BR112014028322B1 - CARRIER-DRUG CONJUGATE; PHARMACEUTICAL COMPOSITIONS AND METHOD OF MANUFACTURING THE PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

CARRIER-DRUG CONJUGATE; PHARMACEUTICAL COMPOSITIONS; METHOD FOR TREATING A PATIENT IN NEED OF A THERAPEUTIC COMPOUND; METHOD OF MANUFACTURING THE PHARMACEUTICAL COMPOSITION; AND PHARMACEUTICAL CARRIER. The invention provides carriers that enhance the absorption, half-life or bioavailability of therapeutic compounds. Carriers comprise targeting groups that bind vitamin D binding protein (DBP), conjugation groups to couple the targeting groups to therapeutic compounds, and, optionally, supporting chemical moieties.

Description

FIELD OF INVENTION

[001] The field of the invention provides the composition and general use of carriers, conjugates, fusions or formulations of therapeutic compounds for the purpose of increasing the potency, absorption, bioavailability or circulating half-life of the compounds by improving their pharmacokinetic properties in vivo .

BACKGROUND OF THE INVENTION

[002] The invention relates to improving the potency, absorption or pharmacokinetic properties of therapeutic compounds. The addition of polyethylene glycol or PEG is a known method to increase the half-life of some compounds by reducing kidney elimination, reducing aggregation, and reducing potentially unwanted immune recognition (Jain, Crit. Rev. Ther. Drug Carrier Syst. 25 :403-447 (2008)). PEG is typically used at a considerably large size (20 to 40 kDa) to maximize circulating half-life. This can be achieved using a single large PEG or multiple smaller PEGs linked to the compound. (Clark et al. J. Biol. Chem. 271:21969-21977 (1996); Fishburn, J. Pharm. Sci. 97:4167-4183 (2008)).

[003] Absorption is an important focus in drug development and medicinal chemistry, as a drug must be absorbed before any medicinal effects occur. The pharmacokinetic profile of a drug can be affected by several factors. Additionally, the absorption properties of therapeutic compounds can vary significantly from compound to compound. Some therapeutic compounds are poorly absorbed after oral or dermal administration. Other therapeutic compounds, such as most peptide and protein-based therapeutic compounds, cannot be administered orally. Alternative routes of administration such as intravenous, subcutaneous, or intramuscular injections are routinely used for some compounds; however, these pathways often result in slow absorption and exposure of therapeutic compounds to enzymes that degrade them, therefore requiring higher doses to achieve efficacy.

[004] Several peptides have been identified as being therapeutically promising. The chemical and biological properties of peptides and proteins make them attractive candidates for use as therapeutic compounds. Peptides and proteins are naturally occurring molecules made up of amino acids and are involved in various physiological processes. Peptides and proteins exhibit a high degree of selectivity and potency, and may not suffer from potential adverse drug-drug interactions or other negative side effects. Therefore, peptides and proteins hold promise as a highly diverse, highly potent, and highly selective class of therapeutic compounds with low toxicity. Peptides and proteins can, however, have short half-lives in vivo. For such peptides, this may be a few minutes. This may make them generally unsuitable in their native form for therapeutic administration. Additionally, peptides may have short duration of action or poor bioavailability.

[005] Fibroblast growth factor 21 (SEQ ID: 2) is a protein that circulates in serum. It is encoded by the FGF21 gene and is a member of a family of atypical fibroblast growth factors (FGFs) that include FGF19 and FGF23. It does not have the conventional heparin-binding FGF domain. Members of the FGF family possess broad mitogenic and cell survival activities and are involved in a variety of biological processes including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth, and tumor invasion. FGF21 is specifically induced by HMGCS2 activity. FGF21 stimulates glucose uptake in adipocytes but not in other cell types. This effect is additive to insulin activity.

[006] In vitro studies indicate that FGF21 prefers to bind to the FGFR1c/b-Klotho receptor complex rather than binding to those containing other FGFR isotypes (Kliewer and Mangelsdorf, Am. J. Clin. Nutr. 91:254S-257S (2010)). FGF21 promotes glucose uptake by adipocytes in vitro. Administration of FGF21 to diabetic animals reduces circulating glucose levels, but an excess of FGF21 does not induce hypoglycemia as is seen with administration of an excess of insulin (Kharitonenkov and Shanafelt, Curr. Opin. Investig. Drugs 10:359-364 (2009)). Therefore, FGF21 is a promising therapeutic protein for the treatment of diabetes. FGF21 has, however, been administered frequently to observe therapeutic benefits in an animal model (Kharitonenkov et al., J. Clin. Invest. 115:1627-1635 (2005)). FGF21 in its natural state has an extremely short half-life in serum (1.1 hours) making the exogenous addition of FGF21 in its natural state clinically unsuitable as a treatment (see WO03/011213). Additionally, FGF21 has poor bioavailability when injected subcutaneously. In a comparative pharmacokinetic study, 1 mg/kg FGF21 was injected intravenously (IV) or subcutaneously (SC) and the concentration of FGF21 was analyzed over time. The results showed a significant reduction in bioavailability using a subcutaneous route of administration (Cmax 73 nM) compared to the intravenous route of administration (Cmax 1890 nM; see Table 1 in Xu J et al., 2009. Am J Physiol Endocrinol Metab 297 : E1105-E1114). The references mentioned above are incorporated herein in their entirety by citation.

[007] The ghrelin peptide (SEQ ID NO:5) is naturally secreted from the stomach in mammals into the circulation to stimulate appetite and the release of growth hormone. Ghrelin stimulates the release of growth hormone (GH) from the pituitary gland through the GHS-R cellular receptor and has important roles in energy homeostasis. Furthermore, ghrelin acts directly on the central nervous system to reduce sympathetic nerve activity. Ghrelin receptors (GHS-Rs) are concentrated in the hypothalamic-pituitary unit. GHS-Rs are distributed in peripheral tissues, including the heart, lung, liver, kidneys, pancreas, stomach, small and large intestines, fat cells, and immune cells.

[008] Ghrelin has been used therapeutically to increase weight and lean body mass in patients suffering from cachexia or involuntary weight loss resulting from a chronic disease such as cancer (Hiura et. al., Cancer Jan 26, 2012, http ://onlinelibrary.wiley.com/doi/10.1002/cncr.27430/abstrac t). However, ghrelin has a naturally short half-life of 11 minutes in humans (Akamizu et al., Eur J Endocrinol 150:447-55 (2004)) and therefore must be dosed frequently to observe therapeutic effects.

[009] Infliximab (Remicade®, Janssen Biotech Inc., U.S. Pat. Nos. US 5,919,452 and US 2002/0141996, incorporated herein in their entirety by reference) is a monoclonal antibody that binds to the tumor necrosis factor alpha (TNF-α, SEQ ID:10), which is used to treat autoimmune diseases. Infliximab is approved by the U.S. Food and Drug Administration (FDA) for the treatment of psoriasis, Crohn's disease, ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, and ulcerative colitis. TNF-α is a chemical messenger (cytokine) and a key part of the autoimmune reaction. Infliximab is administered intravenously by a healthcare professional and is not approved for subcutaneous dosing.

SUMMARY OF THE INVENTION

[010] The invention provides carriers that improve the absorption, stability, half-life, duration of effect, potency or bioavailability of therapeutic compounds. Carriers comprise targeting groups that bind the vitamin D binding protein (DBP), conjugation groups to couple the targeting groups to therapeutic compounds, and optional chemical support moieties.

[011] In one embodiment of the invention, the targeting group is vitamin D, an analogue of vitamin D, a metabolite related to vitamin D, an analogue of a metabolite related to vitamin D, a peptide that binds DBP, a anti-DBP antibody, a derivative of anti-DBP antibody, a nucleotide aptamer that binds DBP, or a small carbon-based molecule that binds DBP.

[012] In another embodiment, the coupling group is an amine-reactive group, a thiol-reactive group, a maleimide group, a thiol group, an aldehyde group, an NHS-ester group, a 4-nitrophenyl ester , an acylimidazole, a haloacetyl group, an iodoacetyl group, a bromoacetyl group, an SMCC group, a SMCC sulfo group, a carbodiimide group and bifunctional crosslinkers such as NHS-Maleimido or combinations thereof. The coupling groups of the invention may promote thiol bonds, amide bonds, oxime bonds, hydrazone bonds, thiazolidinone bonds, or utilize cycloaddition reactions (e.g., click chemistry) to couple the carrier or targeting group to a therapeutic compound.

[013] In another embodiment, the pharmaceutical carrier that additionally comprises a supporting chemical fraction, comprises polyethylene glycol, polylysine, polyethyleneimine, polypropylene glycol, a peptide, serum albumin, thioredoxin, an immunoglobulin, an amino acid, a nucleic acid, a glycan , a modifying group that contains a reactive ligand, a water-soluble polymer, a small carbon chain ligand, or an additional therapeutic chemical moiety.

[014] In another embodiment, the supporting chemical fraction is between about 100 Da. and 200,000 Da. In preferred embodiments, the supporting chemical fraction is between about 100 Da. and 20,000 Da., 200 Da. . and 15,000 Da., 300 Da. and 10,000 Da., 400 Da. and 9,000 Da., 500 Da. and 5,000 Da., 600 Da. and 2,000 Da., 1000 Da. and 200,000 Da., 5000 Da. and 100,000 Da., 10,000 Da. and 80,000 Da., 20,000 Da. and 60,000 Da., or 20,000 Da. and 40,000 Da.

[015] The invention provides a pharmaceutical composition comprising a therapeutic compound conjugated to, fused to, or formulated with a carrier. The carrier comprises a targeting group that binds DBP and increases the absorption, bioavailability, or circulating half-life of the therapeutic compound. The pharmaceutical compositions of the invention may comprise two or more therapeutic compounds conjugated to a single carrier. The pharmaceutical compositions of the invention may comprise two or more carriers conjugated to a therapeutic compound.

[016] In one embodiment, the targeting group in the pharmaceutical composition is vitamin D, an analogue of vitamin D, a metabolite related to vitamin D, an analogue of a metabolite related to vitamin D, a peptide that binds DBP, an anti-DBP antibody, an anti-DBP antibody derivative, a nucleotide aptamer that binds DBP, or a small carbon-based molecule that binds DBP.

[017] In another embodiment, the pharmaceutical composition additionally comprises a supporting chemical fraction. In a preferred embodiment, the supporting chemical moiety is polyethylene glycol, polylysine, polyethyleneimine, polypropylene glycol, a peptide, serum albumin, thioredoxin, an immunoglobulin, an amino acid, a nucleic acid, a glycan, a modifying group containing a reactive linker , a water-soluble polymer, a small carbon chain ligand, or an additional therapeutic compound.

[018] The pharmaceutical compositions of the invention may comprise small molecules, chemical entities, nucleic acids, nucleic acid derivatives, peptides, peptide derivatives, naturally occurring proteins, non-naturally occurring proteins, peptide nucleic acids (PNA), stapled peptides, morpholinos, phosphorodiamidate morpholinos, antisense drugs, RNA-based silencing drugs, aptamers, glycoproteins, enzymes, hormones, cytokines, interferons, growth factors, blood clotting factors, antibodies, antibody fragments, antibody derivatives, toxin-conjugated antibodies, metabolic effectors, analgesics, antipyretics, anti-inflammatory agents, antibiotics, antimicrobial agents, antiviral agents, antifungal drugs, musculoskeletal drugs, cardiovascular drugs, renal drugs, pulmonary drugs, drugs for diseases of the digestive system, hematological drugs, urological drugs, metabolism drugs, hepatic drugs, neurological drugs, antidiabetic drugs, anticancer drugs, drugs to treat stomach pathologies, drugs to treat colon pathologies, drugs to treat skin pathologies, or drugs to treat lymphatic pathologies.

[019] In a preferred embodiment, the pharmaceutical composition comprises a protein that has FGF21 activity comprising an amino acid sequence with at least 90% sequence identity with respect to SEQ ID NO:2. In another preferred embodiment, the targeting group is vitamin D. In another preferred embodiment, the supporting chemical moiety is polyethylene glycol.

[020] In a more preferred embodiment, the invention contemplates a pharmaceutical composition comprising a protein that has FGF21 activity comprising an amino acid sequence with at least 90% sequence identity with respect to SEQ ID NO:2, a supporting chemical fraction which is polyethylene glycol, and a targeting group which is vitamin D. In this embodiment, the targeting group increases the absorption, bioavailability or half-life of the therapeutic compound in circulation. In another more preferred embodiment, the invention contemplates a pharmaceutical composition comprising a protein that has FGF21 activity and the amino acid sequence of SEQ ID NO:2.

[021] In a preferred embodiment, the pharmaceutical composition comprises a protein that has ghrelin activity comprising an amino acid sequence with at least 90% sequence identity with respect to SEQ ID NO:5. In another preferred embodiment, the targeting group is vitamin D. In another preferred embodiment, the supporting chemical moiety is polyethylene glycol.

[022] In a more preferred embodiment, the invention contemplates a pharmaceutical composition comprising a protein that has ghrelin activity comprising an amino acid sequence with at least 90% sequence identity with respect to SEQ ID NO:5, a supporting chemical fraction which is polyethylene glycol, and a targeting group which is vitamin D. In this embodiment, the targeting group increases the absorption, bioavailability or half-life of the therapeutic compound in circulation. In another more preferred embodiment, the invention contemplates a pharmaceutical composition comprising a protein that has ghrelin activity and the amino acid sequence of SEQ ID NO:5.

[023] In one embodiment, the pharmaceutical composition comprises an antibody. In a preferred embodiment, the antibody is an anti-TNF-α antibody that specifically binds to a protein that has an amino acid sequence with at least 90% sequence identity to SEQ ID NO:10. In a more preferred embodiment, the anti-TNF-α antibody specifically binds to a protein having the amino acid sequence of SEQ ID NO:10. In another preferred embodiment, the targeting group is vitamin D. In another preferred embodiment, the supporting chemical moiety is polyethylene glycol.

[024] In a more preferred embodiment, the invention comprises an anti-TNF-α antibody that specifically binds to a protein that has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 10, a supporting chemical moiety which is polyethylene glycol, and a targeting group which is vitamin D. In this embodiment, the targeting group increases the absorption, bioavailability or half-life of the therapeutic compound in circulation.

[025] In certain embodiments, the present invention provides carriers that include those of formula I:

[026] Where:

[027] B is a targeting group selected from vitamin D, a vitamin D analogue, a vitamin D-related metabolite, an analogue of a vitamin D-related metabolite, a peptide that binds DBP, an anti-DBP antibody, a anti-DBP antibody derivative, a nucleotide aptamer that binds DBP, or a small carbon-based molecule that binds DBP;

[028] S is a chemical support fraction comprising polyethylene glycol, polylysine, polyethyleneimine, polypropylene glycol, a peptide, serum albumin, thioredoxin, an immunoglobulin, an amino acid, a nucleic acid, a glycan, a modifying group containing a reactive ligand, a poly(lactic acid), a water-soluble polymer, a small carbon chain ligand, or an additional therapeutic chemical moiety;

[029] C is an amine-reactive group, a thiol-reactive group, a maleimide group, a thiol group, a disulfide group, an aldehyde group, an NHS-ester group, a 4-nitrophenyl ester, an acylimidazole, a haloacetyl group, an iodoacetyl group, a bromoacetyl group, an SMCC group, a sulfo SMCC group, a carbodiimide group and bifunctional crosslinkers such as NHS-Maleimide or combinations thereof;

[030] L1 and L2 are ligands independently selected from OC(O)-, -O-, -S-S-,

[031] L3 is –(CH2)o-;

[032] n is an integer from 0 to 3; It is

[033] -(CH2)n-, -C(O)NH-, -HNC(O)-, -C(O)O-, - -S-, -S(O)-, -S(O) 2- and -NH-. L3 is - (CH2)o-; n is an integer from 0 to 3; and o is an integer from 0 to 3.

[034] In certain embodiments, the present invention provides a carrier method for producing one of formula I:

[035] which comprises the step of reacting a compound of formula Ia:

[036] with a compound of formula Ib:

[037] in the presence of an amide coupling agent,

[038] where B, S, C, L2 and L3 are defined as indicated above and L1 is -C(O)NH-.

[039] In certain other embodiments, the present invention provides a method for producing a carrier of formula I:

[040] which comprises the step of reacting a compound of formula Ia:

[041] with a compound of formula Ic:

[042] in the presence of an amide coupling agent,

[043] hydrolyzing an ester into a carboxylic acid and,

[044] converting a carboxylic acid into an active ester,

[045] where B, S, L2, L3 and n and o are defined as indicated above,

[046] L1 is -C(O)NH- and,

[047] R1 is C1-C6 alkyl.

[048] The invention provides a method for treating a patient in need of a therapeutic compound, comprising administering an effective amount of one or more of the pharmaceutical compositions described herein. Exemplary therapeutic compounds include small molecules, chemical entities, nucleic acids, derivatives of nucleic acids, peptides, derivatives of peptides, naturally occurring proteins, non-naturally occurring proteins, peptide nucleic acids (PNA), stapled peptides, morpholinos, morpholinos phosphorodiamidate, antisense drugs, RNA-based silencing drugs, aptamers, glycoproteins, enzymes, hormones, cytokines, interferons, growth factors, blood clotting factors, antibodies, antibody fragments, antibody derivatives, toxin-conjugated antibodies, effectors metabolic drugs, analgesics, antipyretics, anti-inflammatory agents, antibiotics, antimicrobial agents, antiviral agents, antifungal drugs, musculoskeletal drugs, cardiovascular drugs, renal drugs, pulmonary drugs, drugs for diseases of the digestive system, hematological drugs, urological drugs, metabolism drugs , liver drugs, neurological drugs, antidiabetic drugs, anticancer drugs, drugs to treat stomach pathologies, drugs to treat colon pathologies, drugs to treat skin pathologies, and drugs to treat lymphatic pathologies.

