阅读说明：本技术 用于激活骨祖细胞中细胞信号传导的肽 (Peptides for activating cell signalling in osteoprogenitor cells ) 是由 姚蔚 刘瑞武 基特·S·拉姆 肖文伍 南希·兰尼 于 2019-07-19 设计创作，主要内容包括：本发明提供了拟肽配体的化合物和药物组合物。拟肽配体可以与膦酸盐药物缀合。由于本发明的化合物和药物组合物对间充质干细胞上的α-4β-1整联蛋白和骨表面具有特异性,因此可用于治疗骨质疏松症和用于促进骨生长。(The present invention provides compounds and pharmaceutical compositions of peptidomimetic ligands. The peptidomimetic ligands can be conjugated to phosphonate drugs. Alpha on mesenchymal stem cells due to the compound and the pharmaceutical composition of the invention 4 β 1 Integration of componentsThe proteins and bone surface are specific and therefore useful in the treatment of osteoporosis and in the promotion of bone growth.)

1. A compound according to formula I:

or a pharmaceutically acceptable salt thereof,

wherein:

R¹selected from-L-D, -OH, -NH₂；

R²Selected from H, C_1-6Alkyl and C_3-8A cyclic hydrocarbon group;

each R³Independently selected from H, halogen, C_1-6Alkyl radical, C_1-6Alkoxy and C_1-6A haloalkyl group;

each R⁴Independently selected from C_1-6Alkyl, H, halogen, C_1-6Alkoxy and C_1-6A haloalkyl group;

X¹、X²and X³Independently selected from amino acid residues;

X⁴is selected from (N)⁶-modified) lysineA residue, a citrulline residue, a homocitrulline residue, a leucine residue, an (N-methyl) leucine residue, an isoleucine residue, an (N-methyl) isoleucine residue, and a homophenylalanine residue;

l is a linker;

d is a phosphonate drug;

subscript m is 0,1, 2,3, or 4;

subscript n is 1,2,3,4, or 0;

subscript p is 0,1, 2,3, or 4; and

subscript q is 1,2,3,4, or 0.

2. The compound of claim 1, having a structure according to formula Ia:

or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1 or claim 2, having a structure according to formula II:

or a pharmaceutically acceptable salt thereof, wherein X⁴Selected from citrulline residue and N⁶- (3- (pyridin-3-yl) propanoyl) -lysine residue.

4. A compound according to any one of the preceding claims, wherein X²Selected from the group consisting of negatively charged amino acid residues, hydrophilic amino acid residues, hydroxyproline (Hyp) residues and 1-amino-1-cyclohexanecarboxylic acid (Ach) residues.

5. A compound according to any preceding claim, wherein-X⁴-X³-X²-(X¹)_mis-Cit-Glu-Ser-Val-or-Lys12-Aad-Ach-。

6. The compound of any one of the preceding claims, wherein linker L comprises at least one of N- (8-amino-3, 6-dioxa-octyl) succinamidyl (Ebes) and polyethylene glycol (PEG).

7. The compound of any one of the preceding claims, wherein linker L is selected from the group consisting of:

and

wherein k is 0 to 6.

8. The compound of any preceding claim, wherein D is a phosphonate drug selected from the group consisting of mono-, di-and tri-phosphonates.

9. The compound of any one of the preceding claims, wherein D has the formula:

wherein

R⁵Selected from H, OH and halogen;

R⁶selected from H and C_1-6An alkyl group; and

subscript t is 1 to 6.

10. The compound of any one of the preceding claims, wherein D has a formula selected from:

11. the compound according to any one of the preceding claims, having a formula selected from:

12. the compound according to any one of the preceding claims, having a formula selected from:

13. the compound of claim 1, having a structure according to formula Ib:

14. the compound according to claim 1 or claim 13, having a formula selected from:

15. a pharmaceutical composition comprising a compound of any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

16. A method of treating osteoporosis comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1 to 14 or a pharmaceutical composition of claim 15.

17. A method of promoting bone growth comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1 to 14 or a pharmaceutical composition of claim 15.

18. A method of treating low bone mass comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1 to 14 or a pharmaceutical composition of claim 15.

19. A method of treating a disease or condition characterized by secondary low bone mass, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1 to 14 or a pharmaceutical composition of claim 15.

20. The method of any one of claims 16-19, wherein the therapeutically effective amount is an amount that activates Akt signaling in the individual.

Background

Osteoporosis is a disease in which bone fragility increases due to estrogen deficiency and aging. This is a major public health problem, with nearly 50% of caucasian women and 25% of caucasian men at risk for osteoporotic fractures throughout life (published by the National Osteoporosis Foundation). In addition, over 200 million osteoporotic fractures occur annually in the United states, with 27% of the vertebrae fractured and 14% of the hip fractured. Thus, osteoporosis represents a significant health problem, especially as our population ages rapidly worldwide.

Senescence is associated with a decrease in bone marrow skeletal osteoprogenitors and an appropriate microenvironment that supports the differentiation of these osteoprogenitors into osteoblasts to form bone. With aging, a decrease in the number of Mesenchymal Stem Cells (MSCs) in the bone marrow leads to a decrease in osteogenesis and may be the most important factor in leading to a decrease in bone formation and an increase in bone fragility (Heersche, J.N., C.G.Bellows and Y.Ishida, J Prosthet Dent,1998,79(1): pages 14-6; Ettinger, M.P., Arch Intern Med,2003.163(18): pages 2237-46). Currently, almost all treatments for osteoporosis reduce bone loss by reducing osteoclastic bone resorption, thereby preventing further destruction of bone. Importantly, such antiresorptive drugs cannot restore lost bone structure. Drugs currently available to enhance bone formation include hPTH (1-34) (teriparatide), PTHrp (abalopeptide) and Evenity (Romosozumab), and have been approved by the FDA for the treatment of osteoporosis. Abacavir peptide may be more effective than teriparatide and, in the case of uncoupling of bone remodeling, the onset of fracture reduction is faster than teriparatide. In preclinical studies, treatment with teriparatide and abalopeptide was limited to 2 years, and a black box warning of the risk of osteosarcoma was noted. Teriparatide and abalopeptide are not recommended for patients with high risk of osteosarcoma, a radiation history, primary hyperparathyroidism and any form of secondary hyperparathyroidism. In addition, all current pharmaceutical formulations can reduce the risk of vertebral fractures by about 50% or more, but their efforts to reduce hip fractures remain low.

A therapeutic modality that targets bone formation by increasing the number and/or activity of osteoblasts may be a more attractive approach for enhancing bone formation and promoting bone regeneration. Although bone regeneration by inducing osteogenesis from MSCs is a rational strategy for treating osteoporosis, systemic infusion of MSCs in vivo fails to promote an osteogenic response of bone because MSCs do not migrate to the bone surface, which is a major clinical problem for MSC transplantation (Gao, j., et al, Cells Tissues Organs,2001.169(1): pages 12-20; Meyerrose, t.e., et al, Stem Cells,2007,25(1): pages 220-7). In addition, transplantation of MSCs requires donor ablation using chemotherapy and/or radiation, which may lead to concomitant damage to endogenous mesenchymal cells (Bacigalupo, A., Best practice Res Clin Haematol,2004,17(3): pp 387-99).

Cell adhesion is the process of: through this process the cells associate with each other, migrate toward a specific target or localize within the extracellular matrix. Cell adhesion constitutes one of the fundamental mechanisms behind many biological phenomena. Molecular-based studies of cell adhesion have shown that various cell surface macromolecules (collectively referred to as cell adhesion molecules or receptors) mediate cell-cell and cell-matrix interactions. For example, members of the Cell surface receptor integrin family mediate Cell-Cell and Cell-matrix interactions and regulate Cell motility, migration, survival and proliferation (Hynes, Cell,69:11-25 (1992); Hynes, Cell,1110:673-687 (2002)). Integrins are non-covalent heterodimeric complexes composed of two subunits, alpha and beta. There are at least 18 different alpha subunits and at least 8 different beta subunits.

Mesenchymal stem cells in bone marrow have multilineage potential and represent a mixture of precursors of mesenchymal-derived Cell types including osteoblasts, chondrocytes and adipocytes (Owen, M. et al, Ciba Found Symp,1988,136: pages 42-60; Bruder, S.P., et al, J Cell Biochem,1994,56(3): pages 283-94; Prockop, D.J., Science,1997,276(5309): pages 71-4). All mature stages of Bone cells are heavily dependent on Cell-matrix and Cell-Cell interactions (Mukherjee, S., et al, J Clin Invest,2008,118(2): pp 491-504; Grzesik, W.J. and P.G.Robey, J Bone Miner Res,1994,9(4): pp 487-96; Vukicevic, S., et al, Cell,1990,63(2): pp 437-45; Mbalaville, G., et al, J Bone Miner Res,2006,21(12): pp 1821-7). Bone marrow is the site of committed osteoblast progenitors, and osteogenic differentiation is the default pathway for MSC lineage commitment (Halleux, C., et al, J Musculoskelet neural interaction, 2001,2(1): pages 71-6; Muraglia, A., et al, J Cell Sci,2000,113(Pt 7): pages 1161-6). Mobilization of osteoblast progenitor cells to the surface of Bone is a key step in osteoblast maturation and formation of mineralized tissue (Adams, G.B., et al, Nature,2006,439(7076): pp. 599-603; Chen, X.D., et al, J Bone Miner Res,2007,22(12): pp. 1943-56). Once the osteoblast progenitors are "committed" to the bone surface, they will associate to include osteocalcin, osteopontin, bone sialoprotein, osteonectin, collagenA series of proteins, I and fibronectin, which will further enhance osteoblast adhesion and maturation (Gronthos, S., et al, Periodontol 2000,2006,41: pp. 188-95; Gronthos, S., et al, Bone,2001,28(2): pp. 174-81; Gronthos, S., et al, J Bone Miner Res,1997,12(8): pp. 1189-97). These interactions are primarily mediated by transmembrane integrin receptors, which primarily utilize the arginine-glycine-aspartic acid (RGD) sequence to recognize and bind specific ligands. MSCs express integrins α 1, α 2, α 3, α 4, α 6, α 11, CD51 (integrin α V) and CD29 (integrin β 1) (Brooke, g., et al, Stem Cells Dev, 2008). Integrin alpha is reported₁β₁、α₂β₁、α_vβ₁、α_vβ₅、α₅β₁And alpha₄β₁Expressed in osteoblasts (Grzesik, W.J. and Robey, P.G., J Bone Miner Res,1994,9(4): pages 487-96; Gronthos, S., et al, Bone,2001,28(2): pages 174-81; Gronthos, S., et al, J Bone Miner Res,1997.12(8): pages 1189-97; Cowles, E.A., L.L.Brailey, and G.A.Gronowicz, J Biomed Mater Res,2000,52(4): pages 725-37). Overexpression of alpha on MSCs has been reported₄Integrins increase MSC homing to bone (Mukherjee, S., et al, J Clin Invest,2008,118(2): page 491-504).

Bisphosphonates are widely used for the treatment of osteoporosis. Such drugs are also used as "vehicles" for delivering bone-targeting drugs to bone tissue as prodrugs based on their bisphosphonate moieties. Bisphosphonates have been used to deliver slow-Release diclofenac (a non-steroidal anti-inflammatory drug) into the bones of rats (Hirabayashi, h., et al, J Control Release,2001,70(1-2): pages 183-91). The bisphosphonate dosages required for such drug delivery are typically 10-fold to 100-fold lower than the dosages required to treat osteoporosis, hypocalcemia, paget's disease, or metastatic bone cancer.

It is well known that bone formation is beneficial in the treatment of a variety of different diseases in mammals, including simple aging, bone degeneration and osteoporosis, fracture healing, fusion or joint fixation, osteogenesis imperfecta, etc., and for the successful installation of various medical orthopedic and periodontal implants, such as screws, rods, titanium cages for spinal fusion, hip joints, knee joints, ankle joints, shoulder joints, dental plates and rods, etc.

The use of cathepsin K inhibitors and TGF- β binding proteins and the like to increase bone mineralization for the treatment of diseases characterized at least in part by increased bone resorption (e.g., osteopenia, bone fractures, osteoporosis, arthritis, tumor metastasis, paget's disease, and other metabolic bone disorders) is well known, as shown by U.S. publication No. 2004/0235728 to Selwyn Aubrey Stoch, published on 11/25 of 2004 and U.S. patent No. 6,489,445 to Mary e. brunkow et al, and U.S. publication No. 2004/0009535 to 1/15 of 2004. In Brunkow's 445 patent and 535 publication, TGF- β binding proteins include Sost polypeptide (full-length and short peptide) antibodies that interfere with the interaction between the TGF- β binding protein sclerostin and members of the TGF- β superfamily, particularly bone morphogenic proteins. A novel family of human TGF- β binding proteins and the nucleic acids that encode them are described in the Brunkow's 445 patent. The protein binds to at least human bone morphogenetic protein-5 and human bone morphogenetic protein-6. The foregoing disorders are due to a systemic loss of bone mineral, and thus administration of the antibody therapeutic is for a systemic (systemic) increase in bone mineral density.

U.S. publication No. 2006/0165799, published 2006, 7,27, teaches a bone filler composition for stimulating bone formation and bone consolidation comprising biocompatible calcium sulfate and a viscous biopolymer. The composition is intended to be applied to the missing part of an injured bone without spreading to the surrounding organs.

U.S. publication No. 2005/025604, published on 17.11.2005, shows that bone formation is induced by mechanically inducing an increase in osteoblast activity and increasing systemic blood concentration of bone anabolic agents, including optionally increasing systemic blood concentration of anti-bone resorptive agents.

U.S. patent No. 7,576,175, granted 8/18/2009, shows alpha exhibiting high binding affinity, specificity and stability₄β₁An integrin ligand. The ligands comprise peptides having "n" independently selected amino acids, at least one of which is an unnatural amino acid or a D-amino groupAn acid, and wherein n is an integer from 3 to 20.

U.S. publication No. 2010/0021379, published on 28/1/2010, shows antibody conjugates comprising a targeting agent covalently linked to an antibody or fragment thereof. The targeting agent comprises a ligand comprising an integrin receptor, e.g., alpha₄β₁Integrins have specific peptides or peptidomimetics.

What is needed in the art are new compositions and methods for treating osteoporosis and promoting bone growth.

Disclosure of Invention

In one embodiment, the present invention provides a compound according to formula I:

or a pharmaceutically acceptable salt thereof,

wherein:

R¹selected from-OH, -NH₂and-L-D;

R²selected from H, C_1-6Alkyl and C_3-8A cyclic hydrocarbon group;

each R³And R⁴Independently selected from H, halogen, C_1-6Alkyl radical, C_1-6Alkoxy and C_1-6A haloalkyl group;

X¹、X²and X³Independently selected from amino acid residues;

X⁴is selected from (N)⁶-modified) lysine residues, citrulline residues, homocitrulline residues, leucine residues, (N-methyl) leucine residues, isoleucine residues, (N-methyl) isoleucine residues and homophenylalanine residues;

l is a linker;

d is a phosphonate drug; and

subscripts m, n, p, and q are each independently an integer of 0 to 4.

In another embodiment, the present invention provides a pharmaceutical composition comprising a compound as described herein, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

In another embodiment, the invention provides a method of treating osteoporosis. The method comprises administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof as described herein.

In another embodiment, the present invention provides a method of promoting bone growth. The method comprises administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof as described herein.

In another embodiment, the invention provides a method of treating low bone mass. The method comprises administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof as described herein.

In another embodiment, the invention provides a method of treating a disease or condition characterized by secondary low bone mass. The method comprises administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof as described herein.

Drawings

Figure 1 shows a flow diagram for screening for cell signaling activators with affinity for osteoblast specific transcription factor (osterix) + osteoprogenitor cells.

Fig. 2 shows lymphocyte binding. Peripheral mononuclear cells were extracted from blood and incubated with beads displaying scrambled (scrambles), YLL3 or YLL8 peptides for 1 hour.

FIG. 3 is a comparison of the binding affinities of YLL3, YLL8, and LLP2A peptides for osteoprogenitor cells after 10 days of culture.

FIG. 4 is a comparison of the activation of Akt signaling in osteoprogenitor cells by YLL3, YLL8, and LLP2A peptides after 10 days of culture.

FIG. 5 is a comparison of several YLL peptides with respect to osteogenesis in osteoprogenitor cells. Alkaline phosphatase levels were measured after 14 days. After 21 days, maturation and mineralization of osteoblasts were measured using alizarin red staining.

Fig. 6A-6D show that YLL3 and YLL8 stimulate osteogenesis in vitro. Bone marrow stromal cells were incubated with beads exhibiting autofluorescence quenching of either YLL3 or YLL8 peptide in osteogenic medium. Fig. 6A shows quantitative measurements of alkaline phosphatase (ALP) at day 10 or Alizarin Red (AR) levels at day 21 for the YLL3 and YLL8 beads. Fig. 6B shows representative images of ALP staining in BMSCs cultured with beads displaying either YLL3 or YLL8 on day 10. Fig. 6C shows representative images of AR staining in BMSCs cultured with beads displaying either YLL3 or YLL8 on day 21. FIG. 6D shows the hybridization to 6X10 in osteogenic medium^-8Results of BMSC cultured for 10 days or 21 days with YLL3 and YLL8 peptide of M to measure ALP level on day 10 and AR level on day 21. A, p<0.05 vs control.

