Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—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
  • A61K47/58—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 by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/482—Serine endopeptidases (3.4.21)
  • A61K38/4826—Trypsin (3.4.21.4) Chymotrypsin (3.4.21.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21001—Chymotrypsin (3.4.21.1)

Definitions

• Protein and peptide therapeutic agents comprise an increasing share of the pharmaceutical market with biological drugs making up about $4.5 billion in U.S. sales in 2008.
• biological drugs are monoclonal antibodies, hormones, and therapeutic enzymes.
• therapeutic proteins are susceptible to short half lives, proteolysis, opsonization, and can cause immunogenic responses, each resulting in undesirable pharmacokinetics.
• a variety of technologies have been developed to improve these shortfalls. Included among these technologies are amino acid manipulation, genetic fusion of immunoglobulin domains or serum proteins, and conjugation with natural and synthetic polymers.
• PEG polyethylene glycol
• PEGylation is known to change the physical and chemical properties of the biomolecule, including conformation, electrostatic binding, and hydrophobicity, and can result in improved pharmacokinetic properties for the drug.
• Advantages of PEGylation include improvements in drug solubility and diminution of immunogenicity, increased drug stability and circulatory life once administered, and reductions in proteolysis and renal excretion, which allows for reduced frequency of dosing.
• PEGylation technology has been advantageously applied to therapeutic proteins and oligonucleotides to provide new drugs that have been approved by the U.S. FDA: adensosine deaminase (Pegademenase), asparaginase (Pegaspargase), G-CSF (Pegfilgrastim), interferon- 2a (Peginterferon-a2a), interferon- 2b (Peginterferon-a2b), hGH (Pegvisomant), anti-VEGF aptamer (Pegaptanib), erythropoietin (PEG-EPO), and anti-TNF a Fab' (Certolizumab).
• adensosine deaminase Pegademenase
• asparaginase Pegaspargase
• G-CSF Pegfilgrastim
• interferon- 2a Peginterferon-a2a
• PEGylated therapeutic enzymes presently approved and on the market include depleting enzymes that are used to eliminate certain amino acids from the blood stream to starve growing tumor cells. Examples include PEG-asparaginase, PEG-methioninase, and PEG-arginine deiminase.
• PEG-asparaginase PEG-methioninase
• PEG-arginine deiminase PEG-arginine deiminase.
• their relatively small amino acid substrates are able to diffuse through the PEG layer, enter the active site of the enzyme, and are effectively disposed of as designed.
• biomolecules requiring polymeric protection and that act on larger substrates have not been able to be developed because of the required level of PEGylation to retain the enzyme in the body, which renders the active site inaccessible.
• PEGylation and PEGylated biomolecules are not without their drawbacks. Due to its strong attraction to water, PEG is known to take on a hydrodynamic diameter significantly greater than would be predicted by their molecular weight. This ballooning effect can be attributed to the reduction in the ability of the body to recognize the biomolecule and results in a decrease in the biomolecule's activity.
• a murine A7 monoclonal antibody PEGylated with ten (10) 5 kDa PEG has an activity that is 10% that of the unmodified antibody.
• an interferon-a2a PEGylated with a single 40 kDa PEG has an activity that is 7% that of the unmodified protein.
• modification or hindrance of a biomolecule's active site resulting from PEGylated is a serious problem to be avoided in the development of a biopharmaceutical product.
• the invention provides polymer biomolecule conjugates, reagents and methods for making the conjugates, and methods for using conjugates.
• the invention provides a zwitterionic polymer bioconjugate comprising one or more zwitterionic polymers covalently coupled to a biomolecule.
• the zwitterionic polymer comprises a plurality of repeating units, each repeating unit having the formula:
• Ri is selected from the group consisting of hydrogen, fluorine, trifluoromethyl, C1-C6 alkyl, and C6-C12 aryl groups;
• R 2 and R 3 are independently selected from the group consisting of alkyl and aryl, or taken together with the nitrogen to which they are attached form a cationic center;
• Li is a linker that covalently couples the cationic center [N + (Rs)(R 6 )] to the polymer backbone [-(CH 2 -CR 4 ) n -] ;
• A is C, SO, S0 2 , or PO;
• X " is a counter ion associated with the cationic center
• n is an integer from 1 to about 10,000.
