WO2012075117A2

WO2012075117A2 - Small molecule-polymer conjugates and methods of making same

Info

Publication number: WO2012075117A2
Application number: PCT/US2011/062595
Authority: WO
Inventors: Nicholas Lee Hammond; Lisa Kay Kemp; Tyler Weis Hodges
Original assignee: Ablitech, Inc.
Priority date: 2010-12-01
Filing date: 2011-11-30
Publication date: 2012-06-07
Also published as: WO2012075117A3

Abstract

Disclosed herein are small molecule-polymer conjugates of Formula 1, as well as methods of preparing same, uses of same, and kits for preparing same.

Description

SMALL MOLECULE-POLYMER CONJUGATES AND METHODS OF MAKING

SAME

[0001] This application claims the benefit of U.S. Provisional Application No.

61/418,639, filed December 1, 2010, which is incorporated herein by reference in its entirety.

Background

[0002] Recent advancements in the identification of biologically relevant targets has led to the discovery of pharmaceutically and/or therapeutically useful small molecules, for example small biomolecules (lipids, phospholipids, glycolipids, sterols, vitamin, hormones, neurotransmitters, carbohydrates, sugars, disaccharides, natural products), bimolecular monomers (amino acids, nucleotides, monosaccharides), and bimolecular polymers (peptides, oligopeptides, polypeptides, proteins, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), oligosaccharides, polysaccharides, and lignin). Though the identification of biologically relevant targets has grown considerably, significant issues have plagued the use of such small molecules, particularly the ability to provide stability, deliver increase bioavailability in vivo, and reduce side effects.

[0003] Among the particular problems is the instability or insolubility of small molecules. Such small molecules are generally susceptible to environmental degradation in vitro and enzymatic degradation in vivo. Additionally, solubility issues prevent active small molecule leads from moving forward due to poor pharmacokinetic profiles. Furthermore, the inability to selectively deliver the small molecules to their site of action has limited their therapeutic utility.

[0004] Attempts to solve these pervasive problems have been plagued with further problems. For example, extensive efforts have been made to chemically modify small molecules to improve their pharmacokinetic profile, to provide site-specific activity, and to encapsulate the small molecules within polymer structures. These attempted solutions, however, are not always successful and use significant resources. The solution of this problem would thus address a long felt need in the field of small molecule pharmaceutical development.

Summary

[0005] It is thus an object of the disclosure to provide a method of stabilizing small molecule drugs by modifying the small molecules to provide small molecule conjugates that solve the above-identified problems by, for example, preventing degradation, increasing solubility, increasing half-life, and providing selective targeting. Further, it is an object of the disclosure to provide small molecule conjugates that have enhanced stability, increased circulation times, and selectivity, yet retain their efficacy.

[0006] In one aspect of the disclosure, provided herein is a small molecule-polymer conjugate(s) of Formula

Formula 1

where the linker L is independently a 1 - 20 atom linear or branched linker; the polymer is independently a biocompatible polymer; X is independently an atom of attachment to the small molecule that is O, NH, NR, or S, where R is part of the small molecule; n is an integer from about 1 to about 30; and the X— L bond is degradable. The L— triazole bond can be to either carbon of the triazole ring, and is represented by the loose bond as illustrated in Formula 1. In certain embodiments, the small molecule of Formula 1 is a natural product, nucleic acid, amino acid, peptide, synthetic drug, synthetic drug lead, or sugar.

[0007] In another aspect of the disclosure, provided herein is a method of preparing a small molecule -polymer conjugate(s) of Formula 1. The method comprises: (a) reacting the small molecule with an alkyne-containing electrophilic reagent, and (b) reacting the alkyne- modified small molecule with an azide-containing polymer or mixture of azide-containing polymers. The reaction is illustrated in Scheme 1 below: small molecule -X-L-

polymer

Scheme 1

where the polymer, X, L, and n are as defined above, and Q is a leaving group. In another aspect of the disclosure, provided herein is the use of a small molecule-polymer conjugate(s) of Formula 1, for example for the treatment, prevention, or amelioration of a disease as set forth herein.

[0008] In another aspect of the disclosure, provided herein is a kit suitable for preparing a small molecule -polymer conjugate(s) of the disclosure, the kit comprising an alkyne- containing electrophilic reagent in a first container, an azide-containing biocompatible polymer in a second container, and instructions for their use.

[0009] In various embodiments of the above-described aspects of the disclosure, the small molecule is gemcitabine, vanillin, elvucitabine, prioxicam, amikacin, morphine, dapsone, memantine, temazepam, or raloxifene.

[0010] Those skilled in the art will realize that this invention is capable of embodiments that are different from those shown and details of the devices and methods can be changed in various manners without departing from the scope of this invention. Accordingly, the drawings and descriptions are to be regarded as including such equivalent embodiments as do not depart from the spirit and scope of this invention.

Brief Description of Drawings

[0011] For a more complete understanding and appreciation of this invention, and its many advantages, reference will be made to the following detailed description taken in conjunction with the accompanying drawings.

[0012] FIG. 1 shows the reaction of vanillin with propargyl chloroformate.

[0013] FIG. 2 contains carbon- 13 NMR data for the starting material and reaction product of the reaction shown in FIG. 1.

[0014] FIG. 3 illustrates the reaction of the alkyne-containing vanillin produced in the reaction of FIG. 1 with an azide-containing polymer, to form a small molecule-polymer conjugates of the disclosure.

[0015] FIG. 4 illustrates an embodiment of the second step of the method described herein, illustrating the confirmation of the reaction of alkyne-modified vanillin with the azide containing polymer by carbon NMR.

[0016] FIG. 5 illustrates an embodiment of the first step of the method described herein, wherein one or more groups of gemcitabine are reacted with propargyl chloroformate.

[0017] FIG. 6 illustrates an embodiment of the second step of the method described herein, where one or more alkyne groups of the modified small molecule gemcitabine, the product of the first step of the method, is reacted with an azide-containing polymer, to form a small molecule-polymer conjugate(s) of the disclosure.

Detailed Description

[0018] Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, and pharmacology described herein are those well known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event that there is a plurality of definitions for a term used herein, those in this section prevail unless stated otherwise.

[0019] The term "about" or "approximately" means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or

"approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0020] The term "optionally substituted" is intended to mean that a group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, or alkoxy group, may be substituted with one or more substituents independently selected from, e.g. , (a) alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, each optionally substituted with one or more, in one embodiment, one, two, three, or four, substituents Q; and (b) halo, cyano (-CN), nitro (-N0₂), -C(0)R^a, -C(0)OR^a, -C(0)NR^bR^c, -C(NR^a)NR^bR^c, -OR^a, -OC(0)R^a, -OC(0)OR^a, -OC(0)NR^bR^c, -OC(=NR^a)NR^bR^c, -OS(0)R^a, -OS(0)₂R^a, -OS(0)NR^bR^c, -OS(0)₂NR^bR^c, -NR^bR^c, -NR^aC(0)R^d, -NR^aC(0)OR^d, -NR^aC(0)NR^bR^c,

-NR^aC(=NR^d)NR^bR^c, -NR^aS(0)R^d, -NR^aS(0)₂R^d, -NR^aS(0)NR^bR^c, -NR^aS(0)₂NR^bR^c, -SR^a, -S(0)R^a, -S(0)₂R^a, -S(0)NR^bR^c, and -S(0)₂NR^bR^c, wherein each R^a, R^b, R^c, and R^d is independently (i) hydrogen; (ii) Ci_₆ alkyl, C₂_₆ alkenyl, C₂_₆ alkynyl, C3-7 cycloalkyl, C_6-14 aryl, heteroaryl, or heterocyclyl, each optionally substituted with one or more, in one embodiment, one, two, three, or four, substituents Q; or (iii) R^b and R^c together with the N atom to which they are attached form heterocyclyl, optionally substituted with one or more, in one embodiment, one, two, three, or four, substituents Q. As used herein, all groups that can be substituted are "optionally substituted," unless otherwise specified. [0021] "Biocompatible" refers to being compatible with a living tissue, by virtue of, e.g., low or no toxicity, or no immunological rejection. In certain embodiments, a polymer is biocompatible if it has good safety ratio or therapeutic index or protective index. In certain embodiments, a polymer is biocompatible if it has been approved for use in humans by any regulatory agency, such as the FDA or EMEA. In certain embodiments, whether a conjugate is biocompatible can be determined, for example, using one or more of the following tests: in vitro cytotoxicity testing (ISO 10993-5, USP 87); in vitro and in vivo hemocompatibility testing (ISO 10993-4); in vivo sensitization testing (ISO 10993-10); in vivo irritation testing (ISO 10993-10); in vivo systemic toxicity testing (ISO 10993-11); in vitro and in vivo genotoxicity testing (ISO 10993-3); and in vivo implantation testing (ISO 10993-6).

[0022] "Small molecule" means any organic molecule, including a biological molecule. In certain embodiments, a small molecule has a molecular weight of about 1500 or less, about 1200 or less, about 1000 or less, about 950 or less, about 900 or less, about 850 or less, about 800 or less, about 750 or less, about 700 or less, about 650 or less, about 600 or less, about 550 or less, about 500 or less, about 450 or less, or about 400 or less. In certain

embodiments, a small molecule has a weight of from about 50 to about 1500, about 50 to about 1200, about 50 to about 800, about 50 to about 500, about 100 to about 1500, about 100 to about 1200, about 100 to about 1000, about 200 to about 1000, about 200 to about 500, about 300 to about 1000, about 300 to about 500, about 400 to about 1000, about 50 to about 500, or about 100 to about 500. In a specific embodiment, a small molecule has a molecular weight of 500 or less, 450 or less, 400 or less, 350 or less, 300 or less, 250 or less, 200 or less, or 150 or less. In certain embodiments, a small molecule is a pharmaceutically active molecule, i.e., a molecule that can elicit a therapeutic effect in a living organism. In certain embodiments, a small molecule is, for example, an antibiotic, an antiviral, an antimicrobial, a steroid, a non-steroidal anti-inflammatory agent, an anti-cancer agent. In certain

embodiments, a small molecule is an organic molecule produced by a living organism, or an analog or derivative thereof. Small molecules include, e.g., lipids, phospholipids,

