WO2017210242A1

WO2017210242A1 - Insulin-nuclear hormone conjugates

Info

Publication number: WO2017210242A1
Application number: PCT/US2017/035102
Authority: WO
Inventors: Richard D. Dimarchi; Bin Yang
Original assignee: Indiana University Research And Technology Corporation
Priority date: 2016-06-02
Filing date: 2017-05-31
Publication date: 2017-12-07

Abstract

Disclosed herein are insulin peptides conjugated to a nuclear hormone receptor ligand (NHR ligand) wherein the insulin/NHR ligand conjugate has agonist activity at both the insulin receptor and the corresponding nuclear hormone receptor.

Description

INSULIN-NUCLEAR HORMONE CONJUGATES

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Nos.

62/344,674, filed on June 2, 2016, the disclosure of which is hereby expressly incorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 90 kilobytes acii (text) file named "265655seqlist_ST25.txt," created on May 30, 2017.

BACKGROUND

Insulin is a proven therapy for the treatment of juvenile-onset diabetes and later stage adult-onset diabetes. The peptide is biosynthesized as a larger linear precursor of low potency (approximately 2% to 9% of native insulin), named proinsulin. Proinsulin is proteolytically converted to insulin by the selective removal of a 35-residue connecting peptide (C peptide). The resultant heteroduplex formed by disulfide links between the insulin "A chain" (SEQ ID NO: 1) and "B chain" (SEQ ID NO: 2) chain, representing a total of 51 amino acids, has high potency for the insulin receptor (nM range). Native insulin has approximately one hundredfold selective affinity for the insulin receptor relative to the related insulin-like growth factor 1 receptor, but demonstrates little selectively for the two different insulin receptor isoforms, named A & B.

The insulin-like growth factors 1 and 2 are single chain liner peptide hormones that are highly homologous in their A and B chain sequences, sharing approximately fifty percent homology with native insulin. The IGF A and B chains are linked by a "C-peptide", wherein the C-peptides of the two IGFs differ in size and amino acid sequence, the first being twelve and the second being eight amino acids in length. Human IGF-1 is a 70 aa basic peptide having the protein sequence shown in SEQ ID NO: 3, and has a 43% homology with proinsulin (Rinderknecht et al. (1978) J. Biol. Chem. 253:2769-2776). Human IGF-2 is a 67 amino acid basic peptide having the protein sequence shown in SEQ ID NO: 4. The IGFs demonstrate considerably less activity at the insulin B receptor isoform than the A-receptor isoform.

Applicants have previously identified IGF-1 based insulin peptides analogs, (wherein the native Gln-Phe dipeptide of the B -chain is replaced by Tyr-Leu) that display high activity at the insulin receptor (see PCT/US2009/068713, the disclosure of which is incorporated herein). Such analogs (referred to herein as IGF YL analog peptides) are more readily synthesized than insulin and enable the development of co-agonist analogs for insulin and IGF- 1 receptors, and selective insulin receptor specific analogs. Furthermore, these insulin analogs can also be formulated as single chain insulin agonists in accordance with the present disclosure.

Single chain insulin analogs comprising the insulin A and B chains have been previously prepared (see EP 1,193,272 and US 2007/0129284). However, single chain high potency insulin agonists can also be prepared by insertion of the IGF-1 C-peptide, or analogs thereof, as a connecting peptide linking the insulin B and A peptides. The selective mutation of individual amino acids in the C-peptide sequence yields peptides that are highly selective for insulin relative to IGF- 1 receptor.

Nuclear hormone receptor proteins form a class of ligand activated proteins that, when bound to specific sequences of DNA, serve as on-off switches for transcription within the cell nucleus. These switches control the development and differentiation of skin, bone and behavioral centers in the brain, as well as the continual regulation of reproductive tissues.

Nuclear hormone receptor ligands such as steroids, sterols, retinoids, thyroid hormones, and vitamin D function to activate nuclear hormone receptors. The interaction of the hormone and receptor triggers a conformational change in the receptor, which results in the up-regulation of gene expression. The level of cellular signal transduction activated by the interaction of a ligand and a nuclear hormone receptor is determined by the number of ligands and receptors available for binding, and by the binding affinity between the ligand and the receptor.

Thyroid hormones powerfully influence systemic metabolism through multiple pathways, with profound effects on energy expenditure, fat oxidation, and cholesterol metabolism. Clinical reports revealed sixty years ago that administration of thyroid extracts reduced circulating cholesterol and reversed obesity. However, adverse side effects of thyroid hormone treatment include increased heart rate, cardiac hypertrophy, muscle wasting, and reduced bone density, terminating its clinical use. Discovery of thyromimetics capable of separating lipid metabolism benefits from adverse cardiovascular effects has remained a desire for patients, physicians and the pharmaceutical industry.

Peroxisome proliferator-activated receptors (PPAR) are nuclear hormone receptors. PPAR receptors activate transcription by binding to elements of DNA sequences, known as peroxisome proliferator response elements (PPRE), in the form of a heterodimer with retinoid X receptors (known as RXRs). Three sub-types of human PPAR have been identified and described: PPAR- alpha, PPAR-gamma and PPAR-delta (or NUCI). PPAR- alpha is mainly expressed in the liver, while PPAR-delta is ubiquitous. PPAR-gamma is involved in regulating the differentiation of adipocytes, where it is highly expressed. It also has a key role in systemic lipid homeostasis. A number of compounds that modulate the activity of PPARs have been identified including thiazolidinediones, which have been employed in the treatment of diabetes and metabolic disease. Activation of PPAR gamma is known to reduce blood glucose levels without increasing insulin secretion. Discovery of PPAR agonists capable of separating lipid metabolism benefits from adverse effects has remained a desire for patients, physicians and the pharmaceutical industry

As disclosed herein conjugates are formed between an insulin peptide and a nuclear hormone receptor ligand, wherein the conjugate has agonist activity at both the insulin receptor and the corresponding nuclear hormone receptor. More particularly, the

conjugation of a nuclear hormone receptor ligand is anticipated to produce a beneficial modification of the insulin peptide activity while moderating adverse effects associated with nuclear hormone agonists.

SUMMARY

An insulin agonist/ nuclear hormone receptor ligand conjugate is provided wherein the conjugate has agonist activity at both the insulin receptor and the corresponding nuclear hormone receptor (NHR). These conjugates with plural activities are useful for the treatment of a variety of diseases, including the treatment of diabetes. In accordance with one embodiment the conjugates of the present disclosure can be represented by the following formula:

Q-L-Y

wherein Q is an insulin peptide, Y is a NHR ligand, and L is a linking group or a bond. The insulin peptide component of the conjugate can be native insulin or any known insulin analog that has activity at the insulin receptor including for example any insulin peptide disclosed in published international applications W096/34882, WO 2010/080607, WO 2010/080609, WO 2011/159882, WO/2011/159895 and US Patent No. 6,630,348, the disclosures of which are incorporated herein by reference.

The NHR ligand (Y) is wholly or partly non-peptidic and acts at a nuclear hormone receptor with an activity in accordance with any of the teachings set forth herein. In some embodiments the NHR ligand is an agonist that, in its unbound state, has an EC50 or IC50 of about 1 mM or less, or 100 μΜ or less, or 10 μΜ or less, or 1 μΜ or less. In some embodiments, the NHR ligand has a molecular weight of up to about 5000 daltons, or up to about 2000 daltons, or up to about 1000 daltons, or up to about 500 daltons. The NHR ligand may act at any of the nuclear hormone receptors described herein or have any of the structures described herein. In accordance with one embodiment the NHR ligand component of the conjugate can be a ligand that activates the thyroid hormone receptor or activates the peroxisome proliferator- activated receptors (PPAR).

The conjugates of the present disclosure are anticipated to lessen the amount of insulin that is needed to control hyperglycemia while also simultaneously lowering body weight and triglycerides and/or cholesterol levels. More particularly, the chemical addition of nuclear hormone activity to insulin agonist peptides provides supplemental pharmacology that is envisioned to broaden insulin action such that it could lower triglycerides, cholesterol and body weight in a means that is currently not possible with insulin therapy alone.

Thyroid hormone is recognized for its ability to lower body weight and

independently lower cholesterol through action at the liver. In accordance with one embodiment, applicants anticipate that the insulin-thyroid hormone conjugates will provide new dimensions to insulin action and lessen the amount of insulin needed to control blood glucose leading to less hypoglycemia and less body weight gain. In one embodiment applicants anticipate that insulin can be conjugated to a PPAR agonist to improve insulin sensitivity which should lower exogenous insulin requirements leading to less hypoglycemia and less body weight gain.

In accordance with one embodiment the nuclear hormone receptor ligand is linked to the C-terminus of the insulin B chain or at the N-terminus of the insulin A or B chain, either directly via an ester or amide bond, or indirectly through a spacer. In one embodiment the nuclear hormone receptor ligand is covalently linked to the insulin peptide through the side chain of an amino acid at a position selected from the group consisting of Al, B l, B28 and B29, or the N-terminal alpha amine of the A or B chain, or at the side chain of an amino acid at any position of a linking moiety that links the A chain and B chain of a single chain insulin analog.

In one embodiment the insulin peptide of the conjugate is a two chain insulin analog comprising an A chain and B chain linked to one another via intermolecular disulfide bonds. In a further embodiment the conjugate comprises a two chain insulin analog wherein a first and second nuclear hormone receptor ligand are covalently linked to the insulin peptide at a position selected from the side chain of the B28 or B29 amino acid and the N-terminal alpha amine of the A or B chain.

In one embodiment the NHR ligand-insulin conjugate comprises a hydrophilic moiety linked to the N-terminal alpha amine of the B chain or to the side chain of an amino acid at a position selected from the group consisting of A9, A14 and A15 of the A chain or positions B l, B2, B IO, B22, B28 or B29 of the B chain or to a side chain of an amino acid of the linking moiety in a single chain insulin analog. In one embodiment the NHR ligand is linked to the amino acid side chain of a lysine present at position B28 or B29 and optionally a hydrophilic moiety is linked to the amino terminus of the B chain, optionally through the N-terminal alpha amine of the B chain. In one embodiment the hydrophilic moiety is a polyethylene glycol chain.

In one embodiment a polyethylene glycol chain is covalently bound to the side chain of an amino acid of the linking moiety of the insulin peptide component, when the insulin peptide is a single chain insulin analog. In one embodiment the insulin peptide is a single chain insulin wherein linking moiety joining the B and A chains comprises an amino acid sequence of no more than 17 amino acids in length and comprising the sequence

GYGSSSX₅₇X₅₈ (SEQ ID NO: 21), GAGSSSRR (SEQ ID NO: 22) or

G YGS S S X57X58 APQT ; (SEQ ID NO: 69) wherein X₅₇ and X₅₈ are independently arginine, lysine or ornithine and the amino acid designated by Xs₇ or Xs₈ optionally further comprises a hydrophilic moiety linked to the side chain of the amino acid at that position. In one embodiment the hydrophilic moiety is a polyethylene glycol chain.

Acylation or alkylation can increase the half-life of the NHR ligand-insulin conjugate in circulation. Acylation or alkylation can advantageously delay the onset of action and/or extend the duration of action at the insulin receptors. The insulin peptide of the conjugates may be acylated or alkylated at the same amino acid position where a hydrophilic moiety is linked (including, for example at position 8 of the linking moiety), or at a different amino acid position.

Also encompassed by the present disclosure are pharmaceutical compositions comprising the NHR ligand-insulin conjugates and a pharmaceutically acceptable carrier. In accordance with one embodiment a pharmaceutical composition is provided comprising any of the NHR ligand-insulin conjugates disclosed herein preferably at a purity level of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and a pharmaceutically acceptable diluent, carrier or excipient. Such compositions may contain a conjugate as disclosed herein at a concentration of at least 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, 20 mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml or higher. In one embodiment the pharmaceutical compositions comprise aqueous solutions that are sterilized and optionally stored within various package containers. In other embodiments the pharmaceutical compositions comprise a lyophilized powder. The pharmaceutical compositions can be further packaged as part of a kit that includes a disposable device for administering the composition to a patient. The containers or kits may be labeled for storage at ambient room temperature or at refrigerated temperature.

In accordance with one embodiment an improved method of regulating blood glucose levels in insulin dependent patients is provided. The method comprises the steps of administering to a patient a NHR ligand-insulin conjugate as disclosed herein in an amount therapeutically effective for the control of diabetes. In accordance with one embodiment a method of reducing weight or preventing weight gain is provided wherein the method comprises administering a NHR ligand-insulin conjugate as disclosed herein to a patient in need of such therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. is a schematic overview of the two step synthetic strategy for preparing human insulin. Details of the procedure are provided in Example 1.

Fig. 2 is a graph comparing insulin receptor specific binding of synthetic human insulin relative to purified native insulin. The synthetic insulin was produced by the approach detailed in Fig. 1 where the A 7 -B 7 bond is the first disulfide formed. As indicated by the data presented in the graph, the two molecules have similar binding activities. Fig. 3 shows the structure of an insulin-dexamethasone and two insulin-thyroxine (T3) conjugates, wherein native insulin is modified by linkage of the dexamethasone/T3 to the side chain of the native Lys at position B29. Also provided is the activity of the three conjugates and native insulin at the insulin IR-B receptor using the in vitro assay of Example 3.

Fig. 4 is a graph presenting data for an in vivo study on blood glucose levels in mice administered the insulin-nuclear hormone ligand conjugates CIU-15, CIU-16 and CIU-17 relative to control and native insulin.

Fig. 5 shows the structure of an insulin-Tesaglitazar conjugate, wherein the native insulin heteroduplex (comprising the insulin A chain of SEQ ID NO: 1 and the B chain of SEQ ID NO: 2) is linked to Tesaglitazar via a Benzyl-PEG2-GGG-K linker.

Fig. 6 shows the structure of an insulin- Aleglitazar conjugate, wherein the native insulin heteroduplex (comprising the insulin A chain of SEQ ID NO: 1 and the B chain of SEQ ID NO: 2) is linked to Tesaglitazar via an NHCO linker to the B29 Lys side chain (CIU-164), to the A chain N-terminal amine and the B29 Lys side chain (CIU-165) or the B29 Lys side chain of a B l pegylated insulin analog (CIU-166).

Figs. 7A-7D shows the activity of an insulin- Aleglitazar conjugate (CIU-166) and an insulin-T3 conjugate (CIU-167), wherein the insulin component is native insulin (comprising the insulin A chain of SEQ ID NO: 1 and the B chain of SEQ ID NO: 2) pegylated with a 20K peg at the B l position. Fig. 7A shows the activity of CIU-166 and CIU-167 the insulin IR-B receptor using an in vitro assay. Fig. 7B is a graph presenting data for an in vivo study on blood glucose levels in mice administered CIU-166 and CIU-167. Fig. 7C is a graph presenting data for an in vivo study on body weight in mice administered CIU-166 and CIU- 167; Fig. 7D is a graph presenting data for glucose tolerance test in mice administered CIU- 166 and CIU-167.

DETAILED DESCRIPTION DEFINITIONS

In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.

The term "about" as used herein means greater or lesser than the value or range of values stated by 10 percent, but is not intended to designate any value or range of values to only this broader definition. Each value or range of values preceded by the term "about" is also intended to encompass the embodiment of the stated absolute value or range of values.

As used herein the term "amino acid" encompasses any molecule containing both amino and carboxyl functional groups, wherein the amino and carboxylate groups are attached to the same carbon (the alpha carbon). The alpha carbon optionally may have one or two further organic substituents. For the purposes of the present disclosure designation of an amino acid without specifying its stereochemistry is intended to encompass either the L or D form of the amino acid, or a racemic mixture.

As used herein the term "hydroxyl acid" refers to amino acids that have been modified to replace the alpha carbon amino group with a hydroxyl group.

As used herein the term "non-coded amino acid" encompasses any amino acid that is not an L-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, Tyr.

As used herein a general reference to a peptide is intended to encompass peptides that have modified amino and carboxy termini. For example, an amino acid sequence designating the standard amino acids is intended to encompass standard amino acids at the N- and C- terminus as well as a corresponding hydroxyl acid at the N-terminus and/or a corresponding C-terminal amino acid modified to comprise an amide group in place of the terminal carboxylic acid.

As used herein an "acylated" amino acid is an amino acid comprising an acyl group which is non-native to a naturally-occurring amino acid, regardless by the means by which it is produced. Exemplary methods of producing acylated amino acids and acylated peptides are known in the art and include acylating an amino acid before inclusion in the peptide or peptide synthesis followed by chemical acylation of the peptide. In some embodiments, the acyl group causes the peptide to have one or more of (i) a prolonged half-life in circulation, (ii) a delayed onset of action, (iii) an extended duration of action, (iv) an improved resistance to proteases, such as DPP-IV, and (v) increased potency at the IGF and/or insulin peptide receptors.

As used herein, an "alkylated" amino acid is an amino acid comprising an alkyl group which is non-native to a naturally-occurring amino acid, regardless of the means by which it is produced. Exemplary methods of producing alkylated amino acids and alkylated peptides are known in the art and including alkylating an amino acid before inclusion in the peptide or peptide synthesis followed by chemical alkylation of the peptide. Without being held to any particular theory, it is believed that alkylation of peptides will achieve similar, if not the same, effects as acylation of the peptides, e.g., a prolonged half-life in circulation, a delayed onset of action, an extended duration of action, an improved resistance to proteases, such as DPP-IV, and increased potency at the IGF and/or insulin receptors.

As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.

As used herein the term "pharmaceutically acceptable salt" refers to salts of compounds that retain the biological activity of the parent compound, and which are not biologically or otherwise undesirable. Many of the compounds disclosed herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.

As used herein, the term "hydrophilic moiety" refers to any compound that is readily water-soluble or readily absorbs water, and which are tolerated in vivo by mammalian species without toxic effects (i.e. are biocompatible). Examples of hydrophilic moieties include polyethylene glycol (PEG), polylactic acid, polyglycolic acid, a polylactic- polyglycolic acid copolymer, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxyethyl methacrylate, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatised celluloses such as hydroxymethylcellulose or hydroxyethylcellulose and co-polymers thereof, as well as natural polymers including, for example, albumin, heparin and dextran.

As used herein, the term "treating" includes prophylaxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms. For example, as used herein the term "treating diabetes" will refer in general to maintaining glucose blood levels near normal levels and may include increasing or decreasing blood glucose levels depending on a given situation.

As used herein an "effective" amount or a "therapeutically effective amount" of an insulin analog refers to a nontoxic but sufficient amount of an insulin analog to provide the desired effect. For example one desired effect would be the prevention or treatment of hyperglycemia. The amount that is "effective" will vary from subject to subject, depending on the age and general condition of the individual, mode of administration, and the like. Thus, it is not always possible to specify an exact "effective amount." However, an appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

The term, "parenteral" means not through the alimentary canal but by some other route such as intranasal, inhalation, subcutaneous, intramuscular, intraspinal, or intravenous.

Throughout the application, all references to a particular amino acid position in an insulin analog by letter and number (e.g. position A5) refer to the amino acid at that position of either the A chain (e.g. position A5) or the B chain (e.g. position B5) in the respective native human insulin A chain (SEQ ID NO: 1) or B chain (SEQ ID NO: 2), or the corresponding amino acid position in any analogs thereof. For example, a reference herein to "position B28" absent any further elaboration would mean the corresponding position B27 of the B chain of an insulin analog in which the first amino acid of SEQ ID NO: 2 has been deleted. Similarly, amino acids added to the N-terminus of the native B chain are numbered starting with BO, followed by numbers of increasing negative value (e.g., B-l, B-2...) as amino acids are added to the N-terminus. Alternatively, any reference to an amino acid position in the linking moiety of a single chain analog, is made in reference to the native C chain of IGF 1 (SEQ ID NO: 17). For example, position 9 of the native C chain (or the "position C9") has an alanine residue.

As used herein the term "native insulin peptide" is intended to designate the 51 amino acid heteroduplex comprising the A chain of SEQ ID NO: 1 and the B chain of SEQ ID NO: 2, as well as single-chain insulin analogs that comprise SEQ ID NOS: 1 and 2. The term "insulin peptide" as used herein, absent further descriptive language is intended to encompass the 51 amino acid heteroduplex comprising the A chain of SEQ ID NO: 1 and the B chain of SEQ ID NO: 2, as well as single-chain insulin analogs thereof (including for example those disclosed in published international application W096/34882 and US Patent No. 6,630,348, the disclosures of which are incorporated herein by reference), including heteroduplexes and single-chain analogs that comprise modified analogs of the native A chain and/or B chain and derivatives thereof. Such modified analogs include modification of the amino acid at position A 19, B 16 or B25 to a 4-amino phenylalanine or one or more amino acid substitutions at positions selected from A5, A8, A9, A10, A12, A14, A15, A17, A18, A21, B l, B2, B3, B4, B5, B9, B 10, B 13, B 14, B 17, B20, B21, B22, B23, B26, B27, B28, B29 and B30 or deletions of any or all of positions B l-4 and B26-30. Insulin peptides as defined herein can also be analogs derived from a naturally occurring insulin by insertion or substitution of a non-peptide moiety, e.g. a retroinverso fragment, or incorporation of non- peptide bonds such as an azapeptide bond (CO substituted by NH) or pseudo-peptide bond (e.g. NH substituted with C¾) or an ester bond (e.g., a depsipeptide, wherein one or more of the amide (-CONHR-) bonds are replaced by ester (COOR) bonds).

As used herein, the term "single-chain insulin analog" encompasses a group of structurally-related proteins wherein insulin or IGF A and B chains, or analogs or derivatives thereof, are covalently linked to one another to form a linear polypeptide chain. As disclosed herein the single-chain insulin analog comprises the covalent linkage of the carboxy terminus of the B chain to the amino terminus of the A chain via a linking moiety.

As used herein the term "insulin A chain", absent further descriptive language is intended to encompass the 21 amino acid sequence of SEQ ID NO: 1 as well as functional analogs and derivatives thereof, including insulin analogs known to those skilled in the art, including modification of the sequence of SEQ ID NO: 1 by one or more amino acid insertions, deletions or substitutions at positions selected from A4, A5, A8, A9, A10, A12, A14, A15, A17, A18, A21.

As used herein the term "insulin B chain", absent further descriptive language is intended to encompass the 30 amino acid sequence of SEQ ID NO: 2, as well as modified functional analogs of the native B chain, including one or more amino acid insertions, deletions or substitutions at positions selected from B l, B2, B3, B4, B5, B9, B 10, B 13, B 14, B 17, B20, B21, B22, B23, B25, B26, B27, B28, B29 and B30 or deletions of any or all of positions B l-4 and B26-30.

The term "identity" as used herein relates to the similarity between two or more sequences. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100 to achieve a percentage. Thus, two copies of exactly the same sequence have 100% identity, whereas two sequences that have amino acid deletions, additions, or substitutions relative to one another have a lower degree of identity. Those skilled in the art will recognize that several computer programs, such as those that employ algorithms such as BLAST (Basic Local Alignment Search Tool, Altschul et al. (1993) J. Mol. Biol. 215:403-410) are available for determining sequence identity.

As used herein, the term "selectivity" of a molecule for a first receptor relative to a second receptor refers to the following ratio: EC₅₀ of the molecule at the second receptor divided by the EC₅₀ of the molecule at the first receptor. For example, a molecule that has an EC₅₀ of 1 nM at a first receptor and an EC₅₀ of 100 nM at a second receptor has 100-fold selectivity for the first receptor relative to the second receptor.

As used herein an amino acid "modification" refers to a substitution of an amino acid, or the derivation of an amino acid by the addition and/or removal of chemical groups to/from the amino acid, and includes substitution with any of the 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids.

Commercial sources of atypical amino acids include Sigma-Aldrich (Milwaukee, WI), ChemPep Inc. (Miami, FL), and Genzyme Pharmaceuticals (Cambridge, MA). Atypical amino acids may be purchased from commercial suppliers, synthesized de novo, or chemically modified or derivatized from naturally occurring amino acids.

As used herein an amino acid "substitution" refers to the replacement of one amino acid residue by a different amino acid residue.

As used herein, the term "conservative amino acid substitution" is defined herein as exchanges within one of the following five groups: I. Small aliphatic, nonpolar or slightly polar residues:

Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides:

Asp, Asn, Glu, Gin, cysteic acid and homocysteic acid;

III. Polar, positively charged residues:

His, Arg, Lys; Ornithine (Orn)

IV. Large, aliphatic, nonpolar residues:

Met, Leu, He, Val, Cys, Norleucine (Nle), homocysteine

V. Large, aromatic residues:

Phe, Tyr, Trp, acetyl phenylalanine

As used herein the general term "polyethylene glycol chain" or "PEG chain", refers to mixtures of condensation polymers of ethylene oxide and water, in a branched or straight chain, represented by the general formula H(OCH₂CH₂)_nOH, wherein n is at least 2.

"Polyethylene glycol chain" or "PEG chain" is used in combination with a numeric suffix to indicate the approximate average molecular weight thereof. For example, PEG-5,000 refers to polyethylene glycol chain having a total molecular weight average of about 5,000 Daltons As used herein the term "pegylated" and like terms refers to a compound that has been modified from its native state by linking a polyethylene glycol chain to the compound. A "pegylated polypeptide" is a polypeptide that has a PEG chain covalently bound to the polypeptide.

As used herein a "linker" is a bond, molecule or group of molecules that binds two separate entities to one another. Linkers may provide for optimal spacing of the two entities or may further supply a labile linkage that allows the two entities to be separated from each other. Labile linkages include photocleavable groups, acid-labile moieties, base-labile moieties and enzyme-cleavable groups.

As used herein a "dimer" is a complex comprising two subunits covalently bound to one another via a linker. The term dimer, when used absent any qualifying language, encompasses both homodimers and heterodimers. A homodimer comprises two identical subunits, whereas a heterodimer comprises two subunits that differ, although the two subunits are substantially similar to one another.

The term "Ci-C_n alkyl" wherein n can be from 1 through 6, as used herein, represents a branched or linear alkyl group having from one to the specified number of carbon atoms. Typical Ci-C₆ alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, iso- propyl, butyl, iso-Butyl, sec -butyl, tert-butyl, pentyl, hexyl and the like.

The terms "C₂-C_n alkenyl" wherein n can be from 2 through 6, as used herein, represents an olefinically unsaturated branched or linear group having from 2 to the specified number of carbon atoms and at least one double bond. Examples of such groups include, but are not limited to, 1-propenyl, 2-propenyl (-CH₂-CH=CH₂), 1,3-butadienyl, (- CH=CHCH=CH₂), 1-butenyl (-CH=CHCH₂CH₃), hexenyl, pentenyl, and the like.

The term "C₂-C_n alkynyl" wherein n can be from 2 to 6, refers to an unsaturated branched or linear group having from 2 to n carbon atoms and at least one triple bond.

Examples of such groups include, but are not limited to, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, and the like.

