single monoclonal antibody to a polymer is sufficient to bind the polymers to cells for which the monoclonal antibody is specific.
  • the polymer can be co-conjugated with a pharmaceutically active compound to deliver the compound to the specific cell the monoclonal antibodies function to bind the polymer to.
  • Specific cells can be targeted for treatment by the pharmaceutically active compound, significant quantities of which will not be delivered to other tissues. This is particularly important in applications when the pharmaceutically active compound produces toxic or other undesirable side effects in tissues not intended for treatment. Lower dosage quantities will also be possible because application of the pharmaceutically active compound will be essentially limited to the treatment site.
  • chemotherapeutic compounds can be used to treat cancerous cells that would otherwise be toxic to healthy tissues.
  • the co-conjugates of pharmaceutically active compounds and monoclonal antibodies with the polymers of Formulas I and III are formed by first reacting the pharmaceutically active compound with the polymer according to either the third, fourth or fifth mode of drug conjugation. An excess of polymer is utilized so that pendant functional groups will remain unconjugated for the attachment of the monoclonal antibody.
  • the carbohydrate moieties of the monoclonal antibody are first oxidized to produce aldehyde and ketone groups for conjugation, and the monoclonal antibody is then reacted with the conjugate of the pharmaceutically active compound and the copolymer of Formula I or III having available pendant functional groups according to the third, fourth or fifth mode of drug conjugation, as if the monoclonal antibody were a pharmaceutically active compound.
  • the reaction can be performed in the presence of sodium borohydride or sodium cyanoborohydrate to convert the resulting hydrazone to a hydrazide.
  • the co-conjugates of the pharmaceutically active compound and monoclonal antibody with the polymer of Formula I or III can then be purified by protein chromatography by conventional methods.
  • a number of useful combinations of pharmaceutically active compounds and monoclonal antibodies are available for the treatment of specific cell types in need thereof with suitable pharmaceutically active compounds.
  • chlorin e g a photosensitizer
  • an anti-T cell monoclonal antibody can be co-conjugated with an anti-T cell monoclonal antibody to bind the polymer-drug conjugation to T-cell leukemia cells.
  • the T-cells are rendered photosesitive and subsequent treatment with ultraviolet light substantially reduces or eliminates the T-cell leukemia cells without affecting other types of cells.
  • cytotoxic drugs such as daunomycin, metotrexate, cytorhodin-S, adriamycin, mitomycin, doxorubicin, melphalan and the like.
  • Metal chelating compounds such as EDTA can be co-conjugated with monoclonal antibodies to form complexes with radioative isotpes for the treatment of cells in need thereof, to which the monoclonal antibody is capable of binding.
  • radioactive isotopes include, but are not limited to 99 Tc and 123 I, which can be used, for example in the treatment of cancerous cells.
  • a large number of pharmaceutically active compounds may be conjugated with the polymers of Formulas I and III, including antibiotics, anti- neoplastic agents, antiviral agents, cytotoxic drugs, metal chelators, hormones, and the like.
  • the resulting conjugate can be prepared for administration by incorporating the same into a suitable pharmaceutical formulation.
  • suitable pharmaceutical formulations are well-known in the art and may include, but are not limited to, phosphate buffered saline solutions, water, emulsions such as oil/water emulsion, and various types of wetting agents.
  • Other suitable pharmaceutical formulations include sterile solutions, tablets, coated tablets and capsules.
  • such phar aceutic formulations contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, such as magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, and the like.
  • excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, such as magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, and the like.
  • Such formulations may also include flavor and color additives or other ingredients.
  • Compositions of such formulations are prepared by well-known conventional methods.
  • the invention also provides a method for treating a pathological condition in a subject in need thereof by administering to the subject the composition of the present invention. Administration of the medication may occur in one of several ways, including oral, intravenus, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • polymers of Formula I form an ionically conductive material when combined with an alkali metal electrolyte salt.
  • the polymers of Formula I are described above, and include polymers specifically enumerated as preferred.