[049] In preferred methods, the therapeutic compound is a protein that has FGF21 activity comprising an amino acid sequence with at least 90% sequence identity to SEQ ID NO:2. In other preferred methods, the therapeutic compound is a protein that has ghrelin activity comprising an amino acid sequence with at least 90% sequence identity to SEQ ID NO:5. In other preferred methods, the therapeutic compound is an anti-TNF-α antibody that specifically binds to a protein that has at least 90% sequence identity to SEQ ID NO:10. In other preferred methods, the targeting group is vitamin D or the support is polyethylene glycol.

[050] In other embodiments and methods, the pharmaceutical compositions of the invention are in pharmaceutically acceptable formulations. The pharmaceutical compositions can be provided to patients by a transdermal, oral, parenteral, subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial injection, infusion, inhalation, in a form of administration for ocular, topical, rectal, nasal, buccal, sublingual, vaginal or implanted reservoir administration.

[051] The invention provides the use of the disclosed pharmaceutical compositions for the manufacture of medicines for the treatment of patients in need of the medicines.

[052] The invention provides methods of manufacturing the pharmaceutical compositions disclosed in this document comprising the conjugation of a targeting group and a drug in a carrier-drug compound using coupling groups. The coupling groups can be amine-reactive coupling groups, maleimide coupling groups, cysteine coupling groups, aldehyde coupling groups, or thiol-reactive coupling groups. Maleimide is a useful coupling group for use in coupling with sulfhydryl groups such as a free cysteine residue that can be inserted in a site-specific manner into a peptide or protein at a desired position. Other coupling groups such as NHS- that target amine or aldehyde groups that can be used for site-specific binding to the N-terminal end of a therapeutic compound are well known to those skilled in the art. Other more specialized coupling groups are contemplated and could be replaced by one skilled in the art.

[053] In some methods, the targeting group is vitamin D, a vitamin D analogue, a vitamin D-related metabolite, an analogue of a vitamin D-related metabolite, a peptide that binds DBP, an anti-DBP antibody , an anti-DBP antibody derivative, a nucleotide aptamer that binds DBP, or a small carbon-based molecule that binds DBP.

[054] In other embodiments, methods of manufacturing pharmaceutical compositions further comprise the conjugation of a supporting chemical moiety to the targeting group or drug. The supporting chemical moiety may be polyethylene glycol, polylysine, polyethyleneimine, polypropylene glycol, a peptide, serum albumin, thioredoxin, an immunoglobulin, an amino acid, a nucleic acid, a glycan, a modifying group containing a reactive ligand, a water-soluble polymer, a small carbon chain ligand, or an additional therapeutic compound.

DESCRIPTION OF THE DRAWINGS

[055] Figure 1: A schematic diagram showing the general structure of a carrier coupled to a drug. The carrier comprises a steering group, a support and, optionally, a coupling group.

[056] Figure 2: Reaction scheme showing the chemical structures and synthesis reactions used to generate a carrier, a Vitamin D3-PEG-Maleimide adduct. The carrier was generated by conjugating 1) a Vitamin D analog (the targeting group), 2) a PEG support, and 3) a maleimide coupling group.

[057] Figure 3: The bioavailability and pharmacokinetics of an FGF21-carrier conjugate. FGF21 alone (SEQ ID NO:3) or conjugated to the carrier Vitamin D3-PEG-Maleimide were injected subcutaneously into Sprague Dawley rats at 0.1 mg/kg. Plasma samples were analyzed for FGF21 concentration by ELISA in duplicate and the means of 3 to 5 animals per time point were plotted on the semi-logarithmic plot graph.

[058] Figure 4: The pharmacokinetics of the ghrelin-carrier conjugate. Ghrelin (SEQ ID NO:6) alone or conjugated to the carrier Vitamin Da-PEG-Maleimide were injected subcutaneously into Sprague Dawley rats at 0.1 mg/kg. Plasma samples were analyzed for Ghrelin concentration by ELISA in duplicate and the means of 3 to 5 animals per time point were plotted on the semi-logarithmic plot graph.

[059] Figure 5: Reaction scheme showing the chemical structures and synthesis reactions used to generate another carrier, a Vitamin D3-PEG-NHS adduct. The carrier was generated by conjugating 1) a Vitamin D analog (the targeting group), 2) a PEG support, and 3) an NHS coupling group.

[060] Figure 6: The bioavailability and pharmacokinetics of an infliximab-carrier conjugate. Infliximab alone or conjugated to the carrier Vitamin Da-PEG-NHS were injected subcutaneously into Sprague Dawley rats at 1 mg/kg. Plasma samples were analyzed for infliximab concentration by an infliximab-specific ELISA and means from 3 animals per time point were plotted on a linear plot graph.

DETAILED DESCRIPTION OF THE INVENTION

[061] The invention provides carrier molecules that are covalently linked to, fused to, or formulated with therapeutic proteins, peptides, nucleic acids or small molecules for the purpose of increasing the potency, absorption, bioavailability, circulating half-life or pharmacokinetic properties of therapeutic compounds. In certain embodiments, the carriers comprise a steering group, a support and a coupling group. In other embodiments, the carriers do not have a support, which acts, among other things, as a "spacer" between the targeting group and the therapeutic compound.

[062] Carriers are designed to be suitable for use in humans and animals. Carriers are intended to enhance the pharmacokinetic properties of a biological or chemical entity that is coupled to, conjugated to, fused to, or formulated with the carrier. This occurs through the interaction of the targeting group with vitamin D-binding protein (DBP), which can actively transport molecules quickly and efficiently from the site of administration to circulating plasma, thereby reducing drug exposure to degradative enzymes. By binding to DBP, carriers also improve the circulating half-life of the drug, thereby increasing the potency and therapeutic efficacy of the drug by preventing renal filtration. Methods for conjugating the carrier to therapeutic compounds described herein are known in the art. By way of example, conjugation using the coupling groups of the invention can be carried out using the compositions and methods described in WO93/012145 (Atassi et al.) and 7,803,777 (Defrees et al.), each of which is incorporated herein in its entirety by citation.

[063] When describing and claiming one or more embodiments of the present invention, the following terminology will be used in accordance with the definitions described below.

[064] The term "absorption" is the movement of a drug into the bloodstream. A drug must be introduced by some route of administration (e.g., oral, topical, or dermal) or in a specific dosage form such as a tablet, capsule, or liquid. Intravenous therapy, intramuscular injection, and enteral nutrition provide less variability in absorption and bioavailability is often close to 100%. The fastest route of absorption is inhalation.

[065] An "antagonist" refers to a molecule capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the activities of a particular or specified protein, including its binding to one or more receptors in the case of a ligand, or binding to one or more ligands in the case of a receptor. Antagonists include antibodies and their antigen-binding fragments, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bio-organic molecules, peptidomimetics, pharmacological agents and their metabolites, transcriptional and translational control sequences, and similar. Antagonists also include inhibitors which are small molecules that inhibit proteins, hormones or other bioactive molecules. Antagonists can be fusion proteins, receptor molecules, antisense molecules, aptamers, ribozymes or derivatives that specifically bind to proteins, hormones or other bioactive molecules and thus inhibit their binding to their target.

[066] "Antibodies" (Abs) and "immunoglobulins" (Igs) refer to glycoproteins that have similar structural characteristics. Antibodies show binding specificity to a specific antigen, while immunoglobulins include antibodies and other antibody-like molecules that generally do not show antigen specificity. Polypeptides of the second type are, for example, produced at low levels by the lymphatic system and at increased levels by myelomas.

[067] "Aptamers" are nucleic acid-based compounds that have been selected for binding to a specific target. An example of an aptamer-based therapeutic compound can be found in WO07/035922, which is incorporated herein in its entirety by reference.

[068] The term "bioavailability" refers to the fraction of an administered dose of unchanged drug that reaches systemic circulation, one of the main pharmacokinetic properties of drugs. When a medication is administered intravenously, its bioavailability is 100%. When a drug is administered by other routes (e.g., oral), its bioavailability usually decreases (due to incomplete absorption and first-pass metabolism) or may vary from patient to patient. Bioavailability is an important parameter in pharmacokinetics that is taken into consideration when dosages are calculated for non-intravenous routes of administration.

[069] "Carriers" are compounds that can be conjugated to, fused with, coupled to or formulated with therapeutic compounds to improve the absorption, half-life, bioavailability, pharmacokinetic or pharmacodynamic properties of drugs. They comprise a directing group, a coupling group and, optionally, a chemical support moiety.

[070] An "effective amount" refers to an amount of the therapeutic compound that is effective, in the necessary dosages and for periods of time, to obtain the desired therapeutic or prophylactic result. A "therapeutically effective amount" of a therapeutic compound may vary depending on factors such as the disease state, age, sex and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount may be measured, for example, by better survival rate, faster recovery, or by the improvement, enhancement or elimination of symptoms, or other acceptable biomarkers or surrogate markers. A therapeutically effective amount is also an amount with which any toxic or harmful effects of the therapeutic compound are offset by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount of the therapeutic compound that is effective, in the required dosages and for periods of time, to obtain the desired prophylactic result. Typically, but not necessarily, because a prophylactic dose is used in subjects before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[071] "Half-life" is a scientific term known in the art that refers to the period of time that elapses until half the amount of a test molecule ceases to be detected. An in vivo half-life refers to the time that elapses when half of the test molecule is no longer detected in circulating serum or tissues of a human or animal.

[072] A "hormone" is a biological or chemical messenger from one cell (or group of cells) to another cell that has signaling capacity. As described herein, hormones for use in the invention may be peptides, steroids, pheromones, interleukins, lymphokines, cytokines, or members of other classes of hormones known in the art.

[073] "Homologues" are bioactive molecules that are similar to a reference molecule at the level of nucleotide sequence, peptide sequence, functional or structural. Homologues may include derivatives of sequences that share a certain percentage identity with the reference sequence. Therefore, in one embodiment, the homologous or derived sequences share at least 70 percent sequence identity. In a preferred embodiment, the homologous or derived sequences share at least 80 or 85 percent sequence identity. In a more preferred embodiment, the homologous or derived sequences share at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity. . Homologous or derived nucleic acid sequences can also be defined by their ability to remain linked to a reference nucleic acid sequence under high stringency hybridization conditions. Homologs that have structural or functional similarity to a reference molecule may be chemical derivatives of the reference molecule. Methods for detecting, generating and tracking structural and functional homologues as well as derivatives are known in the art.

[074] "Hybridization" generally depends on the ability of denatured DNA to reanneal itself when complementary strands are present in an environment below its melting temperature. The greater the degree of homology desired between the probe and the hybridization sequence, the higher the relative temperature that can be used. Consequently, higher relative temperatures would make reaction conditions more stringent, while lower temperatures less stringent. For more details and an explanation of the stringency of hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

[075] An "individual", "subject" or "patient" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, primates (including human and non-human primates) and rodents (e.g., mice, hamsters, guinea pigs, and rats). In certain embodiments, the mammal is a human being. A "control subject" refers to a healthy subject who has not been diagnosed as having a disease, dysfunction or pathology that has been identified in an individual, subject or patient. A control subject does not suffer from any signs or symptoms associated with the disease, dysfunction or pathology.

[076] A "medicine" is an active drug that has been manufactured for the treatment of a disease, disorder or pathology.

[077] "Morpholinos" are synthetic molecules that are non-natural variants of natural nucleic acids that utilize a phosphorodiamidate linkage, described in U.S. Patent No. 8,076,476, incorporated herein in its entirety by reference.

[078] "Nucleic acids" are any group of macromolecules of DNA, RNA or variants thereof, which carry genetic information that can control cellular functions. Nucleic acids can have enzyme-like activity (e.g., ribozymes) or can be used to inhibit gene expression in a subject (e.g., RNAi). The nucleic acids used in the inventions described herein may be single-stranded, double-stranded, linear or circular. The inventions further incorporate the use of nucleic acid variants including, but not limited to, aptamers, PNA, Morfolino or other non-natural nucleic acid variants. By way of example, nucleic acids useful for the invention are described in U.S. Patent No. 8,076,476, incorporated herein in its entirety by reference.

[079] "Patient response" or "response" can be evaluated using any end result that indicates a benefit to the patient, including, without limitation, (1) inhibition, to some degree, of disease progression, including slowing and inhibiting total progression; (2) reduction in the number of attacks and/or symptoms of the disease; (3) inhibition (i.e., reduction, decrease or complete inhibition) of the infiltration of pathological cells into adjacent peripheral organs and/or tissues; (4) inhibition (i.e., reduction, diminution, or complete inhibition) of the spread of the disease; (5) reduction of an autoimmune pathology; (6) favorable change in the expression of a biomarker associated with the disorder; (7) relief, to some extent, of one or more symptoms associated with a disorder; (8) increase in the period without disease onset after treatment; or (9) reduced mortality at a given time point after treatment.

[080] As used herein, the term "peptide" is any peptide that comprises two or more amino acids. The term peptide includes short peptides (e.g., peptides comprising between 2 and 14 amino acids), medium-sized peptides (15 to 50), or long-chain peptides (e.g., proteins). The terms peptide, medium-sized peptide and protein may be used interchangeably herein. As used herein, the term "peptide" is interpreted to mean a polymer composed of amino acid residues, related naturally occurring structural variants, and their synthetic analogues that are not naturally occurring linked through peptide bonds, related structural variants of natural occurrence. natural and its synthetic analogues that are not natural. Synthetic peptides can be synthesized, for example, using an automated peptide synthesizer. Peptides can also be synthesized by other means, such as by cells, bacteria, yeast or other living organisms. Peptides can contain amino acids other than the 20 genetically encoded amino acids. Peptides include those modified by natural processes, such as splicing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, and are well known to those skilled in the art. Modifications occur in any region of a peptide, including the main peptide chain, the amino acid side chains, and the terminal amino or carboxyl ends.

[081] As used herein, a "pharmaceutically acceptable carrier" or "a therapeutically effective carrier" is (solid) aqueous or non-aqueous, for example, alcoholic or oleaginous, or a mixture thereof, and may contain a surfactant, emollient , lubricant, stabilizer, colorant, perfume, preservative, pH-adjusting acid or base, a solvent, emulsifier, gelling agent, moisturizer, stabilizer, wetting agent, extended release agent, humectant, or other component customarily included in a particular form of a pharmaceutical composition. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol and oils such as olive oil or injectable organic esters. A pharmaceutically acceptable carrier may contain physiologically acceptable compounds that act, for example, to stabilize or enhance the absorption of a specific inhibitor, for example, carbohydrates such as glucose, sucrose or dextrans, antioxidants such as ascorbic acid or glutathione, chelating agents, proteins of low molecular weight or other stabilizers or excipients.

[082] The term "pharmacokinetics" is currently defined as the time course of absorption, distribution, metabolism and excretion of a therapeutic compound. Enhanced "pharmacokinetic properties" are defined as: the enhancement of one or more of the pharmacokinetic properties as desired for a particular therapeutic compound. Examples include but are not limited to: reduced elimination by metabolism or secretion, increased drug absorption, increased half-life and/or increased bioavailability.

[083] "PNA" refers to peptide nucleic acids with a chemical structure similar to DNA or RNA. Peptide bonds are used to link nucleotides or nucleosides together.

[084] "Supports" are molecules with which other molecules can be covalently or non-covalently linked or formulated. The supports of the invention can act as "spacers" or "linkers" between the targeting group and the drug. The supports may also contain a reactive ligand or may have beneficial therapeutic properties in addition to those of the drug. Therefore, the supports of the invention may be, for example, PEG, serum albumin, thioredoxin, an immunoglobulin, a modifying group containing a reactive ligand, a water-soluble polymer, or a therapeutic compound.

[085] The "stringency" of hybridization reactions is easily determined by one skilled in the art, and is generally an empirical calculation that depends on probe length, washing temperature and salt concentration. In general, longer probes require higher temperatures for correct annealing, while shorter probes require lower temperatures.

[086] "Stringent conditions" or "high stringency conditions", as defined herein, can be identified as those that: (1) employ low ionic strength and high temperature in washing, for example, 0.015 M/M sodium chloride sodium citrate 0.0015 M/sodium dodecyl sulfate 0.1% at 50 °C; (2) employ a denaturing agent during hybridization, such as formamide, e.g., 50% (v/v) formamide containing 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/ 50 mM sodium phosphate buffer pH 6.5 containing 750 mM sodium chloride, 75 mM sodium citrate at 42 °C; or (3) hybridization overnight in a solution employing 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), pyrophosphate 0.1% sodium, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μl/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with a 10-minute wash at 42°C in 0.2x SSC (sodium chloride/sodium citrate) followed by a 10 minute high stringency wash consisting of 0.1x SSC containing EDTA at 55°C.

[087] The "therapeutic compounds" disclosed herein refer to small molecules, chemical entities, nucleic acids, nucleic acid derivatives, peptides, peptide derivatives, naturally occurring proteins, non-naturally occurring proteins, glycoproteins , and steroids that are administered to subjects to treat diseases or disorders or to otherwise affect the health of subjects. Non-limiting examples of therapeutic compounds include polypeptides such as enzymes, hormones, cytokines, antibodies or antibody fragments, antibody derivatives, drugs that affect metabolic function, as well as organic compounds such as analgesics, antipyretics, anti-inflammatory agents, antibiotics, antiviral compounds , antifungal compounds, cardiovascular drugs, drugs that affect renal function, electrolyte metabolism, drugs that act on the central nervous system, chemotherapy compounds, receptor agonists and receptor antagonists. Therapeutic compounds include, for example, extracellular molecules such as serum factors including, but not limited to, plasma proteins such as serum albumin, immunoglobulins, apolipoproteins or transferrin, or proteins found on the surface of erythrocytes or lymphocytes. Therefore, exemplary therapeutic compounds include small molecules, chemical entities, nucleic acids, derivatives of nucleic acids, peptides, derivatives of peptides, naturally occurring proteins, non-naturally occurring proteins, peptide nucleic acids (PNA), stapled peptides, morpholinos phosphorodiamidate, antisense drugs, RNA-based silencing drugs, aptamers, glycoproteins, enzymes, hormones, cytokines, interferons, growth factors, blood clotting factors, antibodies, antibody fragments, antibody derivatives, toxin-conjugated antibodies, effectors metabolic drugs, analgesics, antipyretics, anti-inflammatory agents, antibiotics, antimicrobial agents, antiviral agents, antifungal drugs, musculoskeletal drugs, cardiovascular drugs, renal drugs, pulmonary drugs, drugs for diseases of the digestive system, hematological drugs, urological drugs, metabolism drugs , liver drugs, neurological drugs, antidiabetic drugs, anticancer drugs, drugs to treat stomach pathologies, drugs to treat colon pathologies, drugs to treat skin pathologies, and drugs to treat lymphatic pathologies. The term "therapeutic compound", as used herein, has essentially the same meaning as the terms "drug" or "therapeutic agent".