glycolipids, sterols, vitamins, hormones, neurotransmitters, carbohydrates, sugars, disaccharides, amino acids, nucleotides, nucleosides, saccharides, peptides, nucleic acids, and mixed groups thereof. In certain embodiments, a small molecule is a peptide, lipid, sugar, nucleic acid, or nucleotides, or a derivative or analog thereof. In specific embodiments, the small molecules contains a hydroxyl or an amino group. [0023] In certain embodiments, a small molecule is not a nucleic acid. In certain embodiments, a small molecule is not an miRNA, mirtron, shRNA, siRNA, piRNA, svRNA, antisense RNA , antisense DNA, and locked nucleic acid. In certain embodiments, a small molecule is not a lipid, phospholipid, glycolipid, sterol, vitamin, hormone, neurotransmitter, carbohydrate, sugar, disaccharide, amino acid, nucleotide, nucleoside, polynucleotide, saccharide (mono-, poly-, or oligo-saccharide), peptide, polypeptide, protein, nucleic acid (e.g., ribonucleic acid (RNA), deoxyribonucleic acid (DNA), as well as nucleic acid analogs thereof and polymeric forms thereof), lignin, or a mixed groups thereof. In certain embodiments, a small molecule is not a peptide, polypeptide, protein, lipid, sugar, polysaccharide, nucleic acid, nucleotides, or polynucleotides, as well as well as derivatives of the above comprising amino acid or nucleotide analogs or other non-nucleotide groups. In certain embodiments, a small molecule is not a nucleic acid analog including, for example and without limitation, a phosphorothioate, phosphorodithioate, phosphorotriester, phosphoramidate, boranophosphate, methylphosphonate, chiral-methyl phosphonate, 2-0- methyl ribonucleotide, peptide-nucleic acid (PNA), locked-nucleic acid (LNA), and the like. In certain embodiments, a small molecule is not a cDNA, siRNA, micro RNA, short hairpin RNA, piwi-interacting RNA (piRNs), mitron, antisense molecule, or another oligonucleotide. In certain embodiments, a small molecule is not PYY, chitosan, or ATP.

[0024] "Degradable" means covalent bonds capable of being broken via hydrolysis (reaction with water) under basic or acid conditions, via metabolic pathways, enzymatic degradation (by environmental and/or physiological enzymes), or other biological processes (such as those under physiological conditions in a vertebrate, such as a mammal). A degradable bond includes, but is not limited to, carboxylate esters, phosphate esters, carbamates, anhydrides, acetals, ketals, imines, orthoesters, thioesters, or carbonates.

[0025] "Targeting group" means those moieties that have been shown to influence the accumulation of a small molecule in specific cells. Targeting groups can be comprised of a variety of proteins, peptides, small molecules, or the like. Non-limiting examples include vitamin D, folate (e.g., for cancer cells), estradiol, and testosterone.

[0026] The term "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," "physiologically acceptable carrier," or "physiologically acceptable excipient" refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. See, Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th Edition, Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition, Ash and Ash Eds., Gower

Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, FL, 2004.

[0027] As used herein, the term "effective amount" in the context of administering a therapy to a subject refers to the amount of a therapy which has a prophylactic and/or therapeutic effect(s). In certain embodiments, an "effective amount" in the context of administration of a therapy to a subject or a population of subjects refers to the amount of a therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of a disease or a symptom associated therewith in the subject or population of subjects; (ii) reduce the duration of a disease or a symptom associated therewith in the subject or population of subjects; (iii) prevent the progression of a disease or a symptom associated therewith in the subject or population of subjects; (iv) cause regression of a disease or a symptom associated therewith in the subject or population of subjects; (v) prevent the development or onset of a disease or a symptom associated therewith in the subject or population of subjects; (vi) prevent the recurrence of a disease or a symptom associated therewith in the subject or population of subjects; (vii) prevent or reduce the spread of a disease from the subject or population of subjects to another subject or population of subjects; (viii) reduce organ failure associated with a disease in the subject or population of subjects; (ix) reduce the incidence of hospitalization of the subject or population of subjects; (x) reduce hospitalization length of the subject or population of subjects; (xi) increase the survival of the subject or population of subjects; (xii) eliminate a disease in the subject or population of subjects; (xiii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy in the subject or population of subjects; (xiv) prevent the spread of a virus or bacteria from a cell, tissue, organ of the subject to another cell, tissue, organ of the subject; and/or (xv) reduce the number of symptoms of a disease in the subject or population of subjects. In a specific embodiment, the therapy is a conjugate described herein. Small Molecule-Polymer Conjugates

[0028] The small molecule-polymer conjugate(s) of the disclosure are illustrated in Formula 1 :

Formula 1

where the linker L is independently a 1 - 20 atom linear or branched linker; the polymer is independently a biocompatible polymer; X is independently an atom of attachment to the small molecule that is O, NH, NR, or S, where X is part of the small molecule; n is an integer; and the X— L bond is degradable. The loose bond between "L" and the triazole in Formula 1 indicates that the linker "L" can be bound to either carbon of the triazole ring.

[0029] In certain embodiments, the small molecule is a pharmaceutically active drug or other agent. In certain embodiments, the pharmaceutically active drug is a cholinergic, adrenergic, serotonergic, anesthetic, hypnotic, antiseizure therapeutic, antipsychotic, anxiolytic, stimulant, opiod, analgesic, spasmolytic, cardiac glycoside, antianginal, antiarrhythmic, diuretic, angiotensin converting enzyme inhibitor, angiotensin converting enzyme antagonist, calcium blocker, central sympatholytic, peripheral sympatholytic, vasodilator, antihyperlipoproteinemic, cholesterol biosynthesis inhibitor, antithrombotic, thrombolytic, coagulant, plasma extender, oral hypoglycemic agent, adrenocorticoid, estrogen, progestin, prostaglandin, androgen, thyroid drug, nonsteroidial anti-inflammatory agent, steroidal anti-inflammatory agent, antihistamine, antiallergenic agent, antiulcer agent, antibiotic, antimicrobial, antiparasitic, antifungal, antimycobacterial agent, cancer

chemotherapeutic, antiviral, protease inhibitors, or selective estrogen receptor modulators. In other embodiments, the small molecule is a sugar, carbohydrate, peptide, pesticide, herbicide, anti-fowling agent, aromatic agent, detergent, sequestering agent, preservative, anti-corrosion agent, or catalyst.

[0030] In certain embodiments, the small molecule is a steroidal anti-inflammatory drug, for example hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, or aldosterone. In certain embodiments, the small molecule is a chemotherapeutic agent, for example melphalan, dacarbazine, temozolomide, streptozotocin, methotrexate, pemetrexed, tioguanine, fludarabine, pentostatin, cladribine, floxuridine, gemcitabine, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, irinotecan, topotecan, paclitaxel, or docetaxel. In certain embodiments, the small molecule is an anticoagulant, for example warfarin, acenocoumarol, phenprocoumon, argatroban, or ximelagatran.

[0031] In certain embodiments, the small molecule is an anticancer agent, for example gemcitabine. In certain embodiments, the small molecule is an antimicrobial, for example vanillin or dapsone. In certain embodiments, the small molecule is an antiviral, for example elvucitabine. In certain embodiments, the small molecule is a non-steroidal antiinflammatory drug (NSAID), for example piroxicam. In certain embodiments, the small molecule is an aminoglycoside antibiotic, for example amikacin. In certain embodiments, the small molecule is an analgesic, for example morphine. In certain embodiments, the small molecule is an anti-Alzheimer's drug, for example memantine. In certain embodiments, the small molecule is an anxiolytic, for example temazepam. In certain embodiments, the small molecule is a selective estrogen receptor modulator (SERM), for example raloxifene.

[0032] In certain embodiments, the polymer of Formula 1 is a biocompatible polymer. In certain embodiments, the polymer imparts a stabilizing effect on the small molecule. When n is greater than 1 , the various polymers of Formula 1 can be the same or different. In various embodiments, the polymer is independently anionically charged, cationically charged, or uncharged; hydrophobic, hydrophilic, or amphiphilic; or combinations thereof. In various embodiments, the polymer is a homopolymer, a block copolymer, or a random copolymer. In certain embodiments, the polymer is polydisperse or monodisperse. In various embodiments, the polydispersity index of the polymer is from 1 to about 30, from 1 to about 10, from 1 to about 5, from 1 to about 3, or is a discrete polymer with an index of 1. In certain

embodiments the polymer is linear. In certain embodiments the polymer is branched.

[0033] In certain embodiments, the polymer is a polyethylene glycol (PEG), a polyether, a poly(lactide), a poly(glycolide), a poly(lactide-co-glycolide), a poly(lactic acid), a poly(glycolic acid), a poly(lactic acid-co-glycolic acid), a polyanhydride, a polyorthoester, a polycarbonate, a polyetherester, a polycaprolactone, a polyesteramide, a polyester, a polyacrylate, a polymer of ethylene-vinyl acetate or another acyl substituted cellulose acetate, a polyurethane, a polyamide, a polystyrene, a silicone based polymer, a polyolefm, a polyvinyl chloride, a polyvinyl fluoride, a fluoropolymer, a polypropylene, a polyethylene, a cellulosic, a starch, a naturally occurring polymer, a poly( vinyl imidazole), a polyacetal, a polysulfone, a chlorosulphonate polyolefin, or a blend or copolymer thereof. In a particular embodiment, the polymer is PEG.

[0034] In various embodiments, the polymer of Formula 1 has an average molecular weight of from about 200 to about 50,000, from about 200 to about 40,000, from about 200 to 30,000, from about 200 to about 20,000, from about 200 to about 10,000, from about 200 to about 5,000, from about 200 to about 4,000, from about 200 to about 3,000, from about 200 to about 2,000, from about 200 to about 1,000, or from about 200 to about 500. In various embodiments, the polymer has an average molecular weight of from about 10,000 to about 50,000, from about 10,000 to about 40,000, from about 10,000 to about 30,000, or from about 10,000 to about 20,000. In a particular embodiment, the polymer has an average molecular weight of from about 200 to about 2,000.

[0035] In certain embodiments, the polymer is independently terminated with a nonfunctional group, such as methyl or methoxy, or a small molecule functional group, such as a targeting group. In a specific embodiment, the polymer is terminated with a targeting group that targets the conjugate to certain types of cells, tissues, or organs. In a particular embodiment, the targeting group is a folate, a vitamin D or an analog, a testosterone, or an estradiol.

[0036] The linker "L" can be of varying lengths and composition. In certain

embodiments, the linker is from about 1 to about 20 atoms in length, from about 1 to about 15 atoms in length, from about 1 to about 10 atoms in length, or from about 1 to about 5 atoms in length. In certain embodiments, the linker is 1, 2, 3, 4, 5, or 6 atoms in length. In a particular embodiment, the linker is 3 atoms in length. In various embodiments, the atoms comprising the linker backbone are independently carbon, oxygen, nitrogen, or sulfur. In a particular embodiment, the linker L is:— C(0)0(CH₂)_q— , where q is an integer from 0 to about 20, from about 0 to about 10, from about 1 to about 10, from about 2 to about 10, from about 2 to about 8, from about 2 to about 5, or from about 2 to about 4. In various sub- embodiments, q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a particular sub-embodiment, q is 2. In various sub-embodiments, each methylene group may be optionally substituted, or may itself be a different atom, such as NH, O, or S.