As used herein the term "aryl" refers to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl,

tetrahydronaphthyl, indanyl, indenyl, and the like. The size of the aryl ring and the presence of substituents or linking groups are indicated by designating the number of carbons present. For example, the term "(Ci-C₃ alkyl)(C6-Cio aryl)" refers to a 5 to 10 membered aryl that is attached to a parent moiety via a one to three membered alkyl chain. The term "heteroaryl" as used herein refers to a mono- or bi- cyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring. The size of the heteroaryl ring and the presence of substituents or linking groups are indicated by designating the number of carbons present. For example, the term "(Ci-C_n alkyl)(Cs-C6 heteroaryl)" refers to a 5 or 6 membered heteroaryl that is attached to a parent moiety via a one to "n" membered alkyl chain.

As used herein, the term "halo" refers to one or more members of the group consisting of fluorine, chlorine, bromine, and iodine.

As used herein the term "patient" without further designation is intended to encompass any warm blooded vertebrate domesticated animal (including for example, but not limited to livestock, horses, cats, dogs and other pets) and humans.

The term "isolated" as used herein means having been removed from its natural environment. In some embodiments, the analog is made through recombinant methods and the analog is isolated from the host cell.

The term "purified," as used herein relates to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment and means having been increased in purity as a result of being separated from other components of the original composition. The term "purified polypeptide" is used herein to describe a polypeptide which has been separated from other compounds including, but not limited to nucleic acid molecules, lipids and carbohydrates.

A "peptidomimetic" refers to a chemical compound having a structure that is different from the general structure of an existing peptide, but that functions in a manner similar to the existing peptide, e.g., by mimicking the biological activity of that peptide. Peptidomimetics typically comprise naturally-occurring amino acids and/or unnatural amino acids, but can also comprise modifications to the peptide backbone. For example a peptidomimetic may include a sequence of naturally-occurring amino acids with the insertion or substitution of a non-peptide moiety, e.g. a retroinverso fragment, or

incorporation of non-peptide bonds such as an azapeptide bond (CO substituted by NH) or pseudo-peptide bond (e.g. NH substituted with CH2), or an ester bond (e.g., depsipeptides, wherein one or more of the amide (-CONHR-) bonds are replaced by ester (COOR) bonds). Alternatively the peptidomimetic may be devoid of any naturally-occurring amino acids. ABBREVIATIONS:

Insulin analogs will be abbreviated as follows:

The insulin A and B chains will be designated generically by a capital A for the A chain and a capital B for the B chain. When present, a superscript 0 (e.g., A⁰ or B°) will designate the base sequence is an insulin sequence (A chain: SEQ ID NO: 1, B chain SEQ ID NO: 2) and a superscript 1 (e.g., A¹ or B¹) will designate the base sequence is an IGF-1 sequence (A chain: SEQ ID NO: 5, B chain SEQ ID NO: 6). Modifications that deviate from the native insulin and IGF sequence are indicated in parenthesis following the designation of the A or B chain (e.g., [Β^Ή,ΗΙΟ,ΥΙό,ίΠ) : A¹(H8,N18,N21)]) with the single letter amino acid abbreviation indicating the substitution and the number indicating the position of the substitution in the respective A or B chain, using native insulin numbering. A colon between the A and B chain indicates a two chain insulin whereas a dash will indicate a covalent bond and thus a single chain analog. In single chain analogs a linking moiety will be included between the A and B chains and the designation C¹ refers to the native IGF 1 C peptide, SEQ ID NO: 17. The designation "position C8" in reference to the linking moiety designates an amino acid located at the position corresponding to the eighth amino acid of SEQ ID NO: 17.

EMBODIMENTS

Disclosed herein are conjugates of an insulin peptide and a NHR ligand. In one embodiment the NHR ligand is an NHR agonist. In one embodiment the insulin

agonist/NHR ligand conjugate comprises the structure Q-L-Y, wherein Q is an insulin peptide, Y is a NHR ligand and L is a linking group or a bond, wherein said conjugate has activity at both the corresponding nuclear hormone receptor of Y and an insulin receptor, including for example the insulin subtype B receptor. In one embodiment the NHR ligand is selected from the group consisting of a steroid that exhibits an EC₅₀ of about 1 μΜ or less when unconjugated to Q-L, and has a molecular weight of up to about 1000 daltons. In one embodiment the NHR ligand is a ligand that activates the thyroid hormone receptor or a ligand that activates the peroxisome proliferator- activated receptors (PPAR).

In some embodiments the NHR ligand is an NHR agonist. In one embodiment the

NHR agonist has activity at a Type I NHR when bound to Q-L. In one embodiment the NHR agonist has activity at a Type II NHR when bound to Q-L. In one embodiment the NHR ligand is

i) a steroid that exhibits an EC₅₀ of about 1 μΜ or less when unconjugated to Q-L, and further has a molecular weight of up to about 1000 daltons; or

ii) a ligand that activates the thyroid hormone receptor; or iii) a ligand that activates the peroxisome proliferator-activated receptors (PPAR).

In one embodiment the NHR ligand of the conjugate is selected from the group consisting of estradiol and derivatives thereof, estrone and derivatives thereof, testosterone and derivatives thereof, and Cortisol and derivatives thereof. In one embodiment the NHR ligand is dexamethasone.

In accordance with one embodiment the NHR ligand is a thyroid hormone receptor agonist having the general structure

wherein

Ri5 is C1-C4 alkyl, -CH₂(pyridazinone), -CH₂(OH)(phenyl)F, -CH(OH)CH₃, halo or H; R₂o is halo, CH₃ or H;

R₂i is halo, CH₃ or H;

R₂₂ is H, OH, halo, -CH₂(OH)(C₆ aryl)F, or d-C₄ alkyl; and

R₂₃ is -CH₂CH(NH₂)COOH, -OCH₂COOH, -NHC(0)COOH, -CH₂COOH,

-NHC(0)CH₂COOH, -CH₂CH₂COOH, or -OCH₂P0₃ ^2". In one embodiment the thyroid hormone receptor agonist has the general structure of Formula I:

HO

R20, R21, and R22 are independently selected from the group consisting of H, OH, halo and Ci-C₄ alkyl; and

Ri5 is halo or H. In one embodiment the thyroid hormone receptor agonist is selected from the group consisting of thyroxine T4 (3,5,3',5'-tetra-iodothyronine), and 3,5,3'-triiodo L- thyronine.

In one embodiment the NHR ligand is an agonist of a PPAR. In one embodiment the PPAR agonist is selected from the group consisting of Tesaglitazar, Aleglitazar and thiazolidinediones. In one embodiment the PPAR agonist is Tesaglitazar or Aleglitazar.

In one embodiment the insulin peptide of the conjugate Q-L-Y is a native insulin peptide or any insulin receptor agonist known to those skilled in the art. In one embodiment the insulin peptide (Q) comprises an A chain and a B chain wherein said A chain comprises a sequence

GIVX₄X5CCX8X₉XioCXi2LXi₄Xi5LXi7Xi8YCX2i-R53 (SEQ ID NO: 19), and said B chain comprises a sequence R₆2-X₂₅LCGX₂9X₃oLVX₃₃X₃₄LYLVCGX₄iX₄₂GFX₄5 (SEQ ID NO: 20), wherein

X₄ is glutamic acid or aspartic acid;

X5 is glutamine or glutamic acid

X₈ is histidine, threonine or phenylalanine;

X9 is serine, arginine, lysine, ornithine or alanine;

X10 is isoleucine or serine;

X12 is serine or aspartic acid;

Xi₄ is tyrosine, arginine, lysine, ornithine or alanine;

Xi5 is glutamine, glutamic acid, arginine, alanine, lysine, ornithine or leucine;

Xi7 is glutamic acid, aspartic acid, asparagine, lysine, ornithine or glutamine;

Xi₈ is methionine, asparagine, glutamine, aspartic acid, glutamic acid or threonine;

X21 is selected from the group consisting of alanine, glycine, serine, valine, threonine, isoleucine, leucine, glutamine, glutamic acid, asparagine, aspartic acid, histidine, tryptophan, tyrosine, and methionine;

X25 is histidine or threonine;

X29 is selected from the group consisting of alanine, glycine and serine;

X30 is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;

X33 is selected from the group consisting of aspartic acid and glutamic acid; X₃₄ is selected from the group consisting of alanine and threonine;

X₄i is selected from the group consisting of glutamic acid, aspartic acid or asparagine;

X₄2 is selected from the group consisting of alanine, ornithine, lysine and arginine; X₄5 is tyrosine or phenylalanine;

R₆₂ is selected from the group consisting of AYRPSE (SEQ ID NO: 14), FVNQ (SEQ ID NO: 12), PGPE (SEQ ID NO: 11), a tripeptide glycine-proline-glutamic acid, a tripeptide valine-asparagine-glutamine, a dipeptide proline-glutamic acid, a dipeptide asparagine-glutamine, glutamine, glutamic acid and an N-terminal amine; and

R5₃ is COOH or CONH₂. In one embodiment the insulin peptide is a two chain insulin analog. In another embodiment the insulin peptide is a single chain insulin analog wherein the carboxy terminus of the B chain is linked to the amino terminus of the A chain via a peptide linker. Any of the previous disclosed single chain insulin analogs having activity at the insulin receptor and known to those skilled in the art are encompassed by the present disclosure.

In one embodiment the insulin peptide of the conjugate is a two chain insulin wherein the A and B chains are linked by interchain disulfide bonds, wherein the A chain comprises the sequence GIVEQCCX₈X₉ICSLYQLENYCX₂i-R5₃ (SEQ ID NO: 73) and the B chain comprises a sequence R₆₂-X₂₅LCGAX₃oLVDALYLVCGDX₄₂GFY (SEQ ID NO: 75), wherein

X₈ is histidine or threonine;

X9 is serine, lysine, or alanine;

X21 is alanine, glycine or asparagine;

X25 is histidine or threonine;

X₃o is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;

X₄₂ is selected from the group consisting of alanine, ornithine and arginine; and Rs₃ is COOH or CONH₂;

R₆₂ is selected from the group consisting of FVNQ (SEQ ID NO: 12), a tripeptide valine-asparagine-glutamine, a dipeptide asparagine-glutamine, glutamine, and an N- terminal amine; and

R5₃ is COOH or CONH₂. In one embodiment the A chain comprises the sequence GIYEQCCX₈X₉ICSLYQLENYCX₂i-R5₃ (SEQ ID NO: 73) and the B chain comprises the B chain sequence comprises the sequence FVKQX₂₅LCGSHLVEALYLVCGERGFF-R₆₃ (SEQ ID NO: 147), or FVNQX₂₅LCGSHLVEALYLVCGERGFF-R₆₃ (SEQ ID NO: 148), wherein

X₈ is histidine or threonine;

X9 is serine, lysine, or alanine;

X₂₁ is alanine, glycine or asparagine;

X₂₅ is selected from the group consisting of histidine and threonine; and

R₆ is selected from the group consisting of YTX₂₈KT (SEQ ID NO: 149), YTKPT (SEQ ID NO: 150), YTX₂₈K (SEQ ID NO: 152), YTKP (SEQ ID NO: 151), YTPK (SEQ ID NO: 70), YTX₂₈, YT, Y and a bond.

In one embodiment the A chain comprises the sequence

GIVEQCCX₈SICSLYQLENYCX2i-R5₃ (SEQ ID NO: 153) or

GIVEQCCTSICSLYQLENYCN-R₅₃ (SEQ ID NO: 1) and the B chain comprises the sequence FVKQX₂₅LCGSHLVEALYLVCGERGFFYTEKT (SEQ ID NO: 154),

FVNQX₂₅LCGSHLVEALYLVCGERGFFYTDKT (SEQ ID NO: 155),

FVNQX₂₅LCGS HLVE ALYLVC GERGFF YTKPT (SEQ ID NO: 156) or

FVNQX₂₅LCGS HLVE ALYLVC GERGFF YTPKT (SEQ ID NO: 157) wherein

X₈ is histidine or threonine;

X₂₁ is alanine, glycine or asparagine; X₂₅ is selected from the group consisting of histidine and threonine and Rs₃ is COOH or CONH₂. In one embodiment the A chain comprises a sequence GIVEQCCTSICSLYQLENYCN-R₅₃ (SEQ ID NO: 1) and said B chain comprises a sequence F VNQHLC GS HLVE ALYLVCGERGFFYTPKT (SEQ ID NO: 2) wherein R₅₃ is COOH or CONH₂.

In one embodiment the insulin peptide is a single chain insulin analog. In one embodiment the peptide linker joining the B and A chains is selected from the group consisting of SSSSKAPPPSLPSPSRLPGPSDTPILPQR (SEQ ID NO: 158),

SSSSRAPPPSLPSPSRLPGPSDTPILPQK (SEQ ID NO: 159), GAGSSSX₅₇X₅₈ (SEQ ID NO: 76), GYGSSSX₅₇X₅₈ (SEQ ID NO: 21) and GYGSSSX₅₇X₅₈APQT; (SEQ ID NO: 77), wherein X₅₇ and X₅₈ are independently arginine, lysine or ornithine. In one embodiment both X₅₇ and X₅₈ are independently arginines. In one embodiment the peptide linking moiety joining the insulin A and B chains to form a single chain insulin analog is a peptide sequence consisting of GYGSSSRR (SEQ ID NO: 18) GAGSSSRR (SEQ ID NO: 22) or

GAGS S S RR APQT (SEQ ID NO: 23). In accordance with one embodiment, the linker (L in the formula Q-L-Y) is a linking group or a bond that covalently links the insulin peptide to the NHR ligand. In one embodiment the NHR ligand is linked to the side chain of an amino acid at position B28 or B29 of the insulin peptide. In one embodiment the amino acid at position B28 or B29 of the insulin peptide is lysine and the NHR ligand is linked to the side chain of the lysine. In one embodiment the NHR ligand is linked to the insulin peptide via the N-terminal alpha amine of the insulin A or B chain. In one embodiment the NHR ligand is linked to the insulin peptide via an amid bond form between and amino group of the insulin peptide and a carboxy group of the NHR ligand, optionally through a spacer moiety.

In one embodiment the linker (L in the formula Q-L-Y) is a linking group wherein L is stable in vivo, hydrolyzable in vivo, or metastable in vivo. In one embodiment L comprises an ether moiety, or an amide moiety, an ester moiety, an acid-labile moiety, a reduction-labile moiety , an enzyme-labile moiety, a hydrazone moiety, a disulfide moiety, or a cathepsin-cleavable moiety.

Structure of the NHR Ligand (Y)

The NHR ligand of the invention (Y) is partly or wholly non-peptidic and is hydrophobic or lipophilic. In some embodiments, the NHR ligand has a molecular weight that is about 5000 daltons or less, or about 4000 daltons or less, or about 3000 daltons or less, or about 2000 daltons or less, or about 1750 daltons or less, or about 1500 daltons or less, or about 1250 daltons or less, or about 1000 daltons or less, or about 750 daltons or less, or about 500 daltons or less, or about 250 daltons or less. The structure of Y can be in accordance with any of the teachings disclosed herein.

In the embodiments described herein, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Y that is capable of reacting with Q or

L. One skilled in the art could readily determine the position and means of conjugation in view of general knowledge and the disclosure provided herein.

In any of the embodiments described herein wherein Y comprises a tetracyclic skeleton having three 6-membered rings joined to one 5-membered ring or a variation thereof (e.g. a Y that acts at the vitamin D receptor), the carbon atoms of the skeleton are referred to by position number, as shown below:

For example, a modification having a ketone at position-6 refers to the following structure:

NHR Ligand that Acts on a Type I Nuclear Hormone Receptor

In some embodiments of the invention, the NHR ligand (Y) acts on a Type I nuclear hormone receptor. In some embodiments, Y can have any structure that permits or promotes agonist activity upon binding of the ligand to a Type I nuclear hormone receptor, while in other embodiments Y is an antagonist of the Type I nuclear hormone receptor.

In exemplary embodiments, Y comprises a structure as shown in Formula A:

Formula A

wherein R 1 and R 2 , when present, are independently moieties that permit or promote agonist or antagonist activity upon binding of the compound of Formula A to the Type I nuclear hormone receptor; R³ and R⁴ are independently moieties that permit or promote agonist or antagonist activity upon binding of the compound of Formula A to the Type I nuclear hormone receptor; and each dashed line represents an optional double bond. Formula A may further comprise one or more substituents at one or more of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, 15, 16, 17, 18, and 19. Contemplated optional substituents include, but are not limited to, OH, N¾, ketone, and Q-Cis alkyl groups.

In some embodiments, Y comprises a structure of Formula A wherein R¹ is present and is hydrogen, d-C₇ alkyl; (C₀_C₃ alkyl)C(0)Ci-C₇ alkyl, (C₀_C₃ alkyl)C(0)aryl, or S0₃H;

R is present and is hydrogen, halo, OH, or Ci-C₇ alkyl;

R is hydrogen, halo, OH, or Ci-C₇ alkyl;

R⁴ is hydrogen, (Co-C₈ alkyl)halo, Ci_C₈ alkyl, C₂-C₈ alkenyl, C₂-i₈ alkynyl, heteroalkyl, (C₀_C₈ alkyl)aryl, (C₀_C₈ alkyl)heteroaryl, (C₀_C₈ alkyl)OCi_C₈ alkyl, (C₀_C₈ alkyl)OC₂-C₈ alkenyl, (C₀-C₈ alkyl)OC₂-C₈ alkynyl, (C₀-C₈ alkyl)OH, (C₀-C₈ alkyl)SH, (C₀- C₈ alkyl)NR²⁴Ci_C₈ alkyl, (C₀-C₈ alkyl)NR²⁴C₂-C₈ alkenyl, (C₀-C₈ alkyl)NR²⁴C₂-C₈ alkynyl, (Co-C₈ alkyl)NR²⁴H₂, (C₀_C₈ alkyl)C(0)Ci_C₈ alkyl, (C₀_C₈ alkyl)C(0)C₂_C₈ alkenyl, (C₀_C₈ alkyl)C(0)C₂_C₈ alkynyl, (C₀_C₈ alkyl)C(0)H, (C₀_C₈ alkyl)C(0)aryl, (C₀_C₈

alkyl)C(0)heteroaryl, (C₀-C₈ alkyl)C(0)OCi_C₈ alkyl, (C₀-C₈ alkyl)C(0)OC₂-C₈ alkenyl, (C₀- C₈ alkyl)C(0)OC₂-C₈ alkynyl, (C₀-C₈ alkyl)C(0)OH, (C₀-C₈ alkyl)C(0)0 aryl, (C₀-C₈ alkyl)C(0)0 heteroaryl, (C₀_C₈ alkyl)OC(0)Ci_C₈ alkyl, (C₀_C₈ alkyl)OC(0)C₂_C₈ alkenyl, (Co-C₈ alkyl)OC(0)C₂_Ci₈ alkynyl, (C₀_C₈ alkyl)C(0)NR²⁴Ci_C₈ alkyl, (C₀_C₈

alkyl)C(0)NR²⁴C₂-C₈ alkenyl, (C₀-C₈ alkyl)C(0)NR²⁴C₂-C₈ alkynyl, (C₀-C₈

alkyl)C(0)NR²⁴H₂, (C₀-C₈ alkyl)C(0)NR²⁴aryl, (C₀-C₈ alkyl)C(0)NR²⁴heteroaryl, (C₀-C₈ alkyl)NR²⁴C(0)Ci_C₈ alkyl, (C₀_C₈ alkyl)NR²⁴C(0)C₂_C₈ alkenyl, or (C₀_C₈

alkyl)NR²⁴C(0)C₂_C₈ alkynyl, (C₀_C₈ alkyl)NR²⁴C(0)OH, (C₀_C₈ alkyl)OC(0)OCi_C₈ alkyl, (Co-C₈ alkyl)OC(0)OC₂-C₈ alkenyl, (C₀-C₈ alkyl)OC(0)OC₂-C₈ alkynyl, (C₀-C₈

alkyl)OC(0)OH, (C₀-C₈ alkyl)OC(0)NR²⁴Ci_C₈ alkyl, (C₀-C₈ alkyl)OC(0)NR²⁴C₂-C₈ alkenyl, (C₀_C₈ alkyl)OC(0)NR²⁴C₂_C₈ alkynyl, (C₀_C₈ alkyl)OC(0)NR²⁴H₂, (C₀_C₈ alkyl)NR²⁴(0)OCi_C₈ alkyl, (C₀_C₈ alkyl)NR²⁴(0)OC₂_C₈ alkenyl, (C₀_C₈ alkyl)NR²⁴(0)OC₂_ C₈ alkynyl, or (C₀-C₈ alkyl)NR²⁴(0)OH; and,

R 24 is hydrogen or Ci_C₇ alkyl.

In some embodiments, R 1 is hydrogen, propionate, acetate, benzoate, or sulfate; R 2 is hydrogen or methyl; R³ is hydrogen or methyl; and R⁴ is acetate, cypionate, hemisucciniate, enanthate, or propionate.

In embodiments wherein Y comprises a structure of Formula A, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Formula A that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Formula A and means of conjugation of Formula A to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Formula A is conjugated to L or Q at any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of Formula A. In some embodiments, Formula A is conjugated to L or Q at position 1, 3, 6, 7, 12, 10, 13, 16, 17, or 19 of Formula A.

In some embodiments, Y acts at an estrogen receptor (e.g. ERa, ERP). In some embodiments, Y permits or promotes agonist activity at the estrogen receptor, while in other embodiments Y is an antagonist of ER. In exemplary embodiments, Y can have a structure of Formula B:

Formula B

wherein R¹, R⁵ and R⁶ are moieties that permit or promote agonist or antagonist activity upon binding of the compound of Formula B to the estrogen receptor. In some

embodiments, Formula B further comprises one or more substitutents at one or more of positions 1, 2, 4, 6, 7, 8, 9, 11, 12, 14, 15, and 16 (e.g. a ketone at position-6).

In some embodiments when Y comprises a structure of Formula B, wherein

R¹ is hydrogen, C1-C7 alkyl; (C₀-C₃ alkyl)C(0)Ci-C₇ alkyl, (C₀-C₃ alkyl)C(0)aryl, or S0₃H;

R⁵ is hydrogen, (Co-C₈ alkyl)halo, Ci_C₈ alkyl, C₂-C₈ alkenyl, C₂-i₈ alkynyl, heteroalkyl, (C₀-C₈ alkyl)aryl, (C₀-C₈ alkyl)heteroaryl, (C₀-C₈ alkyl)OCi_C₈ alkyl, (C₀-C₈ alkyl)OC₂-C₈ alkenyl, (C₀-C₈ alkyl)OC₂-C₈ alkynyl, (C₀-C₈ alkyl)OH, (C₀-C₈ alkyl)SH, (C₀- C₈ alkyl)NR²⁴Ci_C₈ alkyl, (C₀_C₈ alkyl)NR²⁴C₂_C₈ alkenyl, (C₀_C₈ alkyl)NR²⁴C₂_C₈ alkynyl, (Co-C₈ alkyl)NR²⁴H₂, (C₀_C₈ alkyl)C(0)Ci_C₈ alkyl, (C₀_C₈ alkyl)C(0)C₂_C₈ alkenyl, (C₀_C₈ alkyl)C(0)C₂-C₈ alkynyl, (C₀-C₈ alkyl)C(0)H, (C₀-C₈ alkyl)C(0)aryl, (C₀-C₈

alkyl)C(0)heteroaryl, (C₀-C₈ alkyl)C(0)OCi_C₈ alkyl, (C₀-C₈ alkyl)C(0)OC₂-C₈ alkenyl, (C₀- C₈ alkyl)C(0)OC₂_C₈ alkynyl, (C₀_C₈ alkyl)C(0)OH, (C₀_C₈ alkyl)C(0)0 aryl, (C₀_C₈ alkyl)C(0)0 heteroaryl, (C₀_C₈ alkyl)OC(0)Ci_C₈ alkyl, (C₀_C₈ alkyl)OC(0)C₂_C₈ alkenyl, (Co-C₈ alkyl)OC(0)C₂-Ci₈ alkynyl, (C₀-C₈ alkyl)C(0)NR²⁴Ci_C₈ alkyl, (C₀-C₈

alkyl)C(0)NR²⁴H₂, (C₀_C₈ alkyl)C(0)NR²⁴aryl, (C₀_C₈ alkyl)C(0)NR²⁴heteroaryl, (C₀_C₈ alkyl)NR²⁴C(0)Ci_C₈ alkyl, (C₀_C₈ alkyl)NR²⁴C(0)C₂_C₈ alkenyl, or (C₀_C₈

alkyl)NR²⁴C(0)C₂-C₈ alkynyl, (C₀-C₈ alkyl)NR²⁴C(0)OH, (C₀-C₈ alkyl)OC(0)OCi_C₈ alkyl, (Co-C₈ alkyl)OC(0)OC₂-C₈ alkenyl, (C₀-C₈ alkyl)OC(0)OC₂-C₈ alkynyl, (C₀-C₈ alkyl)OC(0)OH, (C₀_C₈ alkyl)OC(0)NR ⁴Ci_C₈ alkyl, (C₀_C₈ alkyl)OC(0)NR ⁴C₂_C₈ alkenyl, (C₀_C₈ alkyl)OC(0)NR²⁴C₂_C₈ alkynyl, (C₀_C₈ alkyl)OC(0)NR²⁴H₂, (C₀_C₈ alkyl)NR²⁴(0)OCi_C₈ alkyl, (C₀-C₈ alkyl)NR²⁴(0)OC₂-C₈ alkenyl, (C₀-C₈ alkyl)NR²⁴(0)OC₂- C₈ alkynyl, or (C₀-C₈ alkyl)NR²⁴(0)OH;

R⁶ is hydrogen, Ci_C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, heteroalkyl, (Co-C₈ alkyl)aryl, (C₀_C₈ alkyl)heteroaryl, (C₀_C₈ alkyl)C(0)Ci_C₈ alkyl, (C₀_C₈ alkyl)C(0)C₂_C₈ alkenyl, (C₀-C₈ alkyl)C(0)C₂-C₈ alkynyl, (C₀-C₈ alkyl)C(0)H, (C₀-C₈ alkyl)C(0)aryl, (C₀-C₈ alkyl)C(0)heteroaryl, (C₀-C₈ alkyl)C(0)OCi_C₈ alkyl, (C₀-C₈ alkyl)C(0)OC₂-C₈ alkenyl, (C₀- C₈ alkyl)C(0)OC₂_C₈ alkynyl, (C₀_C₈ alkyl)C(0)OH, C₀_C₈ alkyl)C(0)0 aryl, (C₀_C₈ alkyl)C(0)0 heteroaryl, (C₀_C₈ alkyl)C(0)NR²⁴Ci_C₈ alkyl, (C₀_C₈ alkyl)C(0)NR²⁴C₂_C₈ alkenyl, (C₀-C₈ alkyl)C(0)NR²⁴C₂-C₈ alkynyl, (C₀-C₈ alkyl)C(0)NR²⁴H₂, (C₀-C₈

alkyl)C(0)NR²⁴aryl, or (C₀-C₈ alkyl)C(0)NR²⁴heteroaryl; and

R 24 is hydrogen or Ci_C₇ alkyl.