Fig. 7A-7C illustrate activation of Akt signaling using yl 8. Fig. 7A is a layout of an Akt signaling array. FIG. 7B shows the hybridization to 6X10 in osteogenic media^-8M peptide or hPTH (1-34) were incubated with BMSCs for three days, and Akt arrays were performed using cell lysates. Fig. 7C shows the quantitative pixel density from the selected wells in fig. 7B.

Fig. 8 is a comparison of the treatment of bone mass in young mice with the peptides YLL3, YLL8, and LLP 2A.

Fig. 9A to 9E show the in vivo anabolic effects of YLL3 and YLL8 on bone mass in young mice. Two month old female and male mice were treated with PBS control, 50 μ g/kg of either YLL3 or YLL8 or 25 μ g/kg of PTH, subcutaneously 5 times a week for 21 days (n-4-6/group). All mice received calcein-7 days and-2 days before euthanasia. Trabecular bone volume and surface-based bone formation were measured at the distal femur and cortical bone volume and bone formation were measured at the mid-femur (fig. 9A, 9B and 9C). Fig. 9D shows representative images of trabecular bone samples taken from male mice. Fig. 9E shows representative images of cortical bone samples taken from male mice. P <0.05 relative to PBS.

Fig. 10A-10D illustrate the effect of yl 8 on bone formation in vivo. Four month old female and male mice were controlled with PBS, 5. mu.g/kg (. about.6X 10)^-9M) of YLL8 or 25 μ g/kg PTH, injected subcutaneously 5 times a week for 21 days (n-4-6/group). All mice received alizarin red and calcein-7 days and-2 days before euthanasia. Fig. 10A shows trabecular bone volume and surface-based bone formation measured in the distal metaphysis (DFM) of the femur. Fig. 10B shows a representative image of the DFM trabecular region in female mice. Fig. 10C shows representative images of the DFM trabecular region in male mice. FIG. 10D shows cortical bone mass measured in the middle of the femur by micro-CT; bone strength was obtained by three-point bending of the femur. P <0.05 relative to PBS.

FIG. 11 shows the anabolic effects of YLL on trabecular bone volume, osteocalcin and CTX-1 in young mice. Four month old female and male mice were treated with PBS control, 5 μ g/kg of YLL3, 5 μ g/kg of YLL8, or 25 μ g/kg of PTH, subcutaneously 5 times a week for 28 days (n-4-6/group). Trabecular bone volume was obtained by micro-CT scanning of the distal femur. Osteocalcin and CTX-1 were measured in serum.

Fig. 12 shows expression of Prx1 and osteoblast specific transcription factors in bone. Representative distal femurs are shown for Prx1-GFP (green, left) and osteoblast specific transcription factor-mCherry (red, right) mice.

Fig. 13A-13D show callus mineralization in fracture healing using yl 3. Female Prx1-GFP/ERT mice were fractured at two months of age and received tamoxifen at a dose of 10mg/kg for three days. YLL3 or YLL8 were given at 10 μ g/kg, hPTH (1-34) at 50 μ g/kg, injected subcutaneously 5 times a week for 10 or 21 days. Alizarin red was given-1 day before euthanasia. FIG. 13A shows a representative femoral fracture image from a Prx1-GFP/ERT mouse at day 10. Fig. 13B shows representative callus images from the outer edge of the callus in the designated treatment group at day 10 post fracture, and representative micro-CT thickness maps of callus, callus body volume bone mineral content from the designated treatment group at day 10 post fracture. FIG. 13C shows a femoral image of a representative fracture from a Prx1-GFP/ERT mouse on day 21. Fig. 13D shows representative callus images of the outer edges of the callus from the indicated treatment groups on day 21 post fracture, and representative micro-CT thickness maps of the callus, callus body mass, and bone mineral content from the indicated treatment groups on day 21 post fracture.

FIG. 14 shows the synthesis scheme for YLL3-Ale and YLL8-Ale conjugates.

Fig. 15A to 15C show two month old female mice treated with: PBS control; YLL3-Aln or YLL8-Aln at 100. mu.g/kg or 300. mu.g/kg, for subcutaneous injection once every two weeks, 2 doses; or 500 μ g/kg of YLL3-Aln or YLL8-Aln, in one intravenous dose. Mice were euthanized on day 28 (n-4-5/group). Fig. 15A shows the quantified trabecular bone volume and trabecular bone thickness. Fig. 15B shows a representative 3D image of the trabecular region taken from the distal end of the femur in the indicated treatment group. Fig. 15C shows mid-femoral cortical bone volume and cortical bone thickness measured in the femur by micro-CT representative images of the mid-femoral cortex in the indicated treatment groups. P <0.05 relative to PBS.

Figure 16 shows 28 days of two month old female mice treated subcutaneously once every two weeks with PBS control, either 100 μ g/kg or 300 μ g/kg of YLL3-Aln or YLL8-Aln, respectively (n-4-6/group). Trabecular bone formation was measured in the distal metaphysis of the femur. P <0.05 relative to PBS. The arrows show the bone surface in double marking.

Detailed Description

The present invention provides peptidomimetic ligand compounds, pharmaceutical compositions comprising the compounds, and methods of using the compounds. The peptidomimetic ligands can also be conjugated to phosphonate drugs such as alendronate. The compounds and pharmaceutical compositions of the invention are useful for promoting bone growth, treating osteoporosis, treating low bone mass, and treating diseases or conditions characterized by secondary low bone mass due to their ability to activate Akt signaling.

I. Definition of

The abbreviations used herein have their conventional meaning in the chemical and biological arts.

As used herein, the term "Ale", "Aln" or "alendronate" refers to alendronate.

When substituent groups are designated by their conventional formula as written from left to right, they also encompass the chemically identical substituents resulting from writing the structure from right to left, e.g., -CH₂O-is equivalent to-OCH₂-。

As used herein, unless otherwise specified, the term "halo" or "halogen" by itself or as part of another substituent means a fluorine, chlorine, bromine, or iodine atom.

As used herein, the term "alkyl" refers to a straight or branched chain saturated aliphatic group having the indicated number of carbon atoms. E.g. C₁-C₆Alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, and the like.

The term "haloalkyl" as used herein refers to an alkyl group as defined above wherein some or all of the hydrogen atoms are replaced by halogen atoms. For example, haloalkyl includes trifluoromethyl, fluoromethyl, 1,2,3,4, 5-pentafluoro-phenyl and the like. The term "perfluoro" defines a compound or group having at least two available hydrogens substituted with fluorine. For example, perfluorophenyl means 1,2,3,4, 5-pentafluorophenyl, perfluoromethane means 1,1, 1-trifluoromethyl, perfluoromethoxy means 1,1, 1-trifluoromethoxy.

As used herein, the term "heteroalkyl" refers to an alkyl group having 1 to 3 heteroatoms, such as N, O and S. Additional heteroatoms may also be useful, including but not limited to B, Al, Si, and P. Heteroatoms may also be oxidized, such as, but not limited to, -S (O) -and-S (O)₂-. For example, heteroalkyl groups may include ethers, thioethers, alkyl-amines, and alkyl-thiols.

As used herein, the term "alkoxy" refers to an alkyl group containing an oxygen atom, such as methoxy, ethoxy, and the like.

The term "aryl" as used herein refers to a monocyclic or fused bicyclic, tricyclic, or higher aromatic ring assembly containing 6 to 16 ring carbon atoms. For example, aryl may be phenyl, benzyl or naphthylPreferably phenyl. "arylene" means a divalent group derived from an aryl group. The aryl group may be selected from the group consisting of alkyl, alkoxy, aryl, hydroxy, halo, cyano, amino-alkyl, trifluoromethyl, alkylenedioxy and oxy-C₂-C₃-one, two or three groups in the alkylene group are mono-, di-or tri-substituted; all of which are optionally further substituted, e.g. as defined above; or 1-or 2-naphthyl; or 1-or 2-phenanthryl. Alkylenedioxy is a divalent substituent attached to two adjacent carbon atoms of the phenyl group, such as methylenedioxy or ethylenedioxy. oxy-C₂-C₃Alkylene is also a divalent substituent attached to two adjacent carbon atoms of the phenyl group, for example oxyethylene or oxypropylene. oxy-C₂-C₃An example of an-alkylene-phenyl group is 2, 3-dihydrobenzofuran-5-yl.

As used herein, the term "heteroaryl" refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, wherein 1 to 4 ring atoms are heteroatoms. For example, heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other (especially mono-or di-substituted) group substituted with, for example, alkyl, nitro or halo. Pyridyl represents 2-, 3-or 4-pyridyl, advantageously 2-or 3-pyridyl. Thienyl represents 2-or 3-thienyl. The quinolyl group preferably represents a 2-, 3-or 4-quinolyl group. The isoquinolinyl group preferably represents a 1-, 3-or 4-isoquinolinyl group. Benzopyranyl, benzothiopyranyl preferably represent 3-benzopyranyl or 3-benzothiopyranyl, respectively. Thiazolyl preferably represents 2-or 4-thiazolyl, most preferably 4-thiazolyl. The triazolyl group is preferably 1- (1,2, 4-triazolyl), 2- (1,2, 4-triazolyl) or 5- (1,2, 4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl.

The substituents for aryl and heteroaryl are various and are selected from: -halogen, -OR ', -OC (O) R ', -NR ' R ", -SR ', -R ', -CN, -NO₂、-CO₂R’、-CONR’R”、-C(O)R’、-OC(O)NR’R”、-NR”C(O)R’、-NR”C(O)₂R’、-NR’-C(O)NR”R”’、-NH-C(NH₂)＝NH、-NR’C(NH₂)＝NH、-NH-C(NH₂)＝NR’、-S(O)R’、-S(O)₂R’、-S(O)₂NR’R”、-N₃、-CH(Ph)₂Perfluoro (C)₁-C₄) Alkoxy and perfluoro (C)₁-C₄) Alkyl in an amount from zero to the total number of open valences on the aromatic ring system; and wherein R ', R "and R'" are independently selected from hydrogen, C₁-C₈Alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl) - (C)₁-C₄) Alkyl and (unsubstituted aryl) oxy- (C)₁-C₄) An alkyl group.

As used herein, the term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic, fused bicyclic, or bridged polycyclic ring assembly containing from 3 to 12 ring atoms or the indicated number of atoms. E.g. C_3-8Cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and poly-cyclooctyl.

As used herein, each of the above terms (e.g., "alkyl," "aryl," and "heteroaryl") is intended to include both substituted and unsubstituted forms of the indicated group.

Substituents for hydrocarbyl and heterohydrocarbyl groups (including those groups commonly referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to: -OR ', - (O), (NR', - (N-OR ', - (NR' R '), - (SR'), - (halogen), -SiR 'R' ″, - (oc) (O) R ', - (c) (O) R', - (CO) CO₂R’、-CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、-NR’-C(O)NR”R”’、-NR”C(O)₂R’、-NR-C(NR’R”R”’)＝NR””、-NR-C(NR’R”)＝NR”’、-S(O)R’、-S(O)₂R’、-S(O)₂NR’R”、-NR(SO₂) R', -CN and-NO₂From zero to (2m '+1), where m' is the total number of carbon atoms in such a group. R'R ', R ' and R ' are each independently selected from hydrogen, C₁-C₈Alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl) - (C)₁-C₄) Alkyl and (unsubstituted aryl) oxy- (C)₁-C₄) An alkyl group. When a compound of the invention includes more than one R group, for example, each R group is independently selected as each R ', R ", R'" and R "" group (when more than one of these groups is present). When R' and R "are attached to the same nitrogen atom, they may combine with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR' R "is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will appreciate that the term "alkyl" is intended to include groups containing carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF)₃and-CH₂CF₃) And acyl (e.g., -C (O) CH)₃、-C(O)CF₃、-C(O)CH₂OCH₃Etc.).

The term "peptide" as used herein refers to a compound consisting of a single chain of D-or L-amino acids or a mixture of D-or L-amino acids linked by peptide bonds. Typically, peptides are from about 2 to about 50 amino acids in length. Preferably, the peptides of the invention are from about 2 to about 25 amino acids in length, more preferably from 3 to 20 amino acids in length, and most preferably from 3 to 10 amino acids in length.

As used herein, the term "amino acid" refers to naturally occurring, unnatural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

As used herein, the term "naturally occurring amino acids" refers to those amino acids encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ -carboxyglutamate, and O-phosphoserine. Naturally occurring alpha-amino acids include, but are not limited to, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (gin), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of naturally occurring alpha-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

As used herein, the term "unnatural amino acid" includes, but is not limited to, amino acid analogs, amino acid mimetics, synthetic amino acids, N-configuration amino acids that function in a manner similar to a naturally occurring amino acid, in either the L-configuration or the D-configuration⁶-modified lysine and N-methyl amino acid. Unnatural amino acids are not encoded by the genetic code and can, but do not necessarily, have the same basic structure as a naturally occurring amino acid.

As used herein, the term "amino acid analog" refers to a compound having the same basic chemical structure (i.e., a carbon alpha to a hydrogen, a carboxyl group, an amino group, and an R group) as a naturally occurring amino acid, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.

As used herein, the term "amino acid mimetic" refers to a compound that has a structure that is different from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid. Suitable amino acid mimetics include, but are not limited to, beta-amino acids and gamma-amino acids. In a beta-amino acid, the amino group is bonded to the beta-carbon atom of the carboxyl group such that there are two carbon atoms between the amino group and the carboxyl group. In the γ -amino acid, the amino group is bonded to the γ -carbon atom of the carboxyl group, so that three carbon atoms exist between the amino group and the carboxyl group. Suitable R groups for beta-amino acids or gamma-amino acids include, but are not limited to, the side chains found in naturally occurring amino acids and non-natural amino acids.

As used herein, the term "N⁶Modified lysine means a lysine-based unnatural amino acid in which the nitrogen atom H of the R-group side chain of lysine₂N-CH₂-CH₂-CH₂-CH₂Modified by suitable substituents as described above, such as substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted haloalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. For example, the nitrogen atom of the side chain of the lysine R-group may be substituted with 3- (pyridin-3-yl) propionyl or 3- (pyridin-3-yl) prop-2-enoyl.

Amino acids can be characterized by at least one of several properties. For example, an amino acid can be positively charged, negatively charged, hydrophilic, or hydrophobic.

As used herein, the term "positively charged amino acid" refers to those amino acids having a basic or positively charged side chain at pH values below pKa, including but not limited to Lys, Arg, His, HoArg, Agp, Agb, Dab, Dap, and Orn and stereoisomers thereof. Basic amino acids can be generally referred to by the symbol "X⁺"means.

As used herein, the term "negatively charged amino acid" refers to an amino acid having an acidic or negatively charged side chain at pH values above pKa, and includes, but is not limited to, Asp, Glu, Aad, Bec, and stereoisomers thereof. Acidic amino acids can be generally referred to by the symbol "X^-"means. Those skilled in the art will appreciate that other basic and acidic amino acids are known in the art.

As used herein, the term "neutrally charged amino acids" refers to those amino acids having a neutrally charged side chain at a pH equal to the pKa.

As used herein, the term "hydrophilic amino acid" refers to those amino acids having polar and uncharged side chains, including but not limited to Asn, Ser, Thr, and gin.

As used herein, the term "hydrophobic amino acids" refers to those amino acids having a hydrophobic side chain, including but not limited to Val, Leu, Ile, Met, and Phe. In some embodiments, the hydrophobic amino acid is selected from proline, proline analogs, and stereoisomers thereof. In some embodiments, the proline analogue is hydroxyproline.

The term "D-amino acid" as used herein refers to the D stereoisomer of an amino acid. The letters D and L are commonly used in the art to refer to stereoisomers of amino acids. D-amino acids are those amino acids which can be synthesized from the dextrorotatory isomer of glyceraldehyde, i.e., D-glyceraldehyde. Similarly, L-amino acids are those that can be synthesized from the L-isomer of glyceraldehyde, i.e., L-glyceraldehyde.

Amino acids may be referred to herein by the well-known three-letter symbols or one-letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

With respect to amino acid sequences, those skilled in the art will recognize that a single substitution, deletion, or addition to a nucleic acid, peptide, polypeptide, or protein sequence that alters, adds, or deletes a single amino acid or a small portion of amino acids in the coding sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid (i.e., hydrophobic, hydrophilic, positively charged, neutral, negatively charged). Such chemically similar amino acids include, but are not limited to, naturally occurring amino acids such as L-amino acids, stereoisomers of naturally occurring amino acids such as D-amino acids, and unnatural amino acids such as amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids. Exemplary hydrophobic amino acids include valine, leucine, isoleucine, methionine, phenylalanine, and tryptophan. Exemplary aromatic amino acids include phenylalanine, tyrosine, and tryptophan. Exemplary aliphatic amino acids include serine and threonine. Exemplary basic amino acids include lysine, arginine, and histidine. Exemplary amino acids having carboxylate side chains include aspartate and glutamate. Exemplary amino acids having a carboxamide side chain include asparagine and glutamine. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the invention.

Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, such substitutions may be made: wherein an aliphatic amino acid (e.g., G, A, I, L or V) is replaced by another member of the group. Similarly, an aliphatic polar uncharged group, such as C, S, T, M, N or Q, may be replaced by another member of the group; basic residues such as K, R or H may be substituted for each other. In some embodiments, an amino acid having an acidic side chain, e.g., E or D, may be substituted with its uncharged counterpart, e.g., Q or N, respectively; or vice versa. Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for one another:

1) alanine (a), glycine (G);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V);

6) phenylalanine (F), tyrosine (Y), tryptophan (W);

7) serine (S), threonine (T); and

8) cysteine (C), methionine (M)

(see, e.g., Creighton, Proteins (1984)).

As used herein, the term "linker" refers to a moiety having one or more different reactive functional groups that allows for covalent attachment of a moiety, such as a peptide, to a chelator. In some embodiments, the linking moiety has two different reactive functional groups, i.e., heterobifunctional linkers. Suitable linkers include, but are not limited to, those available from Pierce Biotechnology, Inc. In some embodiments, the linker provides a carboxyl group for attachment of the chelator and an amino group for attachment of the peptide. However, it is understood by those skilled in the art that any reactive functional group may be present on the linker so long as it is compatible with the functional group on the moiety to be covalently attached.

Linkers useful in the present invention include those having one or more different reactive functional groups that allow covalent attachment of moieties such as peptides to a chelator. The linking moiety has two or more different reactive functional groups. In some cases, multivalent linkers may be used. Suitable linkers include, but are not limited to, those available from Pierce Biotechnology, Inc. In some embodiments, the linker provides a carboxyl group for attachment of the chelator and an amino group for attachment of the peptide. However, it is understood by those skilled in the art that any reactive functional group may be present on the linker so long as it is compatible with the functional group on the moiety to be covalently attached. As used herein, the term "chelator-linker conjugate" refers to a chelator covalently linked to a linker. Such chelator-linker conjugates may be attached to the peptide via a functional group present on the linker. Some suitable linkers include, but are not limited to, β -alanine, 2,2' -ethylenedioxybis (ethylamine) monosuccinamide (Ebes), and bis (Ebes) -Lys. Other suitable linkers include those with biotin. Additional linkers can be found in Bioconjugate technologies, Greg t. hermanson, Academic Press, 2 nd edition, 2008 (incorporated herein by reference in its entirety).

As used herein, the term "salt" refers to an acid or base salt of a compound used in the method of the present invention. Illustrative of pharmaceutically acceptable salts are inorganic acid (hydrochloric, hydrobromic, phosphoric, and the like) salts, organic carboxylic acid (acetic, propionic, glutamic, citric, and the like), organic sulfonic acid (methanesulfonic acid) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts. It is understood that pharmaceutically acceptable salts are non-toxic. Additional information about suitable pharmaceutically acceptable salts may be exemplifiedAs in R_EMINGTON:T_HE S_{CIENCE AND} P_{RACTICE OF} P_HARMARCY21 st edition, copyright ownership 2006, Lippincott Williams&Wilkins, Philadelphia PA ("Remington"), which is incorporated herein by reference.

Pharmaceutically acceptable salts of the acidic compounds of the invention include salts with bases, i.e., cationic salts, such as alkali metal and alkaline earth metal salts, e.g., sodium, lithium, potassium, calcium, magnesium salts, and ammonium salts, such as ammonium, trimethylammonium, diethylammonium and tris- (hydroxymethyl) -methyl-ammonium salts.

As used herein, the term "hydrate" refers to a compound that is complexed with at least one water molecule. The compounds of the present invention may complex with 1 to 10 water molecules.

As used herein, the terms "pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to a substance that facilitates administration of an active agent to, and absorption by, an individual. By "pharmaceutically acceptable excipient" is meant an excipient that can be included in the compositions of the present invention and that does not produce a significant deleterious toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, physiological saline solution, lactated ringer's solution, conventional sucrose, conventional dextrose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, pigments, and the like. One skilled in the art will recognize that other pharmaceutical excipients may be used in the present invention.

As used herein, the term "osteoporosis" refers to a disease in which increased bone fragility and/or decreased bone density is due to any established or undetermined cause or condition. "Low bone mass" or "osteopenia" is a condition, not a disease, that may progress to osteoporosis once bone density continues to decrease over time. Low bone mass is characterized by a T score of-1 to-2.15. Osteoporosis is characterized by a T score of less than-2.15. Among the definite causes or conditions to which the methods of the present invention are directed are primary osteoporosis associated with menopause (natural, premature or surgical), aging or both, as well as secondary osteoporosis or secondary low bone mass associated with medical conditions such as paget's disease, chronic kidney disease, amenorrhea from eating disorders, transplantation, hyperthyroidism, hyperparathyroidism, or the use of certain drugs such as various cancer chemotherapies, gonadotropin releasing hormone agonists, medroxyprogesterone acetate for fertility control, corticosteroids, anticonvulsants, etc.

As used herein, the phrase "Akt signaling" refers to any one of a number of biochemical pathways involved in the enzymatic activity of the serine/threonine specific protein kinase Akt (also known as protein kinase B or PKB). Akt itself is activated upon phosphorylation by phosphatidylinositol 3-kinase (PI3K) and other kinases. Activated Akt phosphorylates a number of cellular targets, thereby modulating processes including, but not limited to: cell viability, angiogenesis, osteogenesis, and cell metabolism associated with cell viability.

As used herein, the term "isomer" refers to compounds having the same chemical formula but which are structurally distinguishable.

As used herein, the term "tautomer" refers to one of two or more structural isomers that exist in equilibrium and that are readily converted from one isomeric form to another.

As used herein, the term "patient" or "individual" refers to a living organism that has or is predisposed to a condition that can be treated by administration of a pharmaceutical composition provided herein. Non-limiting examples include humans, other mammals, and other non-mammals.

As used herein, the term "therapeutically effective amount" refers to an amount of a conjugated functional agent or pharmaceutical composition that is useful for treating or ameliorating an identified disease or condition or exhibits a detectable therapeutic or inhibitory effect. This effect can be detected by any assay known in the art.

As used herein, the term "treatment" refers to any indication of successful treatment or amelioration of an injury, pathology, or condition, including any objective or subjective parameter, such as decline; (iii) alleviating; alleviating symptoms or making the patient more tolerant to injury, pathology, or condition; a reduced rate of degeneration or decline; reducing the final point of degeneration; improve the physical and mental health of the patients. Treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of physical examination, neuropsychiatric examination, and/or psychiatric evaluation. For example, the methods of the invention successfully treat delirium in patients by reducing the incidence of conscious or cognitive impairment.

As used herein, the term "disorder" or "condition" refers to a state or health condition of a patient or individual that can be treated with a peptidomimetic ligand compound of the invention.

As used herein, the terms "a", "an" or "an" when used in a group of substituents or "substituent group" herein means at least one. For example, when a compound is substituted with an alkyl or aryl group, the compound is optionally substituted with at least one alkyl group and/or at least one aryl group, wherein each alkyl and/or aryl group is optionally different. In another example, when a compound is substituted with a substituent, the compound is substituted with at least one substituent, wherein each substituent is optionally different. When a group can be substituted with one or more of a number of substituents, such substituents are selected to comply with the principles of chemical bonding and to yield a compound: it is not inherently unstable and/or is not known by those of ordinary skill in the art to be potentially unstable under environmental conditions such as aqueous, neutral, or physiological conditions.

Peptidomimetic ligands and phosphonate drug conjugates

In some embodiments, the invention provides peptidomimetic ligand compounds of formula I:

or a pharmaceutically acceptable salt thereof, wherein R¹Selected from-OH, -NH₂and-L-D. R²Selected from H, C_1-6Alkyl and C_3-8A cyclic hydrocarbon group. Each R³And R⁴Independently selected from H, halogen, C_1-6Alkyl radical, C_1-6Alkoxy and C_1-6A haloalkyl group. X¹、X²And X³Independently selected from amino acid residues. X⁴Is selected from (N)⁶-modified) lysine residues, citrulline residues, homocitrulline residues, leucine residues, (N-methyl) leucine residues, isoleucine residues, (N-methyl) isoleucine residues and homophenylalanine residues. L is a linker. D is phosphonate medicine. Subscripts m, n, p, and q are each independently an integer of 0 to 4.

In some embodiments, R²Selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, branched pentyl, n-hexyl, branched hexyl, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane and cyclooctane. In some embodiments, R²Selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, branched pentyl, n-hexyl and branched hexyl. In some embodiments, R²Selected from the group consisting of H, methyl, ethyl, n-propyl and isopropyl. In some embodiments, subscript n is 0,1, or 2. In some embodiments, subscript n is 1 or 2. In some embodiments, subscript n is 1. In some embodiments, subscript n is 1 or 2 and R²Selected from the group consisting of H, methyl, ethyl, n-propyl and isopropyl. In some embodiments, subscript n is 1 or 2 and R²Selected from H and methyl. In some embodiments, subscript n is 1 and R²Is H.

In some embodiments, each R is³And R⁴Independently selected from the group consisting of H, fluoro, chloro, bromo, iodo, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, branched pentyl, n-hexyl and branched hexyl. In some embodiments, each R is³And R⁴Independently selected from the group consisting of H, fluoro, chloro, bromo, iodo, methyl, ethyl, n-propyl and isopropyl. In some embodiments, each R is³And R⁴Independently selected from the group consisting of H, chloro, bromo, iodo and methyl. In some embodiments, R³Is H and R⁴Selected from the group consisting of H, chloro, bromo and methyl. In some embodiments, R³Is H and R⁴Selected from H and methyl. In some embodiments, subscript p is 0 and subscript q is 0,1, 2, or 3. In some embodiments, subscript p is 0 and subscript q is 1 or 2. In some embodiments, subscript p is 0 and subscript q is 0. In some embodiments, subscript p is 0, subscript q is 1, and R⁴Is methyl.

In some embodiments, X¹、X²And X³Are independently selected amino acid residues, including but not limited to naturally occurring amino acids; a D-amino acid; unnatural amino acids, including but not limited to L-or D-configured amino acid analogs, amino acid mimetics, N-substituted glycines, N-methyl amino acids, phenylalanine analogs, and derivatives of lysine (Lys), ornithine (Orn), and α, γ -diaminobutyric acid (Dbu); a hydrophilic amino acid; a hydrophobic amino acid; a positively charged amino acid; and negatively charged amino acids. In some embodiments, subscript m is 0,1, 2, or 3. In some embodiments, subscript m is 0,1, or 2. When the subscript m is 2, the moiety- (X)¹)_m-is-X^1a-X^1b-, wherein X^1aAnd X^1bAlso as described above and below for X¹、X²And X³Defined independently selected amino acid residues.

Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ -carboxyglutamate, and O-phosphoserine. Naturally occurring alpha-amino acids include, but are not limited to, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (gin), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof.

Stereoisomers of naturally occurring alpha-amino acids include, but are not limited to, D-amino acids and L-amino acids. D-amino acids suitable for use in the present invention include, but are not limited to, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof. In some embodiments, the D-amino acid is selected from the group consisting of D-alpha-amino acids, D-beta-amino acids, D-gamma-amino acids, and combinations thereof. In some embodiments, the D-alpha-amino acid is selected from the group consisting of stereoisomers of naturally occurring alpha-amino acids, non-natural D-alpha-amino acids, and combinations thereof.

Unnatural amino acids include, but are not limited to, amino acid analogs in the L-or D-configuration that function in a manner similar to naturally occurring amino acids, amino acid mimetics, N-substituted glycines, N-methyl amino acids, phenylalanine analogs, and derivatives of lysine (Lys), ornithine (Orn), and alpha, gamma-diaminobutyric acid (Dbu). An unnatural amino acid is not encoded by the genetic code, and can, but need not, have the same basic structure or function as a naturally occurring amino acid.

Non-natural amino acids useful in the present invention include, but are not limited to, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β -alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, t-butylglycine, 2, 4-diaminoisobutyric acid, desmosine, 2' -diaminopimelic acid, 2, 3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-aminoisobutyric acid, 2-aminopropionic acid, 2, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline, ornithine, pentylglycine, pipecolic acid, thioproline, aminophenylalanine, hydroxytyrosine and aminotyrosine.

In some other embodiments, the non-dayThe amino acid is selected from the group consisting of 1-aminocyclopentane-1-carboxylic acid (Acp), 1-aminocyclobutane-1-carboxylic acid (Acb), 1-aminocyclopropane-1-carboxylic acid (Acpc), citrulline (Cit), homocitrulline (HoCit), alpha-aminoadipic acid (Aad), 3- (4-pyridyl) alanine (4-Pal), 3- (3-pyridyl) alanine (3-Pal), propargyl glycine (Pra), alpha-aminoisobutyric acid (Aib), alpha-aminobutyric acid (Abu), norvaline (Nva), alpha, beta-diaminopropionic acid (Dpr), alpha, gamma-diaminobutyric acid (Dbu), alpha-tert-butylglycine (Bug), 3, 5-dinitrotyrosine Tyr (3, 5-di-NO).₂) Norleucine (Nle), 3- (2-naphthyl) alanine (Nal-2), 3- (1-naphthyl) alanine (Nal-1), cyclohexylalanine (Cha), di-n-propylglycine (Dpg), cyclopropylalanine (Cpa), homoleucine (Hle), homoserine (HoSer), homoarginine (Har), homocysteine (Hcy), methionine sulfoxide (Met (O)), methionine methyl sulfonium (Met (S-Me)), alpha-cyclohexylglycine (Chg), 3-benzo-thienylalanine (Bta), taurine (Tau), hydroxyproline (Hyp), O-benzyl-hydroxyproline (Hyp (Bzl)), homoproline (HoPro), beta-homoproline (beta HoPro), thiazolidine-4-carboxylic acid (Thz), Piperidinecarboxylic acid (Nip), isopiperidic acid (Isonip), 3-carboxymethyl-1-phenyl-1, 3, 8-triazaspiro [4,5 ]]Decan-4-one (Cptd), tetrahydro-isoquinoline-3-carboxylic acid (3-Tic), 5H-thiazolo [3,2-a]Pyridine-3-carboxylic acid (Btd), 3-aminobenzoic acid (3-Abz), 3- (2-thienyl) alanine (2-Thi), 3- (3-thienyl) alanine (3-Thi), α -aminosuberic acid (Asu), diethylglycine (Deg), 4-amino-4-carboxy-1, 1-dioxo-tetrahydrothiopyran (Acdt), 1-amino-1- (4-hydroxycyclohexyl) carboxylic acid (Ahch), 1-amino-1- (4-ketocyclohexyl) carboxylic acid (Akch), 4-amino-4-carboxytetrahydropyran (Actp), 3-nitrotyrosine (Tyr (3-NO)₂) 1-amino-1-cyclohexanecarboxylic acid (Ach), 1-amino-1- (3-piperidinyl) carboxylic acid (3-Apc), 1-amino-1- (4-piperidinyl) carboxylic acid (4-Apc), 2-amino-3- (4-piperidinyl) propionic acid (4-App), 2-aminoindane-2-carboxylic acid (Aic), 2-amino-2-naphthylacetic acid (Ana), (2S, 5R) -5-phenylpyrrolidine-2-carboxylic acid (Ppca), 4-thiazolylalanine (Tha), 2-aminocaprylic acid (Aoa), 2-aminoheptanoic acid (Aha), ornithine (Orn), azetidine-2-carboxylic acid (Aca), Alpha-amino-3-chloro-4, 5-dihydro-5-isoxazoleacetic acid (Acdi), thiazolidine-2-carboxylic acid (Thz (2-COOH)), allylglycine (Agl)) 4-cyano-2-aminobutyric acid (Cab), 2-pyridylalanine (2-Pal), 2-quinolylalanine (2-quinolylalanine) (2-Qal), cyclobutylalanine (Cba), phenylalanine analogs, lysine derivatives, ornithine (Orn) derivatives, alpha, gamma-diaminobutyric acid Dbu derivatives, stereoisomers thereof, and combinations thereof (see Liu and Lam, anal. biochem.,295:9-16 (2001)). Thus, the non-natural alpha-amino acid is present as a non-natural L-alpha-amino acid, a non-natural D-alpha-amino acid, or a combination thereof.

In some embodiments, the non-natural amino acid residue is selected from the following compounds of table 1:

TABLE 1 unnatural amino acids useful in the invention.

Suitable amino acid analogs include, but are not limited to, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Suitable amino acid mimetics include, but are not limited to, beta-amino acids and gamma-amino acids. Suitable R groups for beta-amino acids or gamma-amino acids include, but are not limited to, the side chains found in naturally occurring amino acids and non-natural amino acids.