• the invention provides a mixed charge copolymer bioconjugate comprises one or more mixed charge polymers covalently coupled to a biomolecule.
• the mixed charge copolymer comprises a plurality of repeating units, each repeating unit having the formula:
• R 4 and R5 are independently selected from hydrogen, fluorine, trifluoromethyl, C1-C6 alkyl, and C6-C12 aryl groups;
• R 6 , R7, and R 8 are independently selected from alkyl and aryl, or taken together with the nitrogen to which they are attached form a cationic center;
• L 3 is a linker that covalently couples the cationic center [N + (R6)(R7)(R8)] to the polymer backbone;
• X " is the counter ion associated with the cationic center
• n is an integer from 1 to about 10,000;
• p is an integer from 1 to about 10,000.
• biomolecules include proteins, nucleic acids, carbohydrates, lipids, and small molecules.
• the biomolecule has increased thermal stability relative to its unconjugated form.
• the conjugate resists denaturation by chaotropes and is suitable for protein loading with anionic radiotherapeutics (e.g., I 12 s " and I 13 5 ⁇ ).
• compositions that include one or more of the conjugates of the invention and a pharmaceutically accepted carrier or diluent.
• FIGURE 1 is a schematic illustration of the preparation of a representative zwitterionic polymer-protein conjugate of the invention.
• a representative NHS-ester terminated zwitterionic polymer is synthesized by atom transfer radical polymerization using a representative zwitterionic monomer (CBMA) and an NHS-ester initiator.
• the NHS-ester terminated zwitterionic polymer is conjugated to the protein by reacting the NHS-ester with available amino groups (e.g., -amino groups of lysine residues).
• FIGURES 2A and 2B compare gel permeation chromatographs for CT-PEG (LD, MD, and HD) (FIGURE 2A) and representative zwitterionic polymer-protein conjugates of the invention, CT-pCB (LD, MD, and HD) (FIGURE 2B).
• CT-pCB conjugates are pCB are M n Eq. Unconjugated CT is included for comparison.
• FIGURES 3A and 3B compare thermal stability for CT-PEG (LD, MD, and HD) and representative zwitterionic polymer-protein conjugates of the invention, CT-pCB (LD, MD, and HD).
• CT-pCB conjugates are pCB M n Eq. (FIGURE 3 A) and pCB R h Eq. (FIGURE 3B). Unconjugated CT is included for comparison.
• FIGURE 4 compares chemical stability threw incubation in urea for CT-PEG
• CT-pCB (LD, MD, and HD) and representative zwitterionic polymer-protein conjugates of the invention, CT-pCB (LD, MD, and HD).
• CT-pCB conjugates are pCB M n Eq. and pCB R h Eq. Unconjugated CT is included for comparison.
• FIGURE 5 compares stability of CT-PEG (LD, MD, and HD) and representative zwitterionic polymer-protein conjugates of the invention, CT-pCB (LD, MD, and HD), in 100% serum over seven days. Unconjugated CT is included for comparison.
• FIGURES 6A and 6B compare the effect of polymer type and degree of conjugation on k cat with a peptide-based substrate (FIGURE 6A) and a small molecule substrate (FIGURE 6B).
• FIGURES 7A and 7B compare the effect of polymer type and degree of conjugation on Michaelis Constant (k m ) with a peptide-based substrate (FIGURE 7A) and a small molecule substrate (FIGURE 7B).
• FIGURES 8A and 8B compare the effect of polymer type and degree of conjugation on catalytic efficiency (k cat /k m ) with a peptide-based substrate (FIGURE 8A) and a small molecule substrate (FIGURE 8B).
• the invention provides polymer biomolecule conjugates, reagents and methods for making the conjugates, and methods for using conjugates.
• the invention provides a zwitterionic polymer biomolecule conjugate.
• zwitterionic polymer biomolecule conjugate or “zwitterionic polymer bioconjugate” refers to a biomolecule that has been modified by conjugation to a zwitterionic polymer.
• the invention provides a mixed charge copolymer biomolecule conjugate.
• mixed charge copolymer biomolecule conjugate or “mixed charge copolymer bioconjugate” refers to a biomolecule that has been modified by conjugation to a mixed charge copolymer.
• the biomolecule can be modified to include one or more zwitterionic polymers, or one or more mixed charge polymers, which are covalently coupled to the biomolecule.