[0037] In certain embodiments, the L— X bond is degradable. In certain sub- embodiments, the degradable L— X bond is a carbonate bond, a carboxylate ester bond, a phosphate ester bond, an anhydride bond, an acetal bond, a ketal bond, an imine bond, an orthoester bond, a thioester bond a carbamate bond, a urea bond, an amide bond. In a particular sub-embodiment, the L— X bond in a carbonate or carbamate bond.

[0038] In certain embodiments, n is from about 1 to about 100, from about 1 to about 75, from about 1 to about 50, from about 1 to about 30 or from about 1 to about 20. In certain embodiments, n is from about 1 to about 10. In various embodiments, n is from about 11 to about 30, from about 13 to about 27, from about 15 to about 25, or from about 17 to about 22. In various other embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In a particular embodiment, n is from about 11 to about 14.

[0039] In certain embodiments, the small molecule-polymer conjugate of the disclosure is degradable. This is advantageous in that the small molecule-polymer conjugate may be initially stable for a period of time when introduced into a living system. This allows time for small molecule-polymer conjugate to traverse harsh environments, such as the intestinal tract, circulatory system and liver, where the small molecule alone could be trapped or degraded. The degradable nature of the small molecule-polymer conjugate allows for the release of the small molecule to the respective site of action in a living system full intact. The delay of degradation of the small molecule-polymer conjugate allows for distribution to a variety of tissues and organs that would be less accessible by the small molecule alone. In addition, the small molecule-polymer conjugate may also allow for slow release of the small molecule dependent on the rate of degradation. In certain embodiments, the small molecule-polymer conjugate degrades in vivo to release the small molecule with a half- life of less than about 2 weeks, less then about 1 week, less than about 2 days, less than about 1 day, less than about 12 hours, less than about 6 hours, or less than about 3 hours. The half-life can be measured both in vitro by known methods, for example by UV-Vis spectroscopy, or in vivo, by sampling blood serum over time and determining the concentration of the metabolites by known methods, for example HPLC.

[0040] In certain embodiments, the small molecule-polymer conjugate of the disclosure has enhanced stability compared to the corresponding unmodified small molecule, for example in vivo stability as evidenced by, for example, circulation half-life.

[0041] In various embodiments, the small molecule is released from the protecting polymer layer via degradation of a bond, e.g., the L— X bond, through which the small molecule is conjugated to the polymer. In various embodiment, the degradation occurs via hydrolysis (reaction with water) under basic or acid conditions, metabolism, enzymatic degradation (by environmental and/or physiological enzymes), and other biological processes (such as those under physiological conditions in a vertebrate, such as a mammal). In embodiments where the degradation of the small molecule-polymer conjugate generated acid functional groups (e.g., when the degradation occurs at an ester or carbonate bond), the degradation process provides an auto-catalytic effect.

[0042] In various embodiments, release of the small molecule may involve the degradation of a biodegradable linker, or digestion of the polymer into smaller, non- polymeric subunits. Without being bound by theory, two different areas of biodegradation may occur: the cleavage of bonds in the polymer backbone which generally results in monomers and oligomers of the polymer; or the cleavage of a bond connecting the polymer to the small molecule. In certain embodiments, the release of the small molecule (e.g., the degradation of a bond linking the small molecule to the polymer) occurs faster than the degradation of the small molecule itself. The rate of release of the small molecule can be measured both in vitro by known methods, for example by UV-Vis spectroscopy, or in vivo, by sampling blood serum over time and determining the concentration of the metabolites by known methods, for example HPLC.

[0043] The degradation rates of a bond linking the small molecule to the polymer (such as the L— X bond) and of the polymer itself may vary.

[0044] In certain embodiments, the small molecule-conjugate of the disclosure is useful for the treatment, prevention and/or amelioration of a disease, or as a diagnostic tool, for example as a fluorescent probe (i.e., delivery of an imaging agent visible during MRI), or as a cancer diagnostic.

[0045] In certain embodiments, the small molecule-polymer conjugate may be prepared as a composition, comprising the small molecule-polymer conjugate in admixture with one or more carriers.

[0046] In certain embodiments, the small molecule-polymer conjugate is for the treatment, prevention and/or amelioration of a disease or conditions for which the small molecule is useful. In specific embodiments, a subject is administered an effective amount of the small molecule-polymer conjugate for the treatment, prevention and/or amelioration of a disease or condition (e.g., infection) for which the small molecule is useful. The small molecule-polymer conjugate can be administered by any route (e.g., intravenous,

subcutaneous, oral, etc.).

[0047] In certain embodiments, the small molecule-polymer conjugate may be prepared as a pharmaceutical composition formulated for the treatment, prevention and/or amelioration of a disease or conditions for which the small molecule is useful. The pharmaceutical composition comprises the small molecule-polymer conjugate in admixture with one or more pharmaceutically acceptable excipients or carriers. In specific embodiments, the

pharmaceutical composition comprises an effective amount of the small molecule-polymer conjugate. In certain embodiments, the pharmaceutical composition comprising the small molecule-polymer conjugate can be formulated in various dosage forms for parenteral, oral, intradermal, transdermal, colorectal, intraperitoneal, or rectal administration. In a specific embodiment, the pharmaceutical composition may be formulated for intravenous, oral, intraperitoneal, intranasal, intratracheal, subcutaneous, intramuscular, topical, intradermal, transdermal or pulmonary administration. The pharmaceutical compositions can also be formulated as modified release dosage forms, including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmed-release, and gastric retention dosage forms. These dosage forms can be prepared according to

conventional methods and techniques known to those skilled in the art (see, E.W. Martin, Remington: The Science and Practice of Pharmacy; Modified-Release Drug Delivery Technology, Rathbone et al., Eds., Drugs and the Pharmaceutical Science, Marcel Dekker, Inc.: New York, NY, 2003; Vol. 126). The pharmaceutical compositions described herein may be suitable for veterinary and/or human administration. The pharmaceutical

compositions provided herein may be in any form that allows for the composition to be administered to a subject. "Subject" means any animal, whether human or non-human. In certain embodiments, the subject is a non-human animal, such as a cow, horse, sheep, pig, fowl, cat, dog, mouse, rat, rabbit, guinea pig, etc. In certain embodiments, the non-human animal is a pet or farm animal. In a specific embodiment, the subject is human.

[0048] The pharmaceutical compositions provided herein can be administered at once, or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the patient being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or diagnostic data. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations.

[0049] The pharmaceutical compositions provided herein can be provided in a unit- dosage form or multiple-dosage form. A unit-dosage form, as used herein, refers to physically discrete a unit suitable for administration to a human and animal subject, and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of an small molecule-polymer conjugate(s) sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carriers or excipients. Examples of a unit-dosage form include an ampoule, syringe, and individually packaged tablet and capsule. A unit-dosage form may be administered in fractions or multiples thereof. A multiple-dosage form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dosage form. Examples of a multiple-dosage form include a vial, bottle of tablets or capsules, or bottle of pints or gallons.

[0050] In some embodiments, a pharmaceutical composition may comprise one or more other therapies in addition to a small molecule -polymer conjugate. As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized

pharmacopeiae for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in E.W. Martin, Remington: The Science and Practice of Pharmacy; Modified-Release Drug Delivery Technology, Rathbone et al., Eds., Drugs and the Pharmaceutical Science, Marcel Dekker, Inc.: New York, NY, 2003; Vol. 126. The formulation should suit the mode of administration. The pharmaceutically acceptable carrier may be particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) can be liquid, with the compositions being, for example, an oral syrup or injectable liquid. In addition, the carrier(s) can be gaseous, so as to provide an aerosol composition useful in, e.g., inhalatory administration.

[0051] Materials used in preparing the pharmaceutical compositions can be non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the small molecule-polymer conjugate(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of subject {e.g., human), the overall health of the subject, the type of disease or disorder the subject is in need of treatment of, the use of the composition as part of a multi-drug regimen, the particular form of the small molecule-polymer conjugate in the composition, the manner of administration of the composition, and the composition employed.

[0052] In certain embodiments, the compositions described herein can be delivered in a controlled release system. In one embodiment, a pump can be used (see Sefton, CRC Crit. Ref. Biomed. Eng. 1987, 14, 201; Buchwald et al, Surgery 1980, 88: 507; Saudek et al., N. Engl. J. Med. 1989, 321 : 574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, FL, 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York, 1984; Ranger and Peppas, J. Macromol. Sci. Rev.

Macromol. Chem. 1983, 23, 61; see also Levy et al., Science 1985, 228, 190; During et al., Ann. Neurol. 1989, 25, 351; Howard et al., J. Neurosurg., 1989, 71, 105). In yet another embodiment, a controlled-release system can be placed in proximity of the target of the small molecule-polymer conjugates described herein, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, 1984, pp. 115-138). Other controlled-release systems discussed in the review by Langer (Science 1990, 249, 1527-1533) can also be used.

[0053] In certain embodiments, biodegradable polymers, such as ethylene vinyl acetate, polyanhydrides, polyethylene glycol, polymethyl methacrylate polymers, polylactides, poly(lactide-co-glycolides), polyglycolic acid, collagen, polyorthoesters, and polylactic acid, may be used as carriers. In some embodiments, the small molecule-polymer conjugates are prepared with carriers that increase the protection of the small molecule-polymer conjugate against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomes or micelles can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. In certain embodiments, the pharmaceutical compositions comprise one or more adjuvants.

[0054] In certain embodiments, a gemcitabime-polymer conjugate or a pharmaceutical composition comprising the same is provided. Such a conjugate or pharmaceutical composition is useful for the treatment, prevention, and/or amelioration of cancer.

[0055] In certain embodiments, an anti-cancer agent (such as melphalan, dacarbazine, temozolomide, streptozotocin, methotrexate, pemetrexed, tioguanine, fludarabine, pentostatin, cladribine, floxuridine, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, irinotecan, topotecan, paclitaxel, or docetaxel)-polymer conjugate or a

pharmaceutical composition comprising the same is provided. Such a conjugate is useful for the treatment, prevention and/or amelioration of cancer.

[0056] In certain embodiments, a vanillin-polymer conjugate or a pharmaceutical composition comprising the same is provided. Such a conjugate or pharmaceutical composition is useful for the treatment, prevention and/or amelioration of a microbial infection (e.g., a bacterial infection), microbial-related disease or condition, for example leprosy, pneumonia, acne.