N-substituted glycines suitable for use in the present invention include, but are not limited to, N- (2-aminoethyl) glycine, N- (3-aminopropyl) glycine, N- (2-methoxyethyl) glycine, N-benzylglycine, (S) -N- (1-phenylethyl) glycine, N-cyclohexylmethylglycine, N- (2-phenylethyl) glycine, N- (3-phenylpropyl) glycine, N- (6-galactosyl) glycine, N- (2- (3' -indolylethyl) glycine, N- (2- (p-methoxyphenylethyl)) glycine, N- (2- (p-chlorophenylethyl) glycine, and N- [2- (p-hydroxyphenylethyl) ] glycine N-substituted glycine oligomers, referred to herein as "peptoids," have been shown to be protease resistant (Miller et al, Drug Dev. Res.,35:20-32 (1995)). Thus, peptoids comprising at least one non-natural alpha-amino acid, D-amino acid, or a combination thereof are within the scope of the present invention.

Suitable N-methyl amino acids include N-methyl-Ala, N-methyl-Cys, N-methyl-Asp, N-methyl-Glu, N-methyl-Phe, N-methyl-Gly, N-methyl-His, N-methyl-Ile, N-methyl-Arg, N-methyl-Lys, N-methyl-Leu, N-methyl-Met, N-methyl-Asn, N-methyl-Gln, N-methyl-Ser, N-methyl-Thr, N-methyl-Val, N-methyl-Trp, N-methyl-Tyr, N-methyl-Acp, N-methyl-Acb, N-methyl-Acpc, N-methyl-Cit, N-methyl-HoCit, N-methyl-Glu, N-methyl-Gly, N-methyl-His, N-methyl-Ile, N-methyl-Arg, N-methyl-Lys, N-methyl-Leu, N-methyl-Aad, N-methyl-4-Pal, N-methyl-3-Pal, N-methyl-Pra, N-methyl-Aib, N-methyl-Abu, N-methyl-Nva, N-methyl-Dpr, N-methyl-Dbu, N-methyl-Nle, N-methyl-Nal-2, N-methyl-Nal-1, N-methyl-Cha, N-methyl-Cpa, N-methyl-Hle, N-methyl-HoSer, N-methyl-Har, N-methyl-Hcy, N-methyl-Chg, N-methyl-Bta, N-methyl-2-Thi, N-methyl-3-Thi, N-methyl-Asu, N-methyl-Acdt, N-methyl-Ahch, N-methyl-Akch, N-methyl-Actp, N-methyl-Tyr (3-NO)₂) N-methyl-Ach, N-methyl-3-Apc, N-methyl-4-App, N-methyl-Tha, N-methyl-Aoa, N-methyl-Aha, N-methyl-Orn, N-methyl-Aca, N-methyl-Agl, N-methyl-Cab, N-methyl-2-Pal, N-methyl-Cba, N-methyl-HoPhe, N-methyl-Phg, N-methyl-Phe (4-NH-Ach)₂) N-methyl-4-Phe (4-Me), N-methyl-Phe (4-F), N-methyl-Phe (4-Cl), N-methyl-Phe (2-Br), N-methyl-Phe (3-Br), N-methyl-Phe (4-Br), N-methyl-Phe (3-CF)₃) N-methyl-Phe (4-CF)₃) N-methyl-Phe (4-NO)₂) N-methyl-Phe (4-CN), N-methyl-Bpa, N-methyl-Phg (4-Cl), N-methyl-Phg (4-Br), N-methyl-Tyr (Me), N-methyl-Lys 38, N-methyl-Lys 27, N-methyl-Lys 73, N-methyl-Lys 55, N-methyl-Lys 28, N-methyl-Lys 72, N-methyl-Lys 12, N-methyl-Lys 123, N-methyl-Lys 63, N-methyl-Lys 35124, N-methyl-Lys 82, N-methyl-Lys 31, N-methyl-Lys 15, N-methyl-Lys 125, N-methyl-Lys 43, N-methyl-Lys 24, N-methyl-Lys 5, N-methyl-Lys 4, N-methyl-Lys 50, N-methyl-Lys 81, N-methyl-Orn 38, N-methyl-Orn 27, N-methyl-Orn 73, N-methyl-Orn 55, N-methyl-Orn 28, N-methyl-Orn 72, N-methyl-Orn 12, N-methyl-Orn 123, N-methyl-Orn 63, N-methyl-Orn 124, N-methyl-Orn 82, N-methyl-Orn 31, N-methyl-Orn 15, N-methyl-Orn 125, N-methyl-Orn 43, N-methyl-Orn 24, N-methyl-Orn 5, N-methyl-Orn 4, N-methyl-Orn 3683, N-methyl-Orn 4, N-methyl-Orn 4, N-Orn-Or,N-methyl-Orn 50, N-methyl-Orn 81, N-methyl-Dbu 38, N-methyl-Dbu 27, N-methyl-Dbu 73, N-methyl-Dbu 55, N-methyl-Dbu 28, N-methyl-Dbu 72, N-methyl-Dbu 12, N-methyl-Dbu 123, N-methyl-Dbu 63, N-methyl-Dbu 124, N-methyl-Dbu 82, N-methyl-Dbu 31, N-methyl-Dbu 15, N-methyl-Dbu 125, N-methyl-Dbu 43, N-methyl-Dbu 24, N-methyl-Dbu 5, N-methyl-Dbu 4, N-methyl-Dbu 50, N-methyl-Dbu 81, stereoisomers thereof, and combinations thereof.

Suitable phenylalanine analogs that may be used in the present invention include, but are not limited to, homophenylalanine (HoPhe), phenylglycine (Phg), 3-diphenylalanine (Dpa), 4-aminophenylalanine (Phe (4-NH)₂) 2-methylphenylalanine (Phe (2-Me)), 3-methylphenylalanine (Phe (3-Me)), 4-methylphenylalanine (Phe (4-Me)), 4-azidophenylalanine (Phe (4-N)), and the like₃) 2-fluorophenylalanine (Phe (2-F)), 3-fluorophenylalanine (Phe (3-F)), 4-fluorophenylalanine (Phe (4-F)), 2-chlorophenylalanine (Phe (2-Cl)), 3-chlorophenylalanine (Phe (3-Cl)), 4-chlorophenylalanine (Phe (4-Cl)), 2-bromophenylalanine (Phe (2-Br)), 3-bromophenylalanine (Phe (3-Br)), 4-bromophenylalanine (Phe (4-Br)), 2-iodophenylalanine (Phe (2-I)), 3-iodophenylalanine (Phe (3-I)), 4-iodophenylalanine (Phe (4-I)), 2-trifluoromethylphenylalanine (Phe (2-CF).₃) 3-trifluoromethylphenylalanine (Phe (3-CF))₃) 4-trifluoromethylphenylalanine (Phe (4-CF))₃) 2-methoxyphenylalanine (Phe (2-OMe)), 3-methoxyphenylalanine (Phe (3-OMe)), 2-nitrophenylalanine (Phe (2-NO)₂) 3-Nitrophenylalanine (Phe (3-NO))₂) 4-Nitrophenylalanine (Phe (4-NO))₂) 2-cyanophenylalanine (Phe (2-CN)), 3-cyanophenylalanine (Phe (3-CN)), 4-cyanophenylalanine (Phe (4-CN)), 3, 4-dimethoxyphenylalanine (Phe (3, 4-diome)), 3, 4-difluorophenylalanine (Phe (3, 4-dif)), 3, 5-difluorophenylalanine (Phe (3, 5-dif)), 2, 4-dichlorophenylalanine (Phe (2, 4-dif-Cl)), 3, 4-dichlorophenylalanine (Phe (3, 4-dif-Cl)), 4-benzoylphenylalanine (Bpa), 4-carboxyphenylalanine (Phe (4-COOH)), 4' -diphenylalanine (Bip), 2,3,4,5, 6-Pentafluorophenylalanine (Phe (F)₅) 3,4, 5-Trifluorophenylalanine (Phe (F))₃) 4-chlorophenyl glycine), 4-chlorophenyl glycineAcid (Phg (4-Cl)), 2-chlorophenylglycine (Phg (2-Cl)), 3-chlorophenylglycine (Phg (3-Cl)), 4-bromophenylglycine (Phg (4-Br)), 2-bromophenylglycine (Phg (2-Br)), 3-bromophenylglycine (Phg (3-Br)), 4-ethylphenylalanine (Phe (4-Et)), 4-ethoxyphenylalanine (Phe (4-OEt)), 4-butoxyphenylalanine (Phe (4-OBu)), O-methyltyrosine (Tyr (Me)), O-benzyltyrosine (Tyr (Bzl)), 3, 5-dibromotyrosine (Tyr dibr)), 3, 5-diiodotyrosine (Tyr (dii)), homotyrosine (HoTyr)), 3-chlorotyrosine (Tyr (3-Cl)), stereoisomers thereof, and combinations thereof.

Suitable derivatives of lysine (Lys), ornithine (Orn), and α, γ -diaminobutyric acid (Dbu) useful in the present invention include, but are not limited to, Lys123, Lys124, Lys, Orn123, Orn, 124, Orn, 125, Orn, Dbu123, Dbu124, Dbu125, Dbu, and stereoisomers thereof, and combinations thereof. The derivatives of Orn and Dbu are analogous to lysine derivatives with the corresponding carboxylic acids attached to the side chains of Orn and Dbu, respectively. Structures of Lys, Orn, and Dbu derivatives are disclosed in U.S. patent No. 7,576,175, which is incorporated herein by reference.

Suitable hydrophobic amino acids for use in the present invention include, but are not limited to, leucine (Leu), leucine analogs, phenylalanine (Phe), phenylalanine analogs, proline (Pro), proline analogs, valine (Val), isoleucine (Ile), glycine (Gly), alanine (Ala), Met, norvaline (Nva), norleucine (Nle), 1-aminocyclopropane-1-carboxylic acid (Acpc), 1-aminocyclobutane-1-carboxylic acid (Acb), α -cyclohexylglycine (Chg), cyclohexylalanine (Cha), propargylglycine (Pra), cyclopropylalanine (Cpa), homoleucine (Hle), α -aminoisobutyric acid (Aib), α -aminobutyric acid (Abu), 3- (2-thienyl) alanine (2-Thi), 3- (3-thienyl) alanine (3-Thi), 3- (3-pyridyl) alanine (3-Pal), 3- (2-naphthyl) alanine (Nal-2), 2-amino-2-naphthylacetic acid (Ana), tyrosine (Tyr), 3, 5-dinitrotyrosine (Tyr (3, 5-di-NO)₂) Diethylglycine (Deg), 4-amino-4-carboxy-1, 1-dioxo-tetrahydrothiopyran (Acdt), 1-amino-1- (4-hydroxycyclohexyl) carboxylic acid (Ahch), 1-amino-1- (4-ketocyclohexyl) carboxylic acid (Akch), 4-amino-4-carboxytetrahydropyran (Actp), 3-nitrotyrosine (Tyr (3-NO)₂) 1-amino-1-cyclohexanecarboxylic acid (Ach), 2-aminoindan-2-carboxylic acid (Aic), (2S, 5R) -5-phenylpyrrolidine-2-carboxylic acid (Ppca), 4-thiazolylalanine (Tha), 2-aminocaprylic acid (Aoa), 2-aminoheptanoic acid (Aha), and stereoisomers thereof. In some embodiments, the proline analogue is hydroxyproline.

In some embodiments, the hydrophobic amino acid is selected from the compounds of table 2:

table 1. hydrophobic amino acids useful in the present invention.

In some embodiments, the hydrophobic amino acid is selected from the compounds of table 3:

table 3. hydrophobic amino acids useful in the present invention.

Suitable hydrophilic amino acids useful in the present invention include, but are not limited to, Asn, Ser, Thr, Gln, and stereoisomers thereof. Suitable positively charged amino acids useful in the present invention include, but are not limited to, Lys, Arg, His, HoArg, Agp, Agb, Dab, Dap, Orn, and stereoisomers thereof. Suitable negatively charged amino acids useful in the present invention include, but are not limited to, aspartic acid, glutamic acid, α -aminoadipic acid, α -aminosuberic acid, homoaspartic acid, γ -carboxy-glutamic acid, 4-carboxyphenylalanine, and stereoisomers thereof. In other embodiments, the negatively charged amino acid is selected from Aad, Bec, and Bmc.

In some embodiments, X¹May be an unnatural amino acid, a hydrophobic amino acid, a positively charged amino acid, or a negatively charged amino acid. In some embodiments, X²May be a hydrophilic amino acid, a hydrophobic amino acid or a negatively charged amino acid. In some embodiments, X²May be a negatively charged amino acid residue, a hydrophilic amino acid residue, a hydroxyproline (Hyp) residue or a 1-amino-1-cyclohexanecarboxylic acid (Ach) residue. In some embodiments, X³May be a hydrophilic amino acid, a hydrophobic amino acid or a negatively charged amino acid. In some embodiments, when subscript m is 2, and part- (X)¹)_m-is-X^1a-X^1bWhen is, X^1aMay be an unnatural amino acid, a hydrophobic amino acid, or a negatively charged amino acid; and X^1bMay be a hydrophilic amino acid, a hydrophobic amino acid or a negatively charged amino acid.

In some embodiments, X⁴Is selected from N⁶- (3- (pyridin-3-yl) propanoyl) -lysine (Lys12), (E) -N⁶- (3- (pyridin-3-yl) acryloyl) -lysine (Lys38), citrulline (Cit), homocitrulline (HoCit), leucine (Leu), N-methyl-leucine (N-MeLeu), isoleucine (Ile), N-methyl-isoleucine (N-MeIle), and homophenylalanine (HoPhe). In some embodiments, X⁴Is citrulline (Cit) or N⁶- (3- (pyridin-3-yl) propanoyl) -lysine (Lys 12).

In some embodiments, the compound has a structure according to formula Ia:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound has a structure according to formula Ib:

in some embodiments, the compound has a structure according to formula Ic:

in some embodiments, the compound has a structure according to formula II:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a compound of formula I, formula Ia, formula Ib, formula Ic, or formula II, wherein X is¹May be an unnatural amino acid, a hydrophobic amino acid, a positively charged amino acid, or a negatively charged amino acid; x²May be a hydrophilic amino acid, a hydrophobic amino acid, or a negatively charged amino acid; x³May be a hydrophilic amino acid, a hydrophobic amino acid, or a negatively charged amino acid; and X⁴May be N⁶- (3- (pyridin-3-yl) propanoyl) -lysine (Lys12), (E) -N⁶- (3- (pyridin-3-yl) acryloyl) -lysine (Lys38), citrulline (Cit), homocitrulline (HoCit), leucine (Leu), N-methyl-leucine (N-MeLeu), isoleucine (Ile), N-methyl-isoleucine (N-MeIle), and homophenylalanine (HoPhe). In some embodiments, when subscript m is 2, and part- (X)¹)_m-is-X^1a-X^1bWhen is, X^1aMay be an unnatural amino acid, a hydrophobic amino acid, or a negatively charged amino acid; and X^1bMay be a hydrophilic amino acid, a hydrophobic amino acid or a negatively charged amino acid.

In some embodiments, the present invention provides a compound of formula I, formula Ia, formula Ib, formula Ic, or formula II, wherein X is¹Can be proline (Pro), alpha-aminoadipic acid (Aad), valine (Val), arginine (Arg), 1-amino-1- (4-piperidinyl) carboxylic acid (4-Apc), D-histidine (D-His), D-O-methyltyrosineAcids (D-Tyr (Me)), ornithine (Orn), alpha-aminoisobutyric acid (Aib), tyrosine (Tyr), homocitrulline (HoCit), norleucine (Nle), aspartic acid (Asp), isoleucine (Ile), or D-leucine (D-Leu); x²Can be aspartic acid (Asp), isoleucine (Ile), serine (Ser), glutamic acid (Glu), D-glutamic acid (D-Glu), D-propargylglycine (D-Pra), 1-amino-1-cyclohexanecarboxylic acid (Ach), 1-aminocyclopropane-1-carboxylic acid (Acpc), 4-methylphenylalanine (Phe (4-Me)), cyclohexylalanine (Cha), alpha-aminoadipic acid (Aad), hydroxyproline (Hyp), 2-aminoindan-2-carboxylic acid (Aic), glutamine (Gln), or D-alpha-cyclohexylglycine (D-Chg); x³May be glutamic acid (Glu), aspartic acid (Asp), alpha-aminoadipic acid (Aad), isoleucine (Ile), serine (Ser) or norleucine (Nle); and X⁴May be N⁶- (3- (pyridin-3-yl) propanoyl) -lysine (Lys12), (E) -N⁶- (3- (pyridin-3-yl) acryloyl) -lysine (Lys38), citrulline (Cit), homocitrulline (HoCit), leucine (Leu), N-methyl-leucine (N-MeLeu), isoleucine (Ile), N-methyl-isoleucine (N-MeIle), and homophenylalanine (HoPhe). In some embodiments, when subscript m is 2, and part- (X)¹)_m-is-X^1a-X^1bWhen is, X^1aCan be homocitrulline (HoCit), isoleucine (Ile), D-leucine (D-Leu) or aspartic acid (Asp); and X^1bCan be aspartic acid (Asp), threonine (Thr) or D-O-methyltyrosine (D-Tyr (Me)).