  • the polymers of Formula I capable of forming ionically conductive materials are those polymers in which Y is -OH or a C-terminus protecting group having the structure -OR 7 , wherein R 7 is an alkyl group and preferably an ethyl group.
  • the alkali metal electrolyte salt is preferably a lithium electrolyte salt.
  • Suitable lithium salts include LiAsFg, LiPFg, Lil, LiBr, LiBFg, LiAlCl 4 , LiCF 3 C0 2 , LiCF 3 S0 3 .
  • Preferred lithium electrolyte salts include LiAsFg, LiPFg, Lil and LiCF 3 S0 3 .
  • the most preferred lithium electrolyte salts are LiAsF g and LiCF 3 S0 3 .
  • the preparation of the ionically conductive materials utilizes an organic solvent in which the polymer and the alkali metal electrolyte salt are soluble, such as acetonitrile.
  • the ratio of polymer to electrolyte salt should be between about 2:1 and about 10:1 and preferably about 4:1.
  • the total solution concentration (w/v%) of both compounds combined is between about 1 percent and about 25 percent, and preferably about 10 percent, depending upon the solubility of the materials.
  • the polymer and electrolyte salt are dissolved in the solvent, which may be heated slightly to dissolve the materials.
  • the mixture is cast into the desired form, and the solvent is removed by drying, first in air and then under vacuum. The mixture may be heated to remove the solvent.
  • the polymers preferably have a molecular weight greater than about 75,000 daltons to provide the mixture with adequate mechanical strength.
  • the polymers are also preferably cross-linked, as set forth below, to provide adequate mechanical strengths to the material.
  • the mixture of polymer and electrolyte salt with solvent removed may also be compression molded to obtain articles having a desired form.
  • the ionically conductive materials of the present invention are useful as electrodes in electrochemical cells.
  • the ionically conductive materials of the present invention instead of being utilized as electrodes, are particularly useful as solid electrolytes for non-aqueous electrochemical cells.
  • the mixture is particularly well suited for use in non-aqueous secondary cells.
  • Non-aqueous electrochemical cells can be assembled utilizing the ionically conductive material of the present invention by combining a cathode, an anode and a solid electrolyte containing the ionically conductive material.
  • suitable anodes include alkali metals such as sodium, potassium and lithium.
  • the alkali metal electrolyte salt would then be a salt of the metal utilized.
  • the preferred alkali metal is lithium.
  • the anode can also be a counter-electrode capable of reversibly intercalating lithium from the cathode.
  • the alkali metal electrolyte salt must be a lithium salt.
  • Anodes that function as counter-electrodes capable of reversibly intercalating lithium are well-known and are prepared from graphitic carbon.
  • the cathode preferably contains a cathode-active material capable of reversibly intercalating lithium.
  • Suitable lithium-intercalable cathode materials include metal-chalcogen combinations, particularly transition metal-chalcogen combinations, metal halides, and the like.
  • Chalcogens are understood by those of ordinary skill in the art to include the chemically-related elements from Group VI of the periodic table, namely oxygen, sulfur, selenium, tellurium and polonium.
  • the preferred chalcogens are oxygen and sulfur.
  • Preferred transition metals include manganese, nickel, iron, chromium, titanium, vanadium, molybdenum and cobalt.
  • Preferred compositions include molybdenum sulfides, vanadium oxides and manganese oxides. MoS 2 , g 0 13 , Mo 6 S g and Mn0 2 are more preferred, with Mn0 2 being most preferred.
  • the cathode is typically fabricated by depositing a slurry of a cathode-active material, ionically conductive binder and a fugitive liquid carrier such as one of the solvents utilized in the preparation of the ionically conductive materials, on a cathode current collector, and then evaporating the carrier to leave a coherent mass in electrical contact with the current collector.
  • the anode may be prepared by depositing a slurry of a carbonaceous anode material, the ionically conductive binder and the fugitive liquid carrier on an electrically-conductive anode support and then evaporating the carrier to leave a coherent mass in electrical contact with the anode support.
  • the cell is then assembled by sandwiching the cathode and anode layers with the solid electrolyte containing the ionically conductive material of the present invention layered therebetween.