For example, R¹ is hydrogen, propionate, acetate, benzoate, or sulfate; R⁵ is hydrogen, ethynyl, hydroxyl; and R⁶ is acetate, cypionate, hemisucciniate, enanthate, or propionate.

Nonlimiting examples of the compound of Formula B include 17P-estradiol, modified forms of estradiol such as β-estradiol 17-acetate, β-estradiol 17-cypionate, β- estradiol 17-enanthate, β-estradiol 17-valerate, β-estradiol 3,17-diacetate, β-estradiol 3,17- dipropionate, β-estradiol 3 -benzoate, β-estradiol 3 -benzoate 17-n-butyrate, β-estradiol 3- glycidyl ether, β-estradiol 3-methyl ether, β-estradiol 6-one, β-estradiol 3-glycidyl, β- estradiol 6-one 6-(0-carboxymethyloxime), 16-epiestriol, 17-epiestriol, 2-methoxy estradiol, 4-methoxy estradiol, estradiol 17-phenylpropionate, and 17β-estradiol 2-methyl ether, 17a- ethynylestradiol, megestrol acetate, estriol, and derivatives thereof. In some embodiments, carbon 17 has a ketone substituent and R⁵ and R⁶ are absent (e.g. estrone). Some of the aforementioned compounds of Formula B are shown below:

Ethylyl estradiol

In embodiments wherein Y comprises a structure of Formula B, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Formula B that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Formula B and means of conjugation of Formula B to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Formula B is conjugated to L or Q at any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of Formula B. In some embodiments, Formula B is conjugated to L or Q at position 3 or 17 of Formula B.

In other embodiments, Y acts at an estrogen receptor but is not encompassed by Formula B. Nonlimiting examples of ligands that act at an estrogen receptor that are not encompassed by Formula B are shown below:

In some embodiments, Y acts at a glucocorticoid receptor (GR). In some

embodiments, Y comprises any structure that permits or promotes agonist activity at the GR, while in other embodiments Y is an antagonist of GR. In exemplary embodiments, Y comprises a structure of Formula C:

Formula C

wherein R 2", R 3^J, 6 7 8 9 10

R , R', R°, R and R^1U are each independently moieties that permit or promote agonist or antagonist activity upon the binding of the compound of Formula C to the GR; and each dash represents an optional double bond. In some embodiments, Formula C further comprises one or more substituents at one or more of positions 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 14, and 15 (e.g. hydroxyl or ketone at position- 11).

In some embodiments, Y comprises a structure of Formula C wherein

R is hydrogen, halo, OH, or C₁-C₇ alkyl;

alkyl)C(0)NR²⁴aryl, or (C₀-C₈ alkyl)C(0)NR²⁴heteroaryl;

R is hydrogen, Ci_C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, heteroalkyl, (Co-C₈ alkyl)aryl, (C₀_C₈ alkyl)heteroaryl, (C₀ alkyl)C(0)Ci_C₈ alkyl, (C₀ alkyl)C(0)C₂_C₈ alkenyl, (Co alkyl)C(0)C₂-C₈ alkynyl, (C₀)C(O)aryl, (C₀)C(O)heteroaryl, (C₀)C(O)OCi_C₈ alkyl, (C₀ alkyl)C(0)OC₂-C₈ alkenyl, (C₀ alkyl)C(0)OC₂-C₈ alkynyl, or (C₀ alkyl)C(0)OH;

R is hydrogen or Ci_C₇ alkyl;

R⁹ is hydrogen or Ci_C₇ alkyl;

R¹⁰ is hydrogen or OH; and

R 24 is hydrogen or Ci_C₇ alkyl. For example, R² is hydrogen or methyl; R³ is hydrogen, fluoro, chloro, or methyl; R⁶ is hydrogen or C(O) C1-C7 alkyl; R⁷ is hydrogen, C(0)CH₃, or C(0)CH₂CH₃; R⁸ is hydrogen or methyl; R⁹ is hydrogen or methyl; and R¹⁰ is hydroxyl.

Nonlimiting examples of structures of Formula C include:

Beclometasone

Cortisol Cortisone acetate

Prednisone Prednisolone Methylprednisolone

Betamethasone Triamcinolone Dexamethasone and derivatives thereof.

In embodiments wherein Y comprises a structure of Formula C, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Formula C that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Formula C and means of conjugation of Formula C to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Formula C is conjugated to L or Q at any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 of Formula C. In some embodiments, Formula C is conjugated to L or Q at position 3, 10, 16 or 17 of Formula C.

In some embodiments, Y acts at a mineralcorticoid receptor (MR). In some embodiments, Y comprises any structure that permits or promotes agonist activity at the MR, while in other embodiments Y is an antagonist of MR. In exemplary embodiments, Y comprises a structure of Formula D:

Formula D

wherein R 2 , R 3 ,R 7 and R 10 are each independently a moiety that permits or promotes agonist or antagonist activity upon binding of the compound of Formula D to the MR; and the dashed line indicates an optional double bond. In some embodiments, Formula D further comprises one or more substituents at one or more of positions 1, 2, 4, 5, 6, 7, 8, 11, 12, 14, 15, 16, and 17.

In some embodiments, Y comprises a structure of Formula D wherein

R is hydrogen, halo, OH, or C₁-C7 alkyl;

R is hydrogen, Ci_C₈ alkyl, C₂-C8 alkenyl, C₂-C8 alkynyl, heteroalkyl, (Co-Cs alkyl)aryl, (C₀_C₈ alkyl)heteroaryl, (C₀ alkyl)C(0)Ci_C₈ alkyl, (C₀ alkyl)C(0)C₂-C₈ alkenyl, (Co alkyl)C(0)C₂_C₈ alkynyl, (C₀)C(O)aryl, (C₀)C(O)heteroaryl, (C₀)C(O)OCi_C₈ alkyl, (C₀ alkyl)C(0)OC₂-C₈ alkenyl, (C₀ alkyl)C(0)OC₂-C₈ alkynyl, or (C₀ alkyl)C(0)OH;

R¹⁰ is hydrogen or OH; and

R 24 is hydrogen or Ci_C₇ alkyl.

For example, R 2 is hydrogen or methyl; R 3 is hydrogen, fluoro, chloro, or methyl; R 7 is hydrogen, C(0)CH₃, or C(0)CH₂CH₃; and R¹⁰ is hydroxyl.

Nonlimiting examples of compounds of Formula D include:

Aldosterone Deoxycorticosterone acetate Deoxycorticosterone acetate and derivatives thereof.

In embodiments wherein Y comprises a structure of Formula D, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Formula D that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Formula D and means of conjugation of Formula D to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Formula D is conjugated to L or Q at any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 of Formula D. In some embodiments, Formula D is conjugated to L or Q at position 3, 10, 13, or 17 of Formula D.

In some embodiments, Y acts at a progesterone receptor (PR). In some

embodiments, Y comprises any structure that permits or promotes agonist activity at the PR, while in other embodiments Y is an antagonist of PR. In exemplary embodiments, Y comprises a structure of Formula E:

Formula E

wherein R 2 , R 3 , R 4 , and R 7 are each independently moieties that permit or promote agonist or antagonist activity upon binding of the compound of Formula E to the PR; and the dashed line indicates an optional double bond. In some embodiments, Formula E further comprises one or more substituents at one or more of positions 1, 2, 4, 5, 6, 7, 8, 11, 12, 14, 15, 16, and 17 (e.g. a methyl group at position 6).

In some embodiments, Y comprises a structure of Formula E wherein

R is hydrogen, halo, OH, or C₁-C₇ alkyl;

R is hydrogen, halo, OH, or C₁-C₇ alkyl; R⁴ is hydrogen, (Co-C₈ alkyl)halo, Ci_C₈ alkyl, C₂-C₈ alkenyl, C₂-i₈ alkynyl, heteroalkyl, (C₀_C₈ alkyl)aryl, (C₀_C₈ alkyl)heteroaryl, (C₀_C₈ alkyl)OCi_C₈ alkyl, (C₀_C₈ alkyl)OC₂-C₈ alkenyl, (C₀-C₈ alkyl)OC₂-C₈ alkynyl, (C₀-C₈ alkyl)OH, (C₀-C₈ alkyl)SH, (C₀- C₈ alkyl)NR²⁴Ci_C₈ alkyl, (C₀-C₈ alkyl)NR²⁴C₂-C₈ alkenyl, (C₀-C₈ alkyl)NR²⁴C₂-C₈ alkynyl, (Co-C₈ alkyl)NR²⁴H₂, (C₀_C₈ alkyl)C(0)Ci_C₈ alkyl, (C₀_C₈ alkyl)C(0)C₂_C₈ alkenyl, (C₀_C₈ alkyl)C(0)C₂_C₈ alkynyl, (C₀_C₈ alkyl)C(0)H, (C₀_C₈ alkyl)C(0)aryl, (C₀_C₈

alkyl)OC(0)OH, (C₀-C₈ alkyl)OC(0)NR²⁴Ci_C₈ alkyl, (C₀-C₈ alkyl)OC(0)NR²⁴C₂-C₈ alkenyl, (C₀_C₈ alkyl)OC(0)NR²⁴C₂_C₈ alkynyl, (C₀_C₈ alkyl)OC(0)NR²⁴H₂, (C₀_C₈ alkyl)NR²⁴(0)OCi_C₈ alkyl, (C₀_C₈ alkyl)NR²⁴(0)OC₂_C₈ alkenyl, (C₀_C₈ alkyl)NR²⁴(0)OC₂_ C₈ alkynyl, or (C₀-C₈ alkyl)NR²⁴(0)OH;

R is hydrogen, Ci_C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, heteroalkyl, (Co-C₈ alkyl)aryl, (C₀_C₈ alkyl)heteroaryl, (C₀ alkyl)C(0)Ci_C₈ alkyl, (C₀ alkyl)C(0)C₂_C₈ alkenyl, (Co alkyl)C(0)C₂_C₈ alkynyl, (C₀)C(O)aryl, (C₀)C(O)heteroaryl, (C₀)C(O)OCi_C₈ alkyl, (C₀ alkyl)C(0)OC₂-C₈ alkenyl, (C₀ alkyl)C(0)OC₂-C₈ alkynyl, or (C₀ alkyl)C(0)OH; and

R 24 is hydrogen or C 1-C7 alkyl.

For example, R 2 is hydrogen or methyl; R 3 is hydrogen or methyl; R 4 is (Ci alkyl)C(0)Ci_C₄ alkyl, acetate, cypionate, hemisucciniate, enanthate, or propionate; and R is hydrogen, C(0)CH₃, or C(0)CH₂CH₃.

Nonlimiting examples of compounds of Formula E include:

Progesterone 19-nor-progesterone Medroxyprogesterone , and derivatives thereof.

In embodiments wherein Y comprises a structure of Formula E, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Formula E that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Formula E and means of conjugation of Formula E to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Formula E is conjugated to L or Q at any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 of Formula E. In some embodiments, Formula E is conjugated to L or Q through position 3 or 17 of Formula E.

In other embodiments, Y acts at a progesterone receptor but is not encompassed by Formula E. For example, Y can comprise the below structure and analogs thereof:

Norethindrone

In some embodiments, Y acts at an androgen receptor (AR). In some embodiments, Y comprises any structure that permits or promotes agonist activity at the AR, while in other embodiments Y is an antagonist of AR. In exemplary embodiments, Y comprises a structure of Formula F:

Formula F

wherein R¹, when present, R², R³ and R⁶ are each independently a moiety that permits or promotes agonist or antagonist activity upon binding of the compound of Formula F to the AR; and each dashed line represents an optional double bond, with the proviso that no more than one of the optional carbon-carbon double bond is present at position 5. In some embodiments, Formula F further comprises one or more substituents at one or more of positions 1, 2, 4, 5, 6, 7, 8, 11, 12, 14, 15, 16, and 17.

In some embodiments, Y comprises a structure of Formula F wherein

R¹ is hydrogen, C₁-C7 alkyl; (C₀-C₃ alkyl)C(0)Ci-C₇ alkyl, (C₀_C₃ alkyl)C(0)aryl, or

S0₃H;

R is hydrogen, halo, OH, or Ci-C₇ alkyl;

R⁶ is hydrogen, Ci_C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, heteroalkyl, (Co-C₈ alkyl)aryl, (C₀-C₈ alkyl)heteroaryl, (C₀-C₈ alkyl)C(0)Ci_C₈ alkyl, (C₀-C₈ alkyl)C(0)C₂-C₈ alkenyl, (C₀-C₈ alkyl)C(0)C₂-C₈ alkynyl, (C₀-C₈ alkyl)C(0)H, (C₀-C₈ alkyl)C(0)aryl, (C₀-C₈ alkyl)C(0)heteroaryl, (C₀_C₈ alkyl)C(0)OCi_C₈ alkyl, (C₀_C₈ alkyl)C(0)OC₂_C₈ alkenyl, (C₀_ C₈ alkyl)C(0)OC₂_C₈ alkynyl, (C₀_C₈ alkyl)C(0)OH, C₀_C₈ alkyl)C(0)0 aryl, (C₀_C₈ alkyl)C(0)0 heteroaryl, (C₀-C₈ alkyl)C(0)NR²⁴Ci_C₈ alkyl, (C₀-C₈ alkyl)C(0)NR²⁴C₂-C₈ alkenyl, (C₀-C₈ alkyl)C(0)NR²⁴C₂-C₈ alkynyl, (C₀-C₈ alkyl)C(0)NR²⁴H₂, (C₀-C₈

alkyl)C(0)NR²⁴aryl, or (C₀-C₈ alkyl)C(0)NR²⁴heteroaryl; and

R 24 is hydrogen or Ci_C₇ alkyl.

For example, R 1 is hydrogen or absent; R2 is hydrogen or methyl; R 3 is hydrogen or methyl; and R⁶ is H or absent.

Nonlimiting examples of compounds of Formula F include:

Testosterone Dehydroepiandrosterone Androstenedione

5-Androstenediol Androsterone Dihydrotestosterone _and derivatives thereof. In embodiments wherein Y comprises a structure of Formula F, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Formula F that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Formula F and means of conjugation of Formula F to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Formula F is conjugated to L or Q at any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 of Formula F. In some embodiments, Formula F is conjugated to L or Q at position 3 or 17 of Formula F.

In some embodiments, the binding of the NHR ligand to the Type I nuclear hormone receptor results in agonist activity (or antagonist activity) in some but not all cells or tissues expressing the Type I nuclear hormone receptor.

NHR Ligand that Acts on a Type II Nuclear Hormone Receptor

In some embodiments of the invention, the NHR ligand (Y) acts on a Type II nuclear hormone receptor. In some embodiments, Y can have any structure that permits or promotes agonist activity upon binding of the ligand to a Type II nuclear hormone receptor, while in other embodiments Y is an antagonist of the Type II nuclear hormone receptor. In exemplary embodiments, Y exhibits agonist (or antagonist) activity at a thyroid hormone receptor (TR), retinoic acid receptor (RAR), peroxisome proliferator activated receptor (PPAR), Liver X Receptor (LXR), farnesoid X receptor (FXR), vitamin D receptor (VDR), and/or pregnane X receptor (PXR).

In some embodiments, Y acts at a thyroid hormone receptor (e.g. TRa, TRP). In some embodiments, Y comprises any structure that permits or promotes agonist activity at the TR, while in other embodiments Y is an antagonist of TR. In one embodiment a thyroid hormone receptor agonist is provided having the general structure of

wherein

Ri5 is Ci-C₄ alkyl, -CH₂(pyridazinone), -CH₂(OH)(phenyl)F, -CH(OH)CH₃, halo or

H; R₂₀ is halo, CH₃ or H;

R₂₁ is halo, CH₃ or H;

R22 is H, OH, halo, -CH₂(OH)(C₆ aryl)F, or C1-C4 alkyl; and

R₂₃ is -CH₂CH(NH₂)COOH, -OCH₂COOH, -NHC(0)COOH, -CH₂COOH, -NHC(0)CH₂COOH, -CH₂CH₂COOH, or -OCH₂P0₃ ^2".

In accordance with one embodiment the thyroid hormone receptor agonist is a compound of the general structure

wherein

Ri₅ is C₁-C4 alkyl, -CH(OH)CH₃, 1 or H

R₂₀ is I, Br, CH₃ or H;

R₂₁ is I, Br, CH₃ or H;

R22 is H, OH, I, or C1-C4 alkyl; and

R₂₃ is -CH₂CH(NH₂)COOH, -OCH₂COOH, -NHC(0)COOH, -CH₂COOH, -NHC(0)CH₂COOH, -CH₂CH₂COOH, or -OCH₂P0₃ ^2". In one embodiment R₂₃ is

CH₂CH(NH₂)COOH.

wherein

Ri₅ is isopropyl, -CH(OH)CH₃, 1 or H

R₂₀ is I, Br, CI, or CH₃;

R₂₁ is I, Br, CI, or CH₃;

R₂₂ is H; and

R₂₃ is -OCH₂COOH, -CH₂COOH, -NHC(0)CH₂COOH, or -CH₂CH₂COOH. In accordance with one embodiment the thyroid hormone receptor agonist is a compound of the general structure of Formula I:

Ri₅ is halo or H. In one embodiment R₂o and R₂i are each CH₃, R15 is H and R₂₂ are independently selected from the group consisting of H, OH, halo and Ci-C₄ alkyl. In one embodiment R₂o, R21 and R₂₂ are each halo and R15 is H or halo. In a further embodiment R20, R21 and R22 are each I, and R15 is H or I. In accordance with one embodiment Y is selected from the group consisting of thyroxine T4 (3,5,3',5'-tetraiodothyronine) and 3,5,3'- triiodo L- thyronine.

In one embodiment, the thyroid hormone receptor ligand (Y) of the Q-L-Y conjugates, is an indole derivative of thyroxine, including for example, compounds disclosed in U.S. Pat. No. 6,794,406 and US published application no. US 2009/0233979, the disclosures of which are incorporated herein. In one embodiment the indole derivative of thyroxine comprises a compound of the general structure of Formula II:

wherein

Ri₃ is H or Ci-C₄ alkyl;

Ri₄ is Ci-Cg alkyl;

Ri₅ is H or Ci-C₄ alkyl; and

Ri₆ and Ri₇ are independently halo or Ci-C₄ alkyl.

In one embodiment, the thyroid receptor ligand (Y) of the Q-L-Y conjugates, is an indole derivative of thyroxine as disclosed in W097/21993 (U. Cal SF), WO99/00353 (KaroBio), GB98/284425 (KaroBio), and U.S. Provisional Application 60/183,223, the disclosures of which are incorporated by reference herein. In one embodiment the thyroid receptor ligand comprises the general structure of Formula III:

wherein X is oxygen, sulfur, carbonyl, methylene, or NH;

Y is (CH2)_n, where n is an integer from 1 to 5, or C=C;

Ri is halogen, trifluoromethyl, or Ci-C₆ alkyl or C3-C7 cycloalkyl;

R₂ and R₃ are the same or different and are hydrogen, halogen, Ci-C₆ alkyl or C₃-C₇ cycloalkyl, with the proviso that at least one of R₂ and R₃ being other than hydrogen;

R₄ is hydrogen or Ci-C₄ alkyl;

R5 is hydrogen or Ci-C₄ alkyl;

R₆ is carboxylic acid, or ester thereof;

R₇ is hydrogen, or an alkanoyl or aroyl group.

Nonlimiting examples of Y include the following compounds:

Thyroxine (T₄) ^ Triiodothyroxine (T₃) , and derivatives thereof.

In embodiments wherein Y comprises a structure that permits or promotes agonist or antagonist activity at a TR, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Y that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Y and means of conjugation of Y to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Y is conjugated to L or Q through any position of Y. In some embodiments, Y is conjugated to L or Q through the carboxylic acid or amine moieties, as indicated below:

In some embodiments, Y acts at a retinoic acid receptor (e.g. RARa, RARp, RARy). In some embodiments, Y comprises any structure that permits or promotes agonist activity at the RAR, while in other embodiments Y is an antagonist of RAR. In exemplary

embodiments, Y comprises a structure of Formula G:

Formula G

wherein R¹¹ is a moiety that permits or promotes agonist or antagonist activity upon the binding of the compound of Formula G to a RAR, and -~w represents either E or Z stereochemistry.

In some embodiments, Y comprises a structure of Formula G wherein R¹¹ is

C(0)OH, CH₂OH, or C(0)H. In some embodiments, Y comprises a structure of Formula G wherein R¹¹ is a carboxylic acid derivative (e.g. acyl chloride, anhydride, and ester).

Nonlimiting examples of the compound of Formula G include:

All-trans-retinoic acid Retinol

Retinal 1 1 -cis-retinoic acid , and derivatives thereof.

In embodiments wherein Y comprises a structure of Formula G, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Formula G that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Y and means of conjugation of Y to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Y is conjugated to L or Q through any position of Y. In some embodiments, Formula G is conjugated to L or Q at R¹¹.

In some embodiments, Y acts at a peroxisome proliferator activated receptor (e.g. PPARa, PPARp/δ, PPARy). In some embodiments, Y acts at PPARy. In some

embodiments, Y comprises any structure that permits or promotes agonist activity at the PPAR, while in other embodiments Y is an antagonist of PPAR. In some embodiments, Y is a saturated or unsaturated, halogenated or nonhalogenated free fatty acid (FFA) as described by Formula H:

Formula H

wherein n is 0-26 and each R 12 , when present, is independently a moiety that permits or promotes agonist or antagonist activity upon binding of the compound of Formula H to a

PPAR.

In some embodiments, Y comprises a structure of Formula H, wherein n is 0-26 and each R 12 , when present, is independently hydrogen, C1-C7 alkyl, or halogen. In some embodiments Formula B is saturated such as, for example, formic acid, acetic acid, n-caproic acid, heptanoic acid, caprylic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadeconoic acid, palmitic acid, heptadecanoic acid, stearic acid, nonadecanoic acid, arachidic acid, heneicosanoic acid, behenic acid, tricosanoic acid, perfluorononanoic acid (see below), perfluorooctanoic acid (see below), and derivatives thereof. In some embodiments Formula H is unsaturated with either cis or trans

stereochemistry such as, for example, mead acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, linoleic acid, a-linolenic acid, elaidic acid, petroselinic acid, arachidonic acid, dihydroxyeicosatetraenoic acid (DiHETE), octadecynoic acid, eicosatriynoic acid, eicosadienoic acid, eicosatrienoic acid, eicosapentaenoic acid, erucic acid, dihomolinolenic acid, docosatrienoic acid, docosapentaenoic acid, docosahexaenoic acid, adrenic acid, and derivatives thereof, including for example:

Perfluorononanoic acid Perfluorooctanoic acid In embodiments wherein Y comprises a structure of Formula H, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Formula H that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Formula H and means of conjugation of Formula H to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Formula H is conjugated to L or Q at any position on Formula H. In some embodiments, Formula H is conjugated to L or Q through the terminal carboxylic acid moiety.

In some of these embodiments, Y is an eiconsanoid. In specific embodiments, Y is a prostaglandin or a leukotriene. In some exemplary embodiments, Y is a prostaglandin having a structure as described by Formulae J1-J6:

wherein each R is independently a moiety that permits or promotes agonist or antagonist activity upon the binding of the compound of Formula J to a PPAR (e.g. PGJ2 as shown below):

H

In some embodiments when Y comprises a structure of any one of Formulae J1-J6, each R 13 is independently C₇-C₈ alkyl, C₇-C₈ alkenyl, C₇-C₈ alkynyl, or heteroalkyl.

In embodiments wherein Y is an eicosanoid, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of the eicosanoid that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Y and means of conjugation of Y to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Y is conjugated to L or Q through any position of Y. In some embodiments, the eicosanoid is conjugated to L or Q through a terminal carboxylic acid moiety or through a pendant alcohol moiety.

In some exemplary embodiments, Y is a leukotriene having a structure as described by Formula K or a derivatized form of Formula K:

Formula K

wherein each R is independently a moiety that permits or promotes agonist or antagonist activity upon the binding of the compound of Formula K to a PPAR (e.g. leukotriene B4 as shown below):

In some embodiments when Y comprises a structure of Formula K, each R is independently C₃-C₁₃ alkyl, C₃-C₁₃ alkenyl, C₃-C₁₃ alkynyl, or heteroalkyl.

In embodiments wherein Y comprises a structure of Formula K, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Formula K that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Formula K and means of conjugation of Formula K to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Formula K is conjugated to L or Q at any position on Formula K. In some embodiments, Formula K is conjugated to L or Q through the terminal carboxylic acid moiety or through a pendant alcohol moiety.

In some exemplary embodiments, Y is a thiazolidinedione comprising a structure as described by Formula L:

Formula L.

Nonlimiting examples of the compound of Formula L include:

Rosiglitazone Pioglitazone

Troghtazone _{^ and} derivatives thereof.

In embodiments wherein Y comprises a structure of Formula L, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Formula L that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Formula L and means of conjugation of Formula L to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Formula L is conjugated to L or Q at any position on Formula L, such as, for example, a pendant alcohol moiety, or through an aromatic substituent.

In one embodiment wherein Y is Tesaglitzar or Aleglitazar:

Aleglitazar.

In embodiments wherein Y comprises Tesaglitzar or Aleglitazar, Y is conjugated L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position that is capable of reacting with Q or L. In one embodiment, Tesaglitzar or Aleglitazar is conjugated to L or Q through the carboxylic acid moiety of the compound. In some embodiments, Y acts at a RAR-related orphan receptor (e.g. RORa, RORp, RORy). In some embodiments, Y comprises any structure that permits or promotes agonist activity at the ROR, while in other embodiments Y is an antagonist of ROR.

Nonlimiting examples of Y include:

Cholesterol Melatonin

CGP 52608 _s All-trans-retinoic acid , and derivatives thereof.

In embodiments wherein Y acts at a ROR, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Y that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Y and means of conjugation of Y to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Y is conjugated to L or Q through any position of Y, such as, for example, any of the positions previously described herein.

In some embodiments, Y acts at a liver X receptor (LXRa, LXRP). In some embodiments, Y comprises any structure that permits or promotes agonist activity at the LXR, while in other embodiments Y is an antagonist of LXR. In exemplary embodiments, Y is an oxysterol (i.e. oxygenated derivative of cholesterol). Nonlimiting examples of Y in these embodiments include 22(R)-hydroxycholesterol (see below), 24(S)-hydroxycholesterol (see below), 27-hydroxycholesterol, cholestenoic acid, and derivatives thereof.

22(R)-Hydroxycholesterol 24(S)-Hydroxycholesterol In embodiments wherein Y acts at a LXR, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Y that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Y and means of conjugation of Y to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Y is conjugated to L or Q at any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 of Formula F. In some embodiments, Formula F is conjugated to L or Q at position 3 or 17 of Formula F.