[088] As used herein, "treatment" refers to clinical intervention to attempt to alter the natural evolution of the individual or cell being treated, and may be carried out before or during the evolution of clinical pathology. Desirable effects of treatment include prevention of the occurrence or recurrence of a disease or pathology or symptom thereof, relief of a pathology or symptom of the disease, lessening any direct or indirect pathological consequences of the disease, decreasing the rate of progression of the disease, improvement or alleviation disease status, and remission or better prognosis. In some embodiments, the methods and compositions of the invention are useful in attempting to delay the development of a disease or disorder.

[089] A "vitamin" is a term recognized in the art and is defined as a fat-soluble or water-soluble organic substance that is essential in small quantities for normal growth and bodily activity and is obtained naturally from plant and animal foods or supplements.

[090] "Vitamin D" is a group of fat-soluble secosteroids. There are several forms (variants) of vitamin D. The two main forms are vitamin D2 or ergocalciferol, and vitamin D3 or cholecalciferol. Vitamin D without a subscript refers to D2 or D3 or both. In humans, vitamin D can be ingested as cholecalciferol (vitamin D3) or ergocalciferol (vitamin D2). Additionally, humans can synthesize it from cholesterol when sunlight exposure is adequate.

[091] "Vitamin D-binding protein" or "DBP" is a naturally circulating serum protein found in all mammals that, among other activities, can bind and transport vitamin D and its analogues to sites in the liver and kidneys where the vitamin is modified to its active form, and retains vitamin D in its various forms in circulation for, on average, 30 days in humans. The DBP protein sequence is disclosed in SEQ ID NO:7 and an exemplary nucleic acid sequence encoding the DBP protein sequence is disclosed in SEQ ID NO:8. DBP has several naturally occurring isoforms. Exemplary isoforms are available in public sequence databases (e.g., accession numbers NM_001204306.1, NM_001204307.1, NM_000583.3, BC036003.1, M12654.1, X03178.1, AK223458, P_001191235.1, NP_00 0574 .2, AAA61704.1, AAD13872.1, NP_001191236.1, AAA19662.2, I54269, P02774.1, EAX05645.1, AAH57228.1, AAA52173.1, AAB29423.1, AAD14249.1, AAD1425 0.1, and BAD97178.1).

[092] The invention contemplates the use of DBP variants and homologues that contain conservative or non-conservative amino acid substitutions that substantially retain DBP activity. DBP-binding molecules or functional variants of DBP can be identified using known techniques and characterized by known methods (Bouillon et al., J Bone Miner Res. 6(10):1051-7 (1991), Teegarden et. al., Anal. Biochemistry 199(2):293-299 (1991), McLeod et al, J Biol Chem. 264(2):1260-7 (1989), Revelle et al., J. Steroid Biochem. 22:469-474 (1985)). The above references are incorporated herein in their entirety by citation.

[093] The term "water-soluble" refers to chemical fractions that have some detectable degree of solubility in water. Methods for detecting and/or quantifying water solubility are well known in the art. Exemplary water-soluble polymers include peptides, saccharides, polyethers, polyamines, poly(carboxylic acids) and the like.

[094] The invention provides effective administration routes for proteins, peptides and other biological compounds, nucleic acids and small molecule drugs. The invention further provides effective drug administration routes by a transdermal, oral, parenteral, subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial injection, infusion, inhalation, by an ocular, topical, rectal, nasal, buccal, sublingual, vaginal or implanted reservoir form of administration.

[095] Furthermore, the inventions described in this document provide compositions and methods for maintaining target binding activity, that is, the pharmacodynamics (PD) of therapeutic compounds. It also provides compositions and methods for improving the pharmacokinetic (PK) profiles of therapeutic compounds as described herein. The invention also provides compositions and methods for improved drug absorption profiles compared to drug absorption profiles employing the same or different routes of administration, but without the inventions described herein. The invention also provides compositions and methods for improved drug bioavailability profiles compared to the bioavailability profiles of drugs employing the same or different routes of administration, but without the inventions described herein. The invention also provides compositions and methods for improved drug half-life profiles compared to drug half-life profiles that employ the same or different routes of administration, but without the inventions described herein.

[096] The invention also provides alternative drug administration routes that are more economically advantageous and favorable to patients compared to drugs without the inventions described herein.

[097] The invention provides compositions and methods for the use of molecules that serve as carriers that can be conjugated to, fused to, or formulated with active therapeutic compounds for the purpose of improving the absorption, half-life, bioavailability or pharmacokinetic properties of pharmaceuticals. Carriers have the properties of binding to the body's natural DBP. One aspect of the invention provides the use of natural DBP to transport the drug-carrier complex from the site of administration to the circulating serum. Another aspect of the invention is the use of natural DBP to maintain a drug in circulation for an extended period of time. This can prevent its excretion from the body and increase the exposure of the therapeutic compound in the body to achieve a longer therapeutic effect. In another aspect of the invention, a lower dose of the drug is required when it is conjugated to, fused to, or formulated with the carrier, compared to the unconjugated, unfused, or unformulated drug. Another aspect of the invention is the use of a carrier to replace the function of a much larger PEG compound when coupled to a therapeutic compound. This can improve the pharmacokinetic profile and efficacy of the conjugated, fused or formulated compound.

[098] The invention provides a carrier molecule that is preferably composed of one or more parts or components. In one embodiment, the carrier comprises a targeting group and a coupling group for linking the targeting group to the therapeutic compound. In another embodiment, the carrier comprises a supporting chemical moiety that is linked to the targeting group and the therapeutic compound. The targeting group is vitamin D, a vitamin D analogue, a vitamin D-related metabolite, an analogue of a vitamin D-related metabolite, or another molecule that can bind or interact with vitamin D-binding protein (DBP) . In one embodiment, the targeting group is an antibody or antibody derivative, a peptide designed to bind DBP or a fragment thereof, a peptide derived from a phage display or other peptide library selected against DBP or a fragment thereof. , a nucleotide aptamer that binds DBP, a small molecule designed to bind DBP, or derived from a chemical library selected against DBP or a fragment thereof.

[099] The therapeutic compound and carrier conjugates of the invention typically have about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 targeting groups individually linked to a therapeutic compound. In one embodiment, the carrier conjugate of the invention will comprise about 4 targeting groups individually linked to a therapeutic compound, or about 3 targeting groups individually linked to a therapeutic compound, or about 2 targeting groups individually linked to a therapeutic compound or about 1 targeting group attached to a therapeutic compound. The structure of each of the targeting groups attached to the therapeutic compound may be the same or different. In a preferred embodiment, one or more targeting groups are stably linked to the therapeutic compound at the N-terminal end of a therapeutic protein. In another preferred embodiment, one or more targeting groups are stably linked to the therapeutic protein at the C-terminal end of a therapeutic protein. In other preferred embodiments, one or more targeting groups may be stably linked to other sites on the therapeutic protein. For example, a therapeutic compound and carrier conjugate may comprise a targeting group attached to the N-terminus and additionally a targeting group attached to a lysine residue. In another embodiment, a therapeutic compound and carrier conjugate has a targeting group linked to a therapeutic protein through a modification such as a sugar residue that is part of a glycosylation site or a protein acylation site. a peptide or linked to a phosphorylation site or other natural or non-natural modifications that are familiar to the person skilled in the art. Binding sites employing a combination of the sites mentioned above are also contemplated. A preferred embodiment of the present invention comprises a targeting group that is linked to the therapeutic compound at a specific site on a therapeutic compound. In another preferred embodiment, the binding site on a protein may be a cysteine, lysine, the N-terminal end or the C-terminal end.

[0100] In another embodiment, the support is a pharmaceutically acceptable carrier. In preferred embodiments, the support is polyethylene glycol, polylysine, polyethyleneimine, polypropylene glycol, a peptide, serum albumin, thioredoxin, an immunoglobulin, an amino acid, a nucleic acid, a glycan, a modifying group containing a reactive ligand, a water-soluble polymer , a small carbon chain ligand, or an additional therapeutic chemical moiety.

[0101] In one embodiment, the water-soluble support refers to chemical fractions that have some detectable degree of solubility in water. Methods for detecting and/or quantifying water solubility are well known in the art. Exemplary water-soluble polymers include peptides, saccharides, polyethers, polyamines, poly(carboxylic acids) and the like.

[0102] Peptides can have mixed sequences or can be composed of a single amino acid, for example, polylysine. An exemplary polysaccharide is poly(sialic acid). An exemplary polyether is polyethylene glycol, for example m-PEG. Polyethyleneimine is an exemplary polyamine and poly(acrylic acid) is a representative poly(carboxylic acid). The backbone of the water-soluble polymer may be polyethylene glycol (e.g., PEG). However, it should be understood that other related polymers are also suitable for use in carrying out the present invention and that the use of the term PEG or polyethylene glycol is intended to be inclusive and not exclusive in relation thereto. The term PEG includes polyethylene glycol in any of its forms, including alkoxy PEG, bifunctional PEG, multi-arm PEG, bifurcated PEG, branched PEG, pendant PEG (i.e., PEG or related polymers that have one or more functional groups pendant relative to to the polymer backbone) or PEG containing degradable linkages. The polymer backbone can be linear or branched.

[0103] Branched polymer backbones are generally known in the art. Typically, a branched polymer has a centrally branched core chemical moiety and a plurality of linear polymer chains attached to the centrally branched core. PEG is commonly used in branched forms that can be prepared by adding ethylene oxide to various polyols such as glycerol, pentaerythritol and sorbitol. The chemical moiety with central branches can also be derived from various amino acids, such as lysine. Branched polyethylene glycol can be generally represented as R(-PEG-OH)m in which R represents the nuclear chemical moiety, such as glycerol or pentaerythritol and m represents the number of arms. Multi-arm PEG molecules, such as those described in U.S. Patent No. 5,932,462, which is incorporated herein in its entirety by reference, can also be used as the polymeric backbone.

[0104] Various other polymers are also suitable for the invention. Non-peptide, water-soluble polymer backbones with 2 to 300 ends are particularly useful in the invention. Examples of suitable polymers include, but are not limited to, other polyalkylene glycols, such as polypropylene glycol ("PPG"), ethylene glycol and propylene glycol copolymers and the like, poly(oxyethylated polyol), poly(olefinic alcohol), polyvinylpyrrolidone, polylysine, polyethyleneimine, poly (hydroxypropylmethacrylamide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), as described in U.S. Patent No. 5,629,384, which is incorporated herein in its entirety by citation, and copolymers, terpolymers and mixtures thereof. Although the molecular weight of each chain of the polymer backbone can vary, it is typically in the range of about 100 Da to about 100,000 Da.

[0105] In other embodiments, the supporting chemical moiety may be a peptide, serum albumin, thioredoxin, an immunoglobulin, an amino acid, a nucleic acid, a glycan, a modifying group containing a reactive ligand, a water-soluble polymer, a small carbon chain ligand, or an additional therapeutic compound. In one embodiment, the supporting chemical fractions are not toxic to humans and animals. In another embodiment, the supports are endogenous serum proteins. In another embodiment, the supporting chemical fractions are water-soluble polymers. In another embodiment, the supports are polymers that are not naturally occurring. In another embodiment, the supports are naturally occurring chemical moieties that are modified by covalent bonding to additional chemical moieties (e.g., PEG, polypropylene glycol, polyaspartate, biomolecules, therapeutic chemical moieties or diagnostic chemical moieties).

[0106] The conjugation of hydrophilic polymers such as PEG is known in the art. In its most common form, PEG is a linear polymer that terminates in hydroxyl groups at each end: HO—CH2CH2O—(CH2CH2O)n—CH2CH2—OH where n typically ranges from about 3 to about 4000. In one embodiment In the preferred embodiment, the PEG has a molecular weight distribution that is essentially homodisperse. In another preferred embodiment, the PEG is a linear polymer. In another preferred embodiment, the PEG is a branched polymer.

[0107] Many branched or end-functionalized derivatives and various sizes are known in the art and are commercially available. By way of example, conjugation of PEG or PEO can be accomplished using the compositions and methods described herein and in U.S. patent numbers 7,803,777 (Defrees et al.) and 4,179,337 (Davis et al.), each one of which is incorporated herein in its entirety by citation.

[0108] In some embodiments, smaller therapeutic compounds are paired with smaller supporting chemical moieties and larger therapeutic compounds are paired with larger supporting chemical moieties. However, it is contemplated that smaller therapeutic compounds could be paired with a larger supporting chemical moiety and vice versa. Minor therapeutic compounds are defined as having molecular weights of 1Da to 10kDa. Larger therapeutic compounds are defined as having molecular weights of 10kDa to 1000kDa.

[0109] The supports of the present invention could have, for example, a molecular weight of 100 Daltons (Da.), 500 Da., 1000 Da., 2000 Da., 5000 Da., 10,000 Da., 15,000 Da., 20,000 Da., 30,000 Da., 40,000 Da. or 60,000 Da. In one embodiment of the invention, the "small" supporting chemical fractions can be between about 100 Da. and 20,000 Da. In another embodiment, the fractions "Large" support chemistries can be greater than about 20,000 Da. to about 200,000 Da. In preferred embodiments, the support chemical fraction is between about 100 Da. and 200,000 Da. In more preferred embodiments , the supporting chemical fraction is between about 100 Da. and 20,000 Da., 200 Da. and 15,000 Da., 300 Da. and 10,000 Da., 400 Da. and 9,000 Da., 500 Da. and 5,000 Da., 600 Da and 2,000 Da, 1000 Da and 200,000 Da, 20,00 Da and 200,000 Da, 100,000 and 200,000 Da, 5000 Da and 100,000 Da, 10,000 Da and 80,000 Da, 20,000 Da . and 60,000 Da., or 20,000 Da. and 40,000 Da.

[0110] Another component of the carrier molecule preferably comprises a coupling group that is used to covalently link the drug to the support or carrier. The coupling groups of the invention include an amine-reactive group, a thiol-reactive group, a maleimide group, a thiol group, an aldehyde group, an NHS-ester group, a haloacetyl group, an iodoacetyl group, a bromoacetyl group, a SMCC group, a SMCC sulfo group, a carbodiimide group and bifunctional crosslinkers such as NHS-Maleimido, combinations thereof, or other coupling groups familiar to those skilled in the art. The coupling groups of the invention can promote thiol bonds, amide bonds, oxime bonds, hydrazone bonds, thiazolidinone bonds or use cycloaddition reactions also called click chemistry to couple the carrier to a therapeutic compound. In another embodiment, the

[0111] composition preferably includes a combination of one or more therapeutic compounds linked to the coupling group of the support molecule.

[0112] NHS groups are known to those skilled in the art as being useful for coupling to native peptides and proteins without requiring the insertion of a binding site. NHS groups allow binding to most proteins and peptides that contain amino acids with amino groups such as a lysine residue. The use of NHS groups allows flexibility in the carrier conjugation site as the protein structure and reaction time can influence the binding site and the number of carrier molecules conjugated to the therapeutic compound. By way of example, by controlling the molar ratio between the NHS-carrier and the therapeutic compound, one skilled in the art can have some control over the number of carrier molecules linked to the therapeutic compound, thus allowing more than one carrier to be conjugated to a given therapeutic compound if desired.

[0113] Conjugation of the carrier to a therapeutic compound is achieved by mixing a solution of the molecules together in a certain molar ratio using compatible solutions, buffers or solvents. For example, a molar ratio of 1:1, 2:1, 4:1, 5:1, 10:1, 20:1, 25:1, 50:1, 100:1, 1000:1, or 1:2, 1:4, 1:5, 1:10, 1:20, 1:25, 1:50, 1:100 or 1:1000 of carrier for therapeutic compound. In certain embodiments, a molar ratio of 1:1, 2:1, 4:1, 5:1, 10:1, 20:1, 25:1 or 1:2, 1:4, 1 could be used. :5, 1:10, 1:20 1:25, 1:50 carrier for therapeutic compound. In preferred embodiments, a molar ratio of 1:1, 2:1, 4:1, 5:1, 10:1 or 1:2, 1:4, 1:5, 1:10 of carrier could be used. for therapeutic compound. By varying the ratio, this could result in different numbers of individual carriers binding to the therapeutic compound, or it could aid in the selection of a particular binding site. The binding of carriers also depends on pH, buffer, salt and temperature and the variation of these parameters, among other parameters, can influence the binding site, the number of linked carriers and the speed of the reaction. For example, selecting a reaction pH at or below pH 6 could help to selectively conjugate an aldehyde version of the carrier to the N-terminal end of the therapeutic protein or peptide.