  • the anode and cathode current collectors are then placed in electrical contact with their respective anode and cathode terminals.
  • the ionically conductive binder may be present in an amount between about 0.5 percent and about 25 percent by weight of the cathode or anode material, and preferably between about 2 percent and about 10 percent by weight.
  • polymers of Formulas I and III can also be cross-linked to form polymer matrices that can be utilized in the preparation of hydrogel membranes and semi-interpenetrating polymer networks (semi-IPN's) .
  • the polymers of Formulas I and III can be cross-linked by way of hydrolytically stable urethane linkages between a trifunctional amine and the poly(alkylene oxide) moiety of the copolymer.
  • the polymers of Formulas I and III can be cross-linked by way of hydrolytically stable urethane linkages between a trifunctional amine and the poly(alkylene oxide) moiety of the copolymer.
  • Formula I having pendant acyl hydrazine groups, can also be cross-linked by way of hydrolytically labile acyl semicarbazide linkages between a diisocyanate and the pendant acyl hydrazine groups of the polymer.
  • the cross-link density of the polymer matrix can be controlled by varying the length of the poly(alkylene oxide) moiety of the polymers of Formulas I and III.
  • the polymers of Formulas I and III are described above and include polymers specifically enumerated as preferred.
  • the polymer matrices cross-linked by way of acyl semicarbazide linkages utilize polymers according to Formula I having pendant acyl hydrazine functional groups that are prepared as described above with respect to the fourth mode of drug conjugation.
  • polymers having terminal poly(alkylene oxide) groups should be used. Such polymers can be obtained from the polymerization processes of the present invention by reacting the amino acids or peptide sequences with an excess of poly(alkylene oxide) .
  • the terminal poly(alkylene oxide) groups should be activated poly(alkylene oxide) groups.
  • the polymers of Formula I produced by the interfacial polymerization process described above will have activated terminal poly(alkylene oxide) groups.
  • the polymers of Formula III are prepared by the solution polymerization processes described above, which do not result in polymers having activated terminal poly(alkylene oxide) groups.
  • the terminal poly(alkylene oxide) groups of the polymers of Formula III can be activated by the methods described above with respect to the interfacial polymerization process for the preparation of the polymers of Formula I.
  • the activation step should not be performed until after the polymerization of the polymers of Formula III.
  • the urethane cross-linking reaction utilizes an solvent in which the reactants are soluble.
  • suitable solvents include methylene chloride, chloroform, THF, dioxane, water, DMF, acetonirile, and the like.
  • Equivalent quantities of the polymer and the trifunctional amine are reacted.
  • Trifunctional amines are defined as any compound having three free amine groups, including aromatic materials.
  • Suitable trifunctional amines include any soluble material having three amines that can be used as a cross-linking agent, preferred trifunctional amines have the structure N(-R g - NH 2 ) 3 , in which R 6 is the same as described above with respect to Formula II.
  • Trifunctional amines, the alkyl moieties of which have between about 1 and about 10 carbon atoms are preferred.
  • Trifunctional amines with alkyl moieties having between about 2 and about 6 carbon atoms are even more preferred.
  • the solvents may be heated slightly to dissolve the reactants.
  • the solution concentration (w/v%) of the polymer solution should be less than about 10 percent so that cross-linking of the polymer does not occur too rapidly.
  • N-hydroxy succinimide is a bi-product of the cross-linking reaction and will remain embedded in the polymer matrix unless removed by washing with water. However, this is readily accomplished by rinsing the membrane with several successive washings of distilled, deionized water. Analysis of the washing has shown that substantially all of the N-hydroxy succinimide is removed by the first washing.
  • Polymer matrices cross-linked by urethane linkages can also be prepared utilizing the poly(alkylene oxide) homopolymers disclosed above as starting materials for the interfacial polymerization process described above. In other words, it is not necessary for this cross-linking method that the poly(alkylene oxide) be copolymerized with an amino acid or peptide sequence.
  • the acyl semicarbazide cross-linking of the polymers of Formulas I having pendant acyl hydrazine groups does not require the use of polymers having terminal alkylene oxide moieties.