In some embodiments, the present invention provides a compound of formula I, formula Ia, formula Ib, formula Ic, or formula II wherein subscript m is 0 and the moiety-X⁴-X³-X²-is-Lys 12-Aad-Ach-, -Ile-Glu-Acpc-or-HoPhe-Nle-Glu-. In some embodiments, the present invention provides a compound of formula I, formula Ia, formula Ib, or formula II, wherein subscript m is 1, and the moiety-X⁴-X³-X²-X¹-is-N-MeIle-Glu-Asp-Pro-, -Lys 12-Glu-Ile-Aad-, -Cit-Glu-Ser-Val-, -Lys 12-Glu-Glu-Arg-, -N-MeLeu-Asp-D-Pra-4-Apc-, -Lys 38-Glu-Ile-Aad-, -Lys 38-Glu-Glu-Arg-, -HoCit-Ile-Phe (4)-Me) -D-His-, -Leu-Ser-Cha-D-Tyr (Me) -, -HoCit-Glu-Glu-Orn-, -Ile-Glu-Aad-Aib-, -Lys 38-Aad-Phe (4-Me) -Tyr-, -Lys 38-Aad-Aic-Nle-, -Ile-Aad-Ser-Asp-, or-Ile-Aad-Ser-Tyr-. In some embodiments, the present invention provides a compound of formula I, formula Ia, formula Ib, formula Ic, or formula II wherein subscript m is 2, and the moiety-X⁴-X³-X²-X^1a-X^1b-is-Lys 12-Aad-Hyp-HoCit-Asp-, -HoCit-Aad-Gln-Ile-Asp-, -HoCit-Aad-D-Glu-D-Leu-Thr-or-HoCit-Aad-D-Chg-Asp-D-Tyr (Me) -. In some embodiments, the present invention provides a compound of formula I, formula Ia, or formula II, or a pharmaceutically acceptable salt thereof, wherein subscript m is 0, and the moiety-X⁴-X³-X²-is-Lys 12-Aad-Ach-. In some embodiments, the present invention provides a compound of formula I, formula Ia, or formula II, or a pharmaceutically acceptable salt thereof, wherein subscript m is 1, and the moiety-X⁴-X³-X²-X¹-is-Cit-Glu-Ser-Val-.

In some embodiments, the compound of formula Ib is selected from:

in some embodiments, the compound of formula Ic is selected from:

the L moiety of formula I, formula Ia and formula II may be any suitable linker, including but not limited to linkers having one or more different reactive functional groups that allow covalent attachment of moieties such as peptides, monomers and polymers. The linking moiety may have two or more different reactive functional groups. In some cases, a multivalent linker may be used, and the plurality of peptides and/or the plurality of active agents of the present invention may be linked via the linker. Suitable linkers include, but are not limited to, those available from Pierce Biotechnology, Inc. One skilled in the art understands that any reactive functional group may be present on the linker so long as it is compatible with the functional group on the moiety to be covalently attached. Some suitable linkers include, but are not limited to, β -alanine, 2' -ethylenedioxybis (ethylamine) monosuccinamide (Ebes), bis (Ebes) -Lys, and polyethylene glycol. Other suitable linkers include affinity-based linkers that bind moieties via non-covalent interactions (e.g., linkers comprising biotin). Other suitable linkers include cleavable linkers, such as disulfide linkers, e.g., 4- ((2- ((2-aminoethyl) disulfanyl) ethyl) amino) -4-oxobutanoic acid, which can be cleaved under reducing conditions, or peptide linkers, which can be cleaved by the action of a protease.

One of ordinary skill in the art will recognize that other linkers are possible for the compounds of the present invention. Many such linkers can be found in "Bioconjugate Techniques" of Greg T Hermanson, Academic Press, San Diego,1996, or prepared by the Techniques described therein, which are incorporated herein by reference. Furthermore, one of ordinary skill in the art will recognize that other linkers can be prepared based on the Click chemical synthesis techniques as described in Kolb, H.C., Finn, M.G., Sharpless, K.B., Angew.chem.int' l.Ed.40(11): 2004-. Linkers useful in the present invention include those based on Ebes and PEG moieties. The linker may comprise 0 to 6 Ebes or PEG groups. Ebes and PEG groups can be conjugated by the techniques cited above. In some embodiments, linker L of formula I, formula Ia, and formula II comprises at least one of N- (8-amino-3, 6-dioxa-octyl) succinamic acid (Ebes) and polyethylene glycol (PEG).

In some other embodiments, the linker L of formula I, formula Ia, and formula II is selected from:

wherein k is 0 to 6.

The moiety D of formula I, formula Ia and formula II may be any suitable phosphonate drug including, but not limited to, mono-, di-and tri-phosphonates. In some embodiments, the moiety D of formula I, formula Ia, and formula II is a phosphonate or bisphosphonate compound. In some embodiments, the moiety D of formula I, formula Ia, and formula II is a bisphosphonate compound. In some embodiments, moiety D is a bisphosphonate compound, such as, but not limited to, etidronate (didrone), clodronate (Bonefos, Loron), tiludronate (Skelid), pamidronate (APD, Aredia), neridronate, olpadronate, alendronate (Fosamax), ibandronate (Boniva), risedronate (Actonel), and zoledronate (zeeta). Additional bisphosphonates are described in more detail below. Those skilled in the art will appreciate that other bisphosphonates may also be used in the present invention.

In some embodiments, the moiety D of formula I, formula Ia, and formula II has the formula:

wherein R is⁵Selected from H, OH and halogen; r⁶Selected from H and C_1-6An alkyl group; and subscript t is 1 to 6.

In some other embodiments, the moiety D of formula I, formula Ia, and formula II has a formula selected from:

in some embodiments, the compounds of formula I, formula Ia, and formula II are selected from:

in some embodiments, the compounds of formula I, formula Ia, and formula II are selected from:

in some embodiments, salts, hydrates, solvates, prodrug forms, isomers, and metabolites of the compounds of formula I, formula Ia, and formula II are provided.

In some embodiments, the present invention provides isomers or pharmaceutically acceptable salts of the compounds of formula I, formula Ia, and formula II. Salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, phosphonic acid, isonicotinate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, gluconate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1' -methylene-bis- (2-hydroxy-3-naphthoate)). Other salts include, but are not limited to, salts with inorganic bases including alkali metal salts such as sodium, lithium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; an aluminum salt; and ammonium salts such as ammonium salts, trimethyl-ammonium salts, diethyl-ammonium salts, and tris- (hydroxymethyl) -methyl-ammonium salts. Other salts with organic bases include salts with diethylamine, diethanolamine, meglumine and N, N' -dibenzylethylenediamine. Acid addition salts, such as mineral acids, organic carboxylic acids and organic sulfonic acids, such as hydrochloric acid, methanesulfonic acid, maleic acid, are also possible, provided that a basic group, such as a pyridyl group, forms part of the structure.

The neutral forms of the compounds of formula I, formula Ia and formula II may be regenerated by contacting the pharmaceutically acceptable salts of formula I, formula Ia or formula II with a base or acid and isolating the parent compound in the conventional manner. The parent forms of the compounds of formula I, formula Ia and formula II differ from the various salt forms in certain physical properties (e.g. solubility in polar solvents), but otherwise for the purposes of the present invention, the salts are equivalent to the parent forms of the compounds.

Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms (including hydrated forms). In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in a variety of crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to fall within the scope of the present invention.

Certain compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms, in terms of absolute stereochemistry, may be defined as (R) -or (S) -or as (D) -or (L) -of amino acids and individual isomers are encompassed within the scope of the present invention. The compounds of the present invention do not include compounds known in the art that are too unstable to be synthesized and/or isolated. The present invention is intended to include compounds in racemic and optically pure forms. Optically active (R) -and (S) -, or (D) -and (L) -isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.

The invention also provides compounds in prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Alternatively, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, a prodrug may be slowly converted to a compound of the invention when placed in a transdermal patch reservoir along with a suitable enzyme or chemical agent.

The compounds of the present invention can be synthesized by various methods known to those skilled in the art (see Comprehensive Organic Transformations, 1989, Richard c. Techniques useful for synthesizing the compounds of the present invention will be apparent to those skilled in the relevant art and are readily available. The following discussion is provided to illustrate certain of the various methods that can be used to make the compounds of the present invention. However, this discussion is not intended to limit the scope of reactions or reaction sequences that may be used to prepare the compounds of the present invention. One skilled in the art will appreciate that other methods of preparing the compounds may be used in the present invention.

The "one-bead one-compound" (OBOC) combinatorial library approach was first reported in 1991 (Lam et al, Nature,1991,354: pages 82-4). Essentially when a "split-mix" synthetic method (Lam et al, id; Houghten et al, Nature,1991,354: pages 84-6; Furka et al, int.J.peptide Protein Res.,1991,37: pages 487-93) is used to generate combinatorial libraries, each bead expresses only one chemical entity (Lam et al, id; Lam et al, chem.Rev.,1997,97: pages 411-48). A random pool of millions of beads can then be screened in parallel for a particular receptor molecule (e.g., receptor, antibody, enzyme, virus, whole cell, etc.). Positive beads were physically separated for structural determination by microsequencing using automated Edman degradation (Lam et al, Nature,1991,354: pages 82-4).

One-bead-one-compound (OBOC) combinatorial library methods synthesize millions of compounds, such that each bead displays only one compound. An example of a compound identified by the OBOC combinatorial library approach is LLP2A, which binds to integrin α₄β₁Specific binding (IC)₅₀2 pM). LLP2A is useful for expressing alpha with high sensitivity and specificity when conjugated to near infrared fluorescent dyes₄β₁To direct the therapeutic compound to express alpha₄β₁Lymphoma of (9) (Peng, L., et al, Nat Chem Biol,2006,2(7): pages 381-9). This ligand is known to direct compounds to express alpha₄β₁Lymphoma of (1) (Peng, L., et al, Nat Chem Biol,2006,2(7): pages 381-9; Peng, L., et al, Mol Cancer Ther,2008,7(2): pages 432-7, Aina, O.H., et al, Mol Pharm,2007.4(5): pages 631-51; Aina, O.H., et al, Mol Cancer Ther,2005.4(5): pages 806-13).

The compounds described herein may be used in combination with other active agents known to be useful in treating osteoporosis or promoting bone growth, or may be used in combination with adjuvants that may be ineffective alone but may contribute to the efficacy of the active agent. In some embodiments, co-administration of a compound herein with other agents includes administration of one agent within 0.5, 1,2,4, 6, 8, 10, 12, 16, 20, or 24 hours of a second agent. Co-administration includes administering two active agents simultaneously, approximately simultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes of each other), or sequentially in any order. In some embodiments, co-administration may be accomplished by co-formulation, i.e., preparing a single pharmaceutical composition comprising both active agents. In other embodiments, the active agents may be formulated separately. In another embodiment, the active agents and/or adjuvants may be linked or conjugated to each other.

Pharmaceutical compositions and methods of administration

In another embodiment, the present invention provides a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable excipient.

The compounds of the present invention may be prepared and administered in a wide variety of oral, parenteral and topical dosage forms. Oral formulations include tablets, pills, powders, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions and the like, suitable for ingestion by a patient. The compounds of the invention may also be administered by injection, i.e. intravenously, intramuscularly, intradermally, subcutaneously, intraduodenally or intraperitoneally. Likewise, the compounds described herein may be administered by inhalation, e.g., intranasally. In addition, the compounds of the present invention may be administered transdermally. The compounds of the invention may also be administered by: intraocular, intravaginal, and intrarectal routes (including suppositories, insufflation, powders, and aerosols) (e.g., steroid inhalants, see Rohatagi, J.Clin.Pharmacol.35: 1187. sup. 1193, 1995; Tjwa, Ann.Allergy Asthma Immunol.75: 107. sup. 111, 1995).

The compositions typically comprise conventional pharmaceutical carriers or excipients, and may additionally comprise other drugs, carriers, adjuvants, diluents, tissue penetration enhancers, solubilizers, and the like. In some embodiments, the composition will comprise from about 0.01% to about 90% (e.g., from about 0.1% to about 75%, or from about 0.1% to about 50%, or from about 0.1% to about 10%) by weight of the peptidomimetic ligand compound, with the remainder consisting of suitable pharmaceutical carriers and/or excipients. Suitable excipients may be tailored for the particular composition and route of administration by methods well known in the art, such as described by Remington, supra.

For oral administration, the compositions may be in the form of tablets, capsules, emulsions, suspensions, solutions, syrups, sprays, lozenges, powders and sustained release preparations. Suitable excipients for oral administration include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.

In some embodiments, the pharmaceutical composition is in the form of a pill, tablet or capsule, and thus, the composition may comprise any one of the following together with the ligand or combination of ligands: diluents such as lactose, sucrose, dicalcium phosphate, and the like; disintegrants, for example starch or derivatives thereof; lubricants, such as magnesium stearate and the like; and binders such as starch, gum arabic, polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof. The ligands may also be formulated as suppositories placed in, for example, polyethylene glycol (PEG) carriers.

The liquid composition may be prepared by: the ligand or combination of ligands and optionally one or more pharmaceutically acceptable excipients are dissolved or dispersed in a carrier such as saline (e.g., 0.9% w/v sodium chloride), aqueous dextrose, glycerol, ethanol, or the like, to form a solution or suspension, e.g., for oral, topical, or intravenous administration. The ligands of the invention may also be formulated as retention enemas.

For topical administration, the compositions of the present invention may be in the form of emulsions, lotions, gels, creams, jellies, solutions, suspensions, ointments, and transdermal patches. For delivery by inhalation, the compositions may be delivered in dry powder or liquid form by a nebulizer. For parenteral administration, the compositions may be in the form of sterile injectable solutions and sterile packaged powders. In some embodiments, the injectable solution may be formulated at a pH of about 4.5 to about 7.5.

The compositions of the present invention may also be provided in lyophilized form. Such compositions may include a buffer, such as bicarbonate, for reconstitution prior to administration, or the buffer may be included in a lyophilized composition for reconstitution, e.g., with water. The lyophilized composition may also comprise a suitable vasoconstrictor, such as epinephrine. The lyophilized composition may be provided in a syringe, optionally packaged with a buffer for reconstitution, such that the reconstituted composition may be administered immediately to a patient.

In powders, the carrier is a fine solid which is mixed with a fine active ingredient. In tablets, the active ingredient is mixed with the carrier having the necessary binding characteristics in suitable proportions and compacted in the shape and size desired. Suitable solid excipients include, but are not limited to, magnesium carbonate; magnesium stearate; calcium phosphate; calcium silicate; talc; pectin; dextran, dextrin and cyclodextrin inclusion complexes; a low melting wax; cocoa butter; a carbohydrate; sugars, including but not limited to lactose, dextrose, sucrose, mannitol, or sorbitol; starches, including but not limited to starches from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose or sodium carboxymethylcellulose; and gums including gum arabic, tragacanth gum, and acacia gum; and proteins including, but not limited to, gelatin, collagen; microcrystalline cellulose, water, saline, syrup, ethylcellulose, and polyacrylic acids, such as Carbopol, e.g., Carbopol 941, Carbopol 980, Carbopol 981, and the like; a lubricant; mineral oil; a wetting agent; an emulsifier; a suspending agent; preservatives, such as methyl-hydroxy-benzoate, ethyl-hydroxy-benzoate, and propyl-hydroxy-benzoate (i.e., p-hydroxybenzoate); pH adjusters, such as inorganic and organic acids and bases; a sweetener; and a flavoring agent; biodegradable polymer beads. If desired, disintegrating or solubilizing agents may be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, alginates or salts thereof, such as sodium alginate.

The pharmaceutically acceptable carrier may include physiologically acceptable compounds that function, for example, to stabilize the compounds of the present invention or to regulate their absorption, or other excipients as desired. Physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. One skilled in the art will appreciate that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, will depend, for example, on the route of administration of the compounds of the invention and the particular physiochemical characteristics of the compounds of the invention. Generally, such carriers are non-toxic to recipients at the dosages and concentrations employed. Typically, the preparation of such compositions requires combining the therapeutic agent with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates (including glucose, maltose, sucrose or dextrins), chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin are exemplary suitable diluents.

Dragee cores are provided with suitable coatings, for example concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbomer gel, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings for product identification or to characterize the amount of active compound (i.e., dose). The pharmaceutical preparation of the present invention can also be orally administered using, for example, push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a coating such as glycerin or sorbitol. Push-fit capsules may contain a compound of the invention in admixture with filler or binder (e.g., lactose or starch), lubricant (e.g., talc or magnesium stearate), and optionally stabilizer. In soft capsules, the compounds of the invention may be dissolved or suspended in suitable liquids (e.g., fatty oils, liquid paraffin, or liquid polyethylene glycols with or without stabilizers).