In some embodiments, Y acts at the farnesoid X receptor (FXR). In some embodiments, Y comprises any structure that permits or promotes agonist activity at the FXR, while in other embodiments Y is an antagonist of FXR. In some of these

embodiments, Y is a bile acid. In exemplary embodiments, Y has a structure of Formula M:

Formula M

wherein each of R¹⁵, R¹⁶, and R¹⁷ are independently moieties that permit or promote agonist or antagonist activity upon binding of the compound of Formula M to a FXR.

In some embodiments when Y comprises a structure of Formula M, each of R¹⁵ and R¹⁶ are independently hydrogen, (Co-C₈ alkyl)halo, Ci_Ci₈ alkyl, C₂-Ci₈ alkenyl, C₂_Ci₈ alkynyl, heteroalkyl, or (C₀-C₈ alkyl)OH; and R¹⁷ is OH, (C₀-C₈ alkyl)NH(Ci-C₄

alkyl)S0₃H, or (C₀-C₈ alkyl)NH(Ci-C₄ alkyl)COOH.

In some embodiments when Y comprises a structure of Formula M, each of R¹⁵ and R¹⁶ are independently hydrogen or OH; and R¹⁷ is OH, NH(C C₂ alkyl)S0₃H, or NH(C C₂ alkyl)COOH.

Nonlimiting examples of the compound of Formula M include:

Tauroc olic acid ^ Glycocholic acid and derivatives thereof.

In embodiments wherein Y comprises a structure of Formula M, Y is conjugated to L

(e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Formula M that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Formula M and means of conjugation of Formula M to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Formula M is conjugated to L or Q at any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of Formula M. In some embodiments, Formula M is conjugated to L or Q at position 3, 7, 12 or 17 of Formula M.

In some embodiments, Y acts at the vitamin D receptor (VDR). In some

embodiments, Y comprises any structure that permits or promotes agonist activity at the VDR, while in other embodiments Y is an antagonist of VDR. In exemplary embodiments, Y has a structure of Formula N:

Formula N

wherein each of R 18 , R 1 , R 20 , R 21 , R 22 , and R 23 are moieties that permit or promote agonist or antagonist activity upon binding of the compound of Formula N to the VDR such as, for example, any of the vitamin D compounds found in Bouillon et al., Endocrine Reviews,

16(2):200-257 (1995).

In some embodiments wherein Y comprises a structure of Formula N,

R¹⁸ and R¹⁹ are each independently hydrogen, (Co-C₈ alkyl)halo, (Co-C₈

alkyl)heteroaryl, or (C₀-C₈ alkyl)OH;

both of R 20 are hydrogen or both of R 20 are taken together to form =CH₂;

each of R 21 and R 22 are independently Ci-C₄ alkyl; and

R 23 is C₄_Cis alkyl, C₄_Ci₈ alkenyl, C₄_Ci₈ alkynyl, heteroalkyl, (C₄_Ci₈ alkyl)aryl, (C₄_Ci8 alkyl)heteroaryl, (C₀_C₈ alkyl)OCi_Ci₈ alkyl, (C₀_C₈ alkenyl)OCi_Ci₈ alkyl, (C₀_C₈ alkynyl)OCi_Ci₈ alkyl, (C₀_C₈ alkyl)OC₂_Ci₈ alkenyl, (C₀_C₈ alkyl)OC₂_Ci₈ alkynyl, (C₆_Ci₈ alkyl)OH, (C₆-Ci₈ alkyl)SH, (C₆-Ci₈ alkenyl)OH, (C₆-Ci₈ alkynyl)OH, (C₀-C₈ alkyl)NR²⁴Ci_ Ci₈ alkyl, (C₀-C₈ alkenyl)NR²⁴Ci_Ci₈ alkyl, (C₀-C₈ alkynyl)NR²⁴Ci_Ci₈ alkyl, (C₀-C₈ alkyl)NR²⁴C₂_Ci₈ alkenyl, (C₀_C₈ alkyl)NR²⁴C₂_Ci₈ alkynyl, (C₀_C₈ alkyl)C(0)Ci_Ci₈ alkyl, (Co-C₈ alkyl)C(0)C₂_Ci₈ alkenyl, (C₀_C₈ alkyl)C(0)C₂_Ci₈ alkynyl, (C₀_C₈ alkyl)C(0)H, (C₀_ C₈ alkyl)C(0)aryl, (C₀-C₈ alkyl)C(0)heteroaryl, (C₀-C₈ alkyl)C(0)OCi_Ci₈ alkyl, (C₀-C₈ alkyl)C(0)OC₂_Ci₈ alkenyl, (C₀-C₈ alkyl)C(0)OC₂_Ci₈ alkynyl, (C₀-C₈ alkyl)C(0)OH, (C₀-C₈ alkyl)C(0)0 aryl, (C₀_C₈ alkyl)C(0)0 heteroaryl, (C₀_C₈ alkyl)OC(0)Ci_Ci₈ alkyl, (C₀_C₈ alkyl)OC(0)C₂_Ci₈ alkenyl, (C₀_C₈ alkyl)OC(0)C₂_Ci₈ alkynyl, (C₀_C₈ alkyl)C(0)NR²⁴Ci_ Ci₈ alkyl, (C₀-C₈ alkyl)C(0)NR²⁴C₂_Ci₈ alkenyl, (C₀-C₈ alkyl)C(0)NR²⁴C₂_Ci₈ alkynyl, (C₀- C₈ alkyl)C(0)NR²⁴H₂, (C₀-C₈ alkyl)C(0)NR²⁴aryl, (C₀-C₈ alkyl)C(0)NR²⁴heteroaryl, (C₀-C₈ alkyl)NR²⁴C(0)Ci_Ci₈ alkyl, (C₀_C₈ alkyl)NR²⁴C(0)C₂_C ₈ alkenyl, or (C₀_C₈

alkyl)NR²⁴C(0)C₂_Ci₈ alkynyl, (C₀_C₈ alkyl)NR²⁴C(0)OH, (C₀_C₈ alkyl)OC(0)OCi_C ₁₈ alkyl, (Co-C₈ alkyl)OC(0)OC₂_C ₁₈ alkenyl, (C₀-C₈ alkyl)OC(0)OC₂_C ₁₈ alkynyl, (C₀-C₈ alkyl)OC(0)OH, (C₀_C₈ alkyl)0C(0)NR ⁴Ci_C ₁₈ alkyl, (C₀_C₈ alkyl)OC(0)NR ⁴C₂_C ₁₈ alkenyl, (C₀_C₈ alkyl)OC(0)NR²⁴C₂_C ₁₈ alkynyl, (C₀_C₈ alkyl)OC(0)NR²⁴H₂, (C₀_C₈ alkyl)NR²⁴(0)OCi_C ₁₈ alkyl, (C₀-C₈ alkyl)NR²⁴(0)OC₂_C ₁₈ alkenyl, (C₀-C₈

alkyl)NR²⁴(0)OC₂_C ₁₈ alkynyl, or (C₀-C₈ alkyl)NR²⁴(0)OH; and

R 24 is hydrogen or Ci_Ci₈ alkyl.

Nonlimiting examples of the compound of Formula N include:

Calcitriol , 25-Hydroxyvitamin D₃ , and derivatives thereof.

In embodiments wherein Y comprises a structure of Formula N, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Formula N that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Formula N and means of conjugation of Formula N to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Formula N is conjugated to L or Q at any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 of Formula N. In some embodiments, Formula N is conjugated to L or Q at position 1, 3, 19, or 25 of Formula N.

In some embodiments, Y acts at the pregnane X receptor (PXR). In some embodiments, Y comprises any structure that permits or promotes agonist activity at the PXR, while in other embodiments Y is an antagonist of PXR. In some embodiments, Y is a steroid, antibiotic, antimycotic, bile acid, hyperforin, or a herbal compound. In exemplary embodiments, Y is compound that is able to induce CYP3A4, such as dexamethasone and rifampicin. In embodiments wherein Y comprises a structure that acts at the PXR, Y is conjugated to L (e.g. when L is a linking group) or Q (e.g. when L is a bond) at any position of Y that is capable of reacting with Q or L. One skilled in the art could readily determine the position of conjugation on Y and means of conjugation of Y to Q or L in view of general knowledge and the disclosure provided herein. In some embodiments, Y is conjugated to L or Q at any of positions on Y. Modification of the NHR Ligand (Y)

In some embodiments, the NHR ligand is derivatized or otherwise chemically modified to comprise a reactive moiety that is capable of reacting with the insulin peptide (Q) or the linking group (L). In the embodiments described herein, Y is derivatized at any position of Y that is capable of reacting with Q or L. The position of derivatization on Y is apparent to one skilled in the art and depends on the type of NHR ligand used and the activity that is desired. For example, in embodiments wherein Y has a structure comprising a tetracyclic skeleton having three 6-membered rings joined to one 5-membered ring or a variation thereof, Y can be derivatized at any of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. Other positions of derivatization can be as previously described herein.

The NHR ligand can be derivatized using any agent known to one skilled in the art or described herein. For example, estradiol can be derivatized with succinic acid, succinic anhydride, benzoic acid, ethyl 2-bromoacetate, or iodoacetic acid to form the below derivatives of estradiol that are capable of conjugating to Q or L.

Similarly, any of the aforementioned NHR ligands can be derivatized by methods known in the art. Additionally, certain derivatized ligands are commercially available and can be purchased from chemical companies such as Sigma-Aldrich. In accordance with one embodiment Y is selected from the group consisting of estradiol and derivatives thereof, estrone and derivatives thereof, testosterone and derivatives thereof, and Cortisol and derivatives thereof. In one embodiment Y is dexamethasone. In one embodiment Y is selected from the group consisting of thyroxine T4 (3,5,3',5'-tetra- iodo thyronine), 3,5,3 '-triiodo L-thyronine, Tesaglitazar, Aleglitazar and thiazolidinediones. In one embodiment Y is selected from the group consisting of thyroxine T4 (3,5,3',5'-tetra- iodo thyronine), and 3,5,3 '-triiodo L-thyronine. In one embodiment Y is selected from the group consisting of Tesaglitazar and Aleglitazar. Structure of the Insulin peptide

In some embodiments, the insulin peptide of the presently disclosed conjugates is native insulin, comprising the A chain of SEQ ID NO: 1 and the B chain of SEQ ID NO: 2, or an analog of native insulin, including for example a single-chain insulin analog comprising SEQ ID NOS: 1 and 2. In accordance with the present disclosure analogs of insulin encompass polypeptides comprising an A chain and a B chain wherein the insulin analogs differ from native insulin by one or more amino acid substitutions at positions selected from A5, A8, A9, A10, A12, A14, A15, A17, A18, A21, B l, B2, B3, B4, B5, B9, B IO, B 13, B 14, B 17, B20, B21, B22, B23, B26, B27, B28, B29 and B30 or deletions of any or all of positions B l-4 and B26-30.

In one embodiment the insulin peptide is an insulin analog wherein:

(a) the amino acid residue at position B28 is substituted with Asp, Lys, Leu, Val, or Ala, and the amino acyl residue at position B29 is Lys or Pro;

(b) the amino acid residues at any of positions B27, B28, B29, and B30 are deleted or substituted with a nonnative amino acid. In one embodiment an insulin analog is provided comprising an Asp substituted at position B28 or a Lys substituted at position 28 and a proline substituted at position B29. Additional insulin analogs are disclosed in Chance, et al., U.S. Pat. No. 5,514,646; Chance, et al., U.S. patent application Ser. No. 08/255,297; Brems, et al., Protein Engineering, 5:527-533 (1992); Brange, et al., EPO Publication No. 214,826 (published Mar. 18, 1987); and Brange, et al., Current Opinion in Structural Biology, 1:934-940 (1991). The disclosures of which are expressly incorporated herein by reference.

Insulin analogs may also have replacements of the amidated amino acids with acidic forms. For example, Asn may be replaced with Asp or Glu. Likewise, Gin may be replaced with Asp or Glu. In particular, Asn(A18), Asn(A21), or Asp(B3), or any combination of those residues, may be replaced by Asp or Glu. Also, Gln(A15) or Gln(B4), or both, may be replaced by either Asp or Glu.

As disclosed herein single chain insulin agonists are provided comprising a B chain and an A chain of human insulin, or analogs or derivative thereof, wherein the carboxy terminus of the B chain is linked to the amino terminus of the A chain via a linking moiety. In one embodiment the A chain is an amino acid sequence selected from the group consisting of GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1),

GIVDECCFRSCDLRRLEMYCA (SEQ ID NO: 5) or GIVEECCFRSCDLALLETYCA (SEQ ID NO: 7) and the B chain comprises the sequence

FVNQHLC GS HLVE AL YLVC GERGFFYTPKT (SEQ ID NO: 2),

GPETLCGAELVDALYLVCGDRGFYFNKPT (SEQ ID NO: 6) or

A YRPS ETLC GGELVDTLYLVC GDRGF YFS RP A (SEQ ID NO: 8), or a carboxy shortened sequence thereof having one to five amino acids corresponding to B26, B27, B28, B29 and B30 deleted, and analogs of those sequences wherein each sequence is modified to comprise one to five amino acid substitutions at positions corresponding to native insulin positions selected from A5, A8, A9, A10, A14, A15, A17, A18, A21, B l, B2, B3, B4, B5, B9, B IO, B 13, B 14, B20, B22, B23, B26, B27, B28, B29 and B30. In one embodiment the amino acid substitutions are conservative amino acid substitutions. Suitable amino acid substitutions at these positions that do not adversely impact insulin's desired activities are known to those skilled in the art, as demonstrated, for example, in Mayer, et al., Insulin Structure and Function, Biopolymers. 2007;88(5):687-713, the disclosure of which is incorporated herein by reference.

Additional amino acid sequences can be added to the amino terminus of the B chain or to the carboxy terminus of the A chain of the single chain insulin agonists of the present invention. For example, a series of negatively charged amino acids can be added to the amino terminus of the B chain, including for example a peptide of 1 to 12, 1 to 10, 1 to 8 or 1 to 6 amino acids in length and comprising one or more negatively charged amino acids including for example glutamic acid and aspartic acid. In one embodiment the B chain amino terminal extension comprises 1 to 6 charged amino acids. In one embodiment the B chain amino terminal extension comprises the sequence GX61X62X63X64X65K (SEQ ID NO: 26) or X61X62X63X64X65RK (SEQ ID NO: 27), wherein X₆₁, X₆₂, X₆3 Χό4 and X₆₅ are independently glutamic acid or aspartic acid. In one embodiment the B chain comprises the sequence GEEEEEKGPEHLCGAHLVDALYLVCGDX₄₂GFY (SEQ ID NO: 28), wherein X₄₂ is selected from the group consisting of alanine lysine, ornithine and arginine.

High potency NHR ligand-insulin conjugates can also be prepared based on using a modified IGF I and IGF II sequence described in published International application no. WO 2010/080607, the disclosure of which is expressly incorporated herein by reference, as the insulin peptide component. More particularly, analogs of IGF I and IGF II that comprise a substitution of a tyrosine leucine dipeptide for the native IGF amino acids at positions corresponding to B 16 and B 17 of native insulin have a tenfold increase in potency at the insulin receptor.

In accordance with one embodiment the insulin peptide for use in the present disclosure comprises a B chain sequence of R₆₂-

X₂₅LCGX₂9X₃oLVX₃₃X₃₄LYLVCGX₄iX₄₂GFX₄5 (SEQ ID NO: 20) and an A chain sequence of GIVX₄X5CCX8X₉XioCXi2LXi₄Xi5LXi7Xi8Xi9CX2i-R53 (SEQ ID NO: 29) wherein

X₄ is glutamic acid or aspartic acid;

X5 is glutamine or glutamic acid

X₈ is histidine, threonine or phenylalanine;

X9 is serine, arginine, lysine, ornithine or alanine;

X10 is isoleucine or serine;

X12 is serine or aspartic acid

Xi₄ is tyrosine, arginine, lysine, ornithine or alanine;

Xi₅ is glutamine, glutamic acid, arginine, alanine, lysine, ornithine or leucine;

Xi₇ is glutamine, glutamic acid, arginine, aspartic acid, lysine or ornithine;

X19 is tyrosine, 4-methoxy-phenylalanine or 4-amino phenylalanine;

X25 is histidine or threonine;

X29 is selected from the group consisting of alanine, glycine and serine;

X₃₀ is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;

X₃₃ is selected from the group consisting of aspartic acid, glutamine and glutamic acid; X₃₄ is selected from the group consisting of alanine and threonine;

X₄₂ is selected from the group consisting of alanine, lysine, ornithine and arginine; X₄5 is tyrosine, histidine, asparagine or phenylalanine;

R₆₂ is selected from the group consisting of AYRPSE (SEQ ID NO: 14), FVNQ (SEQ ID NO: 12), PGPE (SEQ ID NO: 11), a tripeptide glycine-proline-glutamic acid, a tripeptide valine-asparagine-glutamine, a dipeptide proline-glutamic acid, a dipeptide asparagine-glutamine, glutamine, glutamic acid and a bond; and Rs₃ is COOH or CONH₂. In one embodiment the A chain and the B chain are linked to one another by interchain disulfide bonds, including those that form between the A and B chains of native insulin. In an alternative embodiment the A and B chains are linked together as a linear single chain- insulin peptide.

In one embodiment the conjugates comprise an insulin peptide wherein the A chain comprises a sequence of GIVEQCCXiSICSLYQLENX₂CX₃ (SEQ ID NO: 30) and said B chain sequence comprises a sequence of X₄LCGX₅X₆LVEALYLVCGERGFF (SEQ ID NO: 31), wherein

Xi is selected from the group consisting of threonine and histidine;

X₂ is tyrosine, 4-methoxy-phenylalanine or 4-amino phenylalanine;

X₃ is selected from the group consisting of asparagine and glycine;

X₄ is selected from the group consisting of histidine and threonine;

X5 is selected from the group consisting of alanine, glycine and serine;

X₆ is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid.

In accordance with one embodiment an insulin analog is provided wherein the A chain of the insulin peptide comprises the sequence GIVEQCCX₈X9ICSLYQLENYCX₂₁- R₅ (SEQ ID NO: 73) or GIVEQCCX₈SICSLYQLXi₇NYCX₂i (SEQ ID NO: 32) and the B chain comprising the sequence R₆₂-X₂₅LCGX₂9X₃oLVX₃₃X₃₄LYLVCGX₄iX₄₂GFX₄₅YT-Zi- Bi (SEQ ID NO: 142), wherein

X₈ is selected from the group consisting of threonine and histidine;

X9 is serine, lysine, or alanine;

X₁₇ is glutamine or glutamic acid;

X₂₁ is asparagine or glycine; X25 is histidine or threonine;

X29 is selected from the group consisting of alanine, glycine and serine;

X33 is selected from the group consisting of aspartic acid and glutamic acid;

X₃₄ is selected from the group consisting of alanine and threonine;

R₆2 is selected from the group consisting of FVNQ (SEQ ID NO: 12), a tripeptide valine-asparagine-glutamine, a dipeptide asparagine-glutamine, glutamine and an N-terminal amine

Zi is a dipeptide selected from the group consisting of aspartate-lysine, lysine- proline, and proline-lysine; and

Bi is selected from the group consisting of threonine, alanine or a threonine-arginine- arginine tripeptide.

In accordance with one embodiment an insulin analog is provided wherein the A chain of the insulin peptide comprises the sequence GIVEQCCX8SICSLYQLX₁7NX₁₉CX₂₁ (SEQ ID NO: 32) and the B chain comprising the sequence

X₂₅LCGX₂₉X₃oLVEALYLVCGERGFF (SEQ ID NO: 33) wherein

X₈ is selected from the group consisting of threonine and histidine;

Xi7 is glutamic acid or glutamine;

X19 is tyrosine, 4-methoxy-phenylalanine or 4-amino phenylalanine;

X₂₁ is asparagine or glycine;

X25 is selected from the group consisting of histidine and threonine;

X₂₉ is selected from the group consisting of alanine, glycine and serine;

X₃o is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid. In a further embodiment the B chain comprises the sequence X₂₂VNQX₂₅LCGX₂₉X₃oLVEALYLVCGERGFFYT-Zi-B ₁ (SEQ ID NO: 34) wherein

X₂₂ is selected from the group consisting of phenylalanine and desamino- phenylalanine; X25 is selected from the group consisting of histidine and threonine;

X29 is selected from the group consisting of alanine, glycine and serine;

In accordance with some embodiments the A chain comprises the sequence

GrVEQCCX₈SICSLYQLX₁₇NXi9CX23 (SEQ ID NO: 32) or

GIVDECCX₈X9SCDLXi₄Xi₅LXi₇Xi₈ X₁9CX₂₁-R53 (SEQ ID NO: 35), and the B chain comprises the sequence X₂₅LCGX₂9X₃oLVX₃₃X₃₄LYLVCGDX₄₂GFX₄5 (SEQ ID NO: 36) wherein

X₈ is histidine or phenylalanine;

X9 and X₁₄ are independently selected from arginine, lysine, ornithine or alanine;

Xi5 is arginine, lysine, ornithine or leucine;

X₁₇ is glutamic acid or glutamine;

Xi₈ is methionine, asparagine or threonine;

X19 is tyrosine, 4-methoxy-phenylalanine or 4-amino phenylalanine;

X21 is alanine, glycine or asparagine;

X23 is asparagine or glycine;

X25 is selected from the group consisting of histidine and threonine;

X29 is selected from the group consisting of alanine, glycine and serine;

X33 is selected from the group consisting of aspartic acid and glutamic acid;

X3₄ is selected from the group consisting of alanine and threonine;

X₄2 is selected from the group consisting of alanine, lysine, ornithine and arginine;

X₄5 is tyrosine; and

R53 is COOH or CONH₂.

In a further embodiment the A chain comprises the sequence

GIVDECCX₈X₉SCDLXi₄Xi₅LXi₇Xi₈ X19CX21-R53 (SEQ ID NO: 35), and the B chain comprises the sequence X₂₅LCGX₂9X₃oLVX₃₃X₃₄LYLVCGDX₄₂GFX₄5 (SEQ ID NO: 36) wherein

X₈ is histidine;

X9 and X₁₄ are independently selected from arginine, lysine, ornithine or alanine; Xi5 is arginine, lysine, ornithine or leucine;

X₁₇ is glutamic acid, aspartic acid, asparagine, lysine, ornithine or glutamine;

Xi8 is methionine, asparagine or threonine;

X19 is tyrosine, 4-methoxy-phenylalanine or 4-amino phenylalanine;

X₂i is alanine, glycine or asparagine;

X₂3 is asparagine or glycine;

X₂5 is selected from the group consisting of histidine and threonine;

X₂9 is selected from the group consisting of alanine, glycine and serine;

X33 is selected from the group consisting of aspartic acid and glutamic acid;

X3₄ is selected from the group consisting of alanine and threonine;

X₄₂ is selected from the group consisting of alanine, lysine, ornithine and arginine;

X₄5 is tyrosine or phenylalanine and

R₅₃ is COOH or CONH₂. In a further embodiment the A chain comprises the sequence GIVDECCHX₉SCDLXi₄Xi5LXi7MXi₉CX₂i-R53 (SEQ ID NO: 37), and the B chain comprises the sequence X₂₅LCGAX₃oLVDALYLVCGDX₄₂GFX₄5 (SEQ ID NO: 38) wherein

X9, X₁₄ and Xi₅ are independently ornithine, lysine or arginine;

Xi₇ is glutamic acid or glutamine;

X19 is tyrosine, 4-methoxy-phenylalanine or 4-amino phenylalanine;

X₂i is alanine, glycine or asparagine;

X₂5 is selected from the group consisting of histidine and threonine;

X30 is selected from the group consisting of histidine, aspartic acid and glutamic acid; X₄₂ is selected from the group consisting of alanine, lysine, ornithine and arginine; X₄5 is tyrosine or phenylalanine and

R₅₃ is COOH or CONH₂. In one embodiment the B chain is selected from the group consisting of HLCGAELVDALYLVCGDX₄₂GFY (SEQ ID NO: 39),

GPEHLCGAELVDALYLVCGDX₄₂GFY (SEQ ID NO: 40), GPEHLCGAELVDALYLVCGDX₄₂GFYFNPKT (SEQ ID NO: 41) and GPEHLCGAELVDALYLVCGDX₄₂GFYFNKPT (SEQ ID NO: 42), wherein X₄₂ is selected from the group consisting of ornithine, lysine and arginine. In a further embodiment the A chain comprises the sequence GIVDECCHX₉SCDLXi₄Xi₅LQMYCN-R₅3 (SEQ ID NO: 43), wherein X₉, X₁₄ and X15 are independently ornithine, lysine or arginine.

In another embodiment the A chain comprises the sequence

GIVDECCX8RSCDLYQLENXi₉CN-R53 (SEQ ID NO: 44) and the B chain comprises the sequence R₆₂-X₂₅LCGSHLVDALYLVCGDX₄₂GFX₄5 (SEQ ID NO: 45)

wherein

X₈ is threonine, histidine or phenylalanine;

X₁₉ is tyrosine, 4-methoxy-phenylalanine or 4-amino phenylalanine;

X₂5 is histidine or threonine;

X₄₂ is alanine, ornithine or arginine;

X₄5 is tyrosine histidine, asparagine or phenylalanine;

R₆₂ is selected from the group consisting of AYRPSE (SEQ ID NO: 14), FVNQ

(SEQ ID NO: 12), PGPE (SEQ ID NO: 11), a tripeptide glycine-proline-glutamic acid, a tripeptide valine-asparagine-glutamine, a dipeptide proline-glutamic acid, a dipeptide asparagine-glutamine, glutamine, glutamic acid and a bond; and R53 is COOH or CONH₂. and

R53 is COOH or CONH₂. In a further embodiment X₁₉ is Tyr.

In another embodiment the A chain comprises the sequence

GIVEQCCHSICSLYQLENXi₉CX₂i-R₅₃ (SEQ ID NO: 46) or

GIVDECCHRSCDLRRLEMXi₉CX₂i-R₅3 (SEQ ID NO: 47); and the B chain comprises the sequence FVNQHLCGS HLVE ALYLVCGERGFFYTPKT (SEQ ID NO: 2), or

GPETLCGAELVDALYLVCGDRGFYFNPKT (SEQ ID NO: 48)

wherein

X₁₉ is tyrosine, 4-methoxy phenylalanine or 4-amino-phenylalanine; and

X₂i is alanine, glycine or asparagine, optionally wherein X₁₉ is tyrosine, and X₂₁ is alanine or asparagine.