  • the reaction utilizes the same organic solvents utilized in the urethane cross-linking reaction. Equivalent quantities of the polymer and the diisocyanate are reacted.
  • Alkyl diisocyanates the alkyl moieties of which have between 1 and about 10 carbon atoms are preferred.
  • Alkyl diisocyanates with alkyl moieties having between about 2 and about 6 carbon atoms are even more preferred.
  • Aromatic diisocyanaters such as toluene diisocyanate are also suitable for use with the present invention.
  • the solution concentration (w/v%) of the polymer should again be less than 10 percent so that cross-linking does not occur too rapidly. The polymer is dissolved first and the solvent may be heated slightly to dissolve the material.
  • the above-disclosed diisocyanate can be substituted with other bifunctional compounds.
  • suitable bifunctional compounds include diglycidyl ethers, dialdehydes such as glutaraldehyde, aliphatic and aromatic dicyanates such as Bisphenol A dicyanate, and diamines such as ethylene diamine or hexamethylene diamine.
  • Both the polymer matrices cross-linked with urethane linkages and the polymer matrices cross-linked with acyl semicarbazide linkages demonstrate high equilibrium water content and good mechanical strength and are therefore suitable for biomedical applications such as wound dressings and implant materials.
  • the hydrogel membranes from both types of cross-linked linkages are translucent and flexible films in the dry state.
  • the urethane cross-linked membranes are generally more opaque and somewhat abrasive on the surface, from the presence of N-hydroxy succinimide liberated during the cross-linking reaction. In the dry state, the membranes have extremely high tensile strength and elongation.
  • the membranes When equilibrated with water, the membranes begin to swell almost instantaneously, with the equilibrium reached in less than one hour.
  • the membranes are elastic in the swollen state, with tensile strength independent of the molecular weight of the poly(alkylene oxide) used.
  • the mechanical properties of the polymer matrices can be further improved by forming semi-IPN's with the matrices.
  • a linear, preformed second polymer is entrapped within the polymer matrices, which second polymer is chosen to be biocompatible and to contribute to the mechanical properties of the polymer matrix.
  • the second polymer need not be miscible with the polymers of the present invention.
  • the semi-IPN's of the present invention can be formed from polymers that would not be physically blendable by any other means.
  • second polymers suitable for use with the semi-IPN's of the present invention include poly(BPA carbonate) , poly(desaminotyrosyl tyrosine hexyl ester carbonate) , poly(lactic acid) , poly(caprolactone) , cellulose acetate, cellulose nitrate, poly(ethylene terephthaiate) poly(styrene) and poly(methyl methacrylate) , and the like.
  • Semi-IPN's can be prepared by either cross- linking reaction with both the polymers of Formulas I and III.
  • the semi-IPN's are prepared by dissolving an equimolar amount of the second polymer in the organic solvent with the polymer of Formula I or III.
  • the reaction then proceeds as described above, with respect to the preparation of polymer matrices cross-linked by either urethane or acyl semicarbazide linkages.
  • the second polymer is then entrapped within the cross-linked polymer matrix, as the polymer matrix is formed.
  • Both the cross-linked polymer matrices and the semi-IPN's can be used as means for drug delivery when utilized as wound dressings or biomedical implants.
  • the polymer matrices cross-linked by urethane linkages from the polymers of Formulas I and III are not cross-linked by means of their pendant functional groups, which remain available for drug attachment.
  • the trifunctional amine can also be quaternized for the attachment of pharmaceutically active compounds.
  • the polymer matrices that are cross-linked by acyl semicarbazide linkages covalently bond with the diisocyanate by means of their pendant functional groups, not all pendant functional groups participate in the cross-linking, and an excess of polymer can also be utilized, so that pendant functional groups remain uncross-1inked for drug attachment.
  • Wound dressings prepared from hydrogel membranes or semi-IPN's of the polymer matrices can thus incorporate antibiotics to promote wound healing.
  • the poly(alkylene oxide) copolymers of the present invention are versatile drug carriers derived from bioco patible components that are capable of being adapted to conjugate with a number of drug functional groups, so as not to be limited by drug structure or activity.