Some sustained release embodiments include biodegradable and/or slowly dissolving polymeric substances. Such polymeric materials include polyvinylpyrrolidone, low and medium molecular weight hydroxypropyl and hydroxypropyl methylcellulose, cross-linked sodium carboxymethylcellulose, carboxymethyl starch, potassium methacrylate divinyl benzene copolymers, polyvinyl alcohol, starch derivatives, microcrystalline cellulose, ethyl cellulose, methylcellulose and cellulose derivatives, beta-cyclodextrin, poly (methyl vinyl ether/maleic anhydride), dextran, scleroglucan (scierozlucans), mannan, xanthan gum, alginic acid and its derivatives, dextrin derivatives, glyceryl monostearate, semisynthetic glycerides, glyceryl palmitostearate, glyceryl behenate, polyvinylpyrrolidone, gelatin, magnesium stearate, stearic acid, sodium stearate, talc, sodium benzoate, boric acid and colloidal silicon dioxide.

The sustained-release agent of the present invention may further include excipients such as starch, pregelatinized starch, calcium phosphate mannitol, lactose, sucrose, glucose, sorbitol, microcrystalline cellulose, gelatin, polyvinylpyrrolidone, methyl cellulose, starch solution, ethyl cellulose, gum arabic, tragacanth, magnesium stearate, stearic acid, colloidal silicon dioxide, glycerol monostearate, hydrogenated castor oil, waxes, and mono-, di-and tri-substituted glycerides. Sustained release formulations may also be prepared as generally described in WO 94/06416.

To prepare suppositories, a low melting wax such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active ingredient is dispersed homogeneously therein by stirring. The molten homogeneous mixture is then poured into a mold of conventional size, allowed to cool, and then solidified.

Liquid form preparations include solutions, suspensions and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid formulations may be formulated in solution in aqueous polyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired. Aqueous suspensions suitable for oral use can be prepared by dispersing the fine active ingredient in water with: viscous substances such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as naturally-occurring phosphatides (e.g., lecithin), condensation products of an alkylene oxide with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol (e.g., polyoxyethylene sorbitol monooleate) or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents and one or more sweetening agents, for example sucrose, aspartame or saccharin. The osmotic amount of the preparation can be adjusted.

Also included are solid form preparations intended to be converted, shortly before administration, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions and emulsions. Such formulations may contain, in addition to the active ingredient, such ingredients as colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

Oily suspensions may be formulated by suspending a compound of the invention in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin or in a mixture of these. The oily suspending agents may comprise thickening agents, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as glycerol, sorbitol or sucrose may be added to provide a palatable oral preparation. These formulations may be preserved by the addition of an antioxidant such as ascorbic acid. See Minto, J.Pharmacol.Exp.Ther.281:93-102,1997 as an example of an injectable oil carrier. The pharmaceutical formulations of the present invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil or a mineral oil, as described above, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums such as gum arabic and tragacanth; naturally occurring phospholipids such as soybean lecithin; esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate; and condensation products of these partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. Emulsions may also contain sweetening agents and flavoring agents, such as syrups and elixirs. Such formulations may also contain a demulcent, a preservative or a coloring agent.

In some embodiments, the pharmaceutical formulation is in a unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, for example, dogs, each unit containing a predetermined quantity of active material calculated to produce the desired onset, tolerability and/or therapeutic effect in association with a suitable pharmaceutical excipient (e.g., an ampoule). In addition, higher concentrations of the compositions can be prepared, from which more dilute unit dose compositions can then be prepared. Thus, a higher concentration of a composition will comprise substantially more than, for example, at least 1,2,3,4,5, 6, 7, 8, 9, 10 or more times the amount of the ligand or combination of ligands. In this form, the preparation is subdivided into unit doses containing appropriate quantities of the active ingredient. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Likewise, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate amount of any of these in packaged form. The composition may also contain other compatible therapeutic agents, if desired. Certain pharmaceutical formulations may deliver the compounds of the present invention in a sustained release formulation.

Methods of administration and treatment

The ligands of the invention may be administered by any acceptable mode of administration, with suitable pharmaceutical excipients as required. Thus, administration can be, for example, intravenous, topical, subcutaneous, transdermal, intramuscular, oral, intra-articular, parenteral, intra-arteriolar, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, or by inhalation. It may also be applied directly to the bone surface and/or to the tissue surrounding the bone. The formulations may take any of the solid, semi-solid, or liquid dosage forms described above, such as, for example, tablets, pills, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, gels, aerosols, and the like.

Pharmaceutical formulations are typically delivered to mammals, including humans and non-human mammals. Non-human mammals to be treated using the methods of the present invention include domestic animals (i.e., dogs, cats, mice, rodents, and rabbits) and agricultural animals (cows, horses, sheep, pigs). Local augmentation of bone can be used for fracture healing, fusion (arthrodesis), orthopedic reconstruction, and periodontal repair. Systemic increase in bone can be used to treat low bone mass, i.e., decreased bone mass.

Typically, the administered dose will be effective to deliver a picomolar to micromolar concentration of ligand to the appropriate site or sites. However, one of ordinary skill in the art understands that the dose administered will vary depending on many factors, including but not limited to the particular ligand or group of ligands to be administered, the mode of administration, the type of administration (e.g., imaging, therapy), the age of the patient, and the physical condition of the patient. Preferably, the minimum dose and concentration required to produce the desired result should be used. For children, the elderly, infirm patients, and patients with heart and/or liver disease, the dosage should be adjusted appropriately. Further guidance can be obtained from studies known in the art to evaluate dosage using experimental animal models. However, the increased cell binding affinity and specificity associated with the ligands of the invention allow for a greater safety margin of dose concentration and repeat dosing.

In some embodiments, a therapeutically effective amount is an amount that activates Akt signaling in a subject. Akt signaling and its activation can be assessed in bone marrow stromal cells, for example, using the Akt signaling arrays shown in fig. 7A-7C. Typically, the peptidomimetic ligand compound will be administered in a dosage range of about 0.01mg to about 1000mg (i.e., about 0.01mg/kg to 1000mg/kg) per kilogram body weight of the individual. The dose of the compound may be, for example, from about 0.01mg/kg to 1000mg/kg, or from about 0.1mg/kg to 250mg/kg, or from about 0.2mg/kg to 100 mg/kg. The dose of the compound may be about 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 1mg/kg, 5mg/kg, 10mg/kg, 25mg/kg, 50mg/kg, 75mg/kg, 100mg/kg, 200mg/kg, 300mg/kg, 400mg/kg, 500mg/kg, 600mg/kg, 700mg/kg, 800mg/kg, 900mg/kg or 1000 mg/kg. The dosage may vary depending on the requirements of the patient, the severity of the condition being treated and the particular formulation being administered. The size of the dose will also be determined by the presence, nature and extent of any adverse side effects associated with the administration of the drug in a particular patient. The total dose may be divided and administered in portions over a period of time as appropriate to treat the condition or disorder.

The compound may be administered for a period of time that will vary depending on the nature of the particular condition, its severity, and the overall condition of the individual to whom the compound is administered. Compositions comprising a ligand or combination of ligands of the invention may be administered repeatedly, e.g., at least 2,3,4,5,6, 7, 8 or more times, or the compositions may be administered by continuous infusion. Administration may be, for example, hourly, every 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, or twice daily (including every 12 hours), or any interval thereof. Administration may be once daily, or once every 36 or 48 hours, or once monthly or several months. Following treatment, changes in the condition of the individual and alleviation of symptoms of the disorder can be monitored. The dosage of a compound may be increased in the event that the individual does not respond significantly to a particular dosage level, or may be decreased if remission of symptoms of the condition is observed, or if the condition has been treated, or unacceptable side effects occur at a particular dosage.

In practicing the methods of the present invention, the pharmaceutical composition may be used alone or in combination with other therapeutic or diagnostic agents. The additional drugs used in the combination regimen of the present invention may be administered separately or one or more of the drugs used in the combination regimen may be administered together, e.g., in admixture. If one or more drugs are administered separately, the timing and schedule of administration of each drug may vary. Other therapeutic or diagnostic agents may be administered simultaneously, separately or at different times from the compounds of the invention.

In some embodiments, the present invention provides methods of promoting systemic bone growth. Systemic skeletal growth refers to growth throughout the bones of an individual and may affect all bones in the individual. Individuals in need of systemic bone growth may suffer from a variety of diseases and disease states. In some embodiments, the subject has a low bone-grade disease. Low bone mass can be determined by a variety of methods known to those skilled in the art. For example, low bone mass is characterized by a T value of less than about-1. The low bone mass phenotype disease may include osteoporosis, osteopenia, and osteoporosis-pseudoglioma syndrome (OPPG). In some other embodiments, the low bone-scale disease may be osteopenia or osteoporosis-pseudoglioma syndrome (OPPG). In some other embodiments, the present invention provides methods of treating low bone mass by administering to an individual in need thereof a therapeutically effective amount of a compound of the present invention.

Following administration of the compounds of the invention, systemic bone growth can be determined by a variety of methods, such as improvement in bone density. Bone density can be measured by a number of different methods, including T-score and Z-score. The T-score is the standard deviation above or below the mean of healthy 30 year old adults of the same gender as the patient. Low bone mass is characterized by a T score of-1 to-2.15. Osteoporosis is characterized by a T score of less than-2.15. The Z-score is the number of standard deviations above or below the mean of the patient's age and gender. An improvement in the T score or Z score indicates bone growth. Bone density can be measured at various locations in the bone, such as the spine or hip. One skilled in the art will appreciate that other methods of determining bone density may be used with the present invention.

In some embodiments, the present invention provides a method of treating osteoporosis, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, formula Ia, formula Ib, formula II, or a pharmaceutically acceptable salt thereof.

The invention also provides methods of treating diseases characterized by secondary-induced osteoporosis ("secondary low bone mass"), but not limited to, osteomalacia, hypertrophic fibrous dysplasia, paget's disease, rheumatoid arthritis, zero gravity, osteoarthritis, long-term inactivity or immobility, osteomyelitis, celiac disease, crohn's disease, ulcerative colitis, inflammatory bowel disease, gastrectomy, secondary-induced osteoporosis, amenorrhea, cushing's disease, cushing's syndrome, diabetes (diabetes mellitis), diabetes (diabets), eating disorders, hyperparathyroidism, hyperthyroidism, hyperprolactinemia, Kleinefelter syndrome, thyroid disease, turner's syndrome, steroid-induced osteoporosis, epilepsy or depression-induced osteoporosis, immobility, arthritis, cancer-induced secondary osteoporosis, gonadotropin-releasing hormone agonist-induced low bone mass, thyroid drug-induced low bone mass, dilantin (phenytoin), sodium valproate-induced low bone mass, chemotherapy-induced low bone mass, immunosuppressive-induced low bone mass, blood diluent-induced low bone mass, graves' disease, juvenile rheumatoid arthritis, malabsorption syndrome, anorexia nervosa, kidney disease, anticonvulsant therapy (e.g. for epilepsy), corticosteroid therapy (e.g. for rheumatoid arthritis, asthma), immunosuppressive therapy (e.g. for cancer), insufficient nutrition (especially calcium, vitamin D), excessive exercise-induced amenorrhea (no menstruation), smoking and alcohol abuse, osteoporosis related to pregnancy, copper deficiency, type 2 diamido-aciduria, voran syndrome, Hajdu-Cheney syndrome, juvenile proteorfic outer-layer bone hyperplasia (hyperostosis corticoids deformans juveniles), methylmalonic aciduria type 2, cystathionine beta-synase deficiency, exemestane, hyperimmune globulin E (IgE) syndrome, hemochromatosis, Singleton Merten syndrome, beta thalassemia (homozygous), reflex sympathetic dystrophy, sarcoidosis, Winchester syndrome, Harlerman-Sterey syndrome (HSS), cyproterone, glycerol kinase deficiency, Bonnet-Dechaume-Blanc syndrome, prednisolone, heparin, dysplastic senile skin, Torg osteolysis syndrome, orchiectomy, Fabry's disease, pseudoprogeria syndrome (pseudooprogracil syndrome), Woott-Rallison syndrome, ankylosing spondylitis, myeloma, systemic infantile dystrophia (dysostosis), osteodystrophy, autoimmune lymphoproliferative syndrome, brown-seeka syndrome, Diamond-Blackfan anemia, galactorrhea-hyperprolactinemia, gonadal dysgenesis, kidney disease, menkes disease, menopause, neuritis, ovarian dysfunction due to FSH resistance, familial ovarian dysfunction, premature aging, primary biliary cirrhosis, prolactinoma, familial prolactinoma, renal osteodystrophy, ulcerative colitis, underweight, verner syndrome, bone tumors, bone cancer, fragile bone disease, congenital osteogenesis imperfecta, and delayed osteogenesis imperfecta. Other conditions include bone damage, such as fractured or weakened bones, or bones damaged by radiation therapy. One skilled in the art will appreciate that other types of conditions, diseases, and treatments can also lead to osteoporosis.

Glucocorticoids are a class of corticosteroids, a steroid hormone that is commonly used to treat diseases or conditions associated with overactivity of the immune system, such as asthma, allergy, asthma, autoimmune diseases (e.g., graves' disease, rheumatoid arthritis, lupus, inflammatory bowel disease, etc.) and sepsis. However, the use of steroids or glucocorticoids can lead to rapid bone loss and to a high risk of bone fractures. In some cases, a steroid or glucocorticoid (i.e., a glucocorticosteroid) can cause osteonecrosis, the details of which are described below.

In some embodiments, the present invention provides methods of treating steroid induced bone loss by administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, formula Ia, formula Ib, formula II, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides methods for treating glucocorticoid-induced bone loss by administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, formula Ia, formula Ib, formula II, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides methods of treating steroid-induced osteoporosis by administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, formula Ia, formula Ib, formula II, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides methods for treating glucocorticoid-induced osteoporosis by administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, formula Ia, formula Ib, formula II, or a pharmaceutically acceptable salt thereof.

Osteonecrosis, also known as ischemic necrosis or aseptic necrosis, is the death of bone cells in the skeleton due to reduced blood flow. If left untreated, the death of bone cells in the bone may lead to collapse of the bone region, which in turn may lead to degenerative arthritis of the joint near the bone. Osteonecrosis most commonly affects the hips and knees, but may also affect the shoulders, wrists, hands, ankles, feet, and jaw. Osteonecrosis may have a variety of causes, including traumatic and non-traumatic causes. Typically in traumatic osteonecrosis, severe trauma to the bone can interrupt the blood supply to the bone. Non-traumatic osteonecrosis may be caused by: certain drugs, such as corticosteroid drugs (e.g., prednisone, cortisone, dexamethasone, or methylprednisolone), especially when high doses of the drug are administered over an extended period of time; due to excessive drinking; from radiation therapy; or due to a disease or condition. See, e.g., Xie et al, 2015, Journal of Orthopaedic transformation, 3:58-70, which is incorporated herein by reference.

In some embodiments, the present invention provides methods of treating bone necrosis by administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, formula Ia, formula Ib, formula II, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides methods of treating post-traumatic osteonecrosis (e.g., that which occurs following fracture or dislocation of a bone) by administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, formula Ia, formula Ib, formula II, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides methods of treating non-traumatic osteonecrosis by administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, formula Ia, formula Ib, formula II, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides methods of treating steroid-induced osteonecrosis (e.g., high dose steroid-induced osteonecrosis or glucocorticoid-induced osteonecrosis), alcohol-induced osteonecrosis, or smoking-induced osteonecrosis by administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, formula Ia, formula Ib, formula II, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides methods of increasing vascular density in bone necrotic tissue by administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, formula Ia, formula Ib, formula II, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides methods of preventing or reducing cell death in bone necrotic tissue by administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, formula Ia, formula Ib, formula II, or a pharmaceutically acceptable salt thereof.

In some other embodiments, the present invention provides methods of treating secondary induced osteonecrosis by administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, formula Ia, formula Ib, formula II, or a pharmaceutically acceptable salt thereof. Examples of diseases or conditions that can induce osteonecrosis (i.e., secondary osteonecrosis) include, but are not limited to: Legg-Karl-Peltier disease (Legg-Calv-Perthes disease), Karson disease (Caisson disease), sickle cell disease, post-radiation, chemotherapy, arterial disease, gaucher disease, lipid disorders, connective tissue disease, pancreatitis, kidney disease, liver disease, or lupus. In some embodiments, the osteonecrosis is idiopathic osteonecrosis.

The invention also provides methods of treating a patient population characterized by damaged bones, such as fractured bones or bones damaged by radiation, and children with a contraindicated osteoporosis medication.

In some embodiments, the present invention provides methods of promoting bone growth by administering to an individual in need thereof a therapeutically effective amount of a compound of the present invention. Bone growth can be measured in a variety of ways known to those skilled in the art. Methods of measuring bone growth include, but are not limited to Uct (micro-CT), dual X-ray absorption (bone density), ultrasound, QCT, SPA, DPA, DXR, SEXA, QUS, X-ray, using the human eye during surgical procedures, alizarin red S, serum osteocalcin, serum alkaline phosphatase, serum Bone Gla Protein (BGP), bone mineral content, serum calcium, serum phosphorus, tantalum markers, and serum IGF-1.