[0114] In certain embodiments, the present invention provides carriers that include those of formula I:

[0115] Where:

[0116] B is a targeting group selected from vitamin D, a vitamin D analogue, a vitamin D-related metabolite, an analogue of a vitamin D-related metabolite, a DBP-binding peptide, an anti-DBP antibody, a anti-DBP antibody derivative, a nucleotide aptamer that binds DBP, or a small carbon-based molecule that binds DBP;

[0117] S is a chemical support fraction comprising polyethylene glycol, polylysine, polyethyleneimine, polypropylene glycol, a peptide, serum albumin, thioredoxin, an immunoglobulin, an amino acid, a nucleic acid, a glycan, a modifying group containing a reactive ligand, a poly(lactic acid), a water-soluble polymer, a small carbon chain linker, or an additional therapeutic compound;

[0118] C is an amine-reactive group, a thiol-reactive group, a maleimide group, a thiol group, a disulfide group, an aldehyde group, an NHS-ester group, a 4-nitrophenyl ester, an acylimidazole, an haloacetyl group, an iodoacetyl group, a bromoacetyl group, an SMCC group, a sulfo SMCC group, a carbodiimide group and bifunctional crosslinkers such as NHS-Maleimide or combinations thereof;

[0119] L1 and L2 are ligands independently selected from -(CH2)n-, -C(O)NH-, -HNC(O)-, -C(O)O-, -OC(O)-, -O -, -S-S-, -S-, -S(O)-, -S(O)2- and -NH-;

[0120] L3 is - (CH2)o-;

[0121] n is an integer from 0 to 3; It is

[0122] is an integer from 0 to 3.

[0123] In preferred embodiments, the present invention provides carriers that include those of formula I:

[0124] Where:

[0125] B is a targeting group selected from vitamin D, a vitamin D analogue, a vitamin D-related metabolite, an analogue of a vitamin D-related metabolite, or a small carbon-based molecule that binds DBP;

[0126] S is a chemical support fraction comprising polyethylene glycol, polylysine, polypropylene glycol, a peptide, serum albumin, an amino acid, a nucleic acid, a glycan, a poly(lactic acid), a water-soluble polymer, or a chain linker small carbon dioxide;

[0127] C is a maleimide group, a thiol group, a disulfide group, an aldehyde group, an NHS-ester group, an iodoacetyl group, or a bromoacetyl group;

[0128] L1 and L2 are ligands independently selected from -(CH2)n-, -C(O)NH-, -HNC(O)-, -C(O)O-, -OC(O)-, -O -, -S-, and -NH-;

[0129] L3 is -(CH2)o-;

[0130] n is an integer from 0 to 3; It is

[0131] is an integer from 0 to 3.

[0132] In more preferred embodiments, the present invention provides carriers that include those of formula I:

[0133] Where:

[0134] B is a targeting group selected from vitamin D, a vitamin D analogue, or a vitamin D-related metabolite;

[0135] S is a chemical support fraction comprising polyethylene glycol, polylysine, or polypropylene glycol;

[0136] C is a maleimide group, a disulfide group, an aldehyde group, an NHS-ester group, or an iodoacetyl group;

[0137] L1 and L2 are ligands independently selected from -(CH2)n-, -C(O)NH-, -HNC(O)-, -C(O)O- and -OC(O)-;

[0138] L3 is - (CH2)o-;

[0139] n is an integer from 0 to 3; It is

[0140] is an integer from 0 to 3.

[0141] In even more preferred embodiments, the present invention provides carriers that include those of formulas IIa and IIb:

[0142] Where:

[0143] B is a targeting group selected from vitamin D, a vitamin D analogue, or a vitamin D-related metabolite;

[0144] S is a chemical support fraction comprising polyethylene glycol or polypropylene glycol; It is

[0145] C is a maleimide group, a disulfide group, an aldehyde group, an NHS-ester group, or an iodoacetyl group;

[0146] L2 is -(CH2)n-;

[0147] L3 is -(CH2)o-;

[0148] n is 1; It is

[0149] is 2.

[0150] In certain even more preferred embodiments of formula IIa, B is represented by formula III, S is polyethylene glycol and L3-C is represented by formula IVa.

[0151] In certain even more preferred embodiments of formula IIb, B is represented by formula III, S is polyethylene glycol and L2-C is represented by formula IVb.

[0152] In certain even more preferred embodiments, S is between about 100 Da. and 200,000 Da. In other even more preferred embodiments, the supporting chemical fraction is between about 100 Da. and 20,000 Da., 200 Da. and 15,000 Da., 300 Da. and 10,000 Da., 400 Da. and 9,000 Da., 500 Da. and 5,000 Da., 600 Da. and 2,000 Da., 1000 Da. and 200,000 Da., 5000 Da. . and 100,000 Da., 10,000 Da. and 80,000 Da., 20,000 Da. and 60,000 Da., or 20,000 Da. and 40,000 Da.

[0153] In a specific embodiment, the present invention provides a carrier represented by formula V.

[0154] In another specific embodiment, the present invention provides a carrier represented by formula VI.

[0155] In certain embodiments, the present invention provides a method carrying formula I:

[0156] which comprises the step of reacting a compound of formula Ia:

[0157] with a compound of formula Ib:

[0158] in the presence of an amide coupling agent,

[0159] where B, S, Ce L2 are defined as indicated above and L1 is -C(O)NH-.

[0160] One skilled in the art will recognize that a compound of formula Ib can be used in free base form or in the form of an appropriate salt. Suitable salt forms include, but are not limited to, TFA, HCl, HBr, MsOH, TfOH and AcOH.

[0161] Any suitable amide coupling agent can be used to form a compound of formula I. Suitable amide coupling agents include, but are not limited to, 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU and T3P. In certain embodiments, the amide coupling agent is used alone. In certain embodiments, the amide coupling agent is used with a co-agent such as HOBT or DMAP. In certain embodiments, the amide coupling agent is used with a base such as triethylamine or diisopropylethylamine. In certain embodiments, the amide coupling agent is used with a co-agent such as HOBT or DMAP and a base such as triethylamine or diisopropylethylamine. One skilled in the art will recognize that co-agents other than HOBT or DMAP may be used. Furthermore, one skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine can be used.

[0162] In certain embodiments, the carboxylic acid component of formula Ia is produced by treating an ester of formula Id with a hydrolyzing agent:

[0163] wherein, B is defined as indicated above and R is a branched or unbranched C1-C6 alkyl group.

[0164] Any suitable hydrolyzing agent can be used to prepare a compound of formula Ia from a compound of formula Id.

[0165] In certain other embodiments, the present invention provides a method for producing a carrier of the formula Ig:

[0166] which comprises the steps of reacting a compound of formula Ia:

[0167] with a compound of formula Ic:

[0168] in the presence of an amide coupling agent forming a compound of formula Ie;

[0169] hydrolyzing an ester of formula Ie into a carboxylic acid of formula If; It is

[0170] converting a carboxylic acid of formula If into an active ester of formula I;

[0171] where B, S, C, R1, L2, L3, n and o are defined as indicated above and L1 is -C(O)NH-.

[0172] One skilled in the art will recognize that a compound of formula Ic can be used in free base form or in the form of an appropriate salt. Suitable salt forms include, but are not limited to, TFA, HCl, HBr, MsOH, TfOH and AcOH.

[0173] Any suitable amide coupling agent can be used to form a compound of formula le. Suitable amide coupling agents include, but are not limited to, 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU and T3P. In certain embodiments, the amide coupling agent is used alone. In certain embodiments, the amide coupling agent is used with a co-agent such as HOBT or DMAP. In certain embodiments, the amide coupling agent is used with a base such as triethylamine or diisopropylethylamine. In certain embodiments, the amide coupling agent is used with a co-agent such as HOBT or DMAP and a base such as triethylamine or diisopropylethylamine. One skilled in the art will recognize that co-agents other than HOBT or DMAP may be used. Furthermore, one skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine can be used.

[0174] In certain embodiments, the carboxylic acid component of formula Ia is produced by treating an ester of formula Id with a hydrolyzing agent:

[0175] wherein, B is defined as indicated above and R is a branched or unbranched C1-C6 alkyl group.

[0176] Any suitable hydrolyzing agent can be used to prepare a compound of formula Ia from a compound of formula Id. Suitable hydrolyzing agents include, but are not limited to, lithium hydroxide, sodium hydroxide and potassium hydroxide.

[0177] Any suitable hydrolyzing agent can be used to prepare a compound of formula If from a compound of formula Ie. Suitable hydrolyzing agents include, but are not limited to, lithium hydroxide, sodium hydroxide and potassium hydroxide.

[0178] Any suitable leaving group can be coupled to a carboxylic acid of formula If in the presence of an appropriate coupling reagent to form an active ester of formula I. Suitable leaving groups include, but are not limited to, imidazole, HOBT, NHS and 4-nitrophenol. Suitable coupling reagents include, but are not limited to, 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU and T3P.

[0179] In some embodiments, an active ester of formula I is formed from a carboxylic acid of formula If using a combination of an appropriate leaving group and a coupling reagent.

[0180] In some embodiments, an active ester of formula I is formed from a carboxylic acid of formula If using a single reagent that produces a leaving group and also promotes a coupling reaction. Such reagents include, but are not limited to, 1,1'-carbonyldiimidazole, N,N'-disuccinimidyl carbonate, 4-nitrophenyl trifluoroacetate and HBTU. In some embodiments, the single reagent is used alone. In other embodiments, the single reagent is used with an acyl transfer catalyst. Such acyl transfer catalysts include, but are not limited to, DMAP and pyridine. One skilled in the art will recognize that additional acyl transfer catalysts can be used.

[0181] In a specific embodiment, the present invention provides a method for producing a carrier represented by formula V:

[0182] which comprises the step of reacting a compound of formula Va:

[0183] with a compound of formula Vb:

[0184] in the presence of an amide coupling agent. One skilled in the art will recognize that a compound of formula Vb can be used in free base form or in the form of an appropriate salt. Suitable salt forms include, but are not limited to, TFA, HCl, HBr, MsOH, TfOH and AcOH.

[0185] Any suitable amide coupling agent can be used to form a compound of formula V. Suitable amide coupling agents include, but are not limited to, 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU and T3P. In certain embodiments, the amide coupling agent is used alone. In certain embodiments, the amide coupling agent is used with a co-agent such as HOBT or DMAP. In certain embodiments, the amide coupling agent is used with a base such as triethylamine or diisopropylethylamine. In certain embodiments, the amide coupling agent is used with a co-agent such as HOBT or DMAP and a base such as triethylamine or diisopropylethylamine. One skilled in the art will recognize that co-agents other than HOBT or DMAP may be used. Furthermore, one skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine may be used.

[0186] In a specific embodiment, the carboxylic acid component of formula Va is produced by treating a methyl ester of formula Vc with a hydrolyzing agent:

[0187] Any suitable hydrolyzing agent can be used to prepare a compound of formula Va from a compound of formula Vc. Suitable hydrolyzing agents include, but are not limited to, lithium hydroxide, sodium hydroxide and potassium hydroxide.

[0188] In another specific embodiment, the present invention provides a method for producing a carrier represented by formula VI:

[0189] which comprises the steps of reacting a compound of formula Va:

[0190] with a compound of the formula Via:

[0191] in the presence of an amide coupling agent forming a compound of formula VIb;

[0192] hydrolyzing an ester of formula VIb into a carboxylic acid of formula VIc; It is

[0193] converting a carboxylic acid of formula VIc into an active ester of formula VI;

[0194] One skilled in the art will recognize that a compound of formula VIa can be used in free base form or in the form of an appropriate salt. Suitable salt forms include, but are not limited to, TFA, HCl, HBr, MsOH, TfOH and AcOH.

[0195] Any suitable amide coupling agent can be used to form a compound of formula VIb. Suitable amide coupling agents include, but are not limited to, 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU and T3P. In certain embodiments, the amide coupling agent is used alone. In certain embodiments, the amide coupling agent is used with a co-agent such as HOBT or DMAP. In certain embodiments, the amide coupling agent is used with a base such as triethylamine or diisopropylethylamine. In certain embodiments, the amide coupling agent is used with a co-agent such as HOBT or DMAP and a base such as triethylamine or diisopropylethylamine. One skilled in the art will recognize that co-agents other than HOBT or DMAP may be used. Furthermore, one skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine can be used.

[0196] Any suitable hydrolyzing agent can be used to prepare a compound of formula VIc from a compound of formula VIb. Suitable hydrolyzing agents include, but are not limited to, lithium hydroxide, sodium hydroxide and potassium hydroxide.

[0197] NHS can be coupled to a carboxylic acid of formula VIc in the presence of an appropriate coupling reagent to form an active ester of formula VI. Suitable coupling reagents include, but are not limited to, 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU and T3P.

[0198] In some embodiments, an active ester of formula VI is formed from a carboxylic acid of formula VIc using a combination of NHS and a coupling reagent.

[0199] In some embodiments, an active ester of formula VI is formed from a carboxylic acid of formula VIc using a single reagent that produces a leaving group and also promotes a coupling reaction. Such reagents include, but are not limited to, N,N'-disuccinimidyl carbonate. In some embodiments, the single reagent is used alone. In other embodiments, the single reagent is used with an acyl transfer catalyst. Such acyl transfer catalysts include, but are not limited to, DMAP and pyridine. One skilled in the art will recognize that additional acyl transfer catalysts can be used.

[0200] In one embodiment the carboxylic acid component of formula Va is produced by treating a methyl ester of formula Vc with a hydrolyzing agent:

[0201] Any suitable hydrolyzing agent can be used to prepare a compound of formula Va from a compound of formula Vc. Suitable hydrolyzing agents include, but are not limited to, lithium hydroxide, sodium hydroxide and potassium hydroxide.

[0202] If desired, therapeutic compound and carrier conjugates with different molecular weights can be isolated by gel filtration chromatography and/or ion exchange chromatography. Gel filtration chromatography can be used to fractionate different therapeutic compound and carrier conjugates (e.g., 1-mer, 2-mer, 3-mer, and so on, where "1-mer" indicates a group molecule). of targeting per therapeutic compound, "2-mer" indicates two targeting groups attached to the therapeutic compound, and so on) based on their different molecular weights (where the difference essentially corresponds to the average molecular weight of the targeting group).

[0203] Gel filtration columns suitable for carrying out this type of separation include Superdex and Sephadex columns available from Amersham Biosciences (Piscataway, N.J.). Selection of a particular column will depend on the desired fractionation range. Elution is generally carried out using an appropriate buffer, such as phosphate, acetate or similar. The collected fractions can be analyzed by several different methods, for example, (i) optical density (OD) at 280 nm for protein content, (ii) bovine serum albumin (BSA) protein analysis, and (iii) gel electrophoresis of polyacrylamide with sodium dodecyl sulfate (SDS PAGE).

[0204] Separation of therapeutic compound and carrier conjugates can also be performed by reversed-phase chromatography using a C18 reversed-phase high-performance liquid chromatography (RP-HPLC) column (Amersham Biosciences or Vydac) or by exchange chromatography ion exchange using an ion exchange column, for example, a DEAE- or CM-Sepharose ion exchange column available from Amersham Biosciences. The resulting purified compositions are preferably substantially free of the therapeutic compound conjugated to a non-targeting group. Furthermore, the compositions are preferably substantially free of all directing groups that are not covalently linked.

[0205] The invention provides compositions and methods for making a drug more potent by improving its pharmacokinetic properties using vitamin D or another DBP-binding molecule. The natural pathway of vitamin D formation in the skin with exposure to ultraviolet light depends on interaction with DBP to bring UV-activated vitamin D into the circulation where it can be used in cellular processes (Lips, Prog. Biophys. Molec. Biol. 92 :4-8 (2006); DeLuca, Nutr. Rev. 66 (suppl. 2):S73-S78 (2008)). DBP brings vitamin D into circulation quickly and effectively. DBP also maintains active vitamin D in the circulation for, on average, 30 days (Cooke, N.E., and J.G. Haddad. 1989. Endocr. Rev. 10:294-307; Haddad, J.G. et al. 1993. J. Clin. Invest. 91:2552-2555; Haddad, J. G. 1995. J. Steroid Biochem. Molec. Biol. 53:579-582). The invention provides, for the first time, the use of DBP to deliver therapeutic compounds to the body more effectively. In one embodiment, the therapeutic compound is covalently linked or fused to a carrier. In another embodiment, the therapeutic compound is formulated with the carrier but not covalently linked. In one embodiment, the carrier interacts with DBP for the purpose of delivering the drug from the site of administration into the body more effectively. In another embodiment, the carrier keeps the drug in circulation for an extended period of time.

[0206] In one embodiment, the carrier comprises a targeting group and a coupling group for linking the targeting group to the therapeutic compound. In another embodiment, the carrier comprises a supporting chemical moiety that is linked to the targeting group and the therapeutic compound. The targeting group is vitamin D, a vitamin D analogue, a vitamin D-related metabolite, an analogue of a vitamin D-related metabolite, or another molecule that can bind or interact with vitamin D-binding protein (DBP) . In one embodiment, the targeting group is an antibody or antibody derivative, a peptide designed to bind DBP or a fragment thereof, a peptide derived from a phage display or other peptide library selected against DBP or a fragment thereof. , a nucleotide aptamer that binds DBP, a small molecule designed to bind DBP or derived from a chemical library selected against DBP or a fragment thereof or a chemical moiety that can bind DBP as disclosed herein. In another embodiment, the carrier comprises DBP itself or a DBP derivative.

[0207] Vitamin D is a group of fat-soluble secosteroids. There are several forms (variants) of vitamin D. The two main forms are vitamin D2 or ergocalciferol, and vitamin D3 or cholecalciferol, vitamin D without a subscript refers to D2 or D3 or both. In humans, vitamin D can be ingested as cholecalciferol (vitamin D3) or ergocalciferol (vitamin D2). Additionally, humans can synthesize it from cholesterol when sunlight exposure is adequate.