  • the drug carriers can be administered in a variety of forms that are dominated by the desirable properties of the poly(alkylene oxides) from which the carriers are derived.
  • reaction mixture was stirred at 25°C overnight and then evaporated to dryness on a rotary evaporator (water bath temperature maintained at 40°C). Another 100 mL of toluene was added and evaporated to remove all traces of phosgene.
  • To the polymeric chloroformate was added 30 L of dry toluene, 10 mL of methylene chloride, and 1.7 g (14.8 mmol) of N-hydroxy succinimide, and the mixture was stirred vigorously. The reaction flask was then cooled in an ice water bath and 1.5 g (14.9 mmol) of triethylamine was added gradually. Immediate precipitation of triethylamine hydrochloride was seen.
  • the organic layer was then dried over anhydrous MgS0 4 , filtered and concentrated.
  • the polymer was precipitated using cold ether, cooled to 4 ⁇ C and filtered to recover 6.7 g (67 percent) of the polymer.
  • 500 mg of the crude polymer was dissolved in 10 mL of distilled water and dialyzed against distilled water at room temperature for 48 hours using a SPECTRAPORTM membrane with a molecular weight cut-off of 12,000 to 14,000 daltons.
  • the purified polymer was extracted with methylene chloride, washed with saturated NaCl solution, dried and evaporated to obtain 263 mg (53 percent) of pure polymer.
  • Example 7 The procedure of Example 7 was followed substituting 5.5 mmol of hexamethylene diamine (Aldrich) for the 5.5 mmol of the ethylene diamine. Upon purification of the product, TLC in a 2:1 ratio ethanol to ammonia solution showed absence of free diamine.
  • the reaction mixture was neutralized after one hour by adding a few drops at 0.1 N HCl and extracted into methylene chloride. The extract was washed with saturated sodium chloride, dried over anhydrous magnesium sulfate, filtered and concentrated. The polymer was then precipitated with cold ether. After cooling for several hours, the product was collected on a Buchner funnel, washed with cold ether and dried under vacuum overnight. The recovery was 0.355 g, or 71 percent.
  • reaction product was then dissolved in water (50 mg/mL) and dialyzed against distilled water at room temperature using a SPECTRAPORTM membrane having a molecular weight cutoff of 12,000 to 14,000 daltons. After 24 hours the product was isolated by lyophilization.
  • Example 9 was repeated at pH's of 7.2 and 8.5, reaction times of 1.5 and 3 hours, and polymer to drug ratios of 1:1.
  • the mole-percent degree of drug attachment was determined by iodometric assay, which method measures only the active drug. As shown in Table I, the greatest degree of drug attachment was obtained with the conditions of Example 9, namely, a reaction time of 1 hour, a pH of 7.5 and a ratio of polymer to drug of 1:2.
  • Table I shows that decreasing the reaction time from 3 to 1.5 hours had no significant effect on the degree of drug attachment. This is expected because the active ester would not be stable under the conditions of the reaction for a long period of time. Also, the amount of active drug on the polymer is higher when the reaction is done at a lower pH. Because the iodometric assay is specific for active drug, this could mean that at a pH of 8.5 some of the beta-lactam units of the drug may have been hydrolyzed. Thus, the optimum reaction conditions appear to be mild enough to prevent significant cleavage of the beta-lactam ring while at the same time giving a high degree of conjugation.
  • a precipitate of dicyclohexyi urea formed and was removed by filtration.
  • the drug conjugate was precipitated with cold ether.
  • About 0.250 g of crude product was obtained which was purified by reprecipitation twice from isopropanol. TLC in methanol showed absence of free drug.
  • Acyclovir succinate is prepared by heating a solution of 0.2252 g of acyclovir (1 mmol) (Sigma), 0.200 g of succinic anhydride (2 mmoles) (Aldrich) and 0.14 mL of triethylamine in 15 mL of dry dimethylformamide at 60°C in an oil bath for 21 hours. The solution was then cooled and the volatile constituents were evaporated in vacuo, and the residue was taken up in 8 mL of ice water and acidified to pH 2 with 2 N HCl. A white precipitate was formed that was collected by filtration, thoroughly washed with ice water and dried in vacuo over P 2 0 5 at 40°C to yield 0.180 g (54 percent) of the product. The ester was then recrystallized from methanol and characterized by IR and 1 H NMR spectroscopy.