In another embodiment, the A chain comprises the sequence

GIVEQCCHSICSLYQLENYCX₂i-R₅3 (SEQ ID NO: 160) and the B chain comprises the sequence FVKQX₂₅LCGSHLVEALYLVCGERGFF-R63 (SEQ ID NO: 147), or

FVNQX₂₅LCGSHLVEALYLVCGERGFF-R₆3 (SEQ ID NO: 148), wherein X21 is alanine, glycine or asparagine; and

X25 is selected from the group consisting of histidine and threonine;

X₂₈ is proline, aspartic acid or glutamic acid; and

R₆₃ is selected from the group consisting of YTX₂₈KT (SEQ ID NO: 149), YTKPT (SEQ ID NO: 150), YTX₂₈K (SEQ ID NO: 152), YTKP (SEQ ID NO: 151), YTPK (SEQ ID NO: 70), YTX_2S, YT, Y and a bond. In one embodiment the B chain comprises the sequence

F VKQX₂5LCGS HLVE ALYLVC GERGFF YTEKT (SEQ ID NO: 162),

FVNQX₂₅LCGSHLVEALYLVCGERGFFYTDKT (SEQ ID NO: 164),

FVNQX₂₅LCGS HLVE ALYLVC GERGFF YTKPT (SEQ ID NO: 165) or

FVNQX₂₅LCGS HLVE ALYLVC GERGFF YTPKT (SEQ ID NO: 161) wherein

X₂₅ is selected from the group consisting of histidine and threonine.

Single Chain Insulin Peptide Agonists

As disclosed herein linking moieties can be used to link human insulin A and B chains, or analogs or derivatives thereof, wherein the carboxy terminus of the B25 amino acid of the B chain is directly linked to a first end of a linking moiety, wherein the second end of the linking moiety is directly linked to the amino terminus of the Al amino acid of the A chain via the intervening linking moiety.

In accordance with one embodiment the insulin peptide is a single chain insulin agonist that comprises the general structure B-LM-A wherein B represents an insulin B chain, A represents an insulin A chain, and LM represents a linking moiety linking the carboxy terminus of the B chain to the amino terminus of the A chain. Suitable linking moieties for joining the B chain to the A chain are disclosed herein under the header Linking Moieties for Single Chain-Insulin Analogs and the respective subheaders "Peptide linkers". In one embodiment the linking moiety comprises a linking peptide, and more particularly, in one embodiment the peptide represents an analog of the IGF-1 C peptide. Additional exemplary peptide linkers include but are not limited to the sequence XsiX₅₂GSSSX₅₇X₅₈ (SEQ ID NO: 49) or X₅₁X₅₂GSSSX₅₇X₅₈APQT (SEQ ID NO: 50) wherein X₅₁ is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine and proline, X52 is alanine, valine, leucine, isoleucine or proline and X57 or Xs₈ are independently arginine, lysine, cysteine, homocysteine, acetyl-phenylalanine or ornithine, optionally with a hydrophilic moiety linked to the side chain of the amino acid at position 7 or 8 of the linking moiety (i.e., at the X57 or Xs₈ position). Amino acid positions of the linking moiety are designated based on the corresponding position in the native C chain of IGF 1 (SEQ ID NO: 17). In another embodiment the peptide linking moiety comprises a 29 contiguous amino acid sequence having greater than 70%, 80%, 90% sequence identity to

S S S S X50 APPPS LPS PS RLPGPS DTPILPQX₅1 (SEQ ID NO: 68), wherein X₅₀ and X₅₁ are independently selected from arginine and lysine. In one embodiment the linking moiety is a non-peptide linker comprising a relatively short bifunctional non-peptide polymer linker that approximates the length of an 8-16 amino acid sequence. In one embodiment the non- peptide linker has the structure:

wherein m is an integer ranging from

10 to 14 and the linking moiety is linked directly to the B25 amino acid of the B chain. In accordance with one embodiment the non-peptide linking moiety is a polyethylene glycol linker of approximately 4 to 20, 8 to 18, 8 to 16, 8 to 14, 8 to 12, 10 to 14, 10 to 12 or 11 to 13 monomers.

In one embodiment an NHR ligand-insulin conjugate is provided that comprises an insulin peptide having the structure: IB-LM-IA, wherein IB comprises the sequence R₆2- X₂₅LCGX₂9X₃oLVX₃₃X₃₄LYLVCGX₄iX₄₂GFX₄5 (SEQ ID NO: 20), LM is a linking moiety as disclosed herein that covalently links IB to IA, and IA comprises the sequence

GIVX₄X5CCX8X9XioCXi2LXi₄Xi5LXi7Xi8Xi9CX2i-R53 (SEQ ID NO: 29), wherein

X₄ is glutamic acid or aspartic acid;

X5 is glutamine or glutamic acid;

X₈ is histidine or phenylalanine;

X10 is isoleucine or serine;

X12 is serine or aspartic acid;

X₁₄ is tyrosine, arginine, lysine, ornithine or alanine;

Xi₅ is arginine, lysine, ornithine or leucine;

X₁₇ is glutamic acid or glutamine;

X₁₈ is methionine, asparagine or threonine;

X19 is tyrosine, 4-methoxy-phenylalanine or 4-amino phenylalanine;

X21 is alanine, glycine or asparagine;

X25 is selected from the group consisting of histidine and threonine;

X29 is selected from the group consisting of alanine, glycine and serine; X30 is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;

X33 is selected from the group consisting of aspartic acid and glutamic acid;

X₃₄ is selected from the group consisting of alanine and threonine;

R5₃ is COOH or CONH₂, further wherein the amino acid at the designation X₄5 is directly bound to the linking moiety, LM (i.e., the designation IB-LM-IA as used herein is intended to represent that the B chain carboxyl terminus and the amino terminus of the A chain are directly linked to the linking moiety LM without any further intervening amino acids).

In one embodiment the linking moiety (LM) comprises an amino acid sequence of no more than 17 amino acids in length. In one embodiment the linking moiety comprises the sequence X51X52GSSSX57X58 (SEQ ID NO: 49) or X51X52GSSSX57X58APQT (SEQ ID NO: 50) wherein X₅₁ is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine and proline, Xs₂ is alanine, valine, leucine, isoleucine or proline and X57 or X58 are independently arginine, lysine, cysteine, homocysteine, acetyl-phenylalanine or ornithine, optionally with a hydrophilic moiety linked to the side chain of the amino acid at position 7 or 8 of the linking moiety (i.e., at the X57 or X58 position). Amino acid positions of the linking moiety are designated based on the corresponding position in the native C chain of IGF 1 (SEQ ID NO: 17). In one embodiment LM is GAGS S S RR APQT (SEQ ID NO: 23) or GAGSSSRR (SEQ ID NO: 22).

In another embodiment the linking moiety comprises a 29 contiguous amino acid sequence, directly linked to the carboxy terminal amino acid of the B chain, wherein said 29 contiguous amino acid sequence has greater than 70%, 80%, 90% sequence identity to

S S S S X50 APPPS LPS PS RLPGPS DTPILPQX₅₁ (SEQ ID NO: 68), wherein X₅₀ and X₅₁ are independently selected from arginine and lysine. In one embodiment the linking peptide comprises a total of 29 to 158 or 29 to 58 amino acids and comprises the sequence of SEQ ID NO: 68. In another embodiment the linking moiety comprises a 29 contiguous amino acid sequence, directly linked to the carboxy terminal amino acid of the B chain, wherein said 29 contiguous amino acid sequence has greater than 90% sequence identity to

S S S S X50 APPPS LPS PS RLPGPS DTPILPQX₅1 (SEQ ID NO: 68), wherein X₅₀ and X₅₁ are independently selected from arginine and lysine. In one embodiment the linking moiety comprises the sequence SSSSRAPPPSLPSPSRLPGPSDTPILPQK (SEQ ID NO: 51) or SSSSKAPPPSLPSPSRLPGPSDTPILPQR (SEQ ID NO: 52) optionally with one or two amino acid substitutions.

In accordance with one embodiment a single chain insulin agonist polypeptide is provided comprising a B chain and A chain of human insulin, or analogs or derivative thereof, wherein the last five carboxy amino acids of the native B chain are deleted (i.e., B26-B30), and amino acid B25 is linked to amino acid Al of the A chain via an intervening linking moiety. In one embodiment the linking moiety comprises the structure:

wherein m is an integer ranging from 10 to 14 and the linking moiety is linked directly to the B25 amino acid of the B chain.

In one embodiment an NHR ligand-insulin conjugate is provided comprising an insulin peptide having the general formula IB-LM-IA wherein IB comprises the sequence GPEHLCGAX₃oLVDALYLVCGDX₄₂GFYFNX₄₈X₄9 (SEQ ID NO: 163);

LM comprises the sequence SSSSRAPPPSLPSPSRLPGPSDTPILPQK (SEQ ID NO: 51), SSSSKAPPPSLPSPSRLPGPSDTPILPQR (SEQ ID NO: 52), GYGSSSRR (SEQ ID NO: 18), GAGS S S RRAPQT (SEQ ID NO: 23) or GAGSSSRR (SEQ ID NO: 22); and

IA comprises the sequence GIVDECCXsXgSCDLX^XisLXnXisXigCXii-Rss (SEQ ID NO: 35) wherein

X₈ is histidine or phenylalanine;

X9 is arginine, ornithine or alanine;

X₁₄ and Xi₅ are both arginine;

X₁₇ is glutamic acid;

Xis is methionine, asparagine or threonine

X19 is tyrosine, 4-methoxy-phenylalanine or 4-amino phenylalanine;

X21 is alanine or asparagine;

histidine or threonine; X30 is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;

X₄₂ is selected from the group consisting of alanine, ornithine and arginine;

X₄₈ is lysine or aspartic acid;

X₄9 is proline, ornithine or arginine; and

R53 is COOH.

Linking Moieties for Single Chain Insulin Analogs

Peptide linkers

In accordance with one embodiment the linking moiety is a peptide or peptidomimetic of 6-18, 8-18, 8-17, 8-12, 8-10, 13-17 or 13-15 amino acids (or amino acid analogs or derivatives thereof). In one embodiment the linking moiety is 8 to 17 amino acids in length and comprises the sequence X₅₁X₅₂GSSSRR (SEQ ID NO: 53) wherein X51 is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline and methionine, and X52 is a non-aromatic amino acid, including for example, alanine. In one embodiment the linking moiety is 8 to 17 amino acids in length and comprises a sequence that differs from X₅₁X₅₂GSSSRR (SEQ ID NO: 53) by a single amino acid substitution wherein the amino acid substitution is an amino acid that is pegylated at its side chain, further wherein X₅₁ is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline and methionine, and X52 is a non-aromatic amino acid, including for example, alanine.

In accordance with one embodiment the linking moiety is a derivative of the IGF 1 C chain sequence (GYGSSSRRAPQT; SEQ ID NO: 17). In one embodiment the derivative is a peptide that differs from SEQ ID NO: 17 by a single amino acid substitution of a lysine, cysteine ornithine, homocysteine, or acetyl-phenylalanine residue, and in a further embodiment the lysine, cysteine ornithine, homocysteine, or acetyl-phenylalanine amino acid is pegylated. In one further embodiment the linking moiety is a peptide that differs from SEQ ID NO: 17 by a single lysine substitution. In one specific embodiment the substitution is made at position 8 of SEQ ID NO: 17. Applicants have discovered that use of the IGF 1 C chain sequence and analogs thereof as a linking moiety will generate a single chain insulin polypeptide that has near wild type insulin activity. Furthermore, use of a IGF 1 C chain sequence analog as the linking moiety, wherein position 2 of the IGF 1 C chain sequence is modified, or the carboxy terminal four amino acids are deleted from the IGF 1 C chain sequence, produces a single chain insulin polypeptide that is selective for insulin (i.e., has a higher binding and/or activity at the insulin receptor compared to the IGF-1 receptor). In one embodiment the single chain insulin polypeptide has 5x, lOx, 20x, 30x, 40x, or 50x higher affinity or activity at the insulin receptor relative to the IGF- 1 receptor.

In accordance with one embodiment the linking moiety is a derivative of the IGF 1 C chain sequence (GYGSSSRRAPQT; SEQ ID NO: 17) and comprises a non-native sequence that differs from GYGSSSRR (SEQ ID NO: 18) or GAGSSSRRAPQT (SEQ ID NO: 23) by 1 to 3 amino acid substitutions, or 1 to 2 amino acid substitutions. In one embodiment at least one of the amino acid substitutions is a lysine or cysteine substitution, and in one embodiment the amino acid substitutions are conservative amino acid substitutions. In one embodiment the linking moiety is a peptide (or peptidomimetic) of 8 to 17 amino acids comprising a non-native amino acid sequence that differs from GYGSSSRR (SEQ ID NO: 18) or GAGSSSRRAPQT (SEQ ID NO: 23) by 1 amino acid substitution, including for example substitution with a lysine or cysteine. In one embodiment the linking moiety comprises the sequence GYGSSSRR (SEQ ID NO: 18) or GAGSSSRRAPQT (SEQ ID NO: 23). In one embodiment the linking moiety comprises the sequence GAGSSSRXsgAPQT (SEQ ID NO: 54), GYGSSSX₅₇X₅₈APQT (SEQ ID NO: 69), or an amino acid that differs from SEQ ID NO: 54 by a single amino acid substitution, wherein Xs₇ is arginine and Xs₈ is arginine, ornithine or lysine, and in a further embodiment a polyethylene glycol chain is linked to the side chain of the amino acid at position 8 of said linking moiety. In another embodiment the linking moiety comprises the sequence GX₅₂GSSSRX₅₈APQT (SEQ ID NO: 55), wherein Xs₂ is any non-aromatic amino acid, including for example, alanine, valine, leucine, isoleucine or proline, and Xs₈ represents an amino acid that has a

polyethylene chain covalently linked to its side chain. In one embodiment Xs₈ is a pegylated lysine.

In another embodiment, the linking moiety is an 8 to 17 amino acid sequence comprising the sequence GXs₂GSSSRR (SEQ ID NO: 56), wherein Xs₂ is any amino acid, a peptidomimetic of SEQ ID NO: 31, or an analog thereof that differs from SEQ ID NO: 31 by a single amino acid substitution at any of positions 1, 3, 4, 5, 6, 7 or 8 of SEQ ID NO: 31, with the proviso that when the linking peptide is longer than 8 amino acids Xs₂ is other than tyrosine. In accordance with one embodiment the linking moiety comprises an 8-17 amino acid sequence selected from the group consisting of GYGSSSRR (SEQ ID NO: 18), GAGSSSRR (SEQ ID NO: 22), GAGSSSRRA (SEQ ID NO: 57), GAGSSSRRAP (SEQ ID NO: 58), GAGSSSRRAPQ (SEQ ID NO: 59), GAGS S S RRAPQT (SEQ ID NO: 23), PYGSSSRR (SEQ ID NO: 61), PAGSSSRR (SEQ ID NO: 62), PAGSSSRRA (SEQ ID NO: 63), PAGSSSRRAP (SEQ ID NO: 64), PAGSSSRRAPQ (SEQ ID NO: 65),

P AGS S S RRAPQT (SEQ ID NO: 66). In accordance with one embodiment the linking moiety comprises an amino acid sequence that differs from GYGSSSRR (SEQ ID NO: 18), GAGSSSRR (SEQ ID NO: 22), GAGSSSRRA (SEQ ID NO: 57), GAGSSSRRAP (SEQ ID NO: 58), GAGSSSRRAPQ (SEQ ID NO: 59), GAGS S S RRAPQT (SEQ ID NO: 23), PYGSSSRR (SEQ ID NO: 61), PAGSSSRR (SEQ ID NO: 62), PAGSSSRRA (SEQ ID NO: 63), PAGSSSRRAP (SEQ ID NO: 64), PAGSSSRRAPQ (SEQ ID NO: 65),

P AGS S S RRAPQT (SEQ ID NO: 66) by a single pegylated amino acid including for example a pegylated lysine or pegylated cysteine amino acid substitution. In one

embodiment the pegylated amino acid is at position 8 of the linking moiety.

In one embodiment a peptide sequence named C-terminal peptide (CTP:

SSSSKAPPPSLPSPSRLPGPSDTPILPQR; SEQ ID NO: 52), which is prone to O-linked hyperglycosylation when the protein is expressed in a eukaryotic cellular expression system, can be used as a linker peptide. Surprisingly, applicants have discovered that the CTP peptide can be used to connect the B and A chains of insulin to form a single chain insulin analog while still maintaining high in vitro potency in a manner that the native proinsulin C- peptide can not. In one embodiment a NHR ligand-insulin conjugate is prepared comprising an insulin peptide having the carboxy terminus of the B chain linked to the amino terminus of the A chain via a CTP peptide. In another embodiment an insulin analog is provided as a two-chain construct with the CTP covalently linked to the C-terminus of the B-chain and/or the amino terminus of the B chain. In vitro and in vivo characterization reveals the CTP modified insulin analogs to have high potency in the absence of glycosylation, thus providing a mechanism to extend insulin action that is based on glycosylation, a natural approach to longer duration proteins.

Applicants have discovered that the primary sequence of the CTP peptide does not appear to be critical. Accordingly, in one embodiment the linking moiety comprises a peptide having a length of at least 18 amino acids that shares a similar amino acid content. In one embodiment the linking moiety comprises an analog of (SEQ ID NO: 68), wherein said analog differs from (SEQ ID NO: 68) by 1, 2, 3, 4, 5 or 6 amino acid substitutions. In one embodiment the linking peptide comprises a CTP peptide wherein amino acid substitutions are made at one or more positions selected from positions 1, 2, 3, 4, 10, 13, 15, and 21 of (SEQ ID NO: 68). In one embodiment the linking moiety comprises a 29 contiguous amino acid sequence, directly linked to the carboxy terminal amino acid of the B chain, wherein said 29 contiguous amino acid sequence has greater than 60, 80 or 90% sequence identity to SSSSX₅₀APPPSLPSPSRLPGPSDTPILPQX₅i (SEQ ID NO: 68), with the proviso that the sequence does not comprise a 15 amino acid sequence identical to a 15 amino acid sequence contained within SEQ ID NO 53. In another embodiment the linking moiety comprises a 29 contiguous amino acid sequence, directly linked to the carboxy terminal amino acid of the B chain, wherein at least 58% of the amino acids comprising the 29 contiguous amino acid sequence are selected from the group consisting of serine and proline.

In another embodiment the linking moiety comprises a 29 contiguous amino acid sequence, directly linked to the carboxy terminal amino acid of the B chain, wherein said 29 contiguous amino acid sequence has greater than 70%, 80%, 90% sequence identity to S S S S X50 APPPS LPS PS RLPGPS DTPILPQX₅1 (SEQ ID NO: 68), wherein X₅₀ and X₅₁ are independently selected from arginine and lysine, with the proviso that the sequence does not comprise a 15 amino acid sequence identical to a 15 amino acid sequence contained within SEQ ID NO 53. In another embodiment the linking moiety comprises a 29 contiguous amino acid sequence, directly linked to the carboxy terminal amino acid of the B chain, wherein said 29 contiguous amino acid sequence is an analog of (SEQ ID NO: 52), wherein said analog differs from (SEQ ID NO: 52) only by 1, 2, 3, 4, 5 or 6 amino acid modification, and in a further embodiment the amino acid modifications are conservative amino acid substitutions. In another embodiment the linking moiety comprises a 29 contiguous amino acid sequence, directly linked to the carboxy terminal amino acid of the B chain, wherein said 29 contiguous amino acid sequence is an analog of (SEQ ID NO: 52), wherein said analog differs from (SEQ ID NO: 52) only by 1, 2 or 3 amino acid substitutions.

Applicants have also found that multiple copies of the CTP peptide can be used as the linking peptide in single chain analogs and/or linked to the amino terminus of the B chain in single chain or two chain insulin analogs. The multiple copies of the CTP peptide can be identical or can differ in sequence and can be arranged in a head to tail or head to head orientation. In accordance with one embodiment an insulin analog is provided comprising a CTP peptide having the sequence

(SSSSX₅oAPPPSLPSPSRLPGPSDTPILPQX₅i)n (SEQ ID NO: 68), wherein n is an integer selected from the group consisting of 1, 2, 3 and 4 and X50 and X51 are independently selected from arginine and lysine.

In one embodiment the CTP peptide comprises the sequence

S S S S X50 APPPS LPS PS RLPGPS DTPILPQX₅1 (SEQ ID NO: 68), wherein X₅₀ and X₅₁ are independently selected from arginine and lysine. In another embodiment the CTP peptide comprises a sequence selected from the group consisting of

SSSSRAPPPSLPSPSRLPGPSDTPILPQK (SEQ ID NO: 51),

SSSSKAPPPSLPSPSRLPGPSDTPILPQR (SEQ ID NO: 52) or

SSSSRAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 67), and in a further embodiment the CTP peptide comprises the sequence SSSSRAPPPSLPSPSRLPGPSDTPILPQK (SEQ ID NO: 51).

Structure of L

In some embodiments, L is a bond. In these embodiments, Q and Y are conjugated together by reacting a nucleophilic reactive moiety on Q with and electrophilic reactive moiety on Y. In alternative embodiments, Q and Y are conjugated together by reacting an electrophilic reactive moiety on Q with a nucleophilic moiety on Y. In exemplary embodiments, L is an amide bond that forms upon reaction of an amine on Q (e.g. an ε- amine of a lysine residue) with a carboxyl group on Y. In alternative embodiments, Q and or Y are derivatized with a derivatizing agent before conjugation.

In some embodiments, L is a linking group. In some embodiments, L is a bifunctional linker and comprises only two reactive groups before conjugation to Q and Y. In embodiments where both Q and Y have electrophilic reactive groups, L comprises two of the same or two different nucleophilic groups (e.g. amine, hydroxyl, thiol) before conjugation to Q and Y. In embodiments where both Q and Y have nucleophilic reactive groups, L comprises two of the same or two different electrophilic groups (e.g. carboxyl group, activated form of a carboxyl group, compound with a leaving group) before conjugation to Q and Y. In embodiments where one of Q or Y has a nucleophilic reactive group and the other of Q or Y has an electrophilic reactive group, L comprises one nucleophilic reactive group and one electrophilic group before conjugation to Q and Y.

L can be any molecule with at least two reactive groups (before conjugation to Q and Y) capable of reacting with each of Q and Y. In some embodiments L has only two reactive groups and is bifunctional. L (before conjugation to the peptides) can be represented by Formula VI:

W- Linking Group (L) wherein W and J are independently nucleophilic or electrophilic reactive groups. In some embodiments W and J are either both nucleophilic groups or both electrophilic groups. In some embodiments one of W or J is a nucleophilic group and the other of W or J is an electrophilic group.

In some embodiments, L comprises a chain of atoms from 1 to about 60, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms long. In some embodiments, the chain atoms are all carbon atoms. In some embodiments, the chain atoms in the backbone of the linker are selected from the group consisting of C, O, N, and S. Chain atoms and linkers may be selected according to their expected solubility (hydrophilicity) so as to provide a more soluble conjugate. In some embodiments, L provides a functional group that is subject to cleavage by an enzyme or other catalyst or hydrolytic conditions found in the target tissue or organ or cell. In some embodiments, the length of L is long enough to reduce the potential for steric hindrance.

In some embodiments, the linking group is hydrophilic such as, for example, polyalkylene glycol. Before conjugation to the peptides of the composition, the hydrophilic linking group comprises at least two reactive groups (W and J), as described herein and as shown below:

W- Hydrophilic Linking Group

In specific embodiments, the linking group is polyethylene glycol (PEG). The PEG in certain embodiments has a molecular weight of about 100 Daltons to about 10,000 Daltons, e.g. about 500 Daltons to about 5000 Daltons. The PEG in some embodiments has a molecular weight of about 10,000 Daltons to about 40,000 Daltons.

In some embodiments, the hydrophilic linking group comprises either a maleimido or an iodoacetyl group and either a carboxylic acid or an activated carboxylic acid (e.g. NHS ester) as the reactive groups. In these embodiments, the maleimido or iodoacetyl group can be coupled to a thiol moiety on Q or Y and the carboxylic acid or activated carboxylic acid can be coupled to an amine on Q or Y with or without the use of a coupling reagent. Any appropriate coupling agent known to one skilled in the art can be used to couple the carboxylic acid with the amine. In some embodiments, the linking group is maleimido- PEG(20 kDa)-COOH, iodoacetyl-PEG(20 kDa)-COOH, maleimido-PEG(20 kDa)-NHS, or iodoacetyl-PEG(20 kDa)-NHS.

In some embodiments, the linking group is comprised of an amino acid, a dipeptide, a tripeptide, or a polypeptide, wherein the amino acid, dipeptide, tripeptide, or polypeptide comprises at least two activating groups, as described herein. In some embodiments, the linking group (L) comprises a moiety selected from the group consisting of: amino, ether, thioether, maleimido, disulfide, amide, ester, thioester, alkene, cycloalkene, alkyne, trizoyl, carbamate, carbonate, cathepsin B-cleavable, and hydrazone. In some embodiments, the linking group is an amino acid selected from the group Asp, Glu, homoglutamic acid, homocysteic acid, cysteic acid, gamma-glutamic acid. In some embodiments, the linking group is a dipeptide selected from the group consisting of: Ala- Ala, β-Ala- β-Ala, Leu-Leu, Pro-Pro, γ-aminobutyric acid- γ-aminobutyric acid, and γ-Glu- γ-Glu. In one embodiment L comprises gamma-glutamic acid.

In embodiments where Q and Y are conjugated together by reacting a carboxylic acid with an amine, an activating agent can be used to form an activated ester of the carboxylic acid. The activated ester of the carboxylic acid can be, for example, N-hydroxysuccinimide (NHS), tosylate (Tos), mesylate, triflate, a carbodiimide, or a hexafluorophosphate. In some embodiments, the carbodiimide is 1,3-dicyclohexylcarbodiimide (DCC), 1,1'- carbonyldiimidazole (CDI), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), or 1,3-diisopropylcarbodiimide (DICD). In some embodiments, the

hexafluorophosphate is selected from a group consisting of hexafluorophosphate benzotriazol- l-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-l-yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 2-(lH-7- azabenzotriazol-l-yl)-l,l,3,3-tetramethyl uranium hexafluorophosphate (HATU), and o- benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate (HBTU).

In some embodiments, Q comprises a nucleophilic reactive group (e.g. the amino group, thiol group, or hydroxyl group of the side chain of lysine, cysteine or serine) that is capable of conjugating to an electrophilic reactive group on Y or L. In some embodiments, Q comprises an electrophilic reactive group (e.g. the carboxylate group of the side chain of Asp or Glu) that is capable of conjugating to a nucleophilic reactive group on Y or L. In some embodiments, Q is chemically modified to comprise a reactive group that is capable of conjugating directly to Y or to L. In some embodiments, Q is modified at the C-terminal to comprise a natural or nonnatural amino acid with a nucleophilic side chain, such as an amino acid represented by Formula I, Formula II, or Formula III, as previously described herein (see Acylation and alkylatiori). In exemplary embodiments, the C-terminal amino acid of Q is selected from the group consisting of lysine, ornithine, serine, cysteine, and homocysteine. For example, the C-terminal amino acid of Q can be modified to comprise a lysine residue. In some embodiments, Q is modified at the C-terminal amino acid to comprise a natural or nonnatural amino acid with an electrophilic side chain such as, for example, Asp and Glu. In some embodiments, an internal amino acid of Q is substituted with a natural or nonnatural amino acid having a nucleophilic side chain, such as an amino acid represented by Formula I, Formula II, or Formula III, as previously described herein (see Acylation and alkylation). In exemplary embodiments, the internal amino acid of Q that is substituted is selected from the group consisting of lysine, ornithine, serine, cysteine, and homocysteine. For example, an internal amino acid of Q can be substituted with a lysine residue. In some embodiments, an internal amino acid of Q is substituted with a natural or nonnatural amino acid with an electrophilic side chain, such as, for example, Asp and Glu.