[0057] In certain embodiments, a dapsone -polymer conjugate or a pharmaceutical composition comprising the same is provided. Such a conjugate or pharmaceutical composition is useful for the treatment, prevention and/or amelioration of a microbial infection (e.g., a bacterial infection), microbial-related disease or condition, for example leprosy, pneumonia, acne.

[0058] In certain embodiments, an anti-viral-polymer conjugate (such as an elvucitabine- polymer conjugate) or a pharmaceutical composition comprising same is provided. Such a conjugate or pharmaceutical composition useful for the treatment, prevention and/or amelioration of a virus-related disease or a viral infection, for example HIV or AIDS.

[0059] In certain embodiments, an amikacin-polymer conjugate or a pharmaceutical composition comprising the same is provided. Such a conjugate or pharmaceutical composition is useful as an antibiotic.

[0060] In certain embodiments, a steroidal anti-inflammatory agent-polymer conjugate (such as a hydrocortisone-polymer conjugate, prednisone-polymer conjugate, or

prednisolone-polymer conjugate) or a pharmaceutical composition comprising the same is provided. Such a conjugate or pharmaceutical composition is useful for the treatment, prevention and/or amelioration of inflammation or an inflammatory condition (such as asthma, encephilitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentitated

spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacterial infections).

[0061] In certain embodiments, an NSAID-polymer conjugate (such as a piroxicam- polymer conjugate) or a pharmaceutical composition comprising the same is provided. Such a conjugate or pharmaceutical composition is useful for the treatment, prevention and/or amelioration of inflammation of an inflammatory condition (such as asthma, encephilitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentitated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacterial infections).

[0062] In certain embodiments, a morphine-polymer conjugate or a pharmaceutical composition comprising the same is provided. Such a conjugate or pharmaceutical composition is useful as an analgesic.

[0063] In certain embodiments, a memantine-polymer conjugate or a pharmaceutical composition comprising the same is provided. Such a conjugate or pharmaceutical composition is useful for the treatment, prevention and/or amelioration of Alzheimer's disease.

[0064] In certain embodiments, a temazepam-polymer conjugate or a pharmaceutical composition comprising the same is provided. Such a conjugate or pharmaceutical composition is useful for the treatment, prevention and/or amelioration of anxiety or insomnia.

[0065] In certain embodiments, an anticoagulant (e.g., warfarin, acenocoumarol, phenprocoumon, argatroban, or ximelagatran)-polymer conjugate or a pharmaceutical composition comprising the same is provided. Such a conjugate or pharmaceutical composition useful for the treatment, prevention and/or amelioration of coagulation.

[0066] In certain embodiments, a raloxifene -polymer conjugate or a pharmaceutical composition comprising the same is provided. Such a conjugate or pharmaceutical composition useful for the treatment, prevention and/or amelioration of a disease affected by a selective estrogen receptor modulator (SERM), for example osteoporosis.

[0067] In certain embodiments, the prevention of a disease, condition or an infection (e.g., a bacterial, parasitic, or viral infection) prevents the onset or development of one or more symptoms associated with the disease, condition or infection and/or reduces the number of symptoms associated with the disease or infection. In some embodiments, the prevention of a disease or condition prevents the onset or development of the disease or condition. In certain embodiments, the treatment of a disease, condition or an infection (e.g., a bacterial, viral, or parasitic infection) achieves one, two or more of the following: (i) reduces or ameliorates the severity of a disease or an infection, (ii) reduces the duration of a disease or infection or a symptom thereof, (iii) reduces the incidence of hospitalization due to the disease, condition or infection, and/or (iv) reduces the length of hospitalization due to the disease, condition or infection.

[0068] In certain embodiments, the total equivalent dosage or frequency of administration of the small molecule-polymer conjugate is reduced compared to administration of the small molecule alone. In related embodiments, the small molecule-polymer conjugates can provide reduced toxicity at equivalent dosages compared to the small molecule alone. In other related embodiments, the small molecule-polymer conjugates can reduce the number, frequency, and/or severity of the side effects experienced by a subject compared to those experienced by a subject administered the small molecule alone. The term "equivalent dosage," as used herein, means the dosage based on the molar equivalent of small molecule in the small molecule-polymer conjugate.

[0069] In certain embodiments, the total equivalent dosage of the small molecule- polymer conjugate administered is 0.001 to 1000 mg per kg subject body weight per day (mg/kg per day), from about 0.01 to about 500 mg/kg per day, from about 0.1 to about 100 mg/kg per day, from about 0.5 to about 100 mg/kg per day, or from about 1 to about 100 mg/kg per day, which can be administered in single or multiple doses. For oral

administration, the pharmaceutical compositions provided herein can be formulated in the form of tablets containing from about 1.0 to about 1,000 mg equivalent of the small molecule, in one embodiment, about 1, about 5, about 10, about 15, about 20, about 25, about 50, about 75, about 100, about 150, about 200, about 250, about 300, about 400, about 500, about 600, about 750, about 800, about 900, and about 1,000 mg equivalent of the small molecule. The pharmaceutical compositions can be administered on a regimen of 1 to 4 times per day, including once, twice, three times, and four times per day.

[0070] In certain embodiments, the small molecule-polymer conjugates can be used in combination with other drugs, for example other drugs known to be of therapeutic benefit for the disease indication for which the small molecule is useful.

[0071] In certain embodiments, the small molecule-polymer conjugates can be tested in vitro, in vivo, and/or in animal models to assess their efficacy and toxicity with respect to the disease indications for which the small molecule is useful. Techniques known to one skilled in the art can be used to assess the efficacy and toxicity of a small molecule-polymer conjugate or a pharmaceutical composition comprising the same.

[0072] It will be understood, however, that the specific dose level and frequency of dosage for any particular patient can be varied and will depend upon a variety of factors including the activity of the small molecule employed in the small-molecule polymer conjugate, the metabolic stability and length of action of the conjugate, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug

combination, the severity of the particular condition, and the host undergoing therapy.

[0073] In certain embodiments is provided a kit or a pharmaceutical pack comprising the small molecule-polymer conjugate or a pharmaceutical composition comprising the same. Methods of Preparing the Small Molecule-Polymer Conjugate

[0074] In another aspect of the disclosure, a method of preparing the small molecule- polymer conjugates of Formula 1. The method comprises (a) reacting the small molecule with an alkyne-containing electrophilic reagent, and (b) reacting the alkyne-modified small molecule with an azide-containing polymer or mixture of azide-containing polymers. The reaction is illustrated in Scheme 1 below: small molecule -X-L-

Scheme 1

where the small molecule, the polymer, X, L, and n are as defined above, and Q is a leaving group.

[0075] In certain embodiments, steps (a) and (b) are conducted as a "one-pot" synthesis, without isolation and/or purification of the intermediate alkyne-modified small molecule. In other embodiments, steps (a) and (b) are conducted with isolation and/or purification of the intermediate alkyne-modified small molecule.

[0076] In step (a) of the method, the small molecule is reacted with an alkyne-containing electrophilic reagent to yield an L— X bond. In certain embodiments, the alkyne-containing electrophilic reagent is a carboxylic acid, an acid halide, a carboxylic acid anhydride, a carboxylic acid salt, a carboxylic acid ester, an isocyanate, a carbonate, a carbamate, or a chloroformate. In a certain embodiment, the alkyne-containing electrophilic reagent is

wherein q is an integer from 0 to about 20, from about 0 to about 10, from about 1 to about 10, from about 2 to about 10, from about 2 to about 8, from about 2 to about 5, or from about 2 to about 4. In various sub-embodiments, q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a particular sub-embodiment, q is 2. In various sub-embodiments, each methylene group may be optionally substituted, or may itself be a different atom, such as NH, O, or S. In a particular embodiment, the alkyne-containing electrophilic reagent is a chloroformate, such as propargyl chloroformate.

[0077] In various embodiments, step (a) of the method proceeds via one or more of the following reactions (where R is the small molecule, and X is either OH or NH₂):

Alcohol + propargyl Chloride, condensation reaction yields an carbonate bond

Alcohol + carboxylic acid, condensation reaction yields an ester bond

O O

R ,

R-OH + HO ^v T>

Alcohol + acid halide, condensation reaction yields an ester bond

Alcohol + acid anhydride, condensation reaction yields an ester bond

O O ⁰

R

R-OH + ^^^-^ rr ^-^^^ ⁰

Alcohol + acid salts, condensation reaction yields an ester bond

O O

Na ø il »► R

R-OH + © ^U0^^^^ ^{K ^}0

Alcohol + isocyanate, addition reaction yields a urethane bond

Alcohol + ester, transesterification reaction yields an ester bond

Amine + isocyanate, addition reaction yields a urea bond

Amine + carboxylic acid, neutralization and dehydration reaction yields an amide bond

Amine + acid anhydride, substitution reaction yields an amide bond

Amine + acid halide, substitution reaction yields an amide bond

O O

R .

R-NH₂ + Cr ^ N

H

Amine + acid salts, reaction yields an amide bond

O O

Na ø il »- R

R-NH₂ + © O^^^^. N

H

Amine + ester, reaction yields an amide bond

Amine + chloroformate, reaction yields a carbamate bond

[0078] While the above exemplary reactions are illustrated where the alkyne is 3 atoms away from the small molecule atom (either the nitrogen or the oxygen), the disclosure encompasses other embodiments where the alkyne is anywhere from 1 to about 20 atoms away from the small molecule atom. In other embodiments, the alkyne is from about 2 to about 10, or from about 2 to about 5 atoms away from the small molecule atoms. [0079] Step (a) of the can be conducted in a variety of solvents. In various embodiments, the first step of the method is conducted in water, tetraethylene glycol dimethylether, dimethylsulfoxide, dimethylformamide, chloroform, dichloromethane, pyridine, acetone, ether, or a mixture thereof. In a particular embodiment, the first step of the method is conducted in a mixture of water and one or more of tetraethylene glycol dimethylether, dimethylsulfoxide, dimethylformamide, chloroform, dichloromethane, pyridine, acetone, or ether. In certain embodiments, the reaction is conducted in the absence of water. In other embodiments, the reaction is conducted in water.

[0080] In certain embodiments, step (a) of the method is conducted in the presence of a base. In various embodiments, the base is a tertiary alkyl amine, an aromatic amine, a carbonate, or a hydroxide. In particular embodiments, the base is diisopropylethylamine, triethylamine, pyridine, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, or potassium hydroxide.