[0208] Vitamin D is further modified by enzymes found in various organs into a family of "vitamin D metabolites" that are also capable of binding DBP. For example, vitamin D is converted to calcidiol (25OH hydroxy Vitamin D) in the liver. A part of calcidiol is converted by the kidneys into calcitriol (1, 25 (OH)2 dihydroxy-Vitamin D). Calcidiol is also converted to calcitriol outside the kidneys for other purposes. It is also found in the body at 24, 25(OH)2 dihydroxy Vitamin D. Therefore, in one embodiment, the targeting group is a metabolite of vitamin D.

[0209] In another embodiment, the targeting group is a "vitamin D analogue". These compounds are based on the structure of vitamin D and retain partial function of vitamin D. They interact with some of the same proteins as vitamin D (e.g., DBP and the vitamin D receptor), but with different affinities. Exemplary analogues include: OCT, a chemically synthesized analogue of 1,25(OH)2D3 with an oxygen atom at position 22 in the side chain (Abe et.al., FEBS Lett. 226:58-62 (1987)); the Gemini vitamin D analog, 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124) (So et al., Mol Pharmacol. 79(3):360-7 (2011)); Paricalcitol, a sterol derived from vitamin D2 that lacks the methylene group at carbon 19 found in all natural vitamin D metabolites (Slatopolsky et al., Am J. Kidney Dis. 26: 852 (1995)); Doxercalciferol (1α-hydroxyvitamin D2), like alfacalcidol (1α-hydroxyvitamin D3), is a prodrug that is hydroxylated in the liver to 1α,25(OH)2D2. Unlike alfacalcidol, doxercalciferol is also 24-hydroxylated to produce 1α,24(S)-(OH)2D2 (Knutson et al., Biochem Pharmacol 53: 829 (1997)); Dihydrotachysterol2 (DHT2), hydroxylated in vivo to 25(OH)DHT2 and 1,25(OH)2DHT2 (McIntyre et al., Kidney Int. 55: 500 (1999)). See also Erben and Musculoskel, Neuron Interact. 2(1):59-69 (2001) and Steddon et al. Nephrol. Dial. Transplant. 16 (10): 1965-1967 (2001). The references mentioned above are incorporated in their entirety by citation.

[0210] In another embodiment, the carrier additionally comprises a pharmaceutically acceptable supporting chemical moiety that is covalently linked to the targeting group and the therapeutic compound. The supporting chemical fraction of the carriers of the invention does not necessarily participate but may contribute to the function or improve the pharmacokinetic properties of the therapeutic compound. The supports of the invention do not substantially interfere with the binding of the targeting group to DBP. Similarly, the supports of the invention do not substantially interfere with the structure or function of the therapeutic compound. The length of the supporting chemical moiety depends on the character of the targeting group and the therapeutic compound. One skilled in the art will recognize that various combinations of atoms provide molecules of varying size based on known distances between various bonds (Morrison, and Boyd, Organic Chemistry, 3rd Ed, Allyn and Bacon, Inc., Boston, Mass. (1977), here incorporated by citation). Other supports contemplated by the invention include peptide linkers, protein linkers such as human serum albumin, an antibody or fragment thereof, nucleic acid linkers, small carbon chain linkers, carbon linkers with interspersed oxygen or nitrogen, and combinations of these examples are also contemplated. .

[0211] In another embodiment, a peptide was selected as the targeting group that binds DBP. Methods of screening peptide or protein libraries for DBP-binding peptides are known in the art. In a preferred embodiment, a two-hybrid method of identifying DBP-binding peptides is used. In another preferred embodiment, an in vitro screen for DBP binding is used. This targeting peptide can then be covalently linked or alternatively formulated with a drug. In a preferred embodiment, a supporting chemical moiety is used. In another embodiment, the targeting group is an aptamer that has been selected for binding DBP. This aptamer can then be covalently linked or formulated with a drug via a support or fused directly to the drug.

[0212] In one embodiment, the drug is a DNA molecule, an RNA molecule, an aptamer (single-stranded or double-stranded), DNA or RNA oligonucleotides, larger DNA molecules that are linear or circular, oligonucleotides which are used for RNA interference (RNAi), DNA variations such as replacement DNA/RNA hybrid molecules, synthetic DNA-like molecules such as PNA or other nucleic acid-derived molecules (see WO07/035922, incorporated herein in its entirety by reference). In another embodiment, the therapeutic compound is composed of nuclease-resistant DNA or RNA oligonucleotides. In a preferred embodiment, the nuclease-resistant DNA oligonucleotides are Morpholinos (i.e., phosphorodiamidate analogues of nucleic acids that bind nucleic acids in a sequence-specific manner, AVI BioPharma, Bothell, WA).

[0213] In another embodiment, the drug is a small molecule or small chemical entity. In another embodiment, the drug is a peptide or a derivative of a peptide such as a PNA. In another embodiment, the drug is a protein composed of an entire polypeptide or part of a polypeptide, whether full-length or a fragment or a truncated version, whether pegylated, glycosylated or covalently or non-covalently modified, or unmodified.

[0214] Therapeutic compounds include proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids and the like. Exemplary polypeptides include growth factors such as hepatocyte growth factors (HGF), nerve growth factors (NGF), epidermal growth factors (EGF), fibroblast growth factors including FGF21, blood clotting factors, hormones such as growth hormones, follicle stimulating hormone (FSH), cytokines, interferons, tumor necrosis factors, enzymes, bone morphogenetic proteins, neurotrophins and growth and differentiation factors. The compositions and methods of the invention also include conjugates, agonists, antagonists or other effectors of the proteins mentioned above, glycoproteins, small molecules, hormones, growth factors and other molecules found in a patient or subject.

[0215] Also covered by the invention are therapeutic peptides. The term peptide is intended to include a chain of amino acids. The amino acids in the peptides of the invention may be naturally occurring or may not be naturally occurring. The peptides of the invention may be synthesized chemically or biologically and may include cysteine-rich peptides, circular peptides, stapled peptides, peptides that include D or L amino acids and mixtures thereof, peptide mimics, peptide nucleic acids (PNAs), and combinations thereof. Exemplary embodiments include AIDS vaccines, allergy vaccines, anti-inflammatory peptides, anti-integrin peptides, anti-TCR vaccines, antiallergic peptides, anticancer peptides, antifungal peptides, antibacterial peptides, antirheumatic peptides, antithrombin peptides, antiviral peptides , G protein-coupled receptor (GPCR) ligands and related peptides (e.g., the Secretin family), CGRP analogs, GPCR antagonists, CMV peptides, calpain inhibitors, collagenase inhibitors, DAP inhibitors, defensins, oligopeptides dialytics, Enhancins, endorphins, endothelin antagonists, fibronectin inhibitors, gastrin antagonists, ghrelin, glucagon antagonists, gonadorelin analogues, growth factor peptides, hypothalamic hormones, pituitary hormones, peptides that control the function of the digestive system and the appetite, pro-inflammatory adipose tissue products, peptides that stimulate stem cell proliferation, pro-inflammatory peptides, natural products, herpes simplex vaccines, heparin-binding peptides, hepatitis B vaccines, immunomodulatory peptides, influenza vaccines, antagonists LHRH, opioid peptide derivatives, MMP inhibitors, MUC-1 vaccines, malaria vaccines, melanoma vaccines, meningitis vaccines, neuropeptides, opioid peptides, osteogenic growth peptides, osteoporosis peptides, papillomavirus vaccines, vaccines for prostate cancer, RGD peptides, RSV vaccines, T cell receptor peptides and the like. The invention contemplates synthetic analogues thereof that would be improved as clinical products by additional modifications by the methods described herein. Those skilled in the art will recognize many additional commercially important peptides that would be amenable to the modifications described herein to provide increased half-life, duration of action, absorption and/or bioavailability.

[0216] Also contemplated within the scope of the embodiments described herein are peptides that are branched or cyclic, with or without branching. Cyclic, branched and circular branched peptides result from natural post-translational processes and are also prepared by appropriate synthetic methods. In some embodiments, any peptide product described herein comprises a peptide analog described above that is then covalently linked to an alkyl glycoside surfactant chemical moiety.

[0217] Peptide chains that have substitution in an appropriate position through modification of the analogues claimed in this document are also contemplated within the scope of the embodiments presented in this document. For example, acylation occurs at a linker amino acid, for example at the ε position of lysine, with fatty acids such as octanoic, decanoic, dodecanoic, tetradecanoic, hexadecanoic, octadecanoic, 3-phenylpropanoic acids and the like, or with alkyl chains saturated or unsaturated (Zhang, L. and Bulaj, G. (2012) Curr Med Chem 19: 1602-1618, incorporated herein in its entirety by citation).

[0218] Peptide chains that are composed of natural and non-natural amino acids or analogues of natural amino acids are also contemplated within the scope of the embodiments presented in this document. As used herein, peptide and/or protein "analogs" comprise non-natural amino acids based on natural amino acids, such as tyrosine analogs, which include para-substituted tyrosines, ortho-substituted tyrosines and meta-substituted tyrosines, wherein the substituent on tyrosine comprises an acetyl group, a benzoyl group, an amino group, a hydrazine, a hydroxyamine, a thiol group, a carboxy group, a methyl group, an isopropyl group, a C2-C20 straight chain or a branched hydrocarbon, a saturated or unsaturated hydrocarbon, an O-methyl group, a polyether group, a halogen, a nitro group or the like. Examples of Tyr analogs include 2,4-dimethyl-tyrosine (Dmt), 2,4-diethyl-tyrosine, O-4-allyl-tyrosine, 4-propyl-tyrosine, Ca-methyl-tyrosine and the like. Examples of lysine analogs include ornithine (Orn), homo-lysine, Ca-methyl-lysine (CMeLys), and the like. Examples of phenylalanine analogues include, but are not limited to, meta-substituted phenylalanines, wherein the substituent comprises a methoxy group, a C1-C20 alkyl group, for example, a methyl group, an allyl group, an acetyl group or the like. . Specific examples include, but are not limited to, 2,4,6-trimethyl-L-phenylalanine (Tmp), O-methyl-tyrosine, 3-(2-naphthyl)alanine (Nal(2)), 3-(l - naphthyl)alanine (Nal(l)), 3-methyl-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), fluorinated phenylalanines, isopropyl-phenylalanine, p-azido-phenylalanine, p-acyl-phenylalanine, p-benzoyl-phenylalanine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-phenylalanine, and isopropyl-phenylalanine, and the like.

[0219] Also contemplated within the scope of the embodiments presented in this document are peptide chains that contain non-natural or non-typical amino acids known in the art, for example, C-alpha-disubstituted amino acids such as Aib, Ca-diethylglycine (Deg), aminocyclopentane-1-carboxylic acid (Ac4c), aminocyclopentane-1-carboxylic acid (Ac5c), and the like. These amino acids often result in a restricted structure, often tending towards an alpha-helix structure (Kaul, R. and Balaram, P. (1999) Bioorg Med Chem 7: 105 117, incorporated herein in its entirety by citation). Additional examples of such unnatural amino acids useful in the design of analogues are homo-arginine (Har) and the like. Replacement of reduced amide bonds at certain times results in enhanced protection against enzymatic destruction or alters binding to receptors. As an example, the incorporation of a Tic-Phe dipeptide unit with a reduced amide bond between the residues (designated as Tic-F[CH2-NH]A-Phe) decreases enzymatic degradation.

[0220] Also contemplated within the scope of the embodiments presented in this document are modifications at the amino or carboxyl terminal end that can be optionally introduced into the present peptides or proteins (Nestor, J.J., Jr. (2009) Current Medicinal Chemistry 16: 4399 - 4418). For example, the present peptides or proteins may be truncated or acylated at the N-terminus (Gourlet, P., et al. (1998) Eur J Pharmacol 354: 105-111, Gozes, I. and Furman, S. (2003) Curr Pharm Des 9: 483-494), the contents of which are incorporated herein by citation). Other modifications to the N-terminus of peptides or proteins, such as deletions or incorporation of D-amino acids, such as D-Phe, can also produce potent and long-acting agonists or antagonists when substituted using the modifications described herein such as as long-chain alkyl glycosides. These agonists and antagonists also have commercial utility and are within the scope of the contemplated embodiments described herein. The references mentioned above are incorporated herein in their entirety.

[0221] Also contemplated within the scope of the embodiments described herein are carriers covalently linked to, fused to or formulated with analogues of therapeutic compounds, in which the native therapeutic compound is modified by acetylation, acylation, pegylation, ADP-ribosylation, amidation, covalent bonding of a lipid or lipid derivative, covalent bonding of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, cysteine covalent cross-link formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, anchor formation of GPI, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid bonding, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, sulfation, addition of amino acids to proteins mediated by RNA transfer, such as arginylation and ubiquitination. See, for example, (Nestor, J.J., Jr. (2007) Comprehensive Medicinal Chemistry II 2: 573601, Nestor, J.J., Jr. (2009) Current Medicinal Chemistry 16: 4399 - 4418, Creighton, T.E. (1993, Wold, F (1983) Posttranslational Covalent Modification of Proteins 1-12, Seifter, S. and Englard, S. (1990) Methods Enzymol 182: 626646, Rattan, S. I., et al. (1992) Ann N Y Acad Sci 663: 4862). The references mentioned above are incorporated in their entirety by citation.

[0222] Glycosylated therapeutic peptides can be prepared using conventional Fmoc chemistry and solid phase peptide synthesis techniques, e.g., on resin, where the desired protected glycoamino acids are prepared prior to peptide synthesis and then introduced into the peptide chain at the desired position during peptide synthesis. Therefore, therapeutic peptide polymer conjugates can be conjugated in vitro. Glycosylation may occur before deprotection. The preparation of amino acid glycosides is described in U.S. Patent No. 5,767,254, WO 2005/097158, and Doores, K., et al., Chem. Commun., 1401-1403, 2006, which are incorporated herein in their entirety by citation. For example, selective alpha and beta glycosylations of serine and threonine residues are carried out using the Koenigs-Knorr reaction and the Lemieux in situ anomerization methodology with Schiff base intermediates. Deprotection of the Schiff base glycoside is then accomplished using weakly acidic conditions or hydrogenolysis. A composition comprising a glycosylated therapeutic peptide conjugate is prepared by stepwise solid phase peptide synthesis involving placing an extended peptide chain in contact with protected amino acids in a stepwise manner, with at least one of the protected amino acids being glycosylated, followed by water-soluble polymer conjugation. Such compositions may have purity of at least 95%, at least 97%, or at least 98% of a single conjugated and glycosylated therapeutic peptide species.

[0223] Monosaccharides that can be used to be introduced into one or more amino acid residues of the therapeutic peptides defined and/or disclosed herein include glucose (dextrose), fructose, galactose and ribose. Additional monosaccharides suitable for use include glyceraldehydes, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lixose, xylose, ribulose, xylulose, allose, altrose, mannose, N-acetylneuraminic acid, fucose, N-acetylgalactosamine and N-acetylglucosamine, as well as others. Glycosides, such as mono-, di- and trisaccharides, for use in modifying a therapeutic peptide, one or more amino acid residues of the therapeutic peptides defined and/or disclosed herein include sucrose, lactose, maltose, trehalose, melibiose and cellobiose, among others. Trisaccharides include acarbose, raffinose and melezitose.

[0224] In additional embodiments of the invention, the therapeutic compounds defined and/or disclosed herein may be chemically coupled to biotin. The therapeutic compound/biotin can then bind to avidin.

[0225] Polypeptides that are antibodies are also within the scope of the invention. The term antibody is intended to include monoclonal antibodies, polyclonal antibodies, toxin-conjugated antibodies, humanized antibodies, antibody fragments (e.g., Fc domains), Fab fragments, single-chain antibodies, bi- or multispecific antibodies, Llama antibodies, nanobodies, diabodies, Fv, Fab, F(ab')2, Fab', scFv, scFv-Fc, and the like. Also included in the term are antibody fusion proteins, such as Ig chimeras. Preferred antibodies include humanized or fully human monoclonal antibodies or fragments thereof.

[0226] The terms “antibody” and “immunoglobulin” are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., intact or full-length monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g. , bispecific antibodies, provided they have the desired biological activity) and may also include certain antibody fragments (as described in more detail herein). An antibody can be chimeric, human, humanized and/or affinity matured.

[0227] The terms “full-length antibody”, “intact antibody” and “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, and not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains containing the Fc region. "Antibody fragments" comprise a portion of an intact antibody, preferably comprising its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies, linear antibodies, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical with the exception of possible mutations, e.g. naturally occurring mutations, which may be present in low quantities. Therefore, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of distinct antibodies.

[0228] In certain embodiments, such monoclonal antibody typically includes an antibody that comprises a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single sequence target-binding polypeptide from a plurality of polypeptide sequences. For example, the selection process may be selecting just one clone from a plurality of clones, such as a set of hybridoma clones, phage clones or recombinant DNA clones. It should be understood that a selected target binding sequence may be further altered, for example, to enhance its affinity for the target, to humanize the target binding sequence, to enhance its production in cell culture, to reduce its immunogenicity in vivo. , to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of the present invention. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous because they typically are not contaminated by other immunoglobulins.

[0229] Antibodies that specifically bind to an antigen have high affinity for that antigen. Antibody affinities can be measured by a dissociation constant (Kd). In certain embodiments, an antibody provided herein has a dissociation constant (Kd) of ^100 nM, ^10 nM, ^1 nM, ^0.1 nM, ^0.01 nM, or ^0.001 nM ( e.g. 10-8M or less, e.g. 10-8M to 10-13M, e.g. 10-9M to 10-13M).

[0230] In one embodiment, Kd is measured by an immunolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described in the following rehearsal. The in-solution binding affinity of Fabs to antigen is measured by equilibrating the Fab with a minimum concentration of (125I)-labeled antigen in the presence of a titration series of unlabeled antigen, and subsequently capturing the bound antigen with a coated plate. with anti-Fab antibody (see, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg/ml of an anti-Fab capture antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9. 6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (about 23° C). On a non-adsorbent plate (Nunc #269620), 100 μM or 26 μM [125I]-antigen is mixed with serial dilutions of a Fab of interest (e.g., compatible with anti-VEGF antibody assessment, Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, but incubation may continue for a longer period of time (e.g., approximately 65 hours) to ensure that equilibrium is reached. Afterwards, the mixtures are transferred to the capture plate for incubation at room temperature (e.g. for one hour). The solution is subsequently removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plates are dry, 150 μl/well of scintillant (MICROSCINT-20™; Packard) is added and the plates are counted in a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that provide 20% or less of maximum binding are selected for use in competitive binding assays.