In some embodiments, Y comprises a reactive group that is capable of conjugating directly to Q or to L. In some embodiments, Y comprises a nucleophilic reactive group (e.g. amine, thiol, hydroxyl) that is capable of conjugating to an electrophilic reactive group on Q or L. In some embodiments, Y comprises electrophilic reactive group (e.g. carboxyl group, activated form of a carboxyl group, compound with a leaving group) that is capable of conjugating to a nucleophilic reactive group on Q or L.

Stability of L in vivo

In some embodiments, L is stable in vivo. In some embodiments, L is stable in blood serum for at least 5 minutes, e.g. less than 25%, 20%, 15%, 10% or 5% of the conjugate is cleaved when incubated in serum for a period of 5 minutes. In other embodiments, L is stable in blood serum for at least 10, or 20, or 25, or 30, or 60, or 90, or 120 minutes, or 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18 or 24 hours. In these embodiments, L does not comprise a functional group that is capable of undergoing hydrolysis in vivo. In some exemplary embodiments, L is stable in blood serum for at least about 72 hours. Nonlimiting examples of functional groups that are not capable of undergoing significant hydrolysis in vivo include amides, ethers, and thioethers. For example, the following compound is not capable of undergoing significant hydrolysis in vivo:

In some embodiments, L is hydrolyzable in vivo. In these embodiments, L comprises a functional group that is capable of undergoing hydrolysis in vivo. Nonlimiting examples of functional groups that are capable of undergoing hydrolysis in vivo include esters, anhydrides, and thioesters. For example the following compound is capable of undergoing hydrolysis in vivo because it comprises an ester group:

In some exemplary embodiments L is labile and undergoes substantial hydrolysis within 3 hours in blood plasma at 37 °C, with complete hydrolysis within 6 hours. In some exemplary embodiments, L is not labile.

In some embodiments, L is metastable in vivo. In these embodiments, L comprises a functional group that is capable of being chemically or enzymatically cleaved in vivo (e.g., an acid-labile, reduction-labile, or enzyme-labile functional group), optionally over a period of time. In these embodiments, L can comprise, for example, a hydrazone moiety, a disulfide moiety, or a cathepsin-cleavable moiety. When L is metastable, and without intending to be bound by any particular theory, the Q-L-Y conjugate is stable in an extracellular environment, e.g., stable in blood serum for the time periods described above, but labile in the intracellular environment or conditions that mimic the intracellular environment, so that it cleaves upon entry into a cell. In some embodiments when L is metastable, L is stable in blood serum for at least about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, or 48 hours, for example, at least about 48, 54, 60, 66, or 72 hours, or about 24-48, 48-72, 24-60, 36-48, 36-72, or 48-72 hours.

Pegylation of insulin peptides

Applicants have discovered that covalent linkage of a hydrophilic moiety to the insulin single chain analogs disclosed herein provide analogs having slower onset, extended duration and exhibit a basal profile of activity. In one embodiment, the insulin peptides disclosed herein are further modified to comprise a hydrophilic moiety covalently linked to the side chain of an amino acid at a position selected from the group consisting of A9, A14 and A15 of the A chain or at the N-terminal alpha amine of the B chain (e.g. at position B 1 for insulin based B chain or position B2 for IGF-1 based B chain) or at the side chain of an amino acid at position B 1, B2, B 10, B22, B28 or B29 of the B chain or at any position of the linking moiety that links the A chain and B chain. In exemplary embodiments, this hydrophilic moiety is covalently linked to a Lys, Cys, Orn, homocysteine, or acetyl- phenylalanine residue at any of these positions. In one embodiment the hydrophilic moiety is covalently linked to the side chain of an amino acid of the linking moiety.

Exemplary hydrophilic moieties include polyethylene glycol (PEG), for example, of a molecular weight of about 1,000 Daltons to about 40,000 Daltons, or about 20,000 Daltons to about 40,000 Daltons. Additional suitable hydrophilic moieties include, polypropylene glycol, polyoxyethylated polyols (e.g., POG), polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), polyoxyalkylenes, polyethylene glycol propionaldehyde, copolymers of ethylene glycol/propylene glycol, monomethoxy- polyethylene glycol, mono-(Cl-ClO) alkoxy- or aryloxy-polyethylene glycol,

carboxymethylcellulose, polyacetals, polyvinyl alcohol (PVA), polyvinyl pyrrolidone, poly- 1, 3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, poly (beta-amino acids) (either homopolymers or random copolymers), poly(n- vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers (PPG) and other polyakylene oxides, polypropylene oxide/ethylene oxide copolymers, colonic acids or other polysaccharide polymers, Ficoll or dextran and mixtures thereof.

The hydrophilic moiety, e.g., polyethylene glycol chain in accordance with some embodiments has a molecular weight selected from the range of about 500 to about 40,000 Daltons. In one embodiment the hydrophilic moiety, e.g. PEG, has a molecular weight selected from the range of about 500 to about 5,000 Daltons, or about 1,000 to about 5,000 Daltons. In another embodiment the hydrophilic moiety, e.g., PEG, has a molecular weight of about 10,000 to about 20,000 Daltons. In yet other exemplary embodiment the hydrophilic moiety, e.g., PEG, has a molecular weight of about 20,000 to about 40,000 Daltons. In one embodiment the hydrophilic moiety, e.g. PEG, has a molecular weight of about 20,000 Daltons. In one embodiment an insulin peptide is provided wherein one or more amino acids of the analog are pegylated, and the combined molecular weight of the covalently linked PEG chains is about 20,000 Daltons.

In one embodiment dextrans are used as the hydrophilic moiety. Dextrans are polysaccharide polymers of glucose subunits, predominantly linked by ocl-6 linkages. Dextran is available in many molecular weight ranges, e.g., about 1 kD to about 100 kD, or from about 5, 10, 15 or 20 kD to about 20, 30, 40, 50, 60, 70, 80 or 90 kD.

Linear or branched polymers are contemplated. Resulting preparations of conjugates may be essentially monodisperse or polydisperse, and may have about 0.5, 0.7, 1, 1.2, 1.5 or 2 polymer moieties per peptide.

In one embodiment the hydrophilic moiety is a polyethylene glycol (PEG) chain, optionally linked to the side chain of an amino acid at a position selected from the group consisting of A9, A14 and A15 of the A chain, positions B l, B2, B 10, B22, B28 or B29 of the B chain, at the N-terminal alpha amine of the B chain, or at any position of the linking moiety of a single chain insulin analog that links the A chain and B chain, including for example at position C8. In one embodiment the single chain insulin analog comprises a peptide linking moiety of 8 to 12 amino acids, wherein one of the amino acids of the linking moiety has a polyethylene chain covalently bound to its side chain. In one embodiment the single chain insulin analog comprises a peptide linking moiety of 8 to 12 amino acids, wherein an amino acid of the linking moiety is pegylated and one or more amino acid at a position selected from the group consisting of A9, A14 and A15 of the A chain, positions B l, B2, B 10, B22, B28 or B29 of the B chain is also pegylated. In one embodiment the total molecular weight of the covalently linked PEG chain(s) is about 20,000 Daltons.

In one embodiment a single chain insulin analog comprises a linking moiety of 8 to 12 amino acids, wherein one of the amino acids of the linking moiety has a 20,000 Dalton polyethylene chain covalently bound to its side chain. In another embodiment an insulin analog comprises a peptide linking moiety of 8 to 12 amino acids, wherein one of the amino acids of the linking moiety has a polyethylene chain covalently bound to its side chain and a second PEG chain is linked to the N-terminal alpha amine of the B chain (e.g. at position B 1 for insulin based B chain or position B2 for IGF-1 based B chain) or at the side chain of an amino acid at position B 1, B2 and B29 of the B chain. In one embodiment when two PEG chains are linked to the insulin peptide, each PEG chain has a molecular weight of about 10,000 Daltons. In one embodiment when the PEG chain is linked to an 8 to 12 amino acid linking moiety, the PEG chain is linked at position C7 or C8 of the linking moiety and in one embodiment the PEG chain is linked at position C8 of the linking moiety. In one embodiment when two PEG chains are linked to the single chain insulin analog, with one PEG chain linked at position C8 and the second PEG is linked at A9, A14, A15, B l, B2, B 10, B22, B28 or B29. Hydrophilic moieties such as polyethylene glycol can be attached to the NHR ligand- insulin conjugate under any suitable conditions used to react a protein with an activated polymer molecule. Any means known in the art can be used, including via acylation, reductive alkylation, Michael addition, thiol alkylation or other chemoselective

conjugation/ligation methods through a reactive group on the PEG moiety (e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group) to a reactive group on the target compound (e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group). Activating groups which can be used to link the water soluble polymer to one or more proteins include without limitation sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane and 5-pyridyl. If attached to the peptide by reductive alkylation, the polymer selected should have a single reactive aldehyde so that the degree of

polymerization is controlled. See, for example, Kinstler et al., Adv. Drug. Delivery Rev. 54: 477-485 (2002); Roberts et al., Adv. Drug Delivery Rev. 54: 459-476 (2002); and Zalipsky et al., Adv. Drug Delivery Rev. 16: 157-182 (1995).

Acylation

In some embodiments, the insulin analog is modified to comprise an acyl group. The acyl group can be covalently linked directly to an amino acid of the NHR ligand-insulin conjugate, or indirectly to an amino acid of the NHR ligand-insulin conjugate via a spacer, wherein the spacer is positioned between the amino acid of the NHR ligand-insulin conjugate and the acyl group. The NHR ligand-insulin conjugate may be acylated at the same amino acid position where a hydrophilic moiety is linked, or at a different amino acid position. For example, acylation may occur at any position including any of amino acid of the A or B chains as well as a position within the linking moiety, provided that the activity exhibited by the non-acylated NHR ligand-insulin conjugate is retained upon acylation.

Nonlimiting examples include acylation at positions A14 and A15 of the A chain, positions position B l for insulin based B chain or position B2 for IGF-1 based B chain or positions B 10, B22, B28 or B29 of the B chain or at any position of the linking moiety.

In one specific aspect of the invention, the insulin analog is modified to comprise an acyl group by direct acylation of an amine, hydroxyl, or thiol of a side chain of an amino acid of the NHR ligand-insulin conjugate. In some embodiments, the insulin analog is directly acylated through the side chain amine, hydroxyl, or thiol of an amino acid. In some embodiments, acylation is at position B28 or B29 (according to the amino acid numbering of the native insulin A and B chain sequences). In this regard, an insulin analog can be provided that has been modified by one or more amino acid substitutions in the A or B chain sequence, including for example at positions A14, A15, B l, B2, B IO, B22, B28 or B29 (according to the amino acid numbering of the native insulin A and B chain sequences) or at any position of the linking moiety with an amino acid comprising a side chain amine, hydroxyl, or thiol. In some specific embodiments of the invention, the direct acylation of the insulin peptide occurs through the side chain amine, hydroxyl, or thiol of the amino acid at position B28 or B29 (according to the amino acid numbering of the native insulin A and B chain sequences).

In accordance with one embodiment, the acylated insulin analogs comprise a spacer between the peptide and the acyl group. In some embodiments, the NHR ligand-insulin conjugate is covalently bound to the spacer, which is covalently bound to the acyl group. In some exemplary embodiments, the insulin peptide is modified to comprise an acyl group by acylation of an amine, hydroxyl, or thiol of a spacer, which spacer is attached to a side chain of an amino acid at position B28 or B29 (according to the amino acid numbering of the A or B chain of native insulin), or at any position of the spacer moiety. The amino acid of the NHR ligand-insulin conjugate to which the spacer is attached can be any amino acid comprising a moiety which permits linkage to the spacer. For example, an amino acid comprising a side chain -NH₂, -OH, or -COOH (e.g., Lys, Orn, Ser, Asp, or Glu) is suitable.

In some embodiments, the spacer between the NHR ligand-insulin conjugate and the acyl group is an amino acid comprising a side chain amine, hydroxyl, or thiol (or a dipeptide or tripeptide comprising an amino acid comprising a side chain amine, hydroxyl, or thiol). In some embodiments, the spacer comprises a hydrophilic bifunctional spacer. In a specific embodiment, the spacer comprises an amino poly(alkyloxy)carboxylate. In this regard, the spacer can comprise, for example, NH₂(CH₂CH₂0)_n(CH₂)_mCOOH, wherein m is any integer from 1 to 6 and n is any integer from 2 to 12, such as, e.g., 8-amino-3,6-dioxaoctanoic acid, which is commercially available from Peptides International, Inc. (Louisville, KY). In one embodiment, the hydrophilic bifunctional spacer comprises two or more reactive groups, e.g., an amine, a hydroxyl, a thiol, and a carboxyl group or any combinations thereof. In certain embodiments, the hydrophilic bifunctional spacer comprises a hydroxyl group and a carboxylate. In other embodiments, the hydrophilic bifunctional spacer comprises an amine group and a carboxylate. In other embodiments, the hydrophilic bifunctional spacer comprises a thiol group and a carboxylate. In some embodiments, the spacer between peptide the NHR ligand-insulin conjugate and the acyl group is a hydrophobic bifunctional spacer. Hydrophobic bifunctional spacers are known in the art. See, e.g., Bioconjugate Techniques, G. T. Hermanson (Academic Press, San Diego, CA, 1996), which is incorporated by reference in its entirety. In accordance with certain embodiments the bifunctional spacer can be a synthetic or naturally occurring amino acid comprising an amino acid backbone that is 3 to 10 atoms in length (e.g., 6-amino hexanoic acid, 5-aminovaleric acid, 7-aminoheptanoic acid, and 8- aminooctanoic acid). Alternatively, the spacer can be a dipeptide or tripeptide spacer having a peptide backbone that is 3 to 10 atoms (e.g., 6 to 10 atoms) in length. Each amino acid of the dipeptide or tripeptide spacer attached to the NHR ligand-insulin conjugate can be independently selected from the group consisting of: naturally-occurring and/or non- naturally occurring amino acids, including, for example, any of the D or L isomers of the naturally-occurring amino acids (Ala, Cys, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, Tyr), or any D or L isomers of the non-naturally occurring amino acids selected from the group consisting of: β-alanine (β -Ala), N-a-methyl- alanine (Me-Ala), aminobutyric acid (Abu), a-aminobutyric acid (γ-Abu), aminohexanoic acid (ε- Ahx), aminoisobutyric acid (Aib), aminomethylpyrrole carboxylic acid,

aminopiperidinecarboxylic acid, aminoserine (Ams), aminotetrahydropyran-4-carboxylic acid, arginine N-methoxy-N-methyl amide, β-aspartic acid (β-Asp), azetidine carboxylic acid, 3-(2-benzothiazolyl)alanine, a-tert-butylglycine, 2-amino-5-ureido-n-valeric acid (citrulline, Cit), β-Cyclohexylalanine (Cha), acetamidomethyl-cysteine, diaminobutanoic acid (Dab), diaminopropionic acid (Dpr), dihydroxyphenylalanine (DOPA),

dimethylthiazolidine (DMT A), γ-Glutamic acid (γ-Glu), homoserine (Hse), hydroxyproline (Hyp), isoleucine N-methoxy-N-methyl amide, methyl-isoleucine (MeHe), isonipecotic acid (Isn), methyl-leucine (MeLeu), methyl-lysine, dimethyl-lysine, trimethyl-lysine,

methanoproline, methionine- sulfoxide (Met(O)), methionine-sulfone (Met(02)), norleucine (Nle), methyl-norleucine (Me-Nle), norvaline (Nva), ornithine (Orn), para-aminobenzoic acid (PABA), penicillamine (Pen), methylphenylalanine (MePhe), 4-Chlorophenylalanine (Phe(4-Cl)), 4-fluorophenylalanine (Phe(4-F)), 4-nitrophenylalanine (Phe(4-N02)), 4- cyanophenylalanine ((Phe(4-CN)), phenylglycine (Phg), piperidinylalanine,

piperidinylglycine, 3,4-dehydroproline, pyrrolidinylalanine, sarcosine (Sar), selenocysteine (Sec), U-Benzyl-phosphoserine, 4-amino-3-hydroxy-6-methylheptanoic acid (Sta), 4-amino- 5-cyclohexyl-3-hydroxypentanoic acid (ACHPA), 4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA), l,2,3,4,-tetrahydro-isoquinoline-3-carboxylic acid (Tic), tetrahydropyranglycine, thienylalanine (Thi) , U-Benzyl-phosphotyrosine, O- Phosphotyrosine, methoxytyrosine, ethoxytyrosine, O-(bis-dimethylamino-phosphono)- tyrosine, tyrosine sulfate tetrabutylamine, methyl-valine (MeVal), 1 -amino- 1-cyclohexane carboxylic acid (Acx), aminovaleric acid, beta-cyclopropyl-alanine (Cpa), propargylglycine (Prg), allylglycine (Alg), 2-amino-2-cyclohexyl-propanoic acid (2-Cha), tertbutylglycine (Tbg), vinylglycine (Vg), 1 -amino- 1 -cyclopropane carboxylic acid (Acp), 1-amino-l- cyclopentane carboxylic acid (Acpe), alkylated 3-mercaptopropionic acid, 1-amino-l- cyclobutane carboxylic acid (Acb). In some embodiments the dipeptide spacer is selected from the group consisting of: Ala-Ala, β-Ala- β-Ala, Leu-Leu, Pro-Pro, γ-aminobutyric acid- γ-aminobutyric acid, and γ-Glu- γ-Glu.

The peptide the NHR ligand-insulin conjugate can be modified to comprise an acyl group by acylation of a long chain alkane of any size and can comprise any length of carbon chain. The long chain alkane can be linear or branched. In certain aspects, the long chain alkane is a C₄ to C30 alkane. For example, the long chain alkane can be any of a C₄ alkane, C₆ alkane, C₈ alkane, C10 alkane, C₁₂ alkane, C₁₄ alkane, C₁₆ alkane, C₁₈ alkane, C₂o alkane, C₂₂ alkane, C₂₄ alkane, C_¾ alkane, C₂₈ alkane, or a C30 alkane. In some embodiments, the long chain alkane comprises a C₈ to C₂o alkane, e.g., a C₁₄ alkane, C₁₆ alkane, or a C₁₈ alkane.

In some embodiments, an amine, hydroxyl, or thiol group of the NHR ligand-insulin conjugate is acylated with a cholesterol acid. In a specific embodiment, the peptide is linked to the cholesterol acid through an alkylated des-amino Cys spacer, i.e., an alkylated 3- mercaptopropionic acid spacer. Suitable methods of peptide acylation via amines, hydroxyls, and thiols are known in the art. See, for example, Miller, Biochem Biophys Res Commun 218: 377-382 (1996); Shimohigashi and Stammer, Int J Pept Protein Res 19: 54-62 (1982); and Previero et al., Biochim Biophys Acta 263: 7-13 (1972) (for methods of acylating through a hydroxyl); and San and Silvius, J Pept Res 66: 169-180 (2005) (for methods of acylating through a thiol); Bioconjugate Chem. "Chemical Modifications of Proteins: History and Applications" pages 1, 2-12 (1990); Hashimoto et al., Pharmacuetical Res. "Synthesis of Palmitoyl Derivatives of Insulin and their Biological Activity" Vol. 6, No: 2 pp.l71-176 (1989).

The acyl group of the acylated peptide the NHR ligand-insulin conjugate can be of any size, e.g., any length carbon chain, and can be linear or branched. In some specific embodiments of the invention, the acyl group is a C₄ to C30 fatty acid. For example, the acyl group can be any of a C₄ fatty acid, C₆ fatty acid, C₈ fatty acid, C₁₀ fatty acid, C₁₂ fatty acid, C₁₄ fatty acid, C₁₆ fatty acid, C₁₈ fatty acid, C₂o fatty acid, C₂₂ fatty acid, C₂₄ fatty acid, C_¾ fatty acid, C₂₈ fatty acid, or a C30 fatty acid. In some embodiments, the acyl group is a C₈ to C₂o fatty acid, e.g., a C₁₄ fatty acid or a C₁₆ fatty acid.

In certain embodiments, the the NHR ligand-insulin conjugate comprises an amino acid with a side chain covalently attached, optionally through a spacer, to an acyl group or an alkyl group, wherein the acyl group or alkyl group is non-native to a naturally-occurring amino acid. In one embodiment the covalently linked acyl or alkyl group has a carboxylate at its free end. The acyl group in some embodiments is a C4 to C30 fatty acyl group, optionally with carboxylate groups at each end. In one embodiment the the NHR ligand- insulin conjugate comprises a covalently linked C4 to C30 acyl group optionally with a carboxylate at its free end. In specific aspects, the acyl group or alkyl group is covalently attached to the side chain of an amino acid of the insulin peptide at position B28 or B29.

In an alternative embodiment, the acyl group is a bile acid. The bile acid can be any suitable bile acid, including, but not limited to, cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, and cholesterol acid.

Alkylation

In some embodiments, the NHR ligand-insulin conjugate is modified to comprise an alkyl group. The alkyl group can be covalently linked directly to an amino acid of the insulin analog, or indirectly to an amino acid of the NHR ligand-insulin conjugate via a spacer, wherein the spacer is positioned between the amino acid of the NHR ligand-insulin conjugate and the alkyl group. The alkyl group can be attached to the NHR ligand-insulin conjugate via an ether, thioether, or amino linkage. For example, the NHR ligand-insulin conjugate may be alkylated at the same amino acid position where a hydrophilic moiety is linked, or at a different amino acid position.

Alkylation can be carried out at any position within the NHR ligand-insulin conjugate, including for example in the C-terminal region of the B chain or at a position in the linking moiety, provided that insulin activity is retained. In a specific aspect of the invention, the NHR ligand-insulin conjugate is modified to comprise an alkyl group by direct alkylation of an amine, hydroxyl, or thiol of a side chain of an amino acid of the NHR ligand-insulin conjugate. In some embodiments, the NHR ligand-insulin conjugate is directly alkylated through the side chain amine, hydroxyl, or thiol of an amino acid. In some specific embodiments of the invention, the direct alkylation of the NHR ligand-insulin conjugate occurs through the side chain amine, hydroxyl, or thiol of the amino acid at position A14, A15, B l (for insulin based B chains), B2 (for IGF-1 based B chains), B IO, B22, B28 or B29 (according to the amino acid numbering of the A and B chain of native insulin).

In some embodiments of the invention, the NHR ligand-insulin conjugate comprises a spacer between the peptide and the alkyl group. In some embodiments, the NHR ligand- insulin conjugate is covalently bound to the spacer, which is covalently bound to the alkyl group. In some exemplary embodiments, the NHR ligand-insulin conjugate is modified to comprise an alkyl group by alkylation of an amine, hydroxyl, or thiol of a spacer, wherein the spacer is attached to a side chain of an amino acid at position A14, A15, B l (for insulin based B chains), B2 (for IGF-1 based B chains), B IO, B22, B28 or B29 (according to the amino acid numbering of the A and B chains of native insulin). The amino acid of the NHR ligand-insulin conjugate to which the spacer is attached can be any amino acid (e.g., a singly a-substituted amino acid or an α,α-disubstituted amino acid) comprising a moiety which permits linkage to the spacer. An amino acid of the NHR ligand-insulin conjugate comprising a side chain -N¾, -OH, or -COOH (e.g., Lys, Orn, Ser, Asp, or Glu) is suitable. In some embodiments, the spacer between the peptide the NHR ligand-insulin conjugate and the alkyl group is an amino acid comprising a side chain amine, hydroxyl, or thiol or a dipeptide or tripeptide comprising an amino acid comprising a side chain amine, hydroxyl, or thiol.

In the instance in which the alpha amine is alkylated, the spacer amino acid can be any amino acid. For example, the spacer amino acid can be a hydrophobic amino acid, e.g., Gly, Ala, Val, Leu, He, Trp, Met, Phe, Tyr. Alternatively, the spacer amino acid can be an acidic residue, e.g., Asp and Glu. In exemplary embodiments, the spacer amino acid can be a hydrophobic amino acid, e.g., Gly, Ala, Val, Leu, He, Trp, Met, Phe, Tyr, 6-amino hexanoic acid, 5-aminovaleric acid, 7-aminoheptanoic acid, 8-aminooctanoic acid.

Alternatively, the spacer amino acid can be an acidic residue, e.g., Asp and Glu, provided that the alkylation occurs on the alpha amine of the acidic residue. In the instance in which the side chain amine of the spacer amino acid is alkylated, the spacer amino acid is an amino acid comprising a side chain amine, e.g., an amino acid of Formula I (e.g., Lys or Orn). In this instance, it is possible for both the alpha amine and the side chain amine of the spacer amino acid to be alkylated, such that the peptide is dialkylated. Embodiments of the invention include such dialkylated molecules.

In some embodiments, the spacer comprises a hydrophilic bifunctional spacer. In a specific embodiment, the spacer comprises an amino poly(alkyloxy)carboxylate. In this regard, the spacer can comprise, for example, NH₂(CH₂CH₂0)_n(CH₂)_mCOOH, wherein m is any integer from 1 to 6 and n is any integer from 2 to 12, such as, e.g., 8-amino-3,6- dioxaoctanoic acid, which is commercially available from Peptides International, Inc.

(Louisville, KY). In some embodiments, the spacer between peptide the NHR ligand-insulin conjugate and the alkyl group is a hydrophilic bifunctional spacer. In certain embodiments, the hydrophilic bifunctional spacer comprises two or more reactive groups, e.g., an amine, a hydroxyl, a thiol, and a carboxyl group or any combinations thereof. In certain

embodiments, the hydrophilic bifunctional spacer comprises a hydroxyl group and a carboxylate. In other embodiments, the hydrophilic bifunctional spacer comprises an amine group and a carboxylate. In other embodiments, the hydrophilic bifunctional spacer comprises a thiol group and a carboxylate.

The spacer (e.g., amino acid, dipeptide, tripeptide, hydrophilic bifunctional spacer, or hydrophobic bifunctional spacer) is 3 to 10 atoms (e.g., 6 to 10 atoms, (e.g., 6, 7, 8, 9, or 10 atoms)) in length. In more specific embodiments, the spacer is about 3 to 10 atoms (e.g., 6 to 10 atoms) in length and the alkyl is a C₁₂ to C₁₈ alkyl group, e.g., C₁₄ alkyl group, C₁₆ alkyl group, such that the total length of the spacer and alkyl group is 14 to 28 atoms, e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 atoms. In some

embodiments the length of the spacer and alkyl is 17 to 28 (e.g., 19 to 26, 19 to 21) atoms.