[0081] Step (a) of the method can be conducted at a variety of temperatures and times, provided that the small molecule is not degraded. In certain embodiments, the reaction is conducted at a temperature from about -30 °C to about 25 °C, from about 0 °C to about 25 °C, or from about 5 °C to about 20 °C. In certain embodiments, the reaction is conducted for from about 5 minutes to about 8 hours, from about 5 minutes to about 1 hour, from about 20 minutes to about 40 minutes.

[0082] In certain embodiments, the small molecule is treated with from about 0.001 to about 1000 molar equivalents of alkyne-containing electrophilic reagent based on the number of modifiable positions on the small molecule. In various embodiments, the small molecule is treated with from about 0.001 to about 1, from about 0.01 to about 1, from about 0.1 to about 1, or from about 0.5 to about 1 molar equivalent of alkyne-containing electrophilic reagent based on the number of modifiable positions on the small molecule. In other embodiments, the small molecule is treated with from about 1 to about 1000, from about 1 to about 500, from about 1 to about 100, from about 1 to about 10, or from about 1 to about 5 molar equivalents of alkyne-containing electrophilic reagent based on the number of modifiable positions on the small molecule.

[0083] In step (b) of the method, the alkyne-modified small molecule is reacted with one or a mixture of azide-containing polymers. The azide-containing polymer can be any biocompatible polymer with an azide group. In certain embodiments, the azide-containing polymer imparts a stabilizing effect on the small molecule. When n is greater than 1, the various polymers of Formula 1 can be the same or different. In various embodiments, the azide-containing polymer is independently anionically charged, cationically charged, or uncharged; hydrophobic, hydrophilic, or amphiphilic; or combinations thereof. In various embodiments, the azide-containing polymer is a homopolymer, a block copolymer, or a random copolymer. In certain embodiments, the azide-containing polymer is polydisperse or monodisperse. In various embodiments, the polydispersity index of the azide-containing polymer is from 1 to about 30, from 1 to about 10, from 1 to about 5, from 1 to about 3, or is a discrete polymer with an index of 1. In certain embodiments the azide-containing polymer is linear. In certain embodiments the azide-containing polymer is branched.

[0084] In certain embodiments, the azide-containing polymer is a polyethylene glycol (PEG), a polyether, a poly(lactide), a poly(glycolide), a poly(lactide-co-glycolide), a poly(lactic acid), a poly(glycolic acid), a poly(lactic acid-co-glycolic acid), a polyanhydride, a polyorthoester, a polycarbonate, a polyetherester, a polycaprolactone, a polyesteramide, a polyester, a polyacrylate, a polymer of ethylene-vinyl acetate or another acyl substituted cellulose acetate, a polyurethane, a polyamide, a polystyrene, a silicone based polymer, a polyolefm, a polyvinyl chloride, a polyvinyl fluoride, a fluoropolymer, a polypropylene, a polyethylene, a cellulosic, a starch, a naturally occurring polymer, a poly(vinyl imidazole), a polyacetal, a polysulfone, a chlorosulphonate polyolefm, or a blend or copolymer thereof. In particular embodiments, the azide-containing polymer is PEG-azide.

[0085] In various embodiments, the azide-containing polymer has an average molecular weight of from about 200 to about 50,000, from about 200 to about 40,000, from about 200 to 30,000, from about 200 to about 20,000, from about 200 to about 10,000, from about 200 to about 5,000, from about 200 to about 4,000, from about 200 to about 3,000, from about 200 to about 2,000, from about 200 to about 1,000, or from about 200 to about 500. In various embodiments, the azide-containing polymer has an average molecular weight of from about 10,000 to about 50,000, from about 10,000 to about 40,000, from about 10,000 to about 30,000, or from about 10,000 to about 20,000. In a particular embodiment, the azide- containing polymer has an average molecular weight of from about 200 to about 2,000.

[0086] In certain embodiments, the azide-containing polymer is independently terminated with a non-functional group, such as a methyl or methoxy, or a functional group, such as a targeting group. In a particular embodiment, the targeting group is a folate. In certain embodiments, the azide-containing polymer is a mixture of non-functional terminated and functional terminated polymers. In certain embodiments, the mixture is a mixture of methoxy terminated and folate-terminated polymers, for example a mixture of methoxy- terminated PEG and folate-terminated PEG. In certain embodiments, the polymer is terminated with another small molecule. In such an embodiment, the small molecule- polymer conjugate is a networked small molecule-polymer conjugate, each conjugate comprising more than one small molecule.

[0087] Step (b) of the method can be conducted in a variety of solvents. In various embodiments, step (b) of the method is conducted in methanol, ethanol, propanol, isopropanol, tetraethylene glycol, chloroform, tetrahydrofuran, dichloromethane,

dimethylether, dimethylsulfoxide, dimethylformamide, acetone, ether, water, or a mixture thereof. In a particular embodiment, step (b) of the method is conducted in a mixture of water and one or more of methanol, ethanol, propanol, isopropanol, tetraethylene glycol dimethylether, dimethylsulfoxide, dimethylformamide, acetone, or ether. In a particular embodiment, step (b) of the method is conducted in water. In a particular embodiment, step (b) is conducted in the absence of water.

[0088] Step (b) of the method can be conducted at a variety of temperatures and times, provided that the small molecule is not degraded. In certain embodiments, the reaction is conducted at a temperature from about -30 °C to about 70 °C, from about 0 °C to about 65 °C, or from about 25 °C to about 65 °C. In certain embodiments, the reaction is conducted for from about 1 minute to about 8 hours, from about 5 minutes to about 3 hour, or from about 20 minutes to about 60 minutes.

[0089] In certain embodiments, step (b) of the method is conducted in the presence of a catalyst, for example in the presence of a copper catalyst. In a particular sub-embodiment of this embodiment, the copper catalyst is copper bromide or copper iodide. In certain embodiments, step (b) of the method is conducted in presence of a mixture of copper(II), e.g., copper(II) sulfate, and a reducing agent, e.g., sodium ascorbate.

[0090] In certain embodiments, step (b) of the method is conducted in the absence of a catalyst, for example in the absence of a metal catalyst such as copper. In these

embodiments, the absence of a catalyst such as a copper catalyst may be particularly advantageous as the produced small molecule-polymer conjugate is substantially free of copper. The term "substantially free of copper" means that the small molecule-polymer conjugate contains less than about 100 ppm copper, less than about 10 ppm copper, or less than about 1 ppm copper. In certain embodiments, the small molecule -polymer conjugate contains from about 1 ppm to about 100 ppm copper, or from about 1 ppm to about 10 ppm copper. The amount of copper contained in the small molecule -polymer conjugate can be assayed by any known method in the art, for example atomic absorption spectroscopy or inductively coupled plasma atomic emission spectroscopy (see M. Murillo et al, Journal of Analytic Atomic Spectroscopy, 1999, 14, 815-820).

[0091] In certain embodiments, the method further comprises (c) purifying the small molecule-polymer conjugate. In various embodiments, the conjugate is purified by size exclusion chromatography (e.g., gel permeation chromatography), reverse phase

chromatography (e.g., reverse phase HPLC), thin layer chromatography, ion exchange chromatography (e.g., anion exchange HPLC), column chromatography, precipitation, liquid- liquid extraction, or dialysis. In a particular embodiment, the conjugate is purified size exclusion chromatography.

[0092] In certain embodiments, the method further comprises (d) sterilizing the small molecule-polymer conjugate. The sterilization can be performed using any methods known in the art, for example filtration, ethylene oxide sterilization, or high hydrostatic pressure sterilization. In a specific embodiment, the sterilization is performed by filtration using membranes with a rated pore size of, for example, 0.2 μιη or smaller. In various

embodiments, the filter is nylon, cellulose, polyethersulfone, polytetrafluoroethylene, polyamide, polyvinylidene fluoride, polypropylene, or ANOPORE aluminum oxide. In one embodiment, sterilization efficiency can be determined by streaking the sterilized conjugate onto soybean casein digest medium and analyzing for colony formation.

[0093] Specifically, the reactive moieties on the small molecule can be reacted with propargyl chloroformate. This reaction may be undertaken in a variety of different solvents or solvent mixtures. This reaction may also be undertaken in the presence or absence of bases, acids, acid scavengers, water scavenger, or drying reagents.

[0094] In embodiments where the small molecule has multiple reactive moieties and the alkyne-containing electrophilic reagent is propargyl chloroformate or the like, all of the reactive groups can be modified. In other embodiments, the reactive moieties are

incompletely modified. In various embodiments, the small molecule comprises from about 1 to about 25, from about 1 to about 15, from about 1 to about 10, from about 1 to about 5, from about 1 to about 3, from about 1 to about 2, or 1 alkyne groups after step (a) of the method.

vanillin //

Gemcitabine antimicrobial °

anticancer agent natural product Elvucitabine

antiviral

Formula 2. Alkyne-Modified Small Molecule Drugs

[0095] In embodiments where the small molecule is an active agent and the alkyne - containing electrophilic reagent is propargyl chloroformate or the like, the product of the cycloaddition reaction between an azide-containing polymer (e.g., PEG azide terminated with a methoxy group or a targeting group) and the alkyne appended to a small molecule (e.g., those illustrated in Formula 3) is a small molecule-polymer conjugate connected via a triazole. The alkyne group reacts with an azide end group of a polymer chain to form a triazole linkage. As seen in Formula 3, the resulting small molecule-polymer conjugate exhibits a regioisomerism, that is there are two regioisomers formed at the triazole. This regioisomerism is illustrated by the loose bond in Formula 1.

Formula 3. Gemcitabine-polymer conjugate at one position [0096] In embodiments where the small molecule is an active agent and the alkyne - containing electrophilic reagent is propargyl chloroformate or the like, Formula 4 illustrates the small molecule itself and the small molecule-polymer conjugates, prepared for example by the method described herein. The products of the cycloaddition reaction between an azide-containing polymer (e.g., PEG azide terminated with a methoxy group or a targeting group) and one or more alkyne groups appended to the drug are illustrated in Figure 4. Each of the alkyne groups reacts with an azide end group of a polymer chain to form more than one triazole linkage. The number of polymers per small molecule is limited by the number of free hydroxyl and amine groups available on the small molecule, as illustrated below.

Formula 4. Drug-polymer conjugates at one or more positions [0097] In another aspect of the disclosure, a kit suitable for preparing the small molecule- polymer conjugate of the disclosure is provided, the kit comprising an alkyne-containing electrophilic reagent in a first container, an azide-containing biocompatible polymer in a second container, and instructions for their use.