• the zwitterionic polymer bioconjugate comprises one or more zwitterionic polymers covalently coupled to a biomolecule.
• the zwitterionic polymer comprises a plurality of repeating units, each repeating unit having the formula (I):
• Ri is selected from the group consisting of hydrogen, fluorine, trifluoromethyl, C1-C6 alkyl, and C6-C12 aryl groups;
• R 2 and R 3 are independently selected from the group consisting of alkyl and aryl, or taken together with the nitrogen to which they are attached form a cationic center;
• Li is a linker that covalently couples the cationic center [N + (Rs)(R 6 )] to the polymer backbone [-(CH 2 -CR 4 ) n -];
• A is C, SO, S0 2 , or PO;
• X " is a counter ion associated with the cationic center
• n is an integer from 1 to about 10,000;
• the mixed charge copolymer bioconjugate comprising one or more mixed charge copolymers covalently coupled to a biomolecule.
• the term "mixed charge copolymer" refers to a copolymer having a polymer backbone, a plurality of positively charged repeating units, and a plurality of negatively charged repeating units. In the practice of the invention, these copolymers may be prepared by polymerization of an ion-pair comonomer.
• the mixed charge copolymer includes a plurality of positively charged repeating units, and a plurality of negatively charged repeating units.
• the mixed charge copolymer is substantially electronically neutral.
• substantially electronically neutral refers to a copolymer that imparts advantageous nonfouling properties to the copolymer.
• a substantially electronically neutral copolymer is a copolymer having a net charge of substantially zero (i.e., a copolymer about the same number of positively charged repeating units and negatively charged repeating units).
• the ratio of the number of positively charged repeating units to the number of the negatively charged repeating units is from about 1: 1.1 to about 1:0.5.
• the ratio of the number of positively charged repeating units to the number of the negatively charged repeating units is from about 1: 1.1 to about 1:0.7. In one embodiment, the ratio of the number of positively charged repeating units to the number of the negatively charged repeating units is from about 1: 1.1 to about 1:0.9.
• Ion Pair Comonomers In one embodiment, the copolymers are prepared by copolymerization of suitable polymerizable ion pair comonomers.
• the crosslinked polymer e.g., hydrogel
• R 4 and R 5 are independently selected from hydrogen, fluorine, trifluoromethyl, C1-C6 alkyl, and C6-C12 aryl groups;
• R 6 , R 7 , and Rg are independently selected from alkyl and aryl, or taken together with the nitrogen to which they are attached form a cationic center;
• L 3 is a linker that covalently couples the cationic center [N + (R6)(R 7 )(R8)] to the polymer backbone;
• X " is the counter ion associated with the cationic center
• n is an integer from 1 to about 10,000;
• p is an integer from 1 to about 10,000.
• R 7 and R 8 are C1-C3 alkyl.
• R 6 , R7, and Rg are independently selected from alkyl and aryl, or taken together with the nitrogen to which they are attached form a cationic center.
• R 6 , R 7 , and R 8 are C1-C3 alkyl.
• L 4 is a C1-C20 alkylene chain.
• Representative L 4 groups include -(CH 2 ) n -, where n is 1-20 (e.g., 1, 3, or 5)
• A is C or SO.
• n is an integer from 5 to about 5,000.
• the polymer backbones include vinyl backbones (i.e., -C(R')(R")-C(R'")(R"")-, where R', R", R'", and R'" are independently selected from hydrogen, alkyl, and aryl) derived from vinyl monomers (e.g., acrylate, methacrylate, acrylamide, methacrylamide, styrene).
• N + is the cationic center.
• the cationic center is a quaternary ammonium (e.g., N bonded to L 1; R 2 , R 3 , and L 2 ).
• other useful cationic centers include imidazolium, triazaolium, pyridinium, morpholinium, oxazolidinium, pyrazinium, pyridazinium, pyrimidinium, piperazinium, and pyrrolidinium.
• Ri-Rg are independently selected from hydrogen, alkyl, and aryl groups.
• Representative alkyl groups include CI -CIO straight chain and branched alkyl groups.
• the alkyl group is further substituted with one of more substituents including, for example, an aryl group (e.g., -CH 2 C 6 H 5 , benzyl).
• R 2 and R , and R 6 , R 7 , and R 8 are methyl.