In accordance with one embodiment the bifunctional spacer is a synthetic or non- naturally occurring amino acid comprising an amino acid backbone that is 3 to 10 atoms in length (e.g., 6-amino hexanoic acid, 5-aminovaleric acid, 7-aminoheptanoic acid, and 8- aminooctanoic acid). Alternatively, the spacer can be a dipeptide or tripeptide spacer having a peptide backbone that is 3 to 10 atoms (e.g., 6 to 10 atoms) in length. The dipeptide or tripeptide spacer attached to the NHR ligand-insulin conjugate can be composed of naturally-occurring and/or non-naturally occurring amino acids, including, for example, any of the amino acids taught herein. In some embodiments the spacer comprises an overall negative charge, e.g., comprises one or two negatively charged amino acids. In some embodiments the dipeptide spacer is selected from the group consisting of: Ala- Ala, β-Ala- β-Ala, Leu-Leu, Pro-Pro, γ-aminobutyric acid- γ-aminobutyric acid, and γ-Glu- γ-Glu. In one embodiment the dipeptide spacer is γ-Glu- γ-Glu.

Suitable methods of peptide alkylation via amines, hydroxyls, and thiols are known in the art. For example, a Williamson ether synthesis can be used to form an ether linkage between the insulin peptide and the alkyl group. Also, a nucleophilic substitution reaction of the peptide with an alkyl halide can result in any of an ether, thioether, or amino linkage.

The alkyl group of the alkylated peptide the NHR ligand-insulin conjugate can be of any size, e.g., any length carbon chain, and can be linear or branched. In some embodiments of the invention, the alkyl group is a C₄ to C₃o alkyl. For example, the alkyl group can be any of a C₄ alkyl, C₆ alkyl, C₈ alkyl, Cio alkyl, C_i2 alkyl, C_u alkyl, C_l6 alkyl, C_i8 alkyl, C₂₀ alkyl,

C₂₂ alkyl, C₂₄ alkyl, C_¾ alkyl, C₂₈ alkyl, or a C₃o alkyl. In some embodiments, the alkyl group is a C₈ to C₂o alkyl, e.g., a C₁₄ alkyl or a C₁₆ alkyl.

In some specific embodiments, the alkyl group comprises a steroid moiety of a bile acid, e.g., cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, and cholesterol acid.

When a long chain alkane is alkylated by the NHR ligand-insulin conjugate or the spacer, the long chain alkane may be of any size and can comprise any length of carbon chain. The long chain alkane can be linear or branched. In certain aspects, the long chain alkane is a C₄ to C₃o alkane. For example, the long chain alkane can be any of a C₄ alkane, C₆ alkane, C₈ alkane, Cio alkane, C₁₂ alkane, C₁₄ alkane, C₁₆ alkane, C₁₈ alkane, C₂o alkane,

C₂₂ alkane, C₂₄ alkane, C_¾ alkane, C₂₈ alkane, or a C₃o alkane. In some embodiments the long chain alkane comprises a C₈ to C₂o alkane, e.g., a C₁₄ alkane, C₁₆ alkane, or a C₁₈ alkane.

Also, in some embodiments alkylation can occur between the insulin analog and a cholesterol moiety. For example, the hydroxyl group of cholesterol can displace a leaving group on the long chain alkane to form a cholesterol-insulin peptide product.

Self Cleaving Dipeptide Element

In accordance with one embodiment the insulin peptide of the conjugates disclosed herein are further modified to comprise a self cleaving dipeptide element. In one

embodiment the dipeptide element comprises the structure U-J, wherein U is an amino acid or a hydroxyl acid and J is an N-alkylated amino acid. In one embodiment one or more dipeptide elements are linked to the NHR ligand-insulin conjugate through an amide bond formed through one or more amino groups selected from the N-terminal amino group of the A or B chain of the insulin component, or the side chain amino group of an amino acid present in the conjugate. In accordance with one embodiment one or more dipeptide elements are linked to the NHR ligand-insulin conjugate at an amino group selected from the N-terminal amino group of the conjugate, or the side chain amino group of an aromatic amine of a 4-amino-phenylalanine residue present at a position corresponding to position A19, B 16 or B25 of native insulin, or a side chain of an amino acid of the linking moiety of a single chain insulin analog.

In one embodiment the dipeptide prodrug element comprises the general structure of Formula X:

wherein

Ri_, R_2, R₄ and R₈ are independently selected from the group consisting of H, Ci-Ci₈ alkyl, C₂-Ci₈ alkenyl, (Ci-Cis alkyl)OH, (Ci-Cis alkyl)SH, (C₂-C₃ alkyl)SCH₃, (d-C₄ alkyl)CONH₂, (d-C₄ alkyl)COOH, (d-C₄ alkyl)NH₂, (Ci-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-Ci₀ aryl)R₇, (Ci-C₄ alkyl)(C₃-C₉ heteroaryl), and C1-C12 alkyl(W)Ci-Ci₂ alkyl, wherein W is a heteroatom selected from the group consisting of N, S and O, or Ri and R₂ together with the atoms to which they are attached form a C₃-Ci₂ cycloalkyl or aryl; or R₄ and R₈ together with the atoms to which they are attached form a C₃-C₆ cycloalkyl;

R₃ is selected from the group consisting of Ci-Ci₈ alkyl, (Ci-Ci₈ alkyl)OH, (Ci-Ci₈ alkyl)NH₂, (Ci-Cis alkyl)SH, (C₀-C₄ alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-Cio aryl)R₇, and (C₁-C₄ alkyl)(C₃-C9 heteroaryl) or R₄ and R₃ together with the atoms to which they are attached form a pyrrolidine ring;

R₅ is NHR₆ or OH;

R₆ is H, Ci-C₈ alkyl; and

R₇ is selected from the group consisting of H and OH. In one embodiment when the prodrug element is linked to the N-terminal amine of the NHR ligand-insulin conjugate and R₄ and R₃ together with the atoms to which they are attached form a pyrrolidine ring, then at least one of Ri and R₂ are other than H. In one embodiment a complex is provided comprising the general structure U-J-(Q- L-Y), wherein Q-L-Y comprises any of the structures as described elsewhere in this disclosure and U-J is a dipeptide that is linked via an amide bond to an amine of the Q-L-Y conjugate. In one embodiment U-J is linked to amine present on the insulin peptide. In one embodiment U-J is linked to the N-terminal alpha amine of the A or B chain of the insulin peptide of the conjugate.

In one embodiment, a complex of the structure U-J-(Q-L-Y) is provided, wherein Q- L-Y comprises any of the structures as described elsewhere in this disclosure and wherein U is an amino acid or a hydroxy acid;

J is an N-alkylated amino acid linked to Q through an amide bond between a carboxyl moiety of B and an amine of Q. In one embodiment U-J comprises the structure:

wherein

(a) R 1 , R2 , R 4 and R 8 are independently selected from the group consisting of H, CI -CI 8 alkyl, C2-C18 alkenyl, (CI -CI 8 alkyl)OH, (CI -CI 8 alkyl)SH, (C2-C3 alkyl)SCH₃, (C1-C4 alkyl)CONH₂, (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH₂, (C1-C4

alkyl)NHC(NH₂ ⁺)NH₂, (C0-C4 alkyl)(C3-C6 cycloalkyl), (C0-C4 alkyl)(C2-C5

heterocyclic), (C0-C4 alkyl)(C6-C10 aryl)R⁷, (C1-C4 alkyl)(C3-C9 heteroaryl), and C1-C12 alkyl(Wl)Cl-C12 alkyl, wherein Wl is a heteroatom selected from the group consisting of N, S and O, or

(ii) R 1 and R 2 together with the atoms to which they are attached form a C3-C12 cycloalkyl or aryl; or

(iii) R 4 and R 8 together with the atoms to which they are attached form a C3-C6 cycloalkyl;

(b) R³ is selected from the group consisting of C 1 -C 18 alkyl, (C 1 -C 18 alkyl)OH,

(C1-C18 alkyl)NH₂, (C1-C18 alkyl)SH, (C0-C4 alkyl)(C3-C6)cycloalkyl, (C0-C4 alkyl)(C2- C5 heterocyclic), (C0-C4 alkyl)(C6-C10 aryl)R⁷, and (C1-C4 alkyl)(C3-C9 heteroaryl) or R⁴ and R together with the atoms to which they are attached form a 4, 5 or 6 member heterocyclic ring;

(c) R⁵ is NHR⁶ or OH; (d) R is H, Ci-Cg alkyl; and

(e) R is selected from the group consisting of H and OH

wherein the chemical cleavage half-life (ti_/2) of U- J from Q or Y is at least about 1 hour to about 1 week in PBS under physiological conditions.

In a further embodiment, U-J comprises the structure:

wherein

Ri and R₈ are independently H or Ci-C₈ alkyl;

R₂ and R₄ are independently selected from the group consisting of H, Ci-C₈ alkyl, (C₁-C4 alkyl)OH, (C₁-C4 alkyl)SH, (C₂-C₃ alkyl)SCH₃, (C₁-C4 alkyl)CONH₂, (C₁-C4 alkyl)COOH, (C₁-C4 alkyl)NH₂, and (d-C₄ alkyl)(C₆ aryl)R₇;

R₃ is Ci-C₆ alkyl;

R₅ is NH₂; and

R₇ is selected from the group consisting of hydrogen, and OH.

In a further embodiment, U-J comprises the structure:

wherein

Ri is H;

R₂ is H, C1-C4 alkyl, (CH₂ alkyl)OH, (C1-C4 alkyl)NH₂, or (CH₂)(C₆ aryl)R₇; R₃ is Ci-C₆ alkyl;

R₄ is H, C₁-C4 alkyl, or (CH₂)(C₆ aryl)R₇;

R₅ is NH₂;

R₈ is hydrogen; and

R₇ is H or OH.

In a further embodiment, U-J comprises the structure:

wherein

Ri is H or Ci-C₄ alkyl;

R₂ is H, Ci-C₄ alkyl, or (C1-C4 alkyl)NH₂;

R₃ is Ci-C₆ alkyl;

R₄ is H, or C₁-C₄ alkyl;

R₅ is NH₂; and

R₈ is hydrogen. In a further embodiment, Ri is Ci-C₄ alkyl, R₂ is Ci-C₄ alkyl, R₃ is C C₆ alkyl; R₄ and R₈ are each H and R5 is NH₂. In a further embodiment, R₂ is Ci-C₄ alkyl, or (C₁-C4 alkyl)NH₂ alkyl, R₃ is Ci-C₆ alkyl; R_ls R₄ and R₈ are each H and R₅ is NH₂.

Pharmaceutical compositions comprising the NHR ligand-insulin conjugates disclosed herein can be formulated and administered to patients using standard

pharmaceutically acceptable carriers and routes of administration known to those skilled in the art. Accordingly, the present disclosure also encompasses pharmaceutical compositions comprising one or more of the NHR ligand-insulin conjugates disclosed herein or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier. In one embodiment the pharmaceutical composition comprises a lmg/ml concentration of the NHR ligand-insulin conjugate at a pH of about 4.0 to about 7.0 in a phosphate buffer system. The pharmaceutical compositions may comprise the NHR ligand- insulin conjugate as the sole pharmaceutically active component, or the NHR ligand-insulin conjugate peptide can be combined with one or more additional active agents.

All therapeutic methods, pharmaceutical compositions, kits and other similar embodiments described herein contemplate that NHR ligand-insulin conjugate peptides include all pharmaceutically acceptable salts thereof.

In one embodiment the kit is provided with a device for administering the NHR ligand-insulin conjugate to a patient. The kit may further include a variety of containers, e.g., vials, tubes, bottles, and the like. Preferably, the kits will also include instructions for use. In accordance with one embodiment the device of the kit is an aerosol dispensing device, wherein the composition is prepackaged within the aerosol device. In another embodiment the kit comprises a syringe and a needle, and in one embodiment the NHR ligand-insulin conjugate composition is prepackaged within the syringe.

The compounds of this invention may be prepared by standard synthetic methods, recombinant DNA techniques, or any other methods of preparing peptides and fusion proteins. Although certain non-natural amino acids cannot be expressed by standard recombinant DNA techniques, techniques for their preparation are known in the art.

Compounds of this invention that encompass non-peptide portions may be synthesized by standard organic chemistry reactions, in addition to standard peptide chemistry reactions when applicable.

Exemplary Embodiments

In accordance with embodiment 1, an insulin peptide/NHR ligand conjugate is provided wherein the conjugate comprises the structure Q-L-Y;

wherein

Q is an insulin peptide;

Y is a NHR ligand; and

L is a linking group or a bond

wherein said conjugate has activity at both a nuclear hormone receptor and an insulin receptor, wherein said NHR ligand is

i) a steroid that exhibits an EC₅₀ of about 1 μΜ or less when unconjugated to

Q-L, and further has a molecular weight of up to about 1000 daltons; or

ii) a ligand that activates the thyroid hormone receptor; or

iii) a ligand that activates the peroxisome proliferator-activated receptors (PPAR), wherein the nuclear hormone receptor ligand is covalently linked to the insulin peptide through the side chain of a lysine residue located at a position selected from the group consisting of B28 and B29, or the N-terminal alpha amine of the A or B chain, optionally the NHR ligand is linked to the carboxy terminus of the insulin peptide B chain. More particularly, in one embodiment the NHR ligand is a ligand that activates the thyroid hormone receptor or activates the peroxisome proliferator-activated receptors, wherein the NHR ligand is linked via the side chain of a lysine residue located at the C-terminus of the insulin B chain.

In accordance with embodiment 2 the conjugate of embodiment 1 is provided wherein Y is selected from the group consisting of estradiol and derivatives thereof, estrone and derivatives thereof, testosterone and derivatives thereof, and Cortisol and derivatives thereof.

In accordance with embodiment 3 the conjugate of embodiment 2 is provided wherein Y is dexamethasone.

In accordance with embodiment 4 the conjugate of any one of embodiments 1-3 is provided wherein Y is a compound having the general structure

wherein

Ri₅ is Ci-C₄ alkyl, -CH₂(pyridazinone), -CH₂(OH)(phenyl)F, -CH(OH)CH₃, halo or H; R₂o is halo, CH₃ or H;

R₂i is halo, CH₃ or H;

R₂₂ is H, OH, halo, -CH₂(OH)(C₆ aryl)F, or C1-C4 alkyl; and

R₂₃ is -CH₂CH(NH₂)COOH, -OCH₂COOH, -NHC(0)COOH, -CH₂COOH,

-NHC(0)CH₂COOH, -CH₂CH₂COOH, or -OCH₂P0₃ ^2".

In accordance with embodiment 5 the conjugate of embodiments 4 is provided wherein

Ri₅ is C₁-C4 alkyl, -CH(OH)CH₃, 1 or H

R₂₀ is I, Br, CH₃ or H;

R₂i is I, Br, CH₃ or H;

R₂₂ is H, OH, I, or C₁-C4 alkyl; and

R₂₃ is -CH₂CH(NH₂)COOH, -OCH₂COOH, -NHC(0)COOH, -CH₂COOH,

-NHC(0)CH₂COOH, -CH₂CH₂COOH, or -OCH₂P0₃ ^2".

In accordance with embodiment 6 the conjugate of of any one of embodiment 4 is provided wherein

Ri₅ is C₁-C4 alkyl, l or H;

R₂₀ is I, Br, CH₃ or H;

R₂i is I, Br, CH₃ or H;

R₂₂ is H, OH, I, or Ci-C₄ alkyl; and R₂₃ is -CH₂CH(NH₂)COOH, -OCH₂COOH, -NHC(0)COOH, -CH₂COOH,

-NHC(0)CH₂COOH, -CH₂CH₂COOH, or -OCH₂P0₃ ^2".

In accordance with embodiment 7 the conjugate of any one of embodiments 1-6 is provided wherein Y is a compound of the general structure of Formula I:

wherein

R20, R21, and R₂₂ are independently selected from the group consisting of H, OH, halo and Ci-C₄ alkyl; and

Ri5 is halo or H.

In accordance with embodiment 8 the conjugate of any one of embodiments 1-7 is provided wherein Y is selected from the group consisting of 3,5,3',5'-tetra-iodothyronine, and 3,5,3'-triiodo L-thyronine.

In accordance with embodiment 9 the conjugate of embodiment 1 is provided wherein Y is selected from the group consisting of Tesaglitazar, Aleglitazar and thiazolidinediones, optionally wherein Y is Tesaglitazar or Aleglitazar, optionally Y is Tesaglitazar linked to the N-terminal amine of the insulin B chain.

In accordance with embodiment 10 the conjugate of any one of embodiments 1-9 is provided wherein said insulin peptide (Q) comprises an A chain and a B chain wherein said A chain comprises a sequence

GIVX₄X5CCX8X₉XioCXi2LXi₄Xi5LXi7Xi8YCX2i-R5₃ (SEQ ID NO: 19), and said B chain comprises a sequence R₆2-X₂₅LCGX₂9X₃oLVX₃₃X₃₄LYLVCGX₄iX₄₂GFX₄5 (SEQ ID NO: 20), wherein

X₄ is glutamic acid or aspartic acid;

X5 is glutamine or glutamic acid

X₈ is histidine, threonine or phenylalanine;

X9 is serine, arginine, lysine, ornithine or alanine;

X10 is isoleucine or serine;

X12 is serine or aspartic acid;

X₁₄ is tyrosine, arginine, lysine, ornithine or alanine;

Xi5 is glutamine, glutamic acid, arginine, alanine, lysine, ornithine or leucine; X₁₇ is glutamic acid, aspartic acid, asparagine, lysine, ornithine or glutamine;

X₁₈ is methionine, asparagine, glutamine, aspartic acid, glutamic acid or threonine; X21 is selected from the group consisting of alanine, glycine, serine, valine, threonine, isoleucine, leucine, glutamine, glutamic acid, asparagine, aspartic acid, histidine, tryptophan, tyrosine, and methionine;

X25 is histidine or threonine;

X29 is selected from the group consisting of alanine, glycine and serine;

X₃₃ is selected from the group consisting of aspartic acid and glutamic acid;

X₃₄ is selected from the group consisting of alanine and threonine;

R₅₃ is COOH or CONH₂.

In accordance with embodiment 11 the conjugate of any one of embodiments 1-10 is provided wherein said insulin peptide comprises an A chain and a B chain and said A chain comprises the sequence GIVEQCCX₈X₉ICSLYQLENYCX₂i-R53 (SEQ ID NO: 73) said B chain comprises the sequence R₆2-X₂₅LCGX₂9X₃oLVX₃₃X₃₄LYLVCGX₄iX₄₂GFX₄5 (SEQ ID NO: 20), wherein

X₈ is histidine or threonine;

X9 is serine, lysine, or alanine;

X21 is alanine, glycine or asparagine;

X25 is histidine or threonine;

X29 is selected from the group consisting of alanine, glycine and serine;

X₃₃ is selected from the group consisting of aspartic acid and glutamic acid; X₃₄ is selected from the group consisting of alanine and threonine;

X₄₂ is selected from the group consisting of alanine, ornithine, lysine and arginine; X₄5 is tyrosine or phenylalanine;

P 62 is selected from the group consisting of FVNQ (SEQ ID NO: 12), a tripeptide valine-asparagine-glutamine, a dipeptide asparagine-glutamine, glutamine and an N-terminal amine; and

R₅₃ is COOH or CONH₂.

In accordance with embodiment 12 the conjugate of any one of embodiments 1-11 is provided wherein said insulin peptide comprises an A chain and a B chain wherein said A chain comprises a sequence GIVDECCX8X9SCDLRRLEMX19CX21-R53 (SEQ ID NO: 74) and said B chain comprises a sequence R₆₂-X₂₅LCGAX₃oLVDALYLVCGDX₄₂GFY (SEQ ID NO: 75), wherein

X₈ is phenylalanine or histidine;

X9 is arginine, ornithine or alanine;

X₁9 is tyrosine, 4-methoxy-phenylalanine or 4-amino-phenylalanine;

X₂₁ is alanine or asparagine;

X₂₅ is histidine or threonine;

X₄₂ is selected from the group consisting of alanine ornithine and arginine; and Rs₃ is COOH or CONH₂;

R₅ is COOH or CONH₂.

In accordance with embodiment 13 the conjugate of embodiments 12 is provided wherein the B chain sequence comprises the sequence

FVKQX₂₅LCGSHLVEALYLVCGERGFF-R₆₃ (SEQ ID NO: 147), or

FVNQX₂₅LCGSHLVEALYLVCGERGFF-R₆₃ (SEQ ID NO: 148), wherein

X₂₅ is selected from the group consisting of histidine and threonine; and R₆₃ is selected from the group consisting of YTX₂₈KT (SEQ ID NO: 149), YTKPT (SEQ ID NO: 150), YTX₂₈K (SEQ ID NO: 152), YTKP (SEQ ID NO: 151), YTPK (SEQ ID NO: 70), YTX₂₈, YT, Y and a bond, wherein X₂₈ is proline, aspartic acid or glutamic acid.

In accordance with embodiment 14 the conjugate of any one of embodiments 1-13 is provided wherein the insulin peptide comprises an A chain and a B chain wherein the A chain comprises the sequence GIVEQCCX₈X₉ICSLYQLENYCX₂i-R₅₃ (SEQ ID NO: 73), and the B chain sequence comprises the sequence

FVKQX₂₅LCGS HLVE ALYLVC GERGFF YTEKT (SEQ ID NO: 162),

FVNQX₂₅LCGSHLVEALYLVCGERGFFYTDKT (SEQ ID NO: 164),

FVNQX₂₅LCGS HLVE ALYLVC GERGFF YTKPT (SEQ ID NO: 165) or

FVNQX₂₅LCGS HLVE ALYLVC GERGFF YTPKT (SEQ ID NO: 161) wherein

X₈ is histidine or threonine;

X9 is serine, lysine, or alanine;

X₂₁ is alanine, glycine or asparagine;

X₂₅ is selected from the group consisting of histidine and threonine.

In accordance with embodiment 15 the conjugate of embodiment 11 is provided wherein said B chain comprises a sequence R₆₂-

X₂₅LCGX₂9X₃oLVX₃₃X₃₄LYLVCGX₄iX₄₂GFX₄₅YT-Zi-Bi (SEQ ID NO: 142), wherein X₂₅ is histidine or threonine;

X₂9 is selected from the group consisting of alanine, glycine and serine;

X₃₄ is selected from the group consisting of alanine and threonine;

Bi is selected from the group consisting of threonine, alanine or a threonine-arginine- arginine tripeptide. In accordance with embodiment 16 the conjugate of any one of embodiments 1-15 is provided wherein said A chain comprises a sequence GIVEQCCTSICSLYQLENYCN-R53 (SEQ ID NO: 1) and said B chain comprises a sequence

FVNQHLC GS HLVE AL YLVC GERGFFYTPKT (SEQ ID NO: 2), wherein R53 is COOH or CONH₂.

In accordance with embodiment 17 the conjugate of any one of embodiments 1-16 is provided wherein Y is selected from the group consisting of 3,5,3',5'-tetra-iodothyronine, and 3,5,3'-triiodo L-thyronine and Y is linked via the side chain of the lysine residue present at B28 or B29 of said B chain.

In accordance with embodiment 18 the conjugate of embodiment 17 is provided wherein Y is 3,5,3'-triiodo L-thyronine and Y is linked via the side chain of the lysine residue present at B29 of said B chain.

In accordance with embodiment 19 the conjugate of any one of embodiments 1-18 is provided wherein the insulin peptide is a single chain insulin and the peptide linking moiety joining the B and A chains is selected from the group consisting of

SSSSKAPPPSLPSPSRLPGPSDTPILPQR (SEQ ID NO: 52),

SSSSRAPPPSLPSPSRLPGPSDTPILPQK (SEQ ID NO: 51), GAGSSSX₅₇X₅₈ (SEQ ID NO: 76), GYGSSSX₅₇X₅₈ (SEQ ID NO: 21) and G YGS S S X57X58 APQT ; (SEQ ID NO: 77), wherein

X₅₇ and X58 are independently arginine, lysine or ornithine.

In accordance with embodiment 20 the conjugate of embodiment 19 is provided wherein the peptide linking moiety is selected from the group consisting of GYGSSSRR (SEQ ID NO: 18) and GAGSSSRR (SEQ ID NO: 22).

In accordance with embodiment 21 the conjugate of any one of embodiments 1-20 is provided wherein the N-terminus of said B chain is pegylated.

In accordance with embodiment 22 the conjugate of any one of embodiments 1-21 is provided wherein L is stable in vivo, hydrolyzable in vivo, or metastable in vivo.

In accordance with embodiment 23 the conjugate of embodiment 22 is provided wherein L comprises an ether moiety, or an amide moiety, an ester moiety, an acid-labile moiety, a reduction-labile moiety , an enzyme-labile moiety, a hydrazone moiety, a disulfide moiety, or a cathepsin-cleavable moiety.

In accordance with embodiment 24 the conjugate of any one of embodiments 1-23 is provided wherein Y-L is covalently linked to the insulin peptide (Q) through a position independently selected from the side chain of an amino acid at B28 or B29 of the B chain, the N-terminal alpha amine of the B chain, the N-terminal alpha amine of the A chain and at the side chain of an amino acid at any position of a linking moiety that links the A chain and B chain of a single chain insulin peptide.

In accordance with embodiment 25 the conjugate of embodiment 24 is provided wherein Y-L is covalently linked to the insulin peptide (Q) through the side chain of an amino acid at B28 or B29 of the B chain.

In accordance with embodiment 26 the conjugate of any one of embodiments 1-25 is provided wherein the conjugate is derivatized by linking the structure U-J to the conjugate, wherein

U is an amino acid or a hydroxy acid;

J is an N-alkylated amino acid, sad structure being linked to said conjugate through an amide bond between a carboxyl moiety of J and an amine of the conjugate, wherein the chemical cleavage half- life (ti_/2) of U-J from the conjugate is at least about 1 hour to about 1 week in PBS under physiological conditions.

In accordance with embodiment 27 the conjugate of any one of embodiments 1-26 is provided wherein a hydrophilic moiety is covalently linked at one or more positions corresponding to A14, A15, BO, B l, B IO, B22, B28, B29.

In accordance with embodiment 28 the conjugate of embodiment 27 is provided wherein the hydrophilic moiety is a polyethylene glycol.

In accordance with embodiment 29 the conjugate of any one of embodiments 1-28 is provided further comprising an acyl group or alkyl group covalently linked to an amino acid side chain of said conjugate.

In accordance with embodiment 30 the conjugate of embodiment 29 is provided wherein said acyl group or alkyl group is covalently linked to one or more positions selected from A14, A15, B0, B l, B 10, B22, B28, B29 of the insulin peptide.

In accordance with embodiment 31 the conjugate of any one of embodiments 1-30 is provided as a pharmaceutical composition comprising a conjugate of any one of the preceding embodiments, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In accordance with embodiment 32 the conjugate of any one of embodiments 1-31 is provided for treating diabetes or reducing weight gain or enhncing weight loss. In accordance with embodiment 33 the use of the conjugate of the composition of embodiment 31, or pharmaceutically acceptable salt thereof, is provided for the treatment of diabetes.