Many indicators of bone growth may be used to measure bone growth, including bone density. In some embodiments, bone growth may be evidenced by a 0.1% increase in bone density. In other embodiments, bone growth may be evidenced by a 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% or greater increase in bone density. Those skilled in the art recognize that bone growth is localized, systemic, or both.

In some other embodiments, the methods of the invention promote bone growth by administering a compound of the invention, e.g., a compound of formula I. Administration of the compounds of the invention may promote local bone growth and/or systemic bone growth. In some embodiments, administration of a compound of the invention promotes systemic bone growth. Bone growth can be achieved by increasing bone mineral content, increasing bone density, and/or growth of new bone. In other embodiments, topical administration of the compounds and medicaments of the present invention achieves systemic bone growth.

V. examples

Example 1: screening for cell signaling with affinity for osteoblast specific transcription factor + osteoprogenitor cells Activating agent

The method for the discovery of osteoblast-specific peptides targeting bone cells with cell binding affinity and intracellular signaling involves bead screening of focused-on-bead-one-compound (OBOC) peptide libraries, which are designed based on the integrin α 4 β 1 motif. The synthesis method for developing a focused OBOC combinatorial library is shown below in scheme 1.

Scheme 1

Briefly, TentaGel beads (1.0g, 0.26mmol/g loading) were swollen in DMF (20mL) for 3 hours. The resin was divided into 43 equal portions and placed in 43 disposable polypropylene columns with polyethylene frit. 43 different Fmoc-amino acids (Table 4) (4 equiv.) were dissolved in 6-Cl HOBt (4 equiv.) and DIC (4 equiv.) in DMF and added to 43 columns (each column received only one amino acid). The coupling was carried out at room temperature for 2 hours. After filtration, the beads were combined, mixed and washed 3 times with DMF, MeOH and then DMF each. Fmoc-deprotection of the beads was performed with 20% 4-methylpiperidine (5 min, 15 min). After washing with DMF, MeOH and DMF, the beads were mixed with 1g of blank TentaGel beads. Using the same split-mix procedure as described above, 2g of combined beads were mixed in order with the beads at X₂30 Fmoc-amino acids at position (Table 5), at X₃8 Fmoc-amino acids at position (Table 6) and at X₄The 18 Fmoc-amino acids at position (Table 7) were coupled separately. After Fmoc-deprotection, the beads were washed with DMF, MeOH, DCM and dried thoroughly in vacuo.

₁Table 4. amino acid for position X.

₂TABLE 5 amino acids for position X.

₃Table 6. amino acid for position X.

₄TABLE 7 amino acids for position X.

Two-layer beads were then prepared using a biphasic solvent method as described in the references (Liu R, Marik J, Lam KS. J Am Chem Soc.2002Jul 3; 124(26): 7678-80.). Briefly, the dried beads were swelled in water for 1 day. Water was removed by filtration and a solution of Fmoc-OSu (17.5mg, 0.052mmol, 0.1 equivalents relative to the beads) in DCM/diethyl ether (150mL, 55/45) was added to the wet beads followed by DIEA (36. mu.L, 0.208 mmol). The mixture was shaken vigorously at room temperature for 45 minutes. The liquid was removed by filtration. The beads were washed 3 times each with DMF, MeOH, and then DCM. A solution of Boc anhydride (567mg, 2.6mmol) and DIEA (906. mu.L, 5.2mmol) in DCM was added to the beads and rotated for 1 h. The Kaiser test was negative indicating that the coupling was complete. Fmoc on the bead surface was removed with 20% 4-methylpiperidine (5 min, 15 min). The beads were washed and coupled with 4- [ (N' -2-methylphenyl) ureido ] -phenylacetic acid (UPA) (4 equivalents) in DMF in the presence of 6-Cl HOBt (4 equivalents) and DIC (4 equivalents). The coupling was confirmed to be complete within 4 hours by a negative Kaiser test. After washing with DMF, methanol and DCM, the beads were then dried under vacuum for 1 hour. Using 82.5% TFA 5% phenol 5% thioanisole 5% water: a mixture of 2.5% TIS achieved side chain deprotection. After neutralization twice with 2% DIEA/DMF, the resin was washed sequentially with DMF, MeOH, DCM, DMF/water, and PBS. Bead pools were stored in 70% ethanol.

As shown in fig. 1, OBOC combinatorial libraries were screened and peptides were identified that exhibited affinity for target cells and were used as activators or inhibitors of target cell signaling pathways (one bead/one compound/two functions) (see Liu, r. et al Curr Opin Chem biol.,2017,38: 117-26). Bone Marrow Stromal Cells (BMSCs) were obtained from mice and maintained in mesenchymal maintenance medium for 7 days to 2 weeks (about 3 passages), changed to osteogenic medium for 3 days, and then incubated with focused integrin pools for 1 hour. Cells and beads were then fixed and stained for phosphorylation Atk, a signaling transduction pathway that promotes cell survival and growth in response to extracellular signals (see Manning BD et al, cell.2007,129: 1261-74). Positive beads were identified as cell-bound and stained with green fluorescence (p-Akt +) (FIG. 1, step A). Positive beads were manually picked and subjected to microsequencing using Edman degradation (see, Liu and Lam, anal. biochem.2001,295: 9-16). Each peptide on the beads was resynthesized with a fluorescence quencher (nitrotyrosine) residue inside the beads to quench the autofluorescence of the beads. These beads were incubated with osteoprogenitor cells (OPCs) obtained from osteoblast specific transcription factor-mCherry reporter mice. Osteoblast-specific transcription factors are expressed by osteochondral progenitor cells and are generally considered to be early markers of osteoblast maturation, as described in Mizoguchi T et al development cell.2014,29,340-9 and Liu Y et al, PLoS one.2013,8: e 71318. Positive beads screened by the second round were identified by their binding affinity to osteoblast specific transcription factor + cells (fig. 1, step B). Activation of Akt binding was again confirmed (fig. 1, step C). Using this approach, 22 peptides (table 8) were identified as activators of Akt signaling with high affinity for osteoblast specific transcription factor + cells (fig. 1, step D). It was confirmed that the leader peptides YLL3 and YLL8 have high binding affinity to osteoblast-specific transcription factor cells and have an in vitro osteogenesis effect. Beads showing YLL3 and YLL8 also showed low affinity for lymphocytes (fig. 2).

Example 2: synthesis of YLL ligands

The YLL3, YLL8 and other ligands listed in Table 8 were prepared using commercially available starting materials and methods known in the art. Typically, the YLL ligand is prepared via Fmoc solid phase peptide synthesis using 4- [ (N' -2-methylphenyl) ureido ] phenylacetic acid (UPA), Rink Amide MBHA resin and related components to protect the amino acids, as described in international publication No. WO 2012/031228 a 2.

Solid phase Synthesis of YLL3 and YLL8

YLL8 was synthesized on Rink amide MBHA resin (GL Biochem, Shanghai, China) using standard solid phase peptide synthesis methods. Briefly, Rink amide MBHA resin (0.5g, 0.325mmol, loading 0.65mmol/g) was swollen in DMF for 2 hours, followed by Fmoc-deprotection twice (5 min, 15 min) with 20% 4-methylpiperidine in DMF. The beads were washed with DMF (3X 10mL), MeOH (3X 10mL), and DMF (3X 10 mL). Fmoc-Ach-OH (0.365g, 0.975mmol) was dissolved in 6-Cl HOBt (0.165g, 0.975mmol) and DIC (152. mu.L, 0.975mmol) in DMF and added to the beads. The coupling was carried out at room temperature for 2 hours. After filtration, the beads were washed 3 times with DMF (3X 10mL), MeOH (3X 10mL), and DMF (3X 10mL), respectively. Fmoc deprotection was removed twice with 20% 4-methylpiperidine (5 min, 15 min). After washing with DMF, MeOH and DMF, respectively, the beads were then subjected stepwise to additional cycles of coupling and deprotection with Fmoc-Aad (tBu) and Fmoc-Lys (A12) in the same manner as described above. After Fmoc removal, a solution of 4- [ (N' -2-methylphenyl) ureido ] phenylacetic acid (UPA, 0.923g, 3.25mmol), HOBt (0.498g, 3.25mmol) and DIC (509. mu.L, 3.25mmol) in DMF was added to the beads. The reaction was carried out at room temperature overnight. The beads were washed with DMF (5X 5mL), MeOH (3X 5mL), and DCM (3X 5 mL). The beads were then dried in vacuo for 1 hour, and then a lysis mixture of 95% TFA, 2.5% water, 2.5% TIS was added. The cleavage reaction was carried out at room temperature for 2 hours. The liquid was collected and concentrated. The crude product was precipitated with diethyl ether and purified using preparative RP-HPLC. Fractions were collected and lyophilized to give the designed product YLL8, MALDI-TOF MS: 813.30 Dalton.

YLL3 was synthesized using a method similar to YLL8, but the following different building blocks Fmoc-Val-OH, Fmoc-Ser (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cit-OH and UPA were coupled to the beads in this order. Crude YLL3 was cleaved from the beads and purified by RP-HPLC. MALDI-TOF MS: 756.25 Dalton.

Table 8.YLL ligand.

Example 3: in vitro osteogenesis of YLL peptides

The leader peptides YLL3 and YLL8 had high binding affinity for osteoblast-specific transcription factor cells and exhibited an in vitro osteogenesis effect, as shown in fig. 3 and 4. Mouse Bone Marrow Stromal Cells (BMSCs) were obtained from osteoblast specific transcription factor-mcherry transgenic mice and cultured in osteogenic medium for 10 days with focused pool beads encoding the YLL3, YLL8 and LLP2A peptides and fixed. Osteoblast specific transcription factor (OSX +) labeled osteoblast progenitor cells, and positive cells were red (fig. 3). Mouse BMSCs obtained from normal mice were incubated with focused pool beads displaying LLP2A, YLL3, or YLL8 peptides and cultured in osteogenic medium for 10 days. Staining of the cells with beads was used for anti-Akt-Alexa 488. Positive cells showed Atk activation and were stained green (fig. 4). Focused pool beads showing several peptides of table 4 were also incubated with mouse BMSCs obtained from conventional mice and cultured in osteogenic medium for 10 days. The level of alkaline phosphatase corresponding to osteogenic differentiation and osteoblastic maturation was measured on day 14. Alizarin red was measured on day 21 as a measure of osteoblast maturation and mineralization (fig. 5).

In an additional in vitro osteogenesis study, osteoblast differentiation (alkaline phosphatase, ALP activity) was measured for YLL3 and YLL8 on day 10 and osteoblast maturation (alizarin red staining) was measured on day 21. Beads showing peptides YLL3 or YLL8 increased ALP levels (fig. 6A and 6B) and induced higher mineralized nodule formation (fig. 6A and 6C) compared to beads showing scrambled peptides (control (Con)). Note that the cells were localized to or around the beads and deposited minerals (fig. 6C). These results were confirmed by adding the peptide directly to osteoblast differentiation cultures (fig. 6D).

The specificity of YLL to activate Akt signaling was then investigated by western blot analysis using an Akt signaling array. By first peptide (6X 10)^-8M) was incubated with BMSC in osteogenic medium for three days to confirm activation of the Akt pathway (fig. 7A). Human PTH (1-34) (6x 10)^-8M) was used as a positive control. As shown in fig. 7B, YLL8 has very similar characteristics to PTH in activating members of the Akt signaling pathway compared to control and YLL3, including phosphorylation of Akt, phosphoinositide-dependent kinase 1(ERK1/2), P53, PDK1), P53, 4EBP1, and BCL2, the relevant cell death agonists (BAD) and RIK1 (fig. 7B, fig. 7C)) P53 and Akt activated by YLL3 (fig. 7B, fig. 7C). These results indicate that osteoblast targeting peptides (e.g., YLL8) increase osteoblast differentiation and maturation through signaling pathways or pro-survival mechanisms that promote cell growth.

Example 4: in vivo anabolic effects of YLL peptides

In a study to determine whether the YLL peptide affects bone metabolism in vivo, two month old female and male mice were treated on day 1 with PBS control, YLL3, YLL8, or LLP2A intravenously at 50 μ g/kg. hPTH (1-34) was injected subcutaneously at 25 μ g/kg for 21 days 5 times a week (n-4-6/group). Trabecular bone volume and cortical bone volume at the distal femur and cortical bone volume at the middle of the femur were measured by micro-CT (fig. 8).

In an additional study to determine whether YLL affects bone metabolism in vivo, YLL3 and YLL8 were injected 5 times per week at 25 μ g/kg or 50 μ g/kg Subcutaneously (SC) into 2 month old mice for 21 days, respectively. hPTH (1-34) was injected subcutaneously 5 times a week at 25. mu.g/kg as a positive control. The results from the 50. mu.g/kg group are shown in FIG. 9. Injection of both YLL peptides daily for 21 days did not change body weight or cause any visible side effects. In females, YLL3 and YLL8 increased trabecular bone volume by about 13% (p <0.05 relative to PBS) and 8% (fig. 9A), respectively, which correlates with 60% (p <0.05 relative to PBS) and 86% increase in surface-based trabecular bone formation rate (fig. 9B and 9D). In males, YLL3 and YLL8 increased trabecular bone volume by about 15% (p <0.05 relative to PBS) and 6% (fig. 9A), respectively, which correlates with a 53% (p <0.05 relative to PBS) and 50% (p <0.05 relative to PBS) increase in trabecular bone formation rate (fig. 9A and 9D). In males, both YLL3 and YLL8 increased cortical bone volume by about 15%, and higher bone formation was observed at the periosteal surface (fig. 9C and 9E). Note that YLL3 increased intracortical bone remodeling (fig. 9E). These results indicate that both YLL3 and YLL8 stimulate bone formation on the trabecular and cortical surfaces and are comparable to the anabolic effects of daily PTH treatment.

In another similar study to determine whether YLL would affect bone metabolism in vivo, YLL3 and YLL8 were injected 5 times a week for 21 days (n-5-7/group) subcutaneously (sc) at 5 μ g/kg into 4 month old female and male mice. hPTH (1-34) was injected subcutaneously 5 times a week at 25. mu.g/kg as a positive control. Injection of YLL8 daily for 21 days did not change body weight or cause any significant side effects. Both YLL8 and hPTH (1-34) increased femoral trabecular volume in female mice by about 130% compared to PBS-treated mice. In male mice, YLL8 increased the volume of the femoral trabecular bone by about 70%, while hPTH (1-34) increased the volume of the femoral trabecular bone by about 30% (FIG. 10A). The parameter corresponding to osteoblast activity mineral deposition rate increased by more than 70% in females and about 30% in males, resulting in an overall increase in the rate of surface-based bone formation (fig. 10A, 10B). Anabolic effects of YLL were confirmed by increases in trabecular bone mass measured at the distal metaphysis of the femur by micro-CT and serum levels of osteocalcin (fig. 11). Bone resorption measured by serum CTX1 showed that PTH increased bone resorption by 200% from PBS treated group, especially in female mice, while YLLL did not significantly change CTX1 (fig. 11). Daily treatments with YLL3 and YLL8 for 21 days increased cortical bone mass in males and increased bone strength in both females and males, comparable to daily hPTH (1-34) injections (fig. 10D). These results indicate that YLL3 and YLL8 induce uncoupling of bone formation and resorption and increase bone mass.

Example 5: mineralization potential during bone fracture repair using YLL peptides

The following experiment was performed to determine whether the YLL peptide accelerated fracture repair. Recruitment and activation of endogenous osteoblast lineage cells during fracture healing is a critical step in repairing fractures. Because inducible osteoblast-specific transcription factor-reporter mice are not directly commercially available, inducible Prx1-CreERT-GFP mice were used to follow the recruitment and osteogenic differentiation of osteoprogenitor cells following fracture and YLL treatment. In uninjured mice, Prx1 labeled osteoprogenitor cells at the growth plate and alongside the trabecular and intracortical bone surface, similar to the locations in bone where osteocyte-specific transcription factors are expressed (fig. 12). A closed, stable model of bone fracture in the femur was used to track the movement of osteoblast lineage cells in this model that contribute to intramembranous or endochondral bone formation. Human PTH (1-34) was used as a positive control (50. mu.g/kg, 5 times per week). On day 10 post-fracture, the presence of Prx 1/GFP-expressing cells was observed in the callus, some co-localized with alizarin red, indicating active bone deposition (appposition) from those neighboring cells (fig. 13A). In the YLL8 and PTH treated mice, the number of Prx1+ cells was greatly increased, some co-localized with mineral deposition (fig. 13A). Almost all Prx1+ green cells overlapped alizarin red, indicating that osteoblast differentiation and mineral uptake in these cells was high after yl 3 treatment (fig. 13A), yielding 100% higher callus volume compared to day 10 PBS-treated mice (fig. 13B). Further dynamic histomorphometry studies using fluorescent dye labeling for mineralization at day 21 post fracture showed that nearly all Prx1+ and their progeny cells overlapped AR mineral deposits in all groups. In the YLL 3-treated mice, Prx1+ and its progeny cells were activated and promoted callus formation, co-localized within the regenerated callus region (fig. 13C). Quantitative measurements by micro-CT scanning confirmed higher callus mineral content in YLL3 treated mice on day 21 post-fracture (fig. 13D). These data indicate that a similar anabolic mechanism activates osteoblast lineage cells through YLL8 and PTH during fracture healing. Injection of YLL3 greatly activated osteogenic differentiation of osteoprogenitor cells and induced significantly higher callus mineralization, which accelerated fracture repair.