• Ri-Rg are methyl.
• Representative aryl groups include C6-C12 aryl groups including, for example, phenyl. For certain embodiments of the above formulas, R 2 and R , and/or R 6 , R 7 , and R 8 are taken together with N + form the cationic center.
• Li is a linker that covalently couples the cationic center to the polymer backbone.
• Li includes a functional group (e.g., ester or amide) that couples the remainder of Li to the polymer backbone (or polymerizable moiety for the monomers).
• a functional group e.g., ester or amide
• Li can include an C1-C20 alkylene chain.
• L 2 is a linker that covalently couples the cationic center to the anionic group.
• I ⁇ can be a C1-C20 alkylene chain.
• Representative L 2 groups include -(CH 2 ) n -, where n is 1-20 (e.g., 1, 3, or 5).
• L 3 is a linker that covalently couples the cationic center to the polymer backbone.
• L 3 includes a functional group (e.g., ester or amide) that couples the remainder of L 3 to the polymer backbone (or polymerizable moiety for the monomers).
• L can include an C1-C20 alkylene chain.
• L 4 is a linker that covalently couples the anionic group to the polymer backbone.
• L 4 can be a C1-C20 alkylene chain.
• Representative L 4 groups include -(CH 2 ) n -, where n is 1-20 (e.g., 1, 3, or 5).
• alkyl groups include C1-C30 straight chain and branched alkyl groups.
• the alkyl group is further substituted with one of more substituents including, for example, an aryl group (e.g., -CH 2 C 6 H 5 , benzyl).
• aryl groups include C6-C12 aryl groups including, for example, phenyl including substituted phenyl groups (e.g., benzoic acid).
• X " is the counter ion associated with the cationic center.
• the counter ion can be the counter ion that results from the synthesis of the cationic polymers or the monomers (e.g., CI " , Br " , ⁇ ).
• the counter ion that is initially produced from the synthesis of the cationic center can also be exchanged with other suitable counter ions.
• Suitable counter ions include salicylic acid (2-hydroxybenzoic acid), benzoate, and lactate.
• the size (e.g., n or M n ) of the zwitterionic polymer conjugated to the biomolecule can be varied and depends on the nature of the biomolecule.
• the degree of polymerization (DP or n), number average molecular weight (M n ), and the ratio of weight average and number average molecular weights (M w /M n .), also known as polydispersity index can vary.
• the polymers have a degree of polymerization (n) from 1 to about 10,000.
• n is from about 10 to about 5,000.
• n is from about 100 to about 3,500.
• the polymers have a number average molecular weight (M n ) of from about 200 to about 2,000,000 Da. In one embodiment, M n is from about 2,000 to about 100,000 Da. In another embodiment, M n is from about 20,000 to about 80,000 Da. In one embodiment, the polymers have a ratio of weight average and number average molecular weight (M w /M n .) of from about 1.0 to about 2.0. In one embodiment, M w /M n . is from about 1.1 to about 1.5. In another embodiment, M w /M n . is from about 1.2 to about 2.0.
• the zwitterionic polymer bioconjugate comprises one or more zwitterionic polymers covalently coupled to a biomolecule.
• Suitable biomolecules include biopolymers (e.g., proteins, peptides, oligonucleotides, polysaccharides), lipids, and small molecules.
• the biomolecule is a protein or peptide.
• the terms “protein,” “polypeptide,” and “peptide” can be used interchangeably.
• peptides range from about 5 to about 5000, 5 to about 1000, about 5 to about 750, about 5 to about 500, about 5 to about 250, about 5 to about 100, about 5 to about 75, about 5 to about 50, about 5 to about 40, about 5 to about 30, about 5 to about 25, about 5 to about 20, about 5 to about 15, or about 5 to about 10 amino acids in size.
• Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation. In some embodiments, polypeptides may comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof, as described herein.
• the therapeutic agent may be a hormone, erythropoietin, insulin, cytokine, antigen for vaccination, growth factor.
• the therapeutic agent may be an antibody and/or characteristic portion thereof.
• antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric (i.e., "humanized"), or single chain (recombinant) antibodies.
• antibodies may have reduced effector functions and/or bispecific molecules.
• antibodies may include Fab fragments and/or fragments produced by a Fab expression library (e.g. Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments).