In accordance with embodiment 34 a method of treating diabetes and optionally i) lowering body weight;

ii) lowering triglycerides;

ii) lowering cholesterol levels; or

iv) any combination of i), ii) and iii) is provided wherein the method comprises the step of administering a conjugate of any one of embodiments 1-30 to a patient in need thereof.

EXAMPLE 1

Synthesis of Insulin A & B Chains

Insulin A & B chains were synthesized on 4-methylbenzhyryl amine (MB HA) resin or 4-Hydroxymethyl-phenylacetamidomethyl (PAM) resin using Boc chemistry. The peptides were cleaved from the resin using HF/p-cresol 95:5 for 1 hour at 0°C. Following HF removal and ether precipitation, peptides were dissolved into 50% aqueous acetic acid and lyophilized. Alternatively, peptides were synthesized using Fmoc chemistry. The peptides were cleaved from the resin using Trifluoroacetic acid (TFA)/ Triisopropylsilane (TIS)/ H₂0 (95:2.5:2.5), for 2 hour at room temperature. The peptide was precipitated through the addition of an excessive amount of diethyl ether and the pellet solubilized in aqueous acidic buffer. The quality of peptides were monitored by RP-HPLC and confirmed by Mass Spectrometry (ESI or MALDI).

Insulin A chains were synthesized with a single free cysteine at amino acid 7 and all other cysteines protected as acetamidomethyl A-(SH)⁷(Acm)⁶'^U'²⁰. Insulin B chains were synthesized with a single free cysteine at position 7 and the other cysteine protected as acetamidomethyl B-(SH) 7 (Acm) 19. The crude peptides were purified by conventional RP- HPLC.

The synthesized A and B chains were linked to one another through their native disulfide bond linkage in accordance with the general procedure outlined in Fig. 1. The respective B chain was activated to the Cys -Npys analog through dissolution in DMF or DMSO and reacted with 2,2' -Dithiobis (5-nitropyridine) (Npys) at a 1: 1 molar ratio, at room temperature. The activation was monitored by RP-HPLC and the product was confirmed by ESI-MS.

The first B7-A7 disulfide bond was formed by dissolution of the respective A-

(SH) 7 (Acm) 6 ' 11 ' 20 and B-(Npys) 7 (Acm) 19 at 1: 1 molar ratio to a total peptide concentration of 10 mg/ml. When the chain combination reaction was complete the mixture was diluted to a concentration of 50% aqueous acetic acid. The last two disulfide bonds were formed simultaneously through the addition of iodine. A 40 fold molar excess of iodine was added to the solution and the mixture was stirred at room temperature for an additional hour. The reaction was terminated by the addition of an aqueous ascorbic acid solution. The mixture was purified by RP-HPLC and the final compound was confirmed by MALDI-MS. As shown in Fig. 2 and the data in Table 1, the synthetic insulin prepared in accordance with this procedure compares well with purified insulin for insulin receptor binding.

Insulin peptides comprising a modified amino acid (such as 4-amino phenylalanine at position A 19) can also be synthesized in vivo using a system that allows for incorporation of non-coded amino acids into proteins, including for example, the system taught in US Patent Nos. 7,045,337 and 7,083,970.

Table 1: Activity of synthesized insulin relative to native insulin

EXAMPLE 2

Pegylation of Amine Groups (N-Terminus and Lysine) by Reductive Alkylation

a. Synthesis

Insulin (or an insulin analog), mPEG20k-Aldyhyde, and NaB¾CN, in a molar ratio of 1:2:30, were dissolved in acetic acid buffer at a pH of 4.1-4.4. The reaction solution was composed of 0.1 N NaCl, 0.2 N acetic acid and 0.1 N Na₂C0₃. The insulin peptide concentration was approximately 0.5 mg/ml. The reaction occurs over six hours at room temperature. The degree of reaction was monitored by RP-HPLC and the yield of the reaction was approximately 50%.

b. Purification

The reaction mixture was diluted 2-5 fold with 0.1% TFA and applied to a

preparative RP-HPLC column. HPLC condition: C4 column; flow rate 10 ml/min; A buffer 10% ACN and 0.1% TFA in water; B buffer 0.1% TFA in ACN; A linear gradient B% from 0-40% (0-80 min); PEG-insulin or analogues was eluted at approximately 35% buffer B. The desired compounds were verified by MALDI-TOF, following chemical modification through sulftolysis or trypsin degradation.

Pegylation of Amine Groups (N-Terminus and Lysine) by N-Hydroxysuccinimide Acylation. a. Synthesis

Insulin (or an insulin analog) along with mPEG20k-NHS were dissolved in 0.1 N Bicine buffer (pH 8.0) at a molar ratio of 1: 1. The insulin peptide concentration was approximately 0.5 mg/ml. Reaction progress was monitored by HPLC. The yield of the reaction is approximately 90% after 2 hours at room temperature.

b. Purification

The reaction mixture was diluted 2-5 fold and loaded to RP-HPLC.

HPLC condition: C4 column; flow rate 10 ml/min; A buffer 10% ACN and 0.1% TFA in water; B buffer 0.1% TFA in ACN; A linear gradient B% from 0-40% (0-80 min); PEG- insulin or analogues was collected at approximately 35% B. . The desired compounds were verified by MALDI-TOF, following chemical modification through sulftolysis or trypsin degradation. Reductive Aminated Pegylation of Acetyl Group on the Aromatic Ring Of The

Phenylalanine

a. Synthesis

Insulin (or an insulin analogue), mPEG20k-Hydrazide, and NaBH₃CN in a molar ratio of 1:2:20 were dissolved in acetic acid buffer (pH of 4.1 to 4.4). The reaction solution was composed of 0.1 N NaCl, 0.2 N acetic acid and 0.1 N Na₂C0₃. Insulin or insulin analogue concentration was approximately 0.5 mg/ml. at room temperature for 24h. The reaction process was monitored by HPLC. The conversion of the reaction was

approximately 50%. (calculated by HPLC) b. Purification

The reaction mixture was diluted 2-5 fold and loaded to RP-HPLC.

HPLC condition: C4 column; flow rate 10 ml/min; A buffer 10% ACN and 0.1% TFA in water; B buffer 0.1% TFA in ACN; A linear gradient B% from 0-40% (0-80 min); PEG- insulin, or the PEG-insulin analogue was collected at approximately 35%B. . The desired compounds were verified by MALDI-TOF, following chemical modification through sulftolysis or trypsin degradation.

EXAMPLE 3

Insulin Receptor Binding Assay:

The affinity of each peptide for the insulin or IGF-1 receptor was measured in a competition binding assay utilizing scintillation proximity technology. Serial 3-fold dilutions of the peptides were made in Tris-Cl buffer (0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.1% w/v bovine serum albumin) and mixed in 96 well plates (Corning Inc., Acton, MA) with 0.05 nM (3-[125I]-iodotyrosyl) A TyrA14 insulin or (3-[125I]-iodotyrosyl) IGF-1 (Amersham Biosciences, Piscataway, NJ). An aliquot of 1-6 micrograms of plasma membrane fragments prepared from cells over-expressing the human insulin or IGF-1 receptors were present in each well and 0.25 mg/well polyethylene imine-treated wheat germ agglutinin type A scintillation proximity assay beads (Amersham Biosciences, Piscataway, NJ) were added. After five minutes of shaking at 800 rpm the plate was incubated for 12h at room temperature and radioactivity was measured with MicroB eta 1450 liquid scintillation counter (Perkin-Elmer, Wellesley, MA). Non-specifically bound (NSB) radioactivity was measured in the wells with a four- fold concentration excess of "cold" native ligand than the highest concentration in test samples. Total bound radioactivity was detected in the wells with no competitor. Percent specific binding was calculated as following: % Specific

Binding = (Bound-NSB / Total bound-NSB) x 100. IC50 values were determined by using Origin software (OriginLab, Northampton, MA).

EXAMPLE 4

Insulin Receptor Phosphorylation Assay:

To measure receptor phosphorylation of insulin or incretin-insulin conjugate, receptor transfected HEK293 cells were plated in 96 well tissue culture plates (Costar #3596, Cambridge, MA) and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 100 IU/ml penicillin, 100 g/ml streptomycin, 10 mM HEPES and 0.25% bovine growth serum (HyClone SH30541, Logan, UT) for 16-20 hrs at 37 °C, 5% CO₂ and 90% humidity. Serial dilutions of insulin or insulin analogs were prepared in DMEM supplemented with 0.5% bovine serum albumin (Roche Applied Science #100350, Indianapolis, IN) and added to the wells with adhered cells. After 15 min incubation at 37 °C in humidified atmosphere with 5% C0₂ the cells were fixed with 5% paraformaldehyde for 20 min at room temperature, washed twice with phosphate buffered saline pH 7.4 and blocked with 2% bovine serum albumin in PBS for 1 hr. The plate was then washed three times and filled with horseradish peroxidase-conjugated antibody against phospho tyro sine (Upstate biotechnology #16-105, Temecula, CA) reconstituted in PBS with 2% bovine serum albumin per manufacturer's recommendation. After 3 hrs incubation at room temperature the plate was washed 4 times and 0.1 ml of TMB single solution substrate (Invitrogen, #00-2023, Carlbad, CA) was added to each well. Color development was stopped 5 min later by adding 0.05 ml 1 N HC1. Absorbance at 450 nm was measured on Titertek Multiscan MCC340 (ThermoFisher, Pittsburgh, PA). Absorbance vs. peptide concentration dose response curves were plotted and EC 50 values were determined by using Origin software (OriginLab, Northampton, MA).

EXAMPLE 5

Biosynthesis and Purification of Single Chain Insulin Analogs

An insulin-IGF-I minigene comprising a native insulin B and A chain linked via the IGF-I C chain (Β°-^-Α°) was cloned into expression vector pGAPZa A (purchased from Invitrogen) under GAP promoter (promoter of the glyceraldehyde-3-phosphate

dehydrogenase (GAPDH)) for constitutive expression and purification of recombinant protein in yeast Pichia pastoris. The minigene was fused to an N-terminal peptide encoding Saccharomyces cerevisiae a-mating factor leader signal for secretion of the recombinant protein into the medium. A Kex2 cleavage site between the minigene and the leading a- mating factor sequence was used to cleave the leader sequence for secretion of the minigene with native amino termini. Single-site alanine mutations were introduced into C peptide at positions 1 (G1A), 2 (Y2A), 3 (G3A), 4 (S4A), 5 (S5A), 6 (S6A), 7 (R7A), 8 (R8A), 10 (P10A), 11 (QUA), and 12 (T12A) of the BVA⁰ minigene.

The minigenes including B°C¹A°, eleven alanine mutants, and other select derivatives were transformed into yeast Pichia pastoris by electroporation. Positive transformants were selected on minimal methanol plates and a genomic preparation of each Pichia isolate was performed and integration of the constructs into the yeast genome was confirmed by PCR. An 833 base pair PCR product was visualized on an agarose DNA gel. The insulin analogs were produced by fermentation of a corresponding yeast line. The yeast cells were pelleted by centrifugation at 5 K for 20 minutes in 500 ml Beckman centrifuge tubes and the media was kept for subsequent protein purification.

Growth media supernatants were filtered through 0.2 μιη Millipore filter. Acetonitrile (ACN) was added to the supernatant to a final volume of 20%. The supernatant was purified over a Amberlite XAD7HP resin from Sigma, pre-equilibrated with 20% aqueous ACN. The resin was then rinsed twice with 30 ml of 20% aqueous ACN and contaminants were removed with 30% aqueous ACN containing 0.1% TFA. Partially purified insulin analogs were eluted from the column with 54% aqueous ACN containing 0.1% TFA and

lyophilizied. Lyophilized samples were re-suspended in 0.025M NH₃HCO₃ pH 8 and purified on a Luna C18 column (10 μιη particle size, 300A° pore size). Protein was eluted from the column using a linear gradient of 20-60% aqueous ACN. MALDI-MS positive fractions were pooled and transferred to a disposable scintillation vial for subsequent lyophilization. Lyophilized samples were then resuspended in 20% aqueous ACN containing 0.1% TFA, and purified on a Luna C18 column (10 μιη particle size, 300A° pore size). The protein was eluted from the column using a linear gradient of 18-54% aqueous ACN with 0.1% TFA. Protein elution was monitored at an absorbance 280 nm. MALDI-TOF MS positive fractions were analyzed via a C8 analytical column to insure purity.

The B°-C^1_A⁰ analog demonstrated potency that was equally effective at both insulin receptor isoforms and the IGF-1 receptor. Mutation of the tyrosine at position 2 to alanine or the shortening of the C-peptide to eight amino acids through deletion of C9-12 provided a selective enhancement in the specificity of insulin action by significant reduction in the IGF- 1 receptor activity. See also the data provided in Tables 5A and 5B: Table 5A

Insulin Binding & Phosphorylation Analysis

(B°C¹A°)

Peptide Insulin Binding Insulin Phosphorylation

ICso, nM n EC₅₀, nM n

Insulin 0.54±0.02 4 1.67±0.13 1

IGF-1 18.81 ±1.77 3 29.20±8.41 1

010 (B°C¹A°) 2.83±0.52 2 1.93±0.43 1

G1A 1.21 +0.15 1 2.4±0.24 1

Y2A 1.95±0.28 3 1.86±0.42 1

G3A 1.41 ±0.05 2 2.13±0.02 1

S4A 0.84±0.47 2 0.76±0.35 1

S5A 0.93±0.44 ! 2.23±1.27 1

S6A 1.15±0.24 ! 2.33±1.65 2

R7A 6.04±0.82 5.21 ±4.14 1

R8A 0.63±0.09 ! 2.03±0.06 2

P10A 2.86±0.93 ! 2.59±1.2 1

Q11A 1.79±0.47 ! 2.58±0.83 1

T12A 1.2+0.18 1 2.83±1.31 1

Table 5B

IGF-1 Binding & Phosphorylation Analysis

(B C 1A C )

Position 2 and 3 in the C-peptide are most sensitive to modification at the IGF-1 receptor with the insulin receptor proving to be relatively immune to modification. All of the analogs maintained single unit nanomolar activity with certain specific analogs proving to be slightly enhanced in potency (low single unit nanomolar). The most insulin selective analogs were those that we missing the last four residues of the C-peptide, had an alanine mutation at position two of the C-peptide, or a combination of the two changes.

EXAMPLE 6

Insulin Tolerance Tests

Single administration of six- to eight-week-old male C57BL/6 mice were maintained at 23'C, constant humidity, and a 12-h light— dark cycle. The acute in vivo effects of select peptides were evaluated by subcutaneously injecting peptides solubilized in physiologically buffered saline or a vehicle control to normal or diabetic mice. Blood glucose was measured at various time points through the course of a 24hr period following administration of the peptides. Each group of mice contained 8 animals per group. The average body weight was 25 g. Mice were made diabetic by administration of strep tozotocin.

Serial administration

Repeat daily subcutaneous administration of the peptides or vehicle control was administered to the mice for periods of five days to two weeks. The obese mice were given a diabetogenic diet Mice had free access to water and were fed ad libitum with a high fat diet (HFD) comprising 58% of calories from fat (D 12331; Research Diets, New Brunswick, NJ) and each group of mice contained 8 animals per group. The average body weight was -50 g 20 and the mice were— 6 months old males. Body weight and food intake were measured on the days that peptide or vehicle control was administered to the mice. Fasting blood glucose levels were measured repeatedly.

Claims

Claims:

1. An insulin peptide/NHR ligand conjugate comprising the structure

Q L-Y;

wherein

Q is an insulin peptide;

Y is a NHR ligand; and

L is a linking group or a bond

ii) a ligand that activates the thyroid hormone receptor; or

iii) a ligand that activates the peroxisome proliferator-activated receptors (PPAR)

wherein the nuclear hormone receptor ligand is covalently linked to the insulin peptide through the side chain of a lysine residue located at a position selected from the group consisting of B28 and B29, or through the N-terminal alpha amine of the A or B chain.

2. The conjugate of claim 1, wherein Y is selected from the group consisting of estradiol and derivatives thereof, estrone and derivatives thereof, testosterone and derivatives thereof, and Cortisol and derivatives thereof.

3. The conjugate of claim 2, wherein Y is dexamethasone.

4. The conjugate of claim 1 wherein Y is a compound having the general structure

wherein

Ri5 is C1-C4 alkyl, -CH₂(pyridazinone), -CH₂(OH)(phenyl)F, -CH(OH)CH₃,

H;

R20 is halo, CH₃ or H;

R21 is halo, CH₃ or H;

R22 is H, OH, halo, -CH₂(OH)(C₆ aryl)F, or C1-C4 alkyl; and

R₂₃ is -CH₂CH(NH₂)COOH, -OCH₂COOH, -NHC(0)COOH, -CH₂COOH,

-NHC(0)CH₂COOH, -CH₂CH₂COOH, or -OCH₂P0₃ ² .

5. The conjugate of claim 4 wherein

wherein

Ri5 is C1-C4 alkyl, -CH(OH)CH₃, 1 or H

R₂₀ is I, Br, CH₃ or H;

R21 is I, Br, CH₃ or H;

R22 is H, OH, I, or C1-C4 alkyl; and

R₂₃ is -CH₂CH(NH₂)COOH, -OCH₂COOH, -NHC(0)COOH, -CH₂COOH,

-NHC(0)CH₂COOH, -CH₂CH₂COOH, or -OCH₂P0₃ ^2".

6. The conjugate of claim 4 wherein

Ri5 is Ci-C₄ alkyl, l or H;

R₂₀ is I, Br, CH₃ or H;

R21 is I, Br, CH₃ or H;

R22 is H, OH, I, or C1-C4 alkyl; and

R₂ is -CH₂CH(NH₂)COOH, -OCH₂COOH, -NHC(0)COOH, -CH₂COOH,

-NHC(0)CH₂COOH, -CH₂CH₂COOH, or -OCH₂P0₃ ^2".

7. The conjugate of claim 1 wherein Y is a compound of the general structure of Formula I:

wherein

Ri₅ is halo or H.

8. The conjugate of claim 1 wherein Y is selected from the group consisting of 3,5,3',5'-tetra-iodothyronine, and 3,5,3'-triiodo L-thyronine.

9. The conjugate of claim 1 wherein Y is selected from the group consisting of Tesaglitazar, Aleglitazar and thiazolidinediones, optionally wherein Y is Tesaglitazar.

10. The conjugate of any one of claims 1-9 wherein said insulin peptide (Q) comprises an A chain and a B chain wherein said A chain comprises a sequence

X₄ is glutamic acid or aspartic acid;

X5 is glutamine or glutamic acid

X₈ is histidine, threonine or phenylalanine;

X9 is serine, arginine, lysine, ornithine or alanine;

X10 is isoleucine or serine;

X12 is serine or aspartic acid;

Xi₄ is tyrosine, arginine, lysine, ornithine or alanine;

Xi₇ is glutamic acid, aspartic acid, asparagine, lysine, ornithine or glutamine;

X25 is histidine or threonine;

X29 is selected from the group consisting of alanine, glycine and serine;

X30 is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid; X33 is selected from the group consisting of aspartic acid and glutamic acid;

X₃₄ is selected from the group consisting of alanine and threonine;

R₅₃ is COOH or CONH₂.

11. The conjugate of claim 10 wherein said A chain comprises the sequence GIVEQCCX₈X9lCSLYQLENYCX₂i-R5₃ (SEQ ID NO: 73) said B chain comprises the sequence R₆2-X₂₅LCGX₂9X₃oLVX₃₃X₃₄LYLVCGX₄iX₄₂GFX₄5 (SEQ ID NO: 20), wherein

X₈ is histidine or threonine;

X9 is serine, lysine, or alanine;

X21 is alanine, glycine or asparagine;

X25 is histidine or threonine;

X29 is selected from the group consisting of alanine, glycine and serine;

X₃₄ is selected from the group consisting of alanine and threonine;

R₆₂ is selected from the group consisting of FVNQ (SEQ ID NO: 12), a tripeptide valine-asparagine-glutamine, a dipeptide asparagine-glutamine, glutamine and an N-terminal amine; and

R₅ is COOH or CONH₂.

12. The conjugate of any one of claims 1-9 wherein said insulin peptide comprises an A chain and a B chain wherein said A chain comprises a sequence

GIVDECCX8X9SCDLRRLEMX19CX21-R53 (SEQ ID NO: 74) and said B chain comprises a sequence Rei-XisLCGAXsoLVDALYLVCGDX^GFY (SEQ ID NO: 75), wherein

X₈ is phenylalanine or histidine;

X9 is arginine, ornithine or alanine;

X₁9 is tyrosine, 4-methoxy-phenylalanine or 4-amino-phenylalanine;

X₂₁ is alanine or asparagine;

X25 is histidine or threonine;

X₄₂ is selected from the group consisting of alanine ornithine and arginine; and R₅₃ is COOH or CONH₂;

R₅₃ is COOH or CONH₂.

13. The insulin peptide of claim 12 wherein the B chain sequence comprises the sequence FVKQX₂₅LCGSHLVEALYLVCGERGFF-R₆₃ (SEQ ID NO: 147), or

FVNQX₂₅LCGS HLVE ALYLVC GERGFF-R₆₃ (SEQ ID NO: 148), wherein

X25 is selected from the group consisting of histidine and threonine; and

R₆₃ is selected from the group consisting of YTX₂₈KT (SEQ ID NO: 149), YTKPT (SEQ ID NO: 150), YTX₂₈K (SEQ ID NO: 152), YTKP (SEQ ID NO: 151), YTPK (SEQ ID NO: 70), YTX_2S, YT, Y and a bond, wherein X₂₈ is proline, aspartic acid or glutamic acid.

14. The insulin peptide of claim 13 wherein the B chain sequence comprises the sequence FVKQX₂₅LCGSHLVEALYLVCGERGFFYTEKT (SEQ ID NO: 162),

FVNQX₂₅LCGSHLVEALYLVCGERGFFYTDKT (SEQ ID NO: 164),

FVNQX₂₅LCGS HLVE ALYLVC GERGFF YTKPT (SEQ ID NO: 165) or

FVNQX₂₅LCGS HLVE ALYLVC GERGFF YTPKT (SEQ ID NO: 161) wherein

X₂₅ is selected from the group consisting of histidine and threonine.

15. The conjugate of claim 11 wherein said B chain comprises a sequence R₆₂- X₂₅LCGX₂9X₃oLVX₃₃X₃₄LYLVCGX₄iX₄₂GFX₄₅YT-Zi-Bi (SEQ ID NO: 142), wherein

X₂5 is histidine or threonine;

X₂9 is selected from the group consisting of alanine, glycine and serine;

X₃₄ is selected from the group consisting of alanine and threonine;

16. The conjugate of claim 10 wherein said A chain comprises a sequence GIVEQCCTSICSLYQLENYCN-R53 (SEQ ID NO: 1) and said B chain comprises a sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2), wherein R₅₃ is COOH or CONH₂.

17. The conjugate of claim 14 or 16 wherein Y is selected from the group consisting of 3,5,3',5'-tetra-iodothyronine, and 3, 5, 3 '-triiodo L-thyronine and Y is linked via the side chain of the lysine residue present at B28 or B29 of said B chain.

18. The conjugate of claim 17 wherein Y is 3,5,3'-triiodo L-thyronine and Y is linked via the side chain of the lysine residue present at B29 of said B chain.

19. The conjugate of any one of claims 10-18 wherein the insulin peptide is a single chain insulin and the peptide linking moiety joining the B and A chains is selected from the group consisting of SSSSKAPPPSLPSPSRLPGPSDTPILPQR (SEQ ID NO: 52), SSSSRAPPPSLPSPSRLPGPSDTPILPQK (SEQ ID NO: 51), GAGSSSX₅₇X₅8 (SEQ ID NO: 76), GYGSSSX₅₇X₅₈ (SEQ ID NO: 21) and GYGSSSX₅₇X₅₈APQT; (SEQ ID NO: 77), wherein

X5₇ and X₅s are independently arginine, lysine or ornithine.

20. The conjugate of claim 19 wherein the peptide linking moiety is selected from the group consisting of GYGSSSRR (SEQ ID NO: 18) and GAGSSSRR (SEQ ID NO: 22).

21. The conjugate of any one of claims 17-20 wherein the N-terminus of said B chain is pegylated.

22. The conjugate of any one of claims 1-21, wherein L is stable in vivo, hydrolyzable in vivo, or metastable in vivo.

23. The conjugate of claim 21, wherein L comprises an ether moiety, or an amide moiety, an ester moiety, an acid-labile moiety, a reduction-labile moiety , an enzyme- labile moiety, a hydrazone moiety, a disulfide moiety, or a cathepsin-cleavable moiety.

24. The conjugate of any one of claims 1-16 wherein Y-L is covalently linked to the insulin peptide (Q) through a position independently selected from the side chain of an amino acid at B28 or B29 of the B chain, the N-terminal alpha amine of the B chain, the N- terminal alpha amine of the A chain and at the side chain of an amino acid at any position of a linking moiety that links the A chain and B chain of a single chain insulin peptide.

25. The conjugate of claim 24 wherein Y-L is covalently linked to the insulin peptide (Q) through the side chain of an amino acid at B28 or B29 of the B chain.

26. A derivative of the conjugate of any one of the preceding claims further comprising the structure U-J, wherein

U is an amino acid or a hydroxy acid;

J is an N-alkylated amino acid linked to said conjugate through an amide bond between a carboxyl moiety of J and an amine of the conjugate, wherein the chemical cleavage half-life (ti_/2) of U-J from the conjugate is at least about 1 hour to about 1 week in PBS under physiological conditions.

27. The conjugate of any one of claims 1-26 wherein a hydrophilic moiety is covalently linked at one or more positions corresponding to A14, A15, BO, B l, B IO, B22, B28, B29.

28. The conjugate of claim 27, wherein the hydrophilic moiety is a polyethylene glycol.

29. The conjugate of any one of the preceding claims, further comprising an acyl group or alkyl group covalently linked to an amino acid side chain of said conjugate.

30. The conjugate of claim 29 wherein said acyl group or alkyl group is covalently linked to one or more positions selected from A14, A15, BO, B l, B IO, B22, B28, B29 of the insulin peptide.

31. A pharmaceutical composition comprising a conjugate of any one of the preceding claims, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

32. A kit for administering an insulin peptide/incretin conjugate to a patient in need thereof, said kit comprising the pharmaceutical composition of claim 31; and a device for administering said composition to a patient.

33. Use of the conjugate of the composition of claim 31, or pharmaceutically acceptable salt thereof, for the treatment of diabetes.

34. A method of treating diabetes and optionally

i) lowering body weight;

ii) lowering triglycerides;

ii) lowering cholesterol levels; or

iv) any combination of i), ii) and iii);

said method comprising administering a conjugate of any one of embodiments 1-30 to a patient in need thereof.