[0098] In certain embodiments, the alkyne-containing electrophilic reagent is a carboxylic acid, an acid halide, a carboxylic acid anhydride, a carboxylic acid salt, a carboxylic acid ester, an isocyanate, a carbonate, a carbamate, or a chloro formate. In a particular embodiment, the alkyne-containing electrophilic reagent is

where q is an integer from 0 to about 20, from about 0 to about 10, from about 1 to about 10, from about 2 to about 10, from about 2 to about 8, from about 2 to about 5, or from about 2 to about 4. In various sub-embodiments, q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a particular sub-embodiment, q is 2. In various sub-embodiments, each methylene group may be optionally substituted, or may itself be a different atom, such as NH, O, or S. In a particular embodiment, the alkyne-containing electrophilic reagent is a chloroformate, such as propargyl chloroformate

[0099] The azide-containing polymer can be any biocompatible polymer with an azide group. In certain embodiments, the azide-containing polymer imparts a stabilizing effect on the small molecule. When n is greater than 1, the various polymers of Formula 1 can be the same or different. In various embodiments, the azide-containing polymer is independently anionically charged, cationically charged, or uncharged; hydrophobic, hydrophilic, or amphiphilic; or combinations thereof. In various embodiments, the azide-containing polymer is a homopolymer, a block copolymer, or a random copolymer. In certain embodiments, the azide-containing polymer is polydisperse or monodisperse. In various embodiments, the polydispersity index of the azide-containing polymer is from 1 to about 30, from 1 to about 10, from 1 to about 5, or from 1 to about 3. In certain embodiments the azide-containing polymer is linear. In certain embodiments the azide-containing polymer is branched.

[0100] In certain embodiments, the azide-containing polymer is a polyethylene glycol (PEG), a polyether, a poly(lactide), a poly(glycolide), a poly(lactide-co-glycolide), a poly(lactic acid), a poly(glycolic acid), a poly(lactic acid-co-glycolic acid), a polyanhydride, a polyorthoester, a polycarbonate, a polyetherester, a polycaprolactone, a polyesteramide, a polyester, a polyacrylate, a polymer of ethylene-vinyl acetate or another acyl substituted cellulose acetate, a polyurethane, a polyamide, a polystyrene, a silicone based polymer, a polyolefm, a polyvinyl chloride, a polyvinyl fluoride, a fluoropolymer, a polypropylene, a polyethylene, a cellulosic, a starch, a naturally occurring polymer, a poly(vinyl imidazole), a polyacetal, a polysulfone, a chlorosulphonate polyolefm, or a blend or copolymer thereof. In particular embodiments, the azide-containing polymer is PEG-azide.

[0101] In various embodiments, the azide-containing polymer has an average molecular weight of from about 200 to about 50,000, from about 200 to about 40,000, from about 200 to 30,000, from about 200 to about 20,000, from about 200 to about 10,000, from about 200 to about 5,000, from about 200 to about 4,000, from about 200 to about 3,000, from about 200 to about 2,000, from about 200 to about 1,000, or from about 200 to about 500. In various embodiments, the azide-containing polymer has an average molecular weight of from about 10,000 to about 50,000, from about 10,000 to about 40,000, from about 10,000 to about 30,000, or from about 10,000 to about 20,000. In a particular embodiment, the azide- containing polymer has an average molecular weight of from about 500 to about 5,000.

[0102] In certain embodiments, the azide-containing polymer is independently terminated with a non-functional group, such as a methyl or methoxy, or a functional group, such as a targeting group. In a particular embodiment, the targeting group is a folate. In certain embodiments, the azide-containing polymer is a mixture of non-functional terminated and functional terminated polymers. In certain embodiments, the mixture is a mixture of methoxy terminated and folate-terminated polymers, for example a mixture of methoxy- terminated PEG and folate-terminated PEG. In certain embodiments, the polymer is terminated with another biomolecule. In such an embodiment, the small molecule-polymer conjugate is a networked small molecule-polymer conjugate, each conjugate comprising more than one small molecule.

[0103] In certain embodiments, the first container further comprises a first solvent. In certain embodiments, the first solvent is water, tetraethylene glycol dimethylether, dimethylsulfoxide, dimethylformamide, chloroform, dichloromethane, pyridine, acetone, ether, or a mixture thereof. In certain embodiments, the first solvent is a mixture of water and one or more of tetraethylene glycol dimethylether, dimethylsulfoxide, dimethylformamide, chloroform, dichloromethane, pyridine, acetone, or ether. In a particular embodiment, the first container does not comprise water. In a particular embodiment, the first solvent is water. [0104] In certain embodiments, the second container further comprises a second solvent. In various embodiments, the second solvent is methanol, ethanol, propanol, isopropanol, tetraethylene glycol dimethylether, dimethylsulfoxide, dimethylformamide, acetone, ether, water, or a mixture thereof. In a particular embodiment, the second solvent is water and one or more of methanol, ethanol, propanol, isopropanol, tetraethylene glycol dimethylether, dimethylsulfoxide, dimethylformamide, acetone, or ether. In a particular embodiment, the second solvent is water.

[0105] The kit optionally further comprises one or more containers comprising the following: a base, a metal catalyst, a solvent, a purification column, a filter, a drying agent, a mixing vessel, a magnetic stirbar, and a filtration vessel.

[0106] In a particular embodiment, the kit comprises instructions to (1) dissolve the small molecule in a first solvent (optionally provided with the kit); (2) add the alkyne-containing electrophilic reagent to the solution of the small molecule; (3) optionally add a base to the solution of the small molecule and alkyne-containing electrophilic reagent; (4) stir for between 30 minutes and 8 hours, or for between 1 hour and 2 hours; (5) remove solvent, alkyne-containing electrophilic reagent, and optional base; (6) dissolve the alkyne-modified small molecule in a second solvent (optionally provided with the kit); (7) add the azide- containing polymer; (8) optionally add the metal catalyst; (8) stir for between 1 and 24 hours, or for about 2 hours, at room temperature or at a temperature from 35 °C to about 80 °C; (9) optionally concentrate the reaction mixture; (10) optionally purify using filter or column (optionally provided with the kit). The final product of the kit may be used in a cell based assay or as a diagnostic tool for laboratory use.

Example 1 : Conjugation of Alkvne -Modified Vanillin to PEG Azides

[0107] The small molecule vanillin (200 mg) was dissolved in 10 mL of dichloromethane (DCM) at room temperature. To this mixture was added propargyl chloro formate (192 μΕ) and triethylamine (275 μί), and the reaction is stirred for 2 hours. The reaction mixture was then shaken with water and the organic phase was dried over magnesium sulfate and concentrated under nitrogen. The resulting product was confirmed with NMR (FIG. 2) and found to be pure enough to move on to the next reaction without further purification. FIG. 1 illustrates this reaction. The alkyne-modified vanillin (101 mg) was then dissolved in 10 mL of tetrahydrofuran (THF). The PEG azide (94 μΕ) was then added to the reaction and allowed to stir for 2 hours at room temperature. It is understood that the reaction may be increased by gentle heating to 60 degrees Celsius. FIG. 3 illustrates this reaction. The reaction mixture was then concentrated under nitrogen and purified by column chromatography,

methanol: chloroform (5:95). The resulting product was confirmed with NMR (FIG. 4).

Example 2: Conjugation of Alkyne-Modified Gemcitabine to PEG Azides

[0108] The small molecule Gemcitabine (200 mg) is dissolved in 10 mL of DMF at room temperature. To this mixture is added propargyl chloroformate (259 μί) and triethylamine (371 μί), and the reaction is stirred for 2 hours. FIG. 5 illustrates this reaction. It is understood that such modification may occur at every modifiable site on the biological molecule. The reaction mixture is then shaken with water and the organic phase is dried over magnesium sulfate and concentrated under nitrogen. The resulting product is confirmed with NMR. The alkyne-modified gemcitabine (100 mg) is then dissolved in 10 mL of

tetrahydrofuran (THF). The PEG azide (43 μί) is then added to the reaction and allowed to stir for 2 hours at room temperature. It is understood that the reaction may be increased by gentle heating to 60 degrees Celsius. FIG. 6 illustrates this reaction. The reaction mixture is then concentrated under nitrogen and purified by column chromatography,

methanol: chloroform. The resulting product is confirmed with NMR.

Example 3 : Conjugation of Alkyne -Modified Peptides to PEG Azides

[0109] The peptide PYY (0.25 mg) is dissolved in 25μΙ_^ of DMF at room temperature. To this mixture is added propargyl chloroformate (1.8 μί), and the reaction mixture is allowed to stir for 2 hours. The reaction mixture is then concentrated under N₂ and the residue analyzed by NMR spectroscopy. It is understood that such modification may occur at every modifiable site on the biological molecule, for example at a tyrosine phenolic OH. The alkyne-modified peptide is then dissolved in 25 μΐ, of DMF. PEG azide (1 μί) is added to this mixture and the reaction mixture is allowed to stir for 2 hours at room temperature. The reaction mixture is concentrated under N₂ and analyzed by NMR spectroscopy.

[0110] PYY has been implicated in the treatment of obesity, and thus PYY-polymer conjugates of the disclosure may be useful prodrugs of PYY. See Marianne T. Neary et al, Pharmacology & Therapeutics, 2009, 124(\), 44-56.

Example 4: Conjugation of Alkyne-Modified Nucleotide to PEG Azides [0111] The nucleotide adenosine triphosphate (ATP) (1 mg) was dissolved in 30 of DMF at room temperature. To this mixture was added propargyl chloro formate (1 μί), and the reaction was allowed to stir for 2 hours. The reaction mixture was then concentrated under N₂ and the residue analyzed by NMR spectroscopy. It is understood that such modification may occur at every modifiable site on the biological molecule, for example a sugar OH or the adenine NH₂. The alkyne -modified nucleotide from the modification reaction was dissolved in 30 of DMF. PEG azide (6 μί) was added to this mixture and the reaction was allowed to stir for 2 hours at room temperature. The reaction mixture was concentrated under N₂ and analyzed by NMR spectroscopy.

Example 5 : Preparation of Azide-Containing Polymer Terminated with a Targeting Group

[0112] The carboxylic acid of folate (300mg) is first activated by reacting with N- hydroxysuccimide (NHS) and Ν,Ν'-Dicyclohexylcarbodiimide (DCC) in dimethyl sulfoxide (DMSO) stirred for 18 hours, filtered and washed with 30% acetone-ether to give the corresponding activated ester. This activated ester is then dissolved in dry pyridine and stirred with monoamine PEG azide for 18 hours. The pyridine is evaporated and the resulting mixture chromatographed to give the folate functionalized PEG azide. This folate functionalized PEG azide can be attached to a small molecule as previously mentioned.