  • a mold was prepared by clamping two square glass plates together, one of which had a 5 cm diameter circular cavity. The contacting surfaces of the glass plates were coated with tri ethylchlorosilane (Aldrich) to prevent adhesion. The mold was placed on a level surface inside a glove box and further leveled using a carpenter's level. In a 100 mL beaker, 1.5 g of the poly(PEG-Lys) having pendant acyl hydrazine groups (0.67 mmol of hydrazine groups) of Example 5 was dissolved in 40 mL of methylene chloride. To this solution was added 1.5 g finely powdered sodium bicarbonate.
  • the suspension was stirred for one hour and the supernatant was tested for the presence of chloride ions with silver nitrate.
  • a few drops of the methylene chloride solution were placed into a test tube, the methylene chloride was evaporated, and the residue was reacted with a few drops of silver nitrate solution acetified with nitric acid.
  • the absence of any white turbidity indicated the complete neutralization and removal of hydrochloric acid.
  • the membranes obtained were semi-transparent and were somewhat hygroscopic, curling up when exposed to moisture in ambient air. When placed in water, the size of the films doubled in all dimensions, indicating a very large, swelling ratio. The swollen membranes were transparent.
  • the membrane was assayed with trinitrophenyl sulfonic acid (TNBS) (Fluka) to determine the extent of cross-linking. An excess of TNBS was used, and after reacting with the polymer, the unreacted TNBS was allowed to react with an excess of adipic hydrazide. The IR absorbance obtained at 500 nm was then used to calculate the amount of free hydrazides present on the cross-linked membrane. Using this method, it was found that 80-85 percent of all available hydrazides precipitated in cross-linking, leaving only 15- 20 percent of unreacted hydrazides on the cross-linked membrane.
  • Swelling measurements of the membrane were made by two methods. The dimensions of the dry membrane was measured and the membrane was allowed to swell in water. The increase in dimension was taken as a measure of swelling. Alternatively, the membrane was weighed before and after swelling and the increase in weight was taken as a measure of swelling. Both methods indicated that the membrane absorbs about 5 to 8 times its weight of water.
  • the tensile strength of the membrane was measured using strips of membrane 0.07 mm thick, 5 mm wide and 50 mm long. Measurements were made employing both dry and swollen membranes, the results of which are shown in Table II.
  • the membrane behaved like a perfect elastomer.
  • the membrane did not exhibit a yield point and a plot of stress against strain gave a straight line in accordance with Hooke's Law. This elastic behavior should make them ideal materials for wound dressing and use applications.
  • the stability of the membrane was investigated in acidic, basic and neutral media, the results of which are listed in Table III below. Small specimens of the membrane were placed in contact with a number of aqueous solutions of varying pH at room temperature and the time required for the complete disappearance of the membrane was noted. The membrane was generally found to be more stable in weakly acidic media and extremely unstable in alkaline media.
  • a 10 percent solution in freshly distilled tetrahydrofuran was prepared of a mixture of lithium triflate (Aldrich) and the poly(PEG-Lys-OEt) , prepared according to the procedure described in Example 2, in a polymer-electrolyte ratio of 4:1 by weight.
  • the polymer had a weight-average molecular weight of 140,000 daltons.
  • a film was cast from the solution as described above with respect to Example 16. A sticky film was obtained that was scraped from the glass plates, dried under high vacuum, and pressed into pellets at a pressure of 0.15 ton and a temperature of 27°C. This resulted in the formation of clear pellets.
  • Conductivity was measured using a 70 mg pellet having a thickness of 0.5 mm and a 300 mg pellet having a thickness of 2.0 mm. The conductivity of the pellets was evaluated using standard, established techniques.
  • the temperature dependance of the ionic conductivity of the polymer was then measured between room temperature and 40"C, which is near the melting point of the polymer.