• the biomolecule is a nucleic acid (e.g., DNA, RNA, derivatives thereof).
• the nucleic acid agent is a functional RNA.
• a "functional RNA" is an RNA that does not code for a protein but instead belongs to a class of RNA molecules whose members characteristically possess one or more different functions or activities within a cell. It will be appreciated that the relative activities of functional RNA molecules having different sequences may differ and may depend at least in part on the particular cell type in which the RNA is present.
• RNAi-inducing entities e.g., short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and microRNAs
• ribozymes e.g., tRNAs, rRNAs, RNAs useful for triple helix formation.
• the nucleic acid agent is a vector.
• vector refers to a nucleic acid molecule (typically, but not necessarily, a DNA molecule) which can transport another nucleic acid to which it has been linked.
• a vector can achieve extra-chromosomal replication and/or expression of nucleic acids to which they are linked in a host cell.
• a vector can achieve integration into the genome of the host cell.
• vectors are used to direct protein and/or RNA expression.
• the protein and/or RNA to be expressed is not normally expressed by the cell.
• the protein and/or RNA to be expressed is normally expressed by the cell, but at lower levels than it is expressed when the vector has not been delivered to the cell.
• a vector directs expression of any of the functional RNAs described herein, such as RNAi-inducing entities, ribozymes.
• Carbohydrate Agents In some embodiments, the biomolecule is a carbohydrate. In certain embodiments, the carbohydrate is a carbohydrate that is associated with a protein (e.g. glycoprotein, proteogycan). A carbohydrate may be natural or synthetic.
• a carbohydrate may also be a derivatized natural carbohydrate.
• a carbohydrate may be a simple or complex sugar.
• a carbohydrate is a monosaccharide, including but not limited to glucose, fructose, galactose, and ribose.
• a carbohydrate is a disaccharide, including but not limited to lactose, sucrose, maltose, trehalose, and cellobiose.
• a carbohydrate is a polysaccharide, including but not limited to cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), methylcellulose (MC), dextrose, dextran, glycogen, xanthan gum, gellan gum, starch, and pullulan.
• a carbohydrate is a sugar alcohol, including but not limited to mannitol, sorbitol, xylitol, erythritol, malitol, and lactitol.
• the biomolecule is a lipid.
• the lipid is a lipid that is associated with a protein (e.g., lipoprotein).
• Exemplary lipids that may be used in accordance with the present invention include, but are not limited to, oils, fatty acids, saturated fatty acid, unsaturated fatty acids, essential fatty acids, cis fatty acids, trans fatty acids, glycerides, monoglycerides, diglycerides, triglycerides, hormones, steroids (e.g., cholesterol, bile acids), vitamins (e.g., vitamin E), phospholipids, sphingolipids, and lipoproteins.
• the lipid may comprise one or more fatty acid groups or salts thereof.
• the fatty acid group may comprise digestible, long chain (e.g., C8-C50), substituted or unsubstituted hydrocarbons.
• the fatty acid group may be one or more of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, or lignoceric acid.
• the fatty acid group may be one or more of palmitoleic, oleic, vaccenic, linoleic, alpha- linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic, eicosapentaenoic, docosahexaenoic, or erucic acid.
• the therapeutic agent is a small molecule and/or organic compound with pharmaceutical activity.
• the therapeutic agent is a clinically-used drug.
• the drug is an anticancer agent, antibiotic, anti-viral agent, anti-HIV agent, anti-parasite agent, antiprotozoal agent, anesthetic, anticoagulant, inhibitor of an enzyme, steroidal agent, steroidal or non-steroidal anti-inflammatory agent, antihistamine, immunosuppressant agent, anti-neoplastic agent, antigen, vaccine, antibody, decongestant, sedative, opioid, analgesic, anti-pyretic, birth control agent, hormone, prostaglandin, progestational agent, anti-glaucoma agent, ophthalmic agent, anti-cholinergic, analgesic, anti-depressant, anti- psychotic, neurotoxin, hypnotic, tranquilizer, anti-convulsant, muscle relaxant, anti- Parkinson agent, anti-spas
• a small molecule agent can be any drug.
• the drug is one that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regulatory body.