Example 6: synthesis of YLL 3-alendronate (YLL3-Ale) and YLL 8-alendronate (YLL8-Ale)

To ensure that anabolic effects are bone specific, the YLL3 and YLL8 peptides were conjugated to alendronate.

YLL 3-alendronate (YLL3-Ale) and YLL 8-alendronate (YLL8-Ale) were made by linking alendronate-maleimide (Ale-Mal) (produced by conjugation of alendronate and sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC)) to D-cysteine, lysine, two N- (8-amino-3, 6-dioxa-octyl) succinamide linkers and either YLL3 or YLL8, as shown in FIG. 14 and further described in International publication No. WO 2012/031228A 2, or as described below.

In general, the synthesis of YLL8-Aln involves three steps.

Step 1 solid phase Synthesis of YLL8-Lys (D-Cys)

The synthesis method for the synthesis of YLL8-Lys (D-Cys) is shown in scheme 2 below. Rink amide MBHA resin (0.5g, 0.325mmol, loading 0.65mmol/g) was swollen in DMF for 3 hours. Fmoc was deprotected twice with 20% 4-methylpiperidine in DMF (5 min and 15 min, respectively). After filtration, the beads were then washed with DMF (3X 10mL), methanol (MeOH) (3X 10mL), and DMF (3X 10mL), respectively. Fmoc-Lys (Dde) -OH (0.519g, 0.975mmol) was dissolved in 6-Cl HOBt (0.165g, 0.975mmol) and DIC (152. mu.L, 0.975mmol) in DMF (8mL) and added to the suspension of beads. The coupling was carried out overnight at room temperature. After filtration, the beads were washed with DMF (3X 10mL), MeOH (3X 10mL), and DMF (3X 10mL), respectively. Fmoc was deprotected twice with 20% 4-methylpiperidine in DMF (8mL) (5 min and 15 min, respectively). The Fmoc-linker (0.612g, 1.3mmol) was dissolved in a solution of HOBt (0.22g, 1.3mmol) and DIC (201. mu.L, 1.3mmol) in DMF (8mL) and then added to the suspension of beads. Coupling was performed at room temperature until the Kaiser test was negative. After filtration, the beads were washed with DMF (3X 10mL), MeOH (3X 10mL), and DMF (3X 10mL), respectively. Fmoc was deprotected twice with 20% 4-methylpiperidine in DMF (8mL) (5 min and 15 min, respectively). The Fmoc-linker (0.612g, 1.3mmol) was dissolved in a solution of HOBt (0.22g, 1.3mmol) and DIC (201. mu.L, 1.3mmol) in DMF (8mL) and then added to the suspension of beads. Coupling was performed at room temperature until the Kaiser test was negative. After filtration, the beads were washed with DMF (3X 10mL), MeOH (3X 10mL), and DMF (3X 10mL), respectively. Fmoc was deprotected twice with 20% 4-methylpiperidine in DMF (8mL) (5 min and 15 min, respectively). After filtration, the beads were washed with DMF (3X 10mL), MeOH (3X 10mL), and DMF (3X 10mL), respectively. Fmoc-Ach-OH (0.365g, 0.975mmol) was dissolved in 6-Cl HOBt (0.165g, 0.975mmol) and DIC (152. mu.L, 0.975mmol) in DMF and added to the beads. The coupling was carried out at room temperature for 2 hours.

Scheme 2

After filtration, the beads were washed with DMF (3X 10mL), MeOH (3X 10mL), and DMF (3X 10mL), respectively. The Fmoc deprotecting group was removed twice with 20% 4-methylpiperidine (5 min and 15 min, respectively). After washing with DMF, MeOH and DMF, respectively, the beads were then subjected stepwise to additional cycles of coupling and deprotection with Fmoc-Aad (OtBu) -OH, Fmoc-Lys (A12) and UPA in the same manner as described above.

The Dde protecting group was removed twice with 2% hydrazine monohydrate in DMF (5 min and 10 min respectively), the beads were washed with DMF, MeOH and DMF and then Boc-D-Cys (Trt) -OH (4 equivalents for resin, 220mg, 1.3mmol), HOBt (0.176g, 1.3mmol) and DIC (201. mu.L, 1.3mmol) were coupled in DMF (8 mL). The coupling reaction was carried out at room temperature until the Kaiser test was negative (4 hours to overnight). The beads were washed thoroughly with DMF, MeOH, and DCM, respectively, then dried under vacuum for 1 hour, and then a cleavage mixture (8mL) of 82.5% trifluoroacetic acid (TFA): 5% thioanisole: 5% phenol: 5% water: 2.5% Triisopropylsilane (TIS) (v/v) was added. The cleavage reaction is carried out at room temperature for 2-3 hours. A crude product precipitated as an off-white and was washed with cold diethyl ether. The purity was determined by analytical HPLC and the crude product was used in the next step without further purification.

Step 2. Synthesis of Aln-Mal

The synthesis method for synthesizing Aln-Mal is shown in scheme 3 below. Alendronate disodium salt (35.4mg, 0.1208mmol) (lyophilized powder from alendronate in water and 2 equivalents NaOH) was dissolved in 0.1M PBS (15mL) containing 10mM EDTA, pH 7.5. The aqueous solution was then cooled in an ice-water bath and a solution of sulfo-SMCC (58mg, 0.133mmol) in water (14mL) was added dropwise. After the addition was complete, the resulting solution was allowed to warm to room temperature while stirring for 2 hours to yield an Aln-Mal solution, which was used for the next step of conjugation without purification.

Scheme 3

Step 3 conjugation of YLL8-Aln

The conjugation procedure for the synthesis of YLL8-Aln is shown in scheme 4 below. The solution of Aln-Mal prepared in step 2 was cooled in an ice-water bath, followed by addition of acetonitrile (8mL) and dropwise addition of a solution of LLP2A-Lys (D-Cys) (200mg, 0.133mmol) in a small amount (. about.4 mL) of 50% acetonitrile/water. With NaHCO₃The aqueous solution adjusted the pH to 6 to 7. The resulting mixture was stirred for 1 hour, and then warmed to room temperature. After negative Ellman test, the solution was lyophilized. The resulting powder was redissolved in a small amount of 50% acetonitrile/water and purified by reverse phase high performance liquid chromatography (RP-HPLC); vydac C18 column (10 μm, 22X 250mm), buffer A: 0.1% TFA/H₂O, buffer B: 0.1% TFA/acetonitrile. Gradient: 10% ACN, 1 minute; 40% ACN, 50 minutes; 100% ACN, 3 min. The collected eluate was lyophilized to give an off-white powder of YLL8-Aln, MALDI-TOF MS: 1972.75 Dalton. YLL3-Aln was synthesized by conjugation of YLL3-Lys (D-Cys) and Aln-Mal using a method similar to YLL 8-Aln. MALDI-TOF MS: 1915.76 Dalton.

Scheme 4

In some cases, the YLL-Ale compounds are single organic molecules consisting of highly derivatized synthetic peptidomimetic moieties (UPA-YLL moieties) with high affinity and specificity for OPCs linked via a hydrophilic linker to a bone-targeting bisphosphonate alendronate, wherein the bisphosphonate moieties are linked via a copper-free Click reaction of DBCO with N3-modified alendronate (Aln-N3) to a linker moiety prepared from alendronate and 4-azidobutyrate NHS ester (N3-NHS).

Example 7: alendronate conjugated YLL8 increased bone formation and mass

Next, the effect of alendronate-conjugated peptides on bone metabolism was evaluated. Two month old female mice were treated with PBS control, YLL3-Ale or YLL8-Ale, at 100 μ g/kg (25nmol) or 300 μ g/kg (75nmol), subcutaneously, once every other week, or one IV dose of 500 μ g/kg. Two injections were given to each mouse. The total cumulative dose is 200. mu.g/kg/month to 600. mu.g/kg/month (25nmol to 150nmol), respectively. Alendronate concentrations range from about 1/10 to 1/5 of the compound. Injection of YLL-Ale did not change body weight or cause any visible side effects. Although the peptide YLL3 alone showed anabolic effects, the YLL3-Ale at the two doses used failed to achieve a statistically significant difference in bone formation or bone mass compared to the PBS control (fig. 15). In contrast, at 300 μ g/kg × 2SC dose or 500 μ g/kg × 1IV dose, YLL8-Ale increased trabecular body volume by more than 20% (p <0.05 versus PBS), which correlates with increased trabecular thickness (30%, p <0.05 versus PBS) (FIGS. 15A and 15B) and number (28%, p <0.05 versus PBS). Both the 100 μ g/kg and 300 μ g/kg doses of YLL8-Ale increased cortical bone volume by approximately 16% and cortical thickness by approximately 15% (FIG. 15C). YLL8-Aln increased the bone formation rate by increasing osteoblast number and activity (higher dual-labeled surface and mineral deposition rate on both trabecular and cortical bone surfaces, as shown in FIG. 16). These results indicate that bisphosphonate conjugation of the YLL8 peptide reduces dosing frequency and maintains its anabolic effects. More specifically, these results show that a total of 2 doses of 600. mu.g/kg/month or 500. mu.g/kg monthly doses of YLL8-Aln increased osteoblast activity, stimulated bone formation, and increased trabecular and cortical bone mass.

As described herein, "osteogenic specific" peptides are found using a dual affinity and functional screening method. The osteogenic peptide YLL8 has high affinity for osteoblast-specific transcription factor + cells and activates phosphorylation of Akt (a pro-survival signal for osteoblastic progenitor cells). Both YLL3 and YLL8 increased osteoblast differentiation and maturation in vitro. Short-term (3 weeks) daily low dose injections of YLL3 and YLL8 induced bone anabolic effects comparable to PTH (1-34), increasing the rate of mineral deposition (which corresponds to osteoblast activity) and the rate of bone formation. Daily injection of YLL did not significantly affect bone resorption compared to PTH (1-34). This uncoupling of bone remodeling leads to rapid model-dependent bone gain and rapid increase in bone strength in trabecular and cortical bone, which is higher than in hPTH (1-34) treated mice. Daily injection of YLL, particularly YLL3, for 21 days, greatly increased callus formation and mineralization during fracture repair. Conjugation of YLL8 to alendronate reduced the frequency of injections, but showed similar levels of bone augmentation in trabecular and cortical bone. The relatively small size of the YLL peptide (e.g., three to four synthetic amino acids) compared to other bone anabolic drugs (e.g., large molecular weight proteins) makes the manufacture and scale-up of pharmaceutical and/or pharmaceutical formulations based on the YLL peptide easier, as well as its easier metabolic breakdown. YLL can be used as a "therapeutic peptide" and can also be used with bone binding agents (affiliated agents) to enhance their bone specificity.

Current drug treatment options for osteoporosis typically include antiresorptive therapy and anabolic therapy. For example, recombinant human PTH (1-34) (teriparatide), known for its bone anabolic effect, consists of 34 amino acids, which are the biologically active part of the hormone. PTHrP (amolopeptide), which has recently been approved by the FDA for the treatment of osteoporosis, is a 139-173-amino acid protein homologous to the N-terminus of PTH. Both hPTH (1-34) and PTHrp have significant anabolic effects on bone, but also have other broad physiological effects expressed in various tissues. In addition to its effects on bone and joint development, PTH and PTHrp act in an autocrine/paracrine manner to regulate calcein metabolism and organogenesis, such as mammary gland development. Sclerostin antibodies are another bone anabolic agent, which may be more specific for bone cells. Sclerostin is expressed in bone primarily by bone cells. However, sclerostin may also be expressed in cartilage and lymphocytes, which may lead to extra-skeletal side effects. Thus, the studies described herein have focused on the development of osteogenic anabolic drugs that can increase both trabecular and cortical bone mass.

The canonical Wnt/β -catenin signaling pathway plays a key role for bone formation and is an active drug target for the study/discovery of bone anabolic therapeutic drugs. The studies described herein focus on Akt activation as it is one of the lead kinases that is activated by α 4 β 1 integrin upon binding to osteoprogenitor cells. Also, emphasis has been placed on activating osteoprogenitor cells and their osteogenic potential, rather than inducing cellular mitogens or inducing new bone formation, as is commonly observed with Wnt-targeting or growth factors. To verify the effect of osteogenic YLL peptide on osteoblast differentiation, ALP and AR staining were performed, reflecting the initial and final stages of osteoblast differentiation, respectively. ALP and AR staining studies showed that YLL3 and YLL8 enhanced osteogenic differentiation of osteoprogenitor cells in vitro. Activation of the Akt signaling pathway was also demonstrated. Like PTH, yl 8 shows a broader effect on Akt pathway activation, while yl 3 is more specific for Atk activation. The studies described herein also show that injection of YLL3 and YLL8 resulted in increased mineral deposition rates, indicating activated osteoblast function. Injection of YLL3 and YLL8 also resulted in increased bone formation, bone mass and bone strength in young mice. Short-term treatment studies (i.e., 21 days) showed that anabolic effects of YLL were generally higher in male mice than female mice, probably because young males had higher osteogenesis than females, and high estrogen levels during growth may negatively affect bone formation in females.

The YLL peptide was conjugated to the bone targeting drug alendronate. In this case, alendronate is used as a "carrier" for bone-specific delivery of peptides. To further determine the enhanced bone targeting effect of YLL, in vivo experiments were performed and showed that two subcutaneous or one intravenous dose of YLL8-Aln increased trabecular and cortical bone mass. Collectively, these studies indicate that YLL8 can stimulate osteoblast differentiation in vitro and in vivo, demonstrating the efficacy of YLL8-Aln for bone formation. Dynamic bone morphometry and histological analysis confirmed that YLL8 significantly promoted bone formation by enhancing osteoblast activity. Unexpectedly, when YLL3 was conjugated with alendronate, YLL3-Aln lost anabolic effects on bone. This is probably due to a conformational change in the structural configuration of YLL3 following Aln conjugation. Increasing the dose and/or frequency of administration of YLL3-Aln may produce the anabolic effects observed for the unconjugated YLL3 peptide. Alternatively, the conjugation method can be modified so that YLL3 is cleaved from the alendronate conjugation and released upon reaching the bone.

In addition to aging and osteoporosis, nearly 800 million adults experience fractures each year in the united states alone, of which about 5% to 13% cause fractures to fail or to delay healing. Current therapies for treating fracture nonunion mainly include bone grafting and the use of recombinant Bone Morphogenetic Proteins (BMPs). Significant complication rates for bone grafting range from 10% to 25% and are associated with inconsistent results. BMP therapy requires orthopedic surgery prior to BMP delivery. In addition, BMP may cause inflammation, be poorly mineralized and be expensive. It is a continuing effort to develop osteogenic anabolic agents that increase bone mineralization and accelerate fracture repair. For this reason, daily injection of YLL3 resulted in early (day 10 post-fracture) mature callus development and greater callus mineral density in the later (day 21 post-fracture) phase. Since the initial stage of fracture repair is the recruitment and activation of osteoprogenitor cells, the results show that mice treated with YLL3 have a large amount of mineral taken up by osteoprogenitor cells at an early stage, indicating that activation of these endogenous osteoblasts is being recruited to the fracture site. The YLL3 treated mice formed well mineralized lamellar tissue on day 21, as compared to the more pronounced formation of braided bone in the PBS treated control. Fracture healing occurred on day 21 post-fracture, which was not otherwise observed in mice until day 35 to 45, and was superior to daily injections of PTH used at 50 μ g/kg. These fracture studies did not include measurement of overall bone strength, but the increase in bone mineral density measured by micro-CT was the primary determinant in determining bone strength, suggesting that higher bone strength may result from higher bone mineral density following treatment with yl 3.

In summary, screening methods were used to identify osteoprogenitor cell-specific targeting osteogenic peptides by pro-cell survival (pro-cell survival) mechanisms. The same "one-entity three-action" screening principle can be used to screen for antagonist or agonist drugs targeting other specific signals. Two osteoprogenitor-targeting peptides YLL3 and YLL8 increased osteoblast differentiation and maturation in vitro and increased osteoblast activity in vivo. Improved bone specificity is also observed with peptide-bisphosphonate conjugates, which maintain anabolic effects at lower drug dosing frequencies. Osteogenic peptides exhibit bone growth characteristics and can be used as therapeutic agents for treating osteoporosis and/or accelerating fracture repair.

Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.