[0113] The examples set forth above are provided to give those of ordinary skill in the art with a complete disclosure and description of how to make and use the claimed

embodiments, and are not intended to limit the scope of what is disclosed herein.

Modifications that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent or patent application were specifically and individually indicated to be incorporated herein by reference.

[0114] Illustrative embodiments

1. A small molecule -polymer conjugate of Formula 1 : small molecule X-L—

Formula 1

wherein

X is independently O, NH, NR, or S, and X and R are independently atoms of the small molecule;

L is independently a 1 - 20 atom linear or branched linker;

n is an integer;

the polymer is a biocompatible polymer; and

wherein the X— L bond is degradable.

2. The conjugate of paragraph 1, wherein the small molecule is a cholinergic, an adrenergic, a serotonergic, an anesthetic, an hypnotic, an antiseizure therapeutic, an antipsychotic, an anxiolytic, a stimulant, an opiod, an analgesic, a spasmolytic, a cardiac glycoside, an antianginal, an antiarrhythmic, a diuretic, an angiotensin converting enzyme inhibitor, an angiotensin converting enzyme antagonist, a calcium blocker, a central sympatholytic, a peripheral sympatholytic, a vasodilator, an antihyperlipoproteinemic, a cholesterol biosynthesis inhibitor, an antithrombotic, a thrombolytic, a coagulant, a plasma extender, an oral hypoglycemic agent, an adrenocorticoid, an estrogen, a progestin, a prostaglandin, an androgen, a thyroid drug, a nonsteroidial anti-inflammatory agent, a steroidal anti-inflammatory agent, an antihistamine, an antiallergenic agent, an antiulcer agent, an antibiotic, an antimicrobial, an antiparasitic, an antifungal, an antimycobacterial agent, a cancer chemotherapeutic, an antiviral, a protease inhibitor, or a selective estrogen receptor modulator.

3. The conjugate of any one of paragraphs 1-2, wherein the small molecule is a steroidal anti-inflammatory agent.

4. The conjugate of paragraph 3, wherein the steroidal anti-inflammatory agent is hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone,

betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, or aldosterone. 5. The conjugate of paragraph 1 or 2, wherein the small molecule is an anticancer agent.

6. The conjugate of paragraph 5, wherein the anticancer agent melphalan, dacarbazine, temozolomide, streptozotocin, methotrexate, pemetrexed, tioguanine, fludarabine, pentostatin, cladribine, floxuridine, gemcitabine, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, irinotecan, topotecan, paclitaxel, or docetaxel.

7. The conjugate of paragraph 6, wherein the anticancer agent is gemcitabine.

8. The conjugate of paragraph 1 or 2, wherein the small molecule is an anticoagulant.

9. The conjugate of paragraph 8, wherein the anticoagulant is warfarin, acenocoumarol, phenprocoumon, argatroban, or ximelagatran.

10. The conjugate of paragraph 1 or 2, wherein the small molecule is an antimicrobial.

11. The conjugate of paragraph 10, wherein the antimicrobial is vanillin or dapsone.

12. The conjugate of paragraph 1 or 2, wherein the small molecule is an antiviral.

13. The conjugate of paragraph 12, wherein the antiviral is elvucitabine.

14. The conjugate of paragraph 1 or 2, wherein the small molecule is a nonsteroidal anti-inflammatory drug (NSAID).

15. The conjugate of paragraph 14, wherein the NSAID is prioxicam.

16. The conjugate of paragraph 1 or 2, wherein the small molecule is an analgesic.

17. The conjugate of paragraph 16, wherein the analgesic is morphine. 18. The conjugate of paragraph 1 or 2, wherein the small molecule is an anti- Alzheimer's drug.

19. The conjugate of paragraph 18, wherein the anti- Alzheimer's drug is memantine.

20. The conjugate of paragraph 1 or 2, wherein the small molecule is an anxiolytic.

21. The conjugate of paragraph 20, wherein the anxiolytic is temazepam.

22. The conjugate of paragraph 1 or 2, wherein the small molecule is a selective estrogen receptor modulator (SERM).

23. The conjugate of paragraph 22, wherein the SERM is raloxifene.

24. The conjugate of any of any one of paragraphs 1-23, wherein the X— L bond is a carbonate or carbamate bond.

25. The conjugate of any one of paragraphs 1-24, wherein X— L is

wherein Z is O or NH, and q is an integer from 0 to 20.

26. The conjugate of any one of paragraphs 1-25, wherein the biocompatible polymer is a polyethylene glycol (PEG), a polyether, a poly(lactide), a poly(glycolide), a poly(lactide-co-glycolide), a poly(lactic acid), a poly(glycolic acid), a poly(lactic acid-co- glycolic acid), a polyanhydride, a polyorthoester, a polycarbonate, a polyetherester, a polycaprolactone, a polyesteramide, a polyester, a polyacrylate, a polymer of ethylene-vinyl acetate or another acyl substituted cellulose acetate, a polyurethane, a polyamide, a polystyrene, a silicone based polymer, a polyolefm, a polyvinyl chloride, a polyvinyl fluoride, a fluoropolymer, a polypropylene, a polyethylene, a cellulosic, a starch, a naturally occurring polymer, a poly( vinyl imidazole), a polyacetal, a polysulfone, a chlorosulphonate polyolefin, or a blend or copolymer thereof.

27. The conjugate of any one of paragraphs 1-25, wherein the biocompatible polymer is methoxy-terminated polyethylene glycol or folate-terminated polyethylene glycol.

28. The conjugate of any one of paragraphs 1-25, wherein the biocompatible polymer is methoxy-terminated polyethylene glycol or folate-terminated polyethylene glycol having an average molecule weight of from about 500 to about 2000.

29. The conjugate of any one of paragraphs 1-28, wherein n is from about 1 to about 5.

30. The conjugate of any one of paragraphs 1-29, wherein n is 1 or 2.

31. The conjugate of any one of paragraphs 1-30, wherein the conjugate is substantially free of copper.

32. A method of preparing a small molecule-polymer conjugate of Formula 1 :

Formula 1

wherein

L is independently a 1 - 20 atom linear or branched linker;

n is an integer;

the polymer is a biocompatible polymer; and

wherein the X— L bond is degradable;

the method comprising a. reacting the small molecule with an alkyne-containing electrophilic reagent to form a modified small molecule of Formula B: small moleculeh X-L.— =

n

Formula B

wherein the small molecule, X, L, and n are as defined above; and b. reacting the modified small molecule of Formula B with a polymer or mixture of polymers of Formula C:

Formula C

wherein the polymer is as defined above.

33. The method of paragraph 32, wherein the small molecule is a cholinergic, an adrenergic, a serotonergic, an anesthetic, an hypnotic, an antiseizure therapeutic, an antipsychotic, an anxiolytic, a stimulant, an opiod, an analgesic, a spasmolytic, a cardiac glycoside, an antianginal, an antiarrhythmic, a diuretic, an angiotensin converting enzyme inhibitor, an angiotensin converting enzyme antagonist, a calcium blocker, a central sympatholytic, a peripheral sympatholytic, a vasodilator, an antihyperlipoproteinemic, a cholesterol biosynthesis inhibitor, an antithrombotic, a thrombolytic, a coagulant, a plasma extender, an oral hypoglycemic agent, an adrenocorticoid, an estrogen, a progestin, a prostaglandin, an androgen, a thyroid drug, a nonsteroidial anti-inflammatory agent, a steroidal anti-inflammatory agent, an antihistamine, an antiallergenic agent, an antiulcer agent, an antibiotic, an antimicrobial, an antiparasitic, an antifungal, an antimycobacterial agent, a cancer chemotherapeutic, an antiviral, a protease inhibitor, or a selective estrogen receptor modulator.

34. The method of paragraph 32 or 33, wherein the alkyne-containing electrophilic reagent is:

wherein q is an integer from 0 to 20. 35. The method of any one of paragraphs 32-34, wherein the small molecule is treated with from about 1 to about 1000 molar equivalents of alkyne-containing electrophilic reagent based on the number of modifiable positions on the small molecule.

36. The method of any one of paragraphs 32-35, wherein the biocompatible polymer is a polyethylene glycol (PEG), a polyether, a poly(lactide), a poly(glycolide), a poly(lactide-co-glycolide), a poly(lactic acid), a poly(glycolic acid), a poly(lactic acid-co- glycolic acid), a polyanhydride, a polyorthoester, a polycarbonate, a polyetherester, a polycaprolactone, a polyesteramide, a polyester, a polyacrylate, a polymer of ethylene-vinyl acetate or another acyl substituted cellulose acetate, a polyurethane, a polyamide, a polystyrene, a silicone based polymer, a polyolefm, a polyvinyl chloride, a polyvinyl fluoride, a fluoropolymer, a polypropylene, a polyethylene, a cellulosic, a starch, a naturally occurring polymer, a poly( vinyl imidazole), a polyacetal, a polysulfone, a chlorosulphonate polyolefm, or a blend or copolymer thereof.

37. The method of any one of paragraphs 32-36, wherein the polymer or mixture of polymers of Formula C is methoxy-terminated PEG-azide.

38. The method of any one of paragraphs 32-37, wherein the polymer has an average molecular weight of from about 500 to about 2000.

39. The method of any one of paragraphs 32-38, wherein the polymer or mixture of polymers of Formula C is a mixture of methoxy-terminated PEG-azide and folate- terminated PEG-azide.

40. A kit for preparing the small molecule-polymer conjugate of any one of paragraphs 1-31, comprising an alkyne-containing electrophilic reagent in a first container, a polymer or mixture of polymers of Formula C:

Formula C

in a second container, and instructions for use. The kit according to paragraph 40, wherein the alkyne-containing electrophilic reagent is:

wherein q is an integer from 0 to 20.

42. The kit according paragraphs 40 or 41 , wherein the polymer or mixture of polymers of Formula C is methoxy-terminated PEG-azide.

43. The kit according to any one of paragraphs 40-42, wherein the polymer or mixture of polymers of Formula C is a mixture of methoxy-terminated PEG-azide and folate- terminated PEG-azide.

44. A method of treating, preventing, or ameliorating cancer, comprising administering to a human patient in need thereof a pharmaceutically effective amount of the conjugate of paragraph 1, wherein the small molecule is gemcitabine.

45. A method of treating, preventing, or ameliorating a virus-related disease, for example HIV, comprising administering to a human patient in need thereof a

pharmaceutically effective amount of the conjugate of paragraph 1 , wherein the small molecule is elvucitabine.

46. A method of treating, preventing, or ameliorating inflammation or an inflammatory condition, for example, arthritis, comprising administering to a human patient in need thereof a pharmaceutically effective amount of the conjugate of paragraph 1 , wherein the small molecule is an NSAID, for example piroxicam.