[0231] According to another embodiment, Kd is measured using surface plasmon resonance assays using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. with, e.g. , CM5 type chips with antigen immobilized in ~10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier instructions. The antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (~0.2 μM) before being injected at a flow rate of 5 μl/minute to obtain approximately 10 response units (RU) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, serial two-fold dilutions of Fab (0.78 nM to 500 nM) are injected into PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25 °C at a flow rate of ca. of 25 μl/minute. Association rates (kon) and dissociation rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting dissociation-association sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate is greater than 106 M-1 s-1 in the above surface plasmon resonance assay, the association rate can be determined using a fluorescence quenching technique that measures the increase or decrease in emission intensity of fluorescence (excitation=295 nm; emission=340 nm, bandpass 16 nm) at 25 °C from an anti-antigen antibody (Fab form) 20 nM in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a spectrophotometer equipped with a flow switch (Aviv Instruments) or an SLM-AMINCO™ 8000 series spectrophotometer (ThermoSpectronic) with a stirred cuvette. Other chemistries for coupling the targeted antigen to the chip surface (e.g., streptavidin/biotin, hydrophobic interaction, or disulfide chemistry) are also readily available in lieu of the amine coupling methodology (CM5 type chip) described above, as will be understood by one skilled in the art.

[0232] The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be interpreted as the production of the antibody necessarily being by a specific method. For example, the monoclonal antibodies to be used in accordance with the present invention can be prepared by various techniques, including, for example, the hybridoma method (e.g., Kohler et al, Nature, 256: 495 (1975) ; Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, New York, 1981)), recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567), phage display technologies (see, for example, Clackson et al., Nature, 352: 624-628 (1991); Marks et al. al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284( 1-2): 119-132(2004), and technologies for producing human antibodies or antibodies similar to human antibodies in animals that possess parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, for example, W098/24893; WO96/34096; W096/33735; WO91/10741; Jakobovits et al., Proc. Natl. academic Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. patent numbers 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al., Bio. Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996) and Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). The above patents, publications and references are incorporated in their entirety by citation.

[0233] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain a minimal sequence derived from a non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues of a hypervariable region of the receptor are replaced by residues of a hypervariable region of a non-human species (donor antibody) such as a mouse, mouse, rabbit or non-human primate that has the desired specificity, affinity and/or capacity. In some cases, residues from the framework region (FR) of human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or donor antibody. These modifications can be made to further refine the performance of the antibody. In general, a humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a sequence of human immunoglobulin. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant (Fc) region, typically that of a human immunoglobulin. For details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited in them: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994). The references mentioned above are incorporated in their entirety by citation.

[0234] A “human antibody” is an antibody that comprises an amino acid sequence that corresponds to that of an antibody produced by a human and/or that was produced using any of the techniques for preparing human antibodies as disclosed herein. These techniques include screening human-derived combinatorial libraries, such as phage display libraries (see, for example, Marks et al., J. Mol. Biol, 222: 581-597 (1991) and Hoogenboom et al. , Nucl. Acids Res., 19: 4133-4137 (1991)); using human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies (see, for example, Kozbor, J. Immunol, 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pages 55 to 93 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol, 147: 86 (1991)); and the generation of monoclonal antibodies in transgenic animals (e.g., mice) that are capable of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993)). This definition of a human antibody specifically excludes a humanized antibody that comprises antigen-binding residues that originated from a non-human animal.

[0235] All known types of these antibodies are within the scope of the invention. Exemplary antibodies include those that bind to growth factors, cytokines, lymphokines, cell surface receptors, enzymes, vascular endothelial growth factors, fibroblast growth factors, and antibodies against their respective receptors. Other exemplary antibodies include monoclonal antibodies directed against IgG receptor-Fc fusion proteins and glycoproteins. Any modified (e.g., mutant) version of any of the above-mentioned polypeptides is also within the scope of the invention. The therapeutic compounds to be used in the invention are known in the art and are disclosed by way of example in U.S. Patent No. 7,608,681, incorporated herein in its entirety by reference. Additionally, the invention contemplates conjugates of inhibitors or antagonists of antibodies that occur naturally or not naturally in a subject and that cause autoimmune diseases or unwanted inflammatory pathologies.

[0236] Some aspects of carrier assembly utilize chemical methods that are well known in the art. For example, Vitamin E-PEG is manufactured by Eastman Chemical, Biotin-PEG is manufactured by several PEG manufacturers such as Enzon, Naktar and NOF Corporation. The methods of producing PEG molecules with some vitamins and other therapeutic compounds attached to them follow these and other chemical methods known in the art. Binding of PEG to an oligonucleotide or related molecule occurs, for example, as the PEG2-N-hydroxysuccinimide ester coupled to the oligonucleotide via the 5' amine chemical moiety. Various coupling methods are contemplated and include, for example, coupling of NHS to amine groups such as a lysine residue in a peptide, coupling of maleimide to a sulfhydryl group such as in a cysteine residue, coupling of iodoacetyl to a sulfhydryl, pyridyldithiol coupling to a sulfhydryl group, hydrazide for coupling to a carbohydrate group, aldehyde for coupling to the N-terminal end, or tetrafluorophenyl ester coupling known to react with primary or secondary amines. Other possible chemical coupling methods are known to those skilled in the art and can be substituted. By way of example, conjugation using the coupling groups of the invention can be carried out using the compositions and methods described in WO93/012145 (Atassi et al.) and see also 7,803,777 (Defrees et al.), incorporated herein in its in full by citation.

[0237] In one embodiment, the carrier compounds can be covalently or non-covalently linked to the drug. In another embodiment, the carrier compounds are separated from the drugs, but are mixed together in individual concentrations so as to be formulated into functional units. Exemplary drug formulations of the invention include aqueous solutions, organic solutions, powder formulations, solid formulations and mixed phase formulations.

[0238] The pharmaceutical compositions of the present invention comprise any of the compounds of the present invention and their pharmaceutically acceptable salts, with any pharmaceutically acceptable carrier, adjuvant or vehicle. Pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica colloidal, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene and polyoxypropylene block polymers, polyethylene glycol and wool fat.

[0239] Pharmaceutically acceptable salts retain the desired biological activity of the therapeutic composition without toxic side effects. Examples of such salts are (a) acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like, and salts formed with organic acids such as, for example, acid acetic acid, trifluoroacetic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid and the like; (b) base addition salts or complexes formed with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and the like; or with an organic cation formed from N,N'-dibenzylethylenediamine or ethylenediamine; or (c) combinations of (a) and (b), for example, a zinc tannate salt and the like.

[0240] The pharmaceutical compositions of the present invention can be administered by transdermal, oral, parenteral, inhalation, ocular, topical, rectal, nasal, buccal (including sublingual), vaginal or implanted reservoir forms of administration. The pharmaceutical compositions of the present invention may contain any conventional non-toxic, pharmaceutically acceptable carriers, adjuvants or vehicles. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0241] Also contemplated in some embodiments are pharmaceutical compositions that comprise as an active ingredient, therapeutic compounds described in this document, or their pharmaceutically acceptable salts, in a mixture with a non-toxic and pharmaceutically acceptable component. As mentioned above, such compositions can be prepared for parenteral administration, particularly in the form of liquid suspensions or solutions; for oral or buccal administration, particularly in the form of tablets or capsules; for intranasal administration, particularly in the form of powders, nasal drops, evaporating solutions or aerosols; for inhalation particularly in the form of liquid solutions or dry powders with excipients, broadly defined; for transdermal administration, particularly in the form of a skin patch or microneedle patch; and for rectal or vaginal administration, particularly in the form of a suppository.

[0242] The compositions may be conveniently administered in unit galenic form and may be prepared by any of the methods well known in the pharmaceutical art, for example, as defined in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Co., Easton, PA (1985 ), incorporated herein in its entirety by citation. Formulations for parenteral administration may contain as excipients sterile water or saline alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, saccharides, oils of vegetable origin, hydrogenated naphthalenes, serum albumin or other nanoparticles (as used in Abraxane™, American Pharmaceutical Partners, Inc . Schaumburg, IL), and the like. In the case of oral administration, the formulation can be improved by adding bile salts or acylcarnitines. Formulations for nasal administration may be solids or solutions with evaporating solvents such as hydrofluorocarbons, and may contain excipients for stabilization, for example, saccharides, surfactants, submicron alpha-anhydrous lactose or dextran, or may be aqueous or oily solutions for use in in the form of nasal drops or controlled quantity aerosol. For buccal administration, typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch and the like.

[0243] Delivery of modified therapeutic compounds described herein to a subject for extended periods of time, for example, for periods of one week to one year, can be achieved by a single administration of a controlled release system containing active ingredient sufficient for the desired release time period. Various controlled release systems, such as monolithic or reservoir-type microcapsules, depot-type implants, polymeric hydrogels, osmotic pumps, vesicles, micelles, liposomes, transdermal patches, iontophoretic devices and alternative injectable dosage forms can be used for this purpose. Their location at the site where delivery of the active ingredient is desired is an additional feature of some controlled-release devices, which may be beneficial in the treatment of certain disorders.

[0244] In certain embodiments of transdermal administration, delivery across the skin barrier would be enhanced using electrodes (e.g., iontophoresis), electroporation, or the application of short high-voltage electrical pulses to the skin, radiofrequencies, ultrasound (e.g., sonophoresis), microprojections (e.g. microneedles), jet injectors, thermal ablation, magnetophoresis, lasers, velocity or photomechanical waves. The drug may be included in a single-layer drug-in-adhesive system, multi-layer drug-in-adhesive system, reservoir, matrix, or vapor-type patches, or could employ patchless technology. Delivery across the skin barrier could also be improved using encapsulation, a skin lipid fluidizer, or a solid or hollow microstructured transdermal system (MTS, such as that manufactured by 3M), jet injectors. Formulation additives that assist with the passage of therapeutic compounds through the skin include prodrugs, chemicals, surfactants, cell-penetrating peptides, permeation enhancers, encapsulation technologies, enzymes, enzyme inhibitors, gels, nanoparticles, and peptide chaperones. or proteins.

[0245] A form of controlled-release formulation contains the therapeutic compound or its salt dispersed or encapsulated in a non-antigenic, non-toxic and slow-degrading polymer, such as copoly (lactic/glycolic acid), as described in the pioneering work of Kent et al., U.S. Patent No. 4,675,189, incorporated herein by reference. The compounds, or their salts, can also be formulated in cholesterol or other lipid matrix granules, or silastomer-type matrix implants. Other additional injectable formulations or depot-type, slow-release implants will be apparent to the skilled person. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson ed., Marcel Dekker Inc., New York, 1978; and Controlled Release of Biologically Active Agents, RW Baker, John Wiley & Sons, New York, 1987. References noted above are incorporated in their entirety by citation.

[0246] An additional form of controlled release formulation comprises a biodegradable polymer solution, such as copoly (lactic/glycolic acid) or block copolymers of lactic acid and PEG, which is a bioacceptable solvent and injected subcutaneously or intramuscularly to obtain a depot-type formulation. Mixing the therapeutic compounds described in this document with such a polymeric formulation is suitable for obtaining very long-acting formulations.

[0247] When formulated for nasal administration, absorption through the nasal mucous membrane can be increased by surfactants, such as, for example, glycocholic acid, cholic acid, taurocholic acid, ectocholic acid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid, glycodeoxycholic acid, cyclodextrins and the like in an amount in the range of between about 0.1 and 15 weight percent, between about 0.5 and 4 weight percent, or about 2 weight percent. An additional class of absorption enhancers that have been reported to exhibit greater efficacy with reduced irritation is the class of alkyl maltosides, such as tetradecyl maltoside (Arnold, JJ et al., 2004, J Pharm Sci 93: 2205-13; Ahsan, F et al., 2001, Pharm Res 18:1742046) and its references, all of which are incorporated herein by citation.

[0248] The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, in the form of a sterile injectable aqueous or oleaginous suspension. Such a suspension may be formulated according to techniques known in the art using appropriate wetting or dispersing agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally acceptable and non-toxic diluent or solvent, for example, in the form of a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any bland fixed oil can be used including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are pharmaceutically acceptable natural oils such as olive oil or castor oil, particularly in their polyoxyethylated versions. Such oil solutions or suspensions may also contain a long-chain alcoholic dispersant or diluent such as Ph. Helv or a similar alcohol.

[0249] The pharmaceutical compositions of the present invention can be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets and aqueous solutions and suspensions. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dry corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

[0250] The pharmaceutical compositions of the present invention can also be administered in the form of suppositories for rectal administration. Such compositions can be prepared by mixing a compound of the present invention with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and will therefore melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

[0251] Topical administration of the pharmaceutical compositions of the present invention is particularly useful when the desired treatment involves areas or organs easily accessible by topical application. In the case of topical application to the skin, the pharmaceutical composition must be formulated with an appropriate ointment containing the active components in suspension or dissolved in a carrier. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid petroleum, petroleum jelly, propylene glycol, polyoxyethylene and polyoxypropylene compounds, emulsifying wax and water. Alternatively, the pharmaceutical composition may be formulated with an appropriate lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester waxes, cetostearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of the present invention can also be applied topically to the lower gastrointestinal tract by rectal suppository formulation or in an appropriate enema formulation. Topical transdermal patches are also encompassed by the present invention.

[0252] The pharmaceutical compositions of the present invention can be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulations and may be prepared in the form of saline solutions, employing benzyl alcohol or other appropriate preservatives, absorption promoters to improve bioavailability, fluorocarbons and/or other solubilizing agents or dispersants known in the art.

[0253] When formulated for delivery by inhalation, various formulations offer advantages. Adsorption of the therapeutic compound onto easily dispersed solids such as diketopiperazines (e.g., Technosphere-type particles [Pfutzner, A and Forst, T, 2005, Expert Opin Drug Deliv 2:1097-1106]) or similar structures provides a formulation that results in rapid initial uptake of the therapeutic compound. Freeze-dried powders, particularly glassy particles, containing the therapeutic compound and an excipient are useful for delivery to the lung with good bioavailability, for example, see Exubera® (inhaled insulin from Pfizer and Aventis Pharmaceuticals Inc.).

[0254] Dosage levels between about 0.01 and about 100 mg/kg of body weight per day, preferably 0.5 and about 50 mg/kg of body weight per day of the active ingredient compound are useful. in the prevention and treatment of the disease. Typically, the pharmaceutical compositions of the present invention will be administered about 1 to about 5 times daily or, alternatively, as a continuous infusion. This administration can be used as acute or chronic therapy. The amount of active ingredient that can be combined with carrier materials to produce a unit galenic form will vary depending on the host treated and the particular form of administration. A typical preparation will contain about 5% to about 95% active compound (w/w). Preferably, these preparations contain about 20% to about 80% active compound.

[0255] When a patient's pathology improves, a maintenance dose of a compound, composition or combination of the present invention can be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced depending on the symptoms to a level at which improved pathology is maintained and when symptoms have been alleviated to the desired level, treatment should be discontinued. Patients may, however, require long-term intermittent treatment based on any recurrence of disease symptoms.

[0256] As will be appreciated by the expert, lower or higher dosages may be necessary in relation to those mentioned above. Specific treatment regimens and dosages for any particular patient will depend on several factors, including the activity of the specific compound used, age, body weight, general health, sex, diet, timing of administration, rate of excretion, combination of drugs. , severity and course of an infection, the patient's disposition towards the infection and the judgment of the doctor applying the treatment.

[0257] The carrier-drug conjugate, fusion or formulation provides advantages to the drug manufacturer and the patient compared to the unconjugated, unfused or unformulated drug. Specifically, the carrier-drug conjugate or formulation will be a more potent and longer lasting drug, requiring lower and less frequent dosing compared to the unconjugated, unfused or unformulated drug. This results in lower healthcare costs and a more convenient drug administration schedule for the patient. The carrier-drug conjugate or formulation can also influence the injection route of a drug that is normally infused by intravenous injection to now be administered by subcutaneous injection or in a transdermal delivery system. The route of administration by subcutaneous injection or transdermal delivery is more preferred as they can be administered by the patient himself at home. This can improve patient acceptance.

[0258] In yet another aspect of the invention, DBP levels can be increased as part of carrier-drug therapy. It has been reported that estrogen can increase DBP levels (Speeckaert et al., Clinica Chimica Acta 371:33). It is contemplated herein that DBP levels may be increased by administration of estrogen for more effective delivery of carrier-drug conjugates.

[0259] In yet another aspect of the invention, it is contemplated that the carrier can be used to deliver drugs transdermally. Considering that DBP normally transports UV-activated vitamin D from sites close to the skin surface, the use of a transdermal delivery system with the carrier becomes feasible.

[0260] In order for the invention described in this document to be more fully understood, the following examples are provided. It should be understood that these examples are for illustrative purposes only and should not be considered in any way limiting the present invention.

EXAMPLES EXAMPLE 1: PREPARATION OF AN EXAMPLE THYOL REACTIVE CARRIER COMPOSED OF VITAMIN D3-PEG WITH A REACTIVE MALEIMIDE GROUP

[0261] The maleimide in the carrier of this example was used to conjugate a free cysteine to a protein or peptide in Examples 2 and 3. It is contemplated that the size of the PEG on the supports of the invention is from 0.1 kDa to 100 kDa. Therefore, a 2 kDa PEG was selected as support for this example. The starting materials used in this example were purchased from commercial sources: vitamin D analogue (compound 1, from Toronto Research Chemicals no. B691610) and 2 kDa mPEG-maleimide (compound 4, Creative PEGworks catalog no. PHB-940). .