  • An Arrhenius plot of conductivity vs. temperature (°K) is shown in FIG. 2. Conductivity increases with increasing temperature until the polymer becomes molten, at which point conductivity remains constant as temperature increases.
  • Tris(Aminoethyl) Amine In a 100 mL beaker, 1.87 g of the PEG- bis(Succinimidyl Carbonate) of Example 1 was dissolved in 20 ml of methylene chloride. In another beaker, 82 microliters (89 mg) of tris(aminoethylamine) was dissolved in 20 ml of methylene chloride. The triamine solution was added to the PEG solution with vigorous stirring. After about five minutes, films were cast of the solution following the procedure described above with respect to Example 16.
  • the stability of the membrane was investigated in acidic, basic and neutral media, as described above with respect to Example 16.
  • sodium hydroxide (0.01 and 0.1 N) the membrane disintegrated within a few hours.
  • acidic media and in phosphate buffer (pH 7.4) the membrane appeared to be stable for longer periods of time.
  • the accellerated degredation study of Example 16 was also performed, in which the membrane remained in ⁇ tact for more than a week.
  • An analysis of the buffer in which the accellerated stability study was conducted revealed that during the first 24 hours a small amount of PEG chains had leached from the cross-linked membrane, but throughout the following 72 hours, no more PEG was leached.
  • the poly(PEG-Lys) membrane cross-linked by diisocyanetohexane was prepared as in Example 16, using 210 mg of the poly(PEG-Lys) of Example 5 having acyl hydrazine functional groups, dissolved in 10 mL of methylene chloride. The free base was formed with sodium bicarbonate, and the solution was then filtered. Prior to the addition of four microliters (3.9 mg) of the hexamethylene disocyanate, 0.47 g of poly(caprolactone) (Union Carbide); (mw 72,000) was added to the filtrate, which was stirred for 30 minutes to dissolve the polymer completely. The poly(PEG-Lys) was cross-linked and films were cast following the procedure described above with respect to Example 16. The resulting membrane was hydrophilic and absorbed water with an equilibrium water content of 36%, whereas film made of poly(caprolactone) alone is hydrophobic.
  • the present invention is applicable to the production of polymers conjugated with various pharmaceutically active compounds representing a novel form of drug delivery.
  • the present invention is also applicable to the production of wound dressing of the crosslinked polymers and semi-interpenetrating polymer networks of the crosslinked polymers.
  • the present invention is also applicable to the production of electrochemical cells having solid electrodes of polymers combined with electrolyte salts.

Abstract

Copolymères de poly(oxydes d'alkylène) et de séquences d'aminoacides ou de peptides, ces séquences possédant des groupes fonctionnels pendants qui peuvent être conjugués avec des composés pharmaceutiquement actifs pour des systèmes d'apport de médicament et réticulés pour former des matrices polymères fonctionnelles en tant que membranes d'hydrogel. Les copolymères peuvent aussi être transformées en matériaux conducteurs. On décrit des procédés pour préparer les polymères et pour former les conjugués de médicaments, les membranes d'hydrogel et les matériaux conducteurs.
PCT/US1991/004797 1990-07-06 1991-07-08 Copolymeres d'aminoacides et de poly(oxydes d'alkylene), vehicules de medicament et copolymeres charges bases sur lesdits vehicules WO1992000748A1 (fr)

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CA002086528A CA2086528A1 (fr) 1990-07-06 1991-07-08 Copolymeres d'acide amine de type poly(oxyde d'alkylene) et vehicules de medicaments et copolymeres a charge a base de ceux-ci
AU82355/91A AU8235591A (en) 1990-07-06 1991-07-08 Poly(alkylene oxide) amino acid copolymers and drug carriers and charged copolymers based thereon
JP91512668A JPH05508879A (ja) 1990-07-06 1991-07-08 ポリ（アルキレンオキサイド）アミノ酸コーポリマ並びにこれに基づく薬剤キャリアおよび帯電コーポリマ

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US54949490A 1990-07-06 1990-07-06
US549,494 1990-07-06
US726,301 1991-07-05

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