• drugs approved for human use are listed by the FDA under 21 C.F.R. ⁇ 330.5, 331 through 361, and 440 through 460, incorporated herein by reference; drugs for veterinary use are listed by the FDA under 21 C.F.R. ⁇ 500 through 589, incorporated herein by reference. All listed drugs are considered acceptable for use in accordance with the present invention.
• Biomolecules that can be advantageously conjugated with one or more zwitterionic polymer include biopharmaceuticals that have been approved for use.
• Biomolecules and biopharmaceuticals that have been PEGylated exemplify a class of biopharmaceuticals suitable for conjugation with one or more zwitterionic polymers to provide a zwitterionic polymer bioconjugate of the invention.
• the zwitterionic polymer can be conjugated to the biomolecule by covalent coupling of a suitably reactive polymer (e.g., active ester) with a native biomolecule or biomolecule modified to include a suitably reactive group (e.g., amino group of a native lysine residue for a protein or an amino group that has been incorporated into a protein or oligonucleotide).
• a suitably reactive polymer e.g., active ester
• a suitably reactive group e.g., amino group of a native lysine residue for a protein or an amino group that has been incorporated into a protein or oligonucleotide.
• the zwitterionic polymer bioconjugate can be prepared by covalently coupling one or more polymerization initiators to the biomolecule followed by polymerization using a suitable zwitterionic monomer to graft the polymer from the biomolecule (i.e., in situ polymerization).
• the one or more polymerization initiators are coupled to the biomolecule in the same way as described above for the direct conjugation of the polymer to the biomolecule.
• the in situ polymerization method may only be used for biomolecules that are stable to the reaction conditions required for polymerization.
• the invention provides a composition that includes the zwitterionic polymer bioconjugate of the invention and a pharmaceutically acceptable carrier or diluent.
• Suitable carriers and diluents include those known in the art, such as saline and dextrose.
• the invention provides a method for administration a zwitterionic polymer bioconjugate comprising administering a zwitterionic polymer bioconjugate of the invention to a subject in need thereof.
• the invention provides a method for treating a disease or condition treatable by administration of a biomolecule.
• a zwitterionic polymer conjugate of the biomolecule is administered to a subject in need thereof.
• the biomolecule is exemplified by an enzyme, alpha-chymotrypsin (CT).
• CT alpha-chymotrypsin
• Certain properties of the bioconjugate are compared to a corresponding PEGylated conjugate.
• Stability A thermal stability test was performed, measuring the effect of temperature on enzyme activity. N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide, a 625 Da peptide-based substrate was used to measure activity. From the data in FIGURE 3A, it is seen that both pCB and PEG have stabilizing effects at elevated temperatures.
• Urea as a denaturant, is theorized to interact with the backbone of proteins and known to cause unfolding and loss of function.
• a nearly saturated urea solution was used to measure enzyme activity in situ. The results can be seen in FIGURE 4. Time points of 2, 8 and 32 hours were chosen. At early time points no significant differences were seen. At 8 and 32 hours, greater instability was seen in the CT control and lower conjugated PEG samples. All pCB conjugates showed increased stability compared to the control even at lower degrees of conjugation. Only the highly conjugated PEG samples (HD) had activity comparable to all the other pCB conjugates. This shows that CT-pCB conjugates have a very unique ability to chemically stabilize proteins even with very little attached polymer.
• FIGURE 5 shows the serum stability of CT conjugates over a one week period. On day seven it can be seen that all pCB conjugates have a higher retained activity than all the PEG conjugates including the unconjugated control.
• CT-PEG conjugates were compared to CT-pCB conjugates of equivalent molecular weight (pCB M n Eq) and equivalent hydrodynamic size (pCB R h Eq).
• pCB M n Eq equivalent molecular weight
• pCB R h Eq equivalent hydrodynamic size
• Two substrates were used to evaluate the kinetic effects of the polymers: N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide, a 625 Da peptide-based substrate, and resorufin bromoacetate, a smaller (335 Da), non-peptide-based substrate.
• Resorufin bromoacetate was included as a negative control knowing that small, more hydrophobic substrates are much less inhibited to complex with enzymes by conjugated polymer.
• FIGURE 6 shows k cat values for all conjugates tested.
• k cat is the maximum rate of substrate conversion into product. This is independent of the substrate's ability to access the active site, which is represented by K m .
• k cat occurs post-binding. This can be observed when substrate concentrations are very high.