[0262] According to Figure 2, (R)-methyl5- ((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-((tert-butyldimethylsilyl)oxy )-2-methylenecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)hexanoate (compound 1, 7.5 mg, 0.0145 mmol, 1 equiv., purchased from Toronto Research Chemicals) was dissolved in tetrahydrofuran anhydrous (0.4 mL) and purged with nitrogen. Tetrabutylammonium fluoride (22.7 mg, 0.087 mmol, 6 equiv.) was added and the reaction was stirred at room temperature for 3 hours with monitoring by thin layer chromatography (TLC, silica gel, 30% ethyl acetate in hexanes, UV detection, phosphomolybdic acid staining). To the resulting mixture containing compound 2 were added lithium hydroxide monohydrate (4.2 mg, 0.1015 mmol, 7 equiv.), tetrahydrofuran (0.3 mL) and water (0.15 mL). The reaction was purged with nitrogen and stirred at room temperature for 18 hours. Evaluation by TLC and mass spectroscopy (MS) indicated complete reaction with the presence of the expected compound 3. The reaction mixture was diluted with ether (2 x 15 mL) and washed with 10% aqueous citric acid (30 mL), water (30 mL) and brine (30 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated while the temperature was maintained below 20°C. The sample was additionally dried under a stream of nitrogen providing ((R)-5-((1R,3aS,7aR,E)-4-((Z)-2-((S)—5—hydroxy—2— methylenecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)hexanoic acid) (compound 3, 5.3 mg, 95% yield) in the form of a colorless gum. Rf 0.2 (silica gel, 40% EtOAc in hexanes). NMR analysis revealed the presence of about 1.14% THF and about 0.14% ether.

[0263] Compound 3 (5.3 mg, 0.0137 mmol, 1 equiv.), compound 4 (TFA and MAL-PEG-amine salt, 21.9 mg, 0.0109 mmol, 0.8 equiv. ., purchased from Creative Pegworks) and 2-chloro-1-methylpyridinium iodide (8.7 mg, 0.0342 mmol, 2.5 equiv.) were dissolved in anhydrous dichloromethane (0.5 mL). Triethylamine (7.6 μL, 0.0548 mmol, 4 equiv.) was added and the reaction mixture was stirred for 3 hours at room temperature under nitrogen. The reaction was subsequently diluted with dichloromethane (30 mL) and washed with 10% aqueous citric acid (40 mL), saturated aqueous sodium bicarbonate (30 mL) and brine (30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated while the temperature was maintained below 20°C. The sample was further dried under nitrogen flow to provide the targeted compound in the form of a brown gum. Rf 0.6 (silica gel, 20% methanol in dichloromethane). TLC (ninhydrin staining) analysis of the isolated product indicated the absence of compound 4. 1H NMR analysis of the isolated material confirmed its identity and purity. NMR analysis did not show any significant amounts of methylene chloride or other solvents. EXAMPLE 2: PREPARATION AND CHARACTERIZATION OF A CARRIER AND MODIFIED FGF21 PROTEIN CONJUGATE.

[0264] A modified FGF21 was conjugated to the Vitamin D3-PEG-maleimide carrier described in Example 1. As shown below, the composition of FGF21 and carrier provides significantly improved pharmacokinetic properties compared to a naturally occurring FGF21, thus making the molecule conjugated carrier an important therapeutic compound for the treatment of diabetes.

[0265] FGF21 was expressed in E. coli, purified and conjugated to the carrier as follows. A modified FGF21 was designed to incorporate a free cysteine residue near the amino-terminal end of FGF21 to allow site-specific coupling of the protein to the carrier and a Hise tail was added to facilitate purification. The sequence encoding modified FGF21 (SEQ ID NO:4) was computationally optimized for expression in E. coli, and the gene was chemically synthesized by a contract research organization (DNA2.0 Menlo Park, CA) and cloned into the expression pJexpress401 which contains a T5 promoter and a kanamycin resistance gene. The plasmid was transformed into E. coli Origami2 cells (EMD Biosciences Inc.). Expression of FGF21 from the pJexpress401 vector in the Origami strain was achieved as follows. Luria broth containing 1% glucose was inoculated with an overnight culture at a dilution of 1:100 and grown to log phase where the OD600 was 0.6. The culture temperature was then reduced from 37°C to 18°C and the culture was induced using 0.2 mM IPTG and grown overnight at 18°C under shaking at 180 rpm. The cells were harvested, lysed, and the supernatant collected. The protein was affinity purified using immobilized metal ion affinity chromatography (IMAC) resin and polished by ion exchange chromatography.

[0266] The purified FGF21 protein buffer was subsequently exchanged for 10 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA. Conjugation to the Vitamin Da-PEG-maleimide carrier molecule (Example 1) was achieved by mixing the reactive carrier with thiol dissolved in DMSO at 10 mg/mL with the FGF21 protein containing a free cysteine (SEQ ID NO:3) at 0.5 mg/mL at a 2:1 molar ratio of carrier to protein. The conjugated protein was separated from unreacted components by ion exchange chromatography. Conjugation and purity were confirmed by SDS-PAGE. The buffer of FGF21 alone and the conjugate of FGF21 and carrier were exchanged to PBS and sterilized using a 0.22 micron filter for use in the animal study.

[0267] The pharmacokinetics of FGF21 (SEQ ID NO:3) or the conjugate of FGF21 and carrier were studied in Sprague Dawley rats. Briefly, 0.1 mg/kg of each molecule was injected separately into the rats by SC injection. Plasma samples were collected at 30 minutes, 60 minutes, 120 minutes, 240 minutes, 360 minutes, 24 hours and 48 hours. Samples were analyzed using a proven commercial ELISA kit for FGF21 (Millipore, Cat. #EZHFGF21-19K). The results show a significant difference in absorption after SC administration between FGF21 and the FGF21-carrier conjugate at the following times (30, 60, 120, and 240 minutes), after which FGF21 levels began to decrease (Figure 3). These data show that conjugation of the carrier to FGF21 significantly increased the bioavailability of FGF21 after SC injection (380-fold increase, calculated by dividing the mean Cmax of FGF21, 376 pg/mL, and the mean Cmax of the conjugate of FGF21 and carrier, 143526 pg /mL). The area under the curve (AUC) was also calculated to be 1047980 h*pg/mL for the conjugate of FGF21 and carrier and 1551 h*pg/mL for FGF21 alone. Therefore, there was a 675-fold increase in drug exposure when the mean AUC of the FGF21 and carrier conjugate was divided by the mean AUC of FGF21. Taken together, the data demonstrate the utility of the carrier molecule in increasing bioavailability and indicate that conjugated FGF21 has significant pharmacokinetic advantages over native FGF21. EXAMPLE 3: PREPARATION AND CHARACTERIZATION OF A CARRIER AND GHELLIN PEPTIDE CONJUGATE:

[0268] In this example, as a consequence of the conjugation of the Vitamin D3-PEG-maleimide carrier produced in Example 1 to ghrelin, ghrelin presented a significantly prolonged half-life, thus making the conjugated molecule a potentially useful therapeutic compound for the treatment of cachexia, anorexia and/or frailty in the elderly.

[0269] A synthetic rat ghrelin peptide with a cysteine added to the C-terminal end was purchased from Innovagen (Lund, Sweden, SEQ ID NO:6). The Vitamin Ds-PEG-maleimide carrier as described in Example 1 was selected to have a proportional size relative to a 2 to 3 kDa peptide so that conjugation would not significantly affect bioactivity. Carrier conjugation was achieved by mixing a thiol-reactive carrier (from Example 1) dissolved in DMSO at 10 mg/mL with the ghrelin peptide containing a free cysteine at a concentration of 1 mg/mL in 15 mM MES pH 6.0. 1 mM EDTA in a 2:1 molar ratio of carrier to peptide. The conjugated peptide was separated from unreacted components by ion exchange chromatography. Conjugation and purity were confirmed by SDS-PAGE. The buffer of rat ghrelin peptide (rGhrelin) and rGhrelin and carrier conjugates were exchanged to PBS and sterilized using a 0.22 micron filter for use in the animal study.

[0270] The pharmacokinetics of rGhrelin (SEQ ID NO:6) or the rGhrelin and carrier conjugate were studied in Sprague Dawley rats. Briefly, 0.1 mg/kg of each molecule was injected separately into the rats by intravenous (IV) injection. Plasma samples were collected at 30 minutes, 60 minutes, 120 minutes, 240 minutes, 360 minutes, 24 hours and 48 hours. Samples were analyzed using a proven commercial ELISA kit for analysis of rat plasma rGhrelin (Millipore, Cat. #EZRGRT-91K). The results show significant differences in the pharmacokinetic profiles of rGhrelin and the rGhrelin and carrier conjugate (Figure 4). Half-life calculation using WinNonLin revealed a half-life of 0.37 hours for rGhrelin and a half-life of 8 hours for the rGhrelin and carrier conjugate, which was calculated to be a 22-fold improvement. The data demonstrate a second example of the usefulness of the carrier molecule in increasing half-life. Therefore, ghrelin in a conjugated form is a useful therapeutic compound for treating ghrelin-responsive diseases such as cachexia, anorexia and/or frailty in the elderly. EXAMPLE 4: EVALUATION OF GHRELLIN CARRIER PEPTIDE RECEPTOR BINDING ACTIVITY

[0271] The activity of the ghrelin peptide, when conjugated to a carrier, was not negatively affected by the presence of support and targeting groups. To demonstrate this, ghrelin (SEQ ID:5) and ghrelin conjugated to the Vitamin Ds-PEG-maleimide carrier of Example 3 were compared in terms of receptor binding and activation to a ghrelin receptor (agonist activity) using a ghrelin receptor assay. receptor agonist in cells as described below.

[0272] Test compounds were added to cultures of CHO-K1 cells expressing GHS-R. Ghrelin binds and activates the GHS-R receptor and intracellular calcium responses were evaluated as an indicator of agonist activity. Activity was measured by a calcium flux-based assay developed by GenScript USA Inc. for the FLIPR™ ("Fluorescence Imaging Plate Reader") high-throughput cell tracking instrument.

[0273] CHO-K1 cells expressing GHS-R were cultured in Ham's F12 supplemented with 10% fetal bovine serum, 500 mg/mL G418 and subcultured to maintain optimal cellular health. Cells were plated in a 384-well black-sided, clear-bottom plate at a density of 20,000 cells per well in 20 μL of culture medium for 18 hours before assay and maintained at 37 °C/5% CO2. At the beginning of the assay, 20 μL of Calcium-4 loading buffer was added to the wells. The plate was incubated in the dark at 37 °C for 60 minutes and then at room temperature for 15 minutes. The ghrelin and conjugated ghrelin test articles were at an initial concentration of 1 mM in DMSO. Sequential 10-fold dilutions of the test articles were performed in Hank's Buffered Saline Solution (HBSS) with 20 mM HEPES buffer at pH 7.4 before being added to the test plates. The final concentration of each test article was 5x concentration before addition to the cells. Intracellular calcium levels in cells were measured for 20 seconds using the FLIPR™ instrument before adding the test articles. Ten μL of each diluted test article was added to the plate for a final volume of 50 μL. Changes in intracellular calcium levels were measured for an additional 100 seconds (21 to 120 seconds).

[0274] The average value of the 20 second reading (1 to 20 seconds) was calculated as the baseline reading and the relative fluorescence unit intensity (ΔRFU) values were calculated with the average value of the line reading background being subtracted from the maximum fluorescence units (21 to 120 seconds). Data acquisition and analyzes were performed using ScreenWorks® (version 3.1) and exported to Excel.

[0275] The % activation of the compound was calculated using the following equation:

[0276] % activation = (ΔRFUcompound - ΔRFUFund)/(ΔRFUagonist control - ΔRFUFund)}*100% activation was subsequently plotted as a function of the log of the cumulative doses of the compounds. Data represent the average of duplicate determinations. The EC50 was determined using a data analysis program created by GenScript.

[0277] The EC50 value of ghrelin without the carrier was 88.5 nM. In comparison, carrier-conjugated ghrelin had an EC50 value of 85 nM, which is almost identical to the control peptide. Therefore, the agonist activity of the peptide was preserved after conjugation of the carrier to the ghrelin peptide. Note that assays were performed in the presence of 10% DBP-containing serum. No interference with receptor binding and activation was observed for the peptide conjugated to the carrier. EXAMPLE 5: PREPARATION OF AN EXAMPLE AMINE-REACTIVE CARRIER COMPOSED OF VITAMIN D3-PEG WITH A REACTIVE NHS GROUP

[0278] Reactive NHS groups were generated on carriers for conjugation to amine groups on proteins. A 2 kDa PEG was selected as support for this example. The starting materials used in this example were purchased from Toronto Research Chemicals for the Vitamin D analogue (compound 1) and from Creative Pegworks for the 2 kDa mPEG amino acid (compound 5).

[0279] According to Figure 5 (steps 1 and 2): (R)-methyl-5-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5 -((tert-butyldimethylsilyl)oxy)-2-methylenecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)hexanoate (compound 1, 8.2 mg, 0.0159 mmol, 1 equiv.) was dissolved in anhydrous tetrahydrofuran (THF, 0.4 mL) and the mixture was purged with nitrogen. Tetrabutylammonium fluoride solution (25 mg, 0.096 mmol, 6 equiv.) was added and the reaction mixture was stirred at room temperature for 3 hours with monitoring by thin layer chromatography (TLC, silica gel, 30% ethyl acetate in hexanes , UV detection, phosphomolybdic acid staining). To the resulting mixture containing compound 2 were added lithium hydroxide monohydrate (4.6 mg, 0.109 mmol, 7 equiv.), THF (0.3 mL) and water (0.16 mL). The reaction mixture was purged with nitrogen and stirred at room temperature for 18 hours. Evaluation by TLC and mass spectroscopy (MS) indicated complete reaction with the presence of the expected compound 3. The reaction mixture was diluted with ether (10 mL) and washed with 10% aqueous citric acid (10 mL), water (10 mL) and brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated while the temperature was maintained below 20°C. The sample was additionally dried under a stream of nitrogen providing the acid ((R)-5-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-hydroxy-2 - methylenecyclohexylidene)ethyl-indene)-7a-methyloctahydro-1H-inden-1-yl)hexanoic acid (compound 3, 6.1 mg, 99% yield) in the form of a colorless gum. Rf 0.2 (silica gel, ethyl acetate (EtOAc) 40% in hexanes).Nuclear magnetic resonance spectroscopy (NMR) analysis revealed the presence of ~7% ether.

[0280] According to Figure 5 (step 3): to a solution of PEG-amino acid 4 (18.5 mg, 0.0092 mmol, purchased from Creative Pegworks) in anhydrous methanol, HCl in dioxane (4 M , 1.5 mL) and the reaction mixture was heated to 70 oC in a sealed tube for 20 hours. The reaction was monitored by TLC (ninhydrin staining), and once the reaction was complete, it was concentrated in a rotavapor. The residue was co-evaporated with dichloromethane (3 x 5 mL) and ether (3 x 5 mL) to form a pale yellow foam, which was resuspended in ether (5 mL). The liquid was decanted and the solid obtained was dried to isolate the desired product 5 (14 mg, 75%) as a light yellow solid. Rf 0.2 (silica gel, methanol (MeOH) 20% / DCM / NH4OH 0.2%). NMR analysis did not show any significant amounts of methylene chloride or ether.

[0281] According to Figure 5 (step 4): compound 3 (3.4 mg, 0.009 mmol, 1 equiv.), compound 6 (PEG-amine methyl ester salt and HCl, 14 mg, 0.007 mmol, 0.8 equiv.) and 2-chloro-1-methylpyridinium iodide (5.6 mg, 0.022 mmol, 2.5 equiv.) were dissolved in anhydrous dichloromethane (0.6 mL). Triethylamine (5 μL, 0.0356 mmol, 4 equiv.) was added and the reaction mixture was stirred for 3 hours at room temperature under nitrogen. At this time, the reaction was incomplete, therefore, an additional amount of compound 3 (1.7 mg, 0.0045 mmol) was added and the reaction was continued for another 3 hours and further diluted with dichloromethane (10 mL) and washed with 10% aqueous citric acid (10 mL), saturated aqueous sodium bicarbonate (10 mL) and saline solution (10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated while the temperature was maintained below 20°C. The sample was purified by flash chromatography on silica gel (2 g). The column was eluted first with ethyl acetate to remove unreacted compound 3 and then with 10% MeOH/dichloromethane (20 mL of each). The fractions containing the pure product were combined and evaporated in a rotavapor while the temperature was maintained below 20 °C. The sample was dried under a stream of nitrogen to provide compound 7 as a brown gum (10 mg, 60%). Rf 0.3 (silica gel, 5% MeOH in dichloromethane). TLC (ninhydrin staining) analysis of the isolated product indicated the absence of compound 6. 1H NMR analysis of the isolated material confirmed its identity and purity. NMR analysis revealed the presence of 1.1% methylene chloride.

[0282] According to Figure 5 (step 5): compound 7 (10 mg, 0.0042 mmol) was dissolved in a mixture of THF (0.2 mL) and a drop of methanol. To this solution was added lithium hydroxide monohydrate solution (0.9 mg, 0.021 mmol, 5 equiv. in 0.1 mL of water). The reaction mixture was purged with nitrogen and stirred at room temperature for 18 hours. TLC evaluation indicated complete reaction with the presence of compound 8. The reaction mixture was diluted with dichloromethane (10 mL) and washed with 10% aqueous citric acid (10 mL) and saline solution (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated while the temperature was maintained below 20°C. The sample was further dried under nitrogen flow to provide the desired Vitamin-D3-PEG-acid compound (compound 8.7 mg, 71% yield) in the form of a brown gum. Rf 0.2 (silica gel, 10% MeOH/dichloromethane). NMR analysis revealed the presence of ~2% dichloromethane.