47. A method of treating, preventing, or ameliorating bacterial infection, comprising administering to a human patient in need thereof a pharmaceutically effective amount of the conjugate of paragraph 1, wherein the small molecule is an antibiotic, for example an aminoglycoside antibiotic such as amikacin. 48. A method of treating, preventing, or ameliorating Alzheimer's disease, comprising administering to a human patient in need thereof a pharmaceutically effective amount of the conjugate of paragraph 1, wherein the small molecule is memantine.

49. A method of treating, preventing, or ameliorating insomnia or anxiety, comprising administering to a human patient in need thereof a pharmaceutically effective amount of the conjugate of paragraph 1 , wherein the small molecule is temazepam.

50. A method of treating, preventing, or ameliorating a disease affected by a selective estrogen receptor modulator, for example osteoporosis, comprising administering to a human patient in need thereof a pharmaceutically effective amount of the conjugate of paragraph 1, wherein the small molecule is raloxifene.

51. A pharmaceutical composition comprising an effective amount of the small molecule-polymer conjugate of any one of paragraphs 1-31.

Claims

What is claimed is:

1. A small molecule -polymer conjugate of Formula 1 :

Formula 1

wherein

L is independently a 1 - 20 atom linear or branched linker;

n is an integer;

the polymer is a biocompatible polymer;

wherein the X— L bond is degradable;

and wherein the small molecule is gemcitabine, elvucitabine, piroxicam, amikacin, memantine, temazepam, or raloxifene.

2. A small molecule -polymer conjugate of Formula 1 :

Formula 1

wherein

L is independently a 1 - 20 atom linear or branched linker;

n is an integer;

the polymer is a biocompatible polymer;

wherein the X— L bond is degradable;

and wherein the small molecule is melphalan, dacarbazine, temozolomide, streptozotocin, methotrexate, pemetrexed, tioguanine, fludarabine, pentostatin, cladribine, floxuridine, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, irinotecan, topotecan, paclitaxel, or docetaxel.

A small molecule -polymer conjugate of Formula 1

Formula 1

wherein

L is independently a 1 - 20 atom linear or branched linker;

n is an integer;

the polymer is a biocompatible polymer;

wherein the X— L bond is degradable;

and wherein the small molecule is warfarin, acenocoumarol, phenprocoumon, argatroban, or ximelagatran.

4. A small molecule -polymer conjugate of Formula 1 :

Formula 1

wherein

L is independently a 1 - 20 atom linear or branched linker;

n is an integer;

the polymer is a biocompatible polymer;

wherein the X— L bond is degradable;

and wherein the small molecule is vanillin or dapsone.

5. The conjugate of any one of claims 1-4, wherein the X— L bond is a carbonate or carbamate bond.

6. The conjugate of any one of claims 1-5, wherein X-

wherein Z is O or NH, and q is an integer from 0 to 20.

7. The conjugate of any one of claims 1-6, wherein the biocompatible polymer is a polyethylene glycol (PEG), a polyether, a poly(lactide), a poly(glycolide), a poly(lactide- co-glycolide), a poly(lactic acid), a poly(glycolic acid), a poly(lactic acid-co-glycolic acid), a polyanhydride, a polyorthoester, a polycarbonate, a polyetherester, a polycaprolactone, a polyesteramide, a polyester, a polyacrylate, a polymer of ethylene -vinyl acetate or another acyl substituted cellulose acetate, a polyurethane, a polyamide, a polystyrene, a silicone based polymer, a polyolefm, a polyvinyl chloride, a polyvinyl fluoride, a fluoropolymer, a polypropylene, a polyethylene, a cellulosic, a starch, a naturally occurring polymer, a poly( vinyl imidazole), a polyacetal, a polysulfone, a chlorosulphonate polyolefm, or a blend or copolymer thereof.

8. The conjugate of any one of claims 1-7, wherein the biocompatible polymer is methoxy-terminated polyethylene glycol or folate-terminated polyethylene glycol.

9. The conjugate of any one of claims 1-8, wherein the biocompatible polymer is methoxy-terminated polyethylene glycol or folate-terminated polyethylene glycol having an average molecule weight of from about 500 to about 2000.

10. The conjugate of any one of claims 1-9, wherein n is from about 1 to about 5.

11. The conjugate of any one of claims 1-10, wherein the conjugate is

substantially free of copper.

12. A pharmaceutical composition comprising an effective amount of the conjugate of any one of claims 1-11.

13. A method of preparing a small molecule-polymer conjugate of Formula 1 :

Formula 1

wherein

L is independently a 1 - 20 atom linear or branched linker;

n is an integer;

the polymer is a biocompatible polymer;

the X— L bond is degradable; and

wherein the small molecule is gemcitabine, elvucitabine, piroxicam, amikacin, memantine, temazepam, or raloxifene;

the method comprising

a. reacting the small molecule with an alkyne-containing electrophilic reagent to form a modified small molecule of Formula B:

n

Formula B

Formula C wherein the polymer is as defined above.

14. A method of preparing a small molecule-polymer conjugate of Formula 1 : small molecule X-L—

Formula 1

wherein

L is independently a 1 - 20 atom linear or branched linker;

n is an integer;

the polymer is a biocompatible polymer;

the X— L bond is degradable; and

wherein the small molecule is melphalan, dacarbazine, temozolomide, streptozotocin, methotrexate, pemetrexed, tioguanine, fludarabine, pentostatin, cladribine, floxuridine, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, irinotecan, topotecan, paclitaxel, or docetaxel;

the method comprising

a. reacting the small molecule with an alkyne-containing electrophilic reagent to form a modified small molecule of Formula B: small moleculeH-X-L— =

n

Formula B

Formula C wherein the polymer is as defined above.

A method of preparing a small molecule-polymer conjugate of Formula 1

Formula 1

wherein

L is independently a 1 - 20 atom linear or branched linker;

n is an integer;

the polymer is a biocompatible polymer;

the X— L bond is degradable; and

wherein the small molecule is warfarin, acenocoumarol, phenprocoumon, argatroban, or ximelagatran;

the method comprising

Formula B

Formula C wherein the polymer is as defined above.

16. A method of preparing a small molecule-polymer conjugate of Formula 1 :

Formula 1

wherein

L is independently a 1 - 20 atom linear or branched linker;

n is an integer;

the polymer is a biocompatible polymer;

the X— L bond is degradable; and

wherein the small molecule is vanillin or dapsone;

the method comprising

Formula B

N₃— ( polymer

Formula C wherein the polymer is as defined above.

17. The method of any one of claims 13-16, wherein the alkyne-containing electrophilic reagent is:

wherein q is an integer from 0 to 20.

18. The method of any one of claims 13-17, wherein the biocompatible polymer is a polyethylene glycol (PEG), a polyether, a poly(lactide), a poly(glycolide), a poly(lactide- co-glycolide), a poly(lactic acid), a poly(glycolic acid), a poly(lactic acid-co-glycolic acid), a polyanhydride, a polyorthoester, a polycarbonate, a polyetherester, a polycaprolactone, a polyesteramide, a polyester, a polyacrylate, a polymer of ethylene -vinyl acetate or another acyl substituted cellulose acetate, a polyurethane, a polyamide, a polystyrene, a silicone based polymer, a polyolefm, a polyvinyl chloride, a polyvinyl fluoride, a fluoropolymer, a polypropylene, a polyethylene, a cellulosic, a starch, a naturally occurring polymer, a poly( vinyl imidazole), a polyacetal, a polysulfone, a chlorosulphonate polyolefm, or a blend or copolymer thereof.

19. The method of any one of claims 13-18, wherein the polymer or mixture of polymers of Formula C is methoxy-terminated PEG-azide.

20. The method of any one of claims 13-18, wherein the polymer or mixture of polymers of Formula C is a mixture of methoxy-terminated PEG-azide and folate-terminated PEG-azide.

21. The method of any one of claims 13-20, wherein the polymer has an average molecular weight of from about 500 to about 2000.

22. A kit for preparing the small molecule-polymer conjugate of any one of claims 1-11, comprising an alkyne-containing electrophilic reagent in a first container, a polymer or mixture of polymers of Formula C:

Formula C in a second container, and instructions for use.

23. The kit according to claim 22, wherein the alkyne-containing electrophilic reagent is:

wherein q is an integer from 0 to 20.

24. The kit according to claim 22 or 23, wherein the polymer or mixture of polymers of Formula C is methoxy-terminated PEG-azide.

25. The kit according to claim 22 or 23, wherein the polymer or mixture of polymers of Formula C is a mixture of methoxy-terminated PEG-azide and folate-terminated PEG-azide.

26. A method of treating, preventing, or ameliorating cancer, comprising administering to a human patient in need thereof an effective amount of the conjugate of claim 1, wherein the small molecule is gemcitabine.

27. A method of treating, preventing, or ameliorating cancer, comprising administering to a human patient in need thereof an effective amount of the conjugate of claim 2.

28. A method of treating, preventing, or ameliorating a virus-related disease, comprising administering to a human patient in need thereof an effective amount of the conjugate of claim 1, wherein the small molecule is elvucitabine.

29. The method of claim 28, wherein the virus-related disease is HIV.

30. A method of treating, preventing, or ameliorating inflammation or an inflammatory condition, comprising administering to a human patient in need thereof an effective amount of the conjugate of claim 1 , wherein the small molecule is piroxicam.

31. The method of claim 30, wherein the inflammatory condition is arthritis.

32. A method of treating or ameliorating bacterial infection, comprising administering to a human patient in need thereof an effective amount of the conjugate of claim 1 , wherein the small molecule is amikacin.

33. A method of treating, preventing, or ameliorating bacterial infection, comprising administering to a human patient in need thereof an effective amount of the conjugate of claim 4.

34. A method of reducing coagulation, comprising administering to a human patient in need thereof an effective amount of the conjugate of claim 3.

35. A method of treating, preventing, or ameliorating Alzheimer's disease, comprising administering to a human patient in need thereof an effective amount of the conjugate of claim 1, wherein the small molecule is memantine.

36. A method of treating, preventing, or ameliorating insomnia or anxiety, comprising administering to a human patient in need thereof an effective amount of the conjugate of claim 1 , wherein the small molecule is temazepam.

37. A method of treating, preventing, or ameliorating a disease affected by a selective estrogen receptor modulator, comprising administering to a human patient in need thereof an effective amount of the conjugate of claim 1 , wherein the small molecule is raloxifene.

38. The method of claim 37, wherein the disease affected by a selective estrogen receptor modulator is osteoporosis.