• An increase in k cat represents a difference inherent to the enzyme-substrate complex. From FIGURE 6 is can be seen that all PEG and pCB M n Eq conjugates do not show significant change in k cat . For these conjugates, the polymers act passively. However, for the pCB R h Eq samples, there appears to be an increase in k cat . This is evidence of the polymer acting directly on the enzyme- substrate complex.
• the increase in k cat is more apparent and correlates more with the degree of conjugation. This is hypothesized to be due to the substrates amide chemistry, and shows that the polymer's effect on the enzyme and the peptide substrate are additive.
• FIGURE 7 shows the Michaelis Constant (K m ) values for all polymer protein conjugates.
• K m is the substrate concentration at which 50% maximal activity (k cat ) is reached and approximates the affinity of substrate for the enzyme.
• PEG has increasingly adverse effects, shown by an increase in K m with increasing incorporation of polymer.
• CT-pCB conjugates were seen not to have any inhibitory effects.
• K m for the pCB R h Eq conjugates was seen to decrease as more polymers were incorporated onto the protein.
• K m is greatly affected by the size and chemistry of conjugated polymers. As seen in the negative control, there is little trend with the small molecule substrate in comparison. More hydrophobic small molecule substrates are known to diffuse very easily in and out of the polymer shell of enzyme-polymer conjugates.
• FIGURE 8 shows the catalytic efficiency (k cat /K m ) for all polymer protein conjugates. This is a universal term to define the total system's efficiency on catalyzing a substrate. These take into account the enzyme's ability to complex with a substrate and effectively convert it into product. Due to the combination of reasons mentioned above, the increase in K m has resulted in low catalytic efficiencies for PEG conjugates. In contrast, the decrease in K m and increase in k cat for the pCB R h Eq has resulted in a large increase in catalytic efficiency. To note, the highly conjugated PEG conjugate is seen to have a 47.6% reduction in catalytic efficiency, while the highly conjugated pCB R h Eq conjugate is seen to have a 220.4% increase in activity.
• N-Hydroxysuccinimide (2.26 g, 19.6 mmol) and 2-bromopropionic acid (1.45 ml, 16.4 mmol) were dissolved, in 500 ml of anhydrous dichloromethane in a round-bottomed flask, with a magnetic stirrer. The flask was cooled to 0°C and a solution of N,N'-dicyclohexylcarbodiimide (3.35 g, 16.34 mmol) in DCM (25 ml) was added dropwise. After stirring at room temperature overnight the reaction mixture was filtered and the solvent removed under reduced pressure to give a yellow solid.
• Atom transfer radical polymerization was carried out in anhydrous dimethylformamide (DMF) using a Cu( 1 )Br/HMTETA catalyst (FIGURE 1A).
• DMF and the liquid HMTETA ligand are separately purged of oxygen by bubbling with nitrogen, lg (3.67 mmol) of CBMA-l-tBut monomer and 125 mg (0.5 mmol) of NHS-initiator were added to a Schlenk tube.
• To a second Schlenk tube was added 71.7 mg (0.5 mmol) of Cu(l)Br. Both tubes were deoxygenated by cycling between nitrogen and vacuum three times.
• pCB conjugates with a larger pCB polymer were also prepared ("pCB R h Eq") with equivalent size determined by GPC for kinetic analysis. Only the pCB M n Eq was used the stability studies to prove the smaller of the two prepared pCB polymers still had more advantageous effects when compared to 5kDa PEG.
• CT Conjugation to surface exposed lysine ⁇ -amino groups of CT with NHS activated pCB and PEG polymers.
• CT was prepared at 5 mg/mL in 200 mM HEPES buffer at pH 8.0 (FIGURE 1C). Dry polymer was added directly to the enzyme solution. The feed ratios of polymer to protein were adjusted to control the degree of conjugation (number of polymers/CT protein). The reaction solution was stirred for 2 h on ice and then placed in the refrigerator overnight. Conjugates were purified from unreacted polymer by several buffer exchanges with phosphate buffered saline, pH 7.4, using ultrafiltration (30,000 Da MWCO). Glycerol was added at 40% after final filtration and stored at -20°C. The degree of modification (number of polymers/CT protein) was determined by a TNBS assay. The number of polymers per protein are set forth in Table 1. Conjugation was also confirmed in the GPC chromato grams (FIGURE 2).

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