[0283] Stock solutions were prepared: 34 g of N-hydroxysuccinimide in 1 mL of anhydrous dimethylformamide (DMF) and 61 mg of dicyclohexylcarbodiimide (DCC) in 1 mL of anhydrous dichloromethane.

[0284] According to Figure 5 (step 6): to a solution of compound 8 (7 mg, 0.003 mmol, 1 equiv.) in dichloromethane (0.3 mL), a solution of N-hydroxysuccinimide in DMF was added (10 μL, 0.34 mg, 0.003 mmol) followed by a solution of DCC in dichloromethane (10 μL, 0.61 mg, 0.003 mmol) and the reaction mixture was purged with nitrogen and stirred for 20 hours. As the reaction was incomplete as indicated by TLC, additional amounts of N-hydroxysuccinimide in DMF (25 μL, 0.85 mg, 0.0075 mmol) and DCC in dichloromethane (25 μL, 1.53 mg, 0.0075 mmol) were added and the reaction was continued for another 20 hours. Chloroform (10 mL) was added to the reaction mixture and it was washed with water (10 mL). The organic phase was dried over sodium sulfate and removal of the solvent provided the desired bleached Vitamin D3 — PEG — NHS compound (7.0 mg, crude). 1H NMR and TLC (Rf: 0.3, 10% MeOH/chloroform) of this material indicated the presence of the desired material. The carrier Vitamin D3 — PEG — NHS was subsequently used in Example 6. EXAMPLE 6: PREPARATION AND CHARACTERIZATION OF A CARRIER AND ANTIBODY CONJUGATE

[0285] The carrier and infliximab conjugate showed increased serum concentrations and bioavailability in rats compared to infliximab alone.

[0286] Infliximab, sold in the form of a lyophilized powder with the appropriate salts (Hannah Pharmaceuticals), was resuspended to a concentration of 10 mg/mL in water. The Vitamin D3 — PEG - NHS carrier was resuspended at a concentration of 10 mg/mL in DMSO. The Vitamin D3 — PEG - NHS carrier and infliximab were then mixed in a molar ratio of 5:1 and 10:1 of carrier to infliximab. A therapeutic compound and carrier conjugate of the invention typically has at least 1 and could have between 1 and 10 carrier molecules individually linked to a therapeutic compound. When using an NHS version of the carrier, more than one carrier can be linked to a therapeutic protein and this can be experimentally controlled by changing the molar ratio between the carrier and therapeutic compound targeted in the reaction. In this example, a targeted distribution of 2 to 4 carriers was established as the desired parameter. By testing two different molar ratios and examining the resulting conjugates by mass spectrometry, an actual ratio was determined.

[0287] Infliximab and infliximab and carrier-NHS conjugates were separated from unconjugated carrier using a 40 kDa cutoff desalting column (Zeba Spin, Thermo Scientific). Mass spectrometry was used to calculate the intact mass of infliximab in the reactions. The results show that unmodified infliximab had a mass predominantly of 149 kDa. The mass of infliximab conjugated to the carrier had a mass in increasing ratios with an average carrier binding of 3 kDa of 2 to 4 carriers per antibody.

[0288] The purified 5:1 reaction product was subsequently used in a pharmacokinetic study in rats to compare infliximab to the infliximab and carrier conjugate administered subcutaneously (SC). The dose was 1 mg/kg in all test groups with three rats per group. Serum samples were collected before dosing and at various times from 5 minutes to 48 hours after injection. Serum samples were subsequently analyzed by ELISA using a specific ELISA kit for determining serum infliximab levels (Promonitor™, Progenika™). Pharmacokinetic parameters were determined using WinNonLin. The results shown in Figure 6 demonstrate a 1.5-fold improvement in serum concentration and area under the curve (AUC) when the antibody is conjugated to the carrier Vitamin D3 - PEG - NHS compared to the unconjugated antibody. More specifically, at 24 hours and 48 hours, concentrations of 2100 ng/mL and 4800 ng/mL are observed for infliximab. In contrast, at 24 hours and 48 hours, concentrations of 3200 ng/mL and 7000 ng/mL are observed for infliximab conjugated to the carrier. This translates into a 50% bioavailability improvement. The AUC calculation showed a 50% improvement (Figure 6). Therefore, this example shows the utility of the carrier in improving the serum concentration and bioavailability of infliximab compared to the unconjugated antibody. EXAMPLE SEQUENCES SEQ ID NO:1 Protein sequence of human FGF21 MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYLYT DDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYG SLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSP HRDPAPRGPARFLPLPGLPPA LPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS SEQ ID NO:2 Protein sequence of mature human FGF21 HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPES LLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAH GLPLHLPG NKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQ GRSPSYAS SEQ ID NO:3 Modified mature human FGF21 protein sequence (6-his tail at the end N-terminal, tev cleavage site, an additional 182P at the C-terminal end, and modifications of the following residues using mature human FGF21 sequence numbering: I3C and G170E) MGSHHHHHHSSGENLYFQGHPCPDSSPLLQFGGQVRQRYLYTDDAQQTEAH LEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALY GSLHFDPEAC SFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILA PQPPDVGSSDPLSMVEPSQGRSPSYASP SEQ ID NO:4 Coding sequence optimized for FGF21 expression in E. coli (see amino acids in SEQ ID NO:3) ATGGGCTCACATCATCACCACCATCATAGCAGCGGAGAGAAC TTGTATTTT CAGGGACATCCGTGCCCTGACAGCAGCCCGCTGCTGCAGTTCGGTGGTCAAGTCCGTCAGC GTTACCTGTACACTGACGACGCGCAACAGACCGAGGCGCACCTGGAAATTCGCGAAGATGG TACGGTGGGTGGC GCAGCGGACCAAAGCCCGGAGTCCCTGTTGCAGCTGAAGGCCCTGAAG CCGGGTGTCATCCAAATCCTGGGCGTTAAAACCAGCCGTTTTCTGTGCCAACGTCCGGATG GTGCGCTGTACGGTTCCCTGCACTTCGACCCAGAGGCATGTAGCTTTCGTGAACTGCTGCT GGAAGATGGCTATAATGTGTACCAGTCTGAGGCGCACGGTCTGCCGTTGCACTTGCCGGGT AACA AAAGCCCGCACCGCGACCCAGCACCGCGTGGTCCGGCTCGCTTCCTGCCGCTGCCGG GTCTGCCTCCGGCGCTGCCGGAGCCGCCAGGCATTCTGGCTCCGCAACCGCCGGATGTTGG CAGCAGCGATCCGCTGAGCATGGTTGAACCGTCGCAGGGCCGCAGCCCGTCTTATGCCAGC CCGTAA SEQ ID NO:5 Protein sequence of human ghrelin GS(n-octanoyl-S) FLSPEHQRVQQRKESKKPPAKLQPR SEQ ID NO:6 Rat ghrelin with added C-terminal cys GS(n-octanoyl-S)FLSPEHQKAQQRKESKKPPAKLQPRC SEQ ID NO:7 DBP protein sequence MKRVLVLLLAVAFGHALERGRDYEKNKVCKEFSHLGKEDFTSLSLVLYSRK FPSGTFEQVSQLVKEVVSL TEACCAEGADPDCYDTRTSALSAKSCESNSPFPVHPGTAECC TKEGLERKLCMAALKHQPQEFPTYVEPTNDEICEAFRKDPKEYANQFMWEYSTNYGQAPLS LLVSYTKSYLSMVGSCCTSASPTVCFLKERLQLKHLSLTTLSNRVCSQYAAYGEKKSRLS NLIKLAQKVPTADLEDVLPLAEDITNILSKCCESASEDCMAKELPEH TVKLCDNLSTKNSK FEDCCQEKTAMDVFVCTYFMPAAQLPELPDVELPTNKDVCDPGNTKVMDKYTFELSRRTHL PEVFLSKVLEPTLKSLGECCDVEDSTTCFNAKGPLLKKELSSFIDKGQELCADYSENTFTE YKKKLAERLKAKLPDATPTELAKLVNKHSDFASNCCSINSPPLYCDSEIDAELKNIL SEQ ID NO:8 Nucleotide sequence BPD tip TTTAATAATAATTCTGTGTTGCTTCTGAGATTAATAATTGATTAATTCATA GTCAGGAATCTTTGTAAAAAGGAAACCAATTACTTTTGGCTACCACTTTTACATGGTCACC TACAGGAGAGAGGAGGTGCTGCAAGACTCTCTGGTAGAAAAATGAAGAGGGTCCTGGTACT ACTGCTTGCTGTGGCATTTGGACATGCTTTAGAGAGAGGCCGGGATTATGAAAAGAATAAA GTCTGCA AGGAATTCTCCCATCTGGGAAAGGAGGACTTCACATCTCTGTCACTAGTCCTGT ACAGTAGAAAATTTCCCAGTGGCACGTTTGAACAGGTCAGCCAACTTGTGAAGGAAGTTGT CTCCTTGACCGAAGCCTGCTGTGCGGAAGGGGCTGACCCTGACTGCTATGACACCAGGACC TCAGCACTGTCTGCCAAGTCCTGTGAAAGTAATTCTCCATTCCCCGTTCACCCAGGC ACTG CTGAGTGCTGCACCAAAGAGGGCCTGGAACGAAAGCTCTGCATGGCTGCTCTGAAACACCA GCCACAGGAATTCCCTACCTACGTGGAACCCACAAATGATGAAATCTGTGAGGCGTTCAGG AAAGATCCAAAGGAATATGCTAATCAATTTATGTGGGAATATTCCACTAATTACGGACAAG CTCCTCTGTCACTTTTAGTCAGTTACACCAAGAGTTATCTTTCTATGGTAGGG TCCTGCTG TACCTCTGCAAGCC CAACTGTATGCTTTTTGAAAGAGAGACTCCAGCTTAAACATTTATCA CTTCTCACCACTCTGTCAAATAGAGTCTGCTCACAATATGCTGCTTATGGGGAGAAGAAAT CAAGGCTCAGCAATCTCATAAAGTTAGCCCAAAAAGTGCCTACTGCTGATCTGGAGGATGT TTTGCCACTAGCTGAAGATATTACTAACATCCTCTCCAAATGCTGTGAGTC TGCCTCTGAA GATTGCATGGCCAAAGAGCTGCCTGAACACACAGTAAAACTCTGTGACAATTTATCCACAA AGAATTCTAAGTTTGAAGACTGTTGTCAAGAAAAAACAGCCATGGACGTTTTTGTGTGCAC TTACTTCATGCCAGCTGCCCAACTCCCCGAGCTTCCAGATGTAGAGTTGCCCACAAACAAA GATGTGTGTGATCCAGGAAACACCAAAGTCATGGATAAGTATAC ATTTGAACTAAGCAGAA GGACTCATCTTCCGGAAGTATTCCTCAGTAAGGTACTTGAGCCAACCCTAAAAAGCCTTGG TGAATGCTGTGATGTTGAAGACTCAACTACCTGTTTTAATGCTAAGGGCCCTCTACTAAAG AAGGAACTATCTTCTTTCATTGACAAGGGACAAGAACTATGTGCAGATTATTCAGAAAATA CATTTACTGAGTACAAGAAAAAACTGGCAGAGCGACTAAAA GCAAAATTGCCTGATGCCAC ACCCACGGAACTGGCAAAGCTGGTTAACAAGCACTCAGACTTTGCCTCCAACTGCTGTTCC ATAAACTCACCTCCTCTTTACTGTGATTCAGAGATTGATGCTGAATTGAAGAATATCCTGT AGTCCTGAAGCATGTTTATTAACTTTGACCAGAGTTGGAGCCACCCAGGGGAATGATCTCT GATGACCTAACCTAAGCAAAACCACTGAGCTTCTGGGAA GACAACTAGGATACTTTCTACT TTTTCTAGCTACAATATCTTCATACAATGACAAGTATGATGATTTGCTATCAAAATAAATT GAAATATAATGCAAACCATAAAAAAAAAAAAAAAAAAA SEQ ID NO:9 Tumor necrosis factor alpha (TNF-α) ATGAGCACTGAAAGCATGATCCGGGACGTGGAGCTGGCCGAGGAGGCGCTC CCCAAGAAGACAGGGGGGCCCCAGGGCTCCAG GCGGTGCTTGTTCCTCAGCCTCTCTCTCCT TCCTGATCGTGGCAGGCGCCACCACGCTCTTCTGCCTGCTGCACTTTGGGGTGATCGGCCC CCAGAGGGAAGAGTTCCCCAGGGACCTCTCTCTAATCAGCCCTCTGGCCCAGGCAGTCAGA TCATCTTCTCGAACCCCGAGTGACAAGCCTGTAGCCCATGTTGTAGCAAACCCTCAAGCTG AGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTCCTGGCCAATGGCGTGGAGCT GAGAGATAACCAGTTGGTGGTGCCATCAGAGGGCCTGTACCTCATCTACTCCCAGGTCCTC TTCAAGGGCCAAGGCTGCCCCTCCACCCATGTGCTCCTCACCCACACCATC AGCCGCATCG CCGTCTCCTACCAGACCAAGGTCAACCTCCTCTCTGCCATCAAGAGCCCCTGCCAGAGGGA GACCCCAGAGGGGGCTGAGGCCAAGCCCTGGTATGAGCCCATCTATCTGGGAGGGGTCTTC CAGCTGGAGAAGGGTGACCGACTCAGCGCTGAGATCAATCGGCCCGACTATCTCGACTTT GCCGAGTCTGGGCAGGTCTACTTTGGGATCATTGCCCTGTGA SEQ ID NO: 10 Tumor necrosis factor alpha (TNF-α) MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLL HFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANAL LANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAI KS PCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

[0289] All publications and patent documents disclosed or cited in this document are incorporated in their entirety by citation. The above description is presented for illustration and description purposes only. This description is not intended to limit the invention to the precise form disclosed. The scope of the invention is intended to be defined by the claims appended hereto.

Claims (15)

1. CARRIER-DRUG CONJUGATE, characterized in that it comprises a targeting group comprising vitamin D that is not hydroxylated at the carbon 1 position linked to a therapeutic peptide, in which said vitamin D is cholecalciferol, ergocalciferol or calcidiol, and in which said therapeutic peptide has the sequence of SEQ ID NOS:2 or 5. 2. CONJUGATE, according to claim 1, characterized in that said targeting group is linked to said therapeutic peptide by means of a support, which is selected from the group consisting of polyethylene glycol, polylysine, polyethyleneimine, polypropylene glycol, an amino acid, an acid nucleic acid, a glycan, a water-soluble polymer, and a carbon chain linker. 3. CONJUGATE, according to claim 2, characterized in that said support has between about 100 Da and 200,000 Da. 4. PHARMACEUTICAL COMPOSITION, characterized by comprising a carrier-drug conjugate, comprising a vitamin D targeting group that is not hydroxylated at the carbon 1 position, linked to a therapeutic peptide through a support that has between 300 Da and 60,000 Da , wherein said vitamin D is cholecalciferol, ergocalciferol or calcidiol, and wherein said therapeutic peptide has the sequence of SEQ ID NOS: 2 or 5. 5. PHARMACEUTICAL COMPOSITION, according to claim 4, characterized in that said carrier increases the absorption, bioavailability, or half-life of said therapeutic peptide in circulation. 6. PHARMACEUTICAL COMPOSITION, according to claim 5, characterized in that said support is selected from the group consisting of polyethylene glycol, polylysine, polyethyleneimine, polypropylene glycol, an amino acid, a nucleic acid, a glycan, a water-soluble polymer, and a chain linker small carbon dioxide. 7. PHARMACEUTICAL COMPOSITION, according to claim 11, characterized in that said support is polyethylene glycol. 8. METHOD OF MANUFACTURING THE PHARMACEUTICAL COMPOSITION, as defined in claim 5, characterized by comprising the conjugation of said targeting group and said therapeutic peptide, wherein said conjugation step uses a coupling group. 9. METHOD according to claim 8, characterized in that said coupling group is selected from the group consisting of an amine-reactive group, a thiol-reactive group, a maleimide group, a thiol group, an aldehyde group, a NHS-ester, a haloacetyl group, an iodoacetyl group, a bromoacetyl group, an SMCC group, a SMCC sulfo group, a carbodiimide group, bifunctional crosslinkers, NHS-Maleimide and combinations thereof. 10. METHOD, according to claim 8, characterized in that said conjugation step is carried out by cycloaddition reactions. 11. METHOD, according to claim 8, characterized in that said support is selected from the group consisting of polyethylene glycol, polylysine, polyethyleneimine, polypropylene glycol, an amino acid, a nucleic acid, a glycan, a modifying group containing a reactive ligand, a water-soluble polymer, a small carbon chain ligand, and an additional therapeutic chemical moiety. 12. PHARMACEUTICAL COMPOSITION, characterized by comprising: (a) a therapeutic peptide having the sequence SEQ IDNOs: 2 or 5; (b) a stable connection support; (c) a targeting group, which is vitamin D which is cholecalciferol, ergocalciferol or calcidiol; wherein upon administration to a first subject, said therapeutic peptide has a half-life measured by means of a functional assay of blood samples taken at a plurality of time points, which is greater than the half-life of said therapeutic peptide administered to a second subject without said binding support and targeting group as measured by said functional assay from blood samples taken at a plurality of time points. 13. PHARMACEUTICAL COMPOSITION, according to claim 12, characterized in that administration to said first and second individuals is carried out by means of subcutaneous injection. 14. PHARMACEUTICAL COMPOSITION, according to claim 12, characterized in that said pharmaceutical peptide stably linked to said support and targeting group retains substantially the same activity as the therapeutic peptide not linked to said support and targeting group, as measured by the functional test site. 15. PHARMACEUTICAL COMPOSITION, according to claim 12, characterized in that said functional test is an ELISA analysis.