Methods of treating osteonecrosis with LLP2A-bisphosphonate compounds

Abstract

A method for treating osteonecrosis in a subject is provided. In some embodiments, the method comprises administering to a subject having or suspected of having osteonecrosis a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug. In some embodiments, the pharmaceutical composition comprises a conjugate of LLP2A and alendronate (LLP2A-Ale). In some embodiments, the method further comprises administering exogenous mesenchymal stem cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 278,921, filed January 14, 2016, which is incorporated herein in its entirety for all purposes.

[0002] STATEMENT REGARDING RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under Grant No. AR0631366 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. [Background technology]

[0003] Background of the Invention Osteonecrosis, also known as avascular necrosis, is a disorder in which the blood supply in the bone area is impaired, causing the bone to die in that area.There are two types of osteonecrosis: traumatic osteonecrosis and non-traumatic osteonecrosis.In traumatic osteonecrosis, damage to bone, such as fracture or dislocation, damages blood vessels in the bone.In non-traumatic osteonecrosis, the impairment of blood supply and osteonecrosis occur without direct trauma or injury.In some cases, the cause of bone death in patients is unknown (idiopathic osteonecrosis).

[0004] Osteonecrosis can affect any number of bones and joints in the body, including, but not limited to, the hip, shoulder, knee, ankle, wrist, and jaw. If left untreated, or if initial treatment is unsuccessful, osteonecrosis can lead to the collapse of the bone and destruction of the joint around the bone. Summary of the Invention

[0005] In one aspect, a method for treating osteonecrosis in a subject is provided. In some embodiments, the method comprises: administering to a subject a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug. Includes.

[0006] In some embodiments, the subject has or is suspected of having osteonecrosis in at least one bone, e.g., the femur, hip, knee, shoulder, ankle, wrist, or jaw. In some embodiments, prior to the administering step, the method further comprises identifying the subject as having at least one osteonecrotic lesion in at least one bone. In some embodiments, the method further comprises measuring the size of at least one osteonecrotic lesion prior to administering the pharmaceutical composition. In some embodiments, the method further comprises measuring the size of at least one osteonecrotic lesion after administering the pharmaceutical composition and detecting a decrease in the size of the osteonecrotic lesion compared to the size of the osteonecrotic lesion before administering. In some embodiments, a decrease in the size of the osteonecrotic lesion compared to the size of the osteonecrotic lesion before administering the pharmaceutical composition indicates that osteonecrosis has been treated in the subject.

[0007] In another aspect, methods are provided for increasing vascular density in osteonecrotic tissue (e.g., in a subject, e.g., in a subject having or suspected of having osteonecrotic tissue). In some embodiments, the methods include: administering to a subject a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug. Includes.

[0008] In some embodiments, the method further comprises identifying a subject having osteonecrotic tissue (e.g., at least one osteonecrotic lesion in at least one bone) prior to administering. In some embodiments, the method further comprises measuring the amount of vascular density in at least one bone having at least one osteonecrotic lesion (e.g., measuring the amount of vascular density at or around the site of the osteonecrotic lesion) prior to administering the pharmaceutical composition. In some embodiments, the method further comprises measuring the amount of vascular density in at least one bone having at least one osteonecrotic lesion (e.g., measuring the amount of vascular density at or around the site of the osteonecrotic lesion) after administering the pharmaceutical composition. In some embodiments, a higher amount of vascular density (e.g., at or around the site of the osteonecrotic lesion) compared to the amount of vascular density before administering the pharmaceutical composition indicates increased vascular density in the osteonecrotic tissue.

[0009] In yet another aspect, methods are provided for preventing or reducing cell death in osteonecrotic tissue (e.g., in a subject having or suspected of having osteonecrotic tissue). In some embodiments, the methods include: administering to a subject a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug. Includes.

[0010] In some embodiments, the method further comprises identifying a subject having osteonecrotic tissue (e.g., at least one osteonecrotic lesion in at least one bone) prior to administering. In some embodiments, the method further comprises measuring the amount of apoptotic and / or necrotic cells in at least one osteonecrotic tissue (e.g., at or around the site of at least one osteonecrotic lesion) prior to administering the pharmaceutical composition. In some embodiments, the method further comprises measuring the amount of apoptotic and / or necrotic cells in at least one osteonecrotic tissue (e.g., at or around the site of at least one osteonecrotic lesion) after administering the pharmaceutical composition. In some embodiments, a decrease in the amount of apoptotic and / or necrotic cells in at least one osteonecrotic tissue (e.g., at or around the site of at least one osteonecrotic lesion) compared to the amount of apoptotic and / or necrotic cells before administering the pharmaceutical composition indicates that cell death is prevented or reduced in the osteonecrotic tissue.

[0011] In some embodiments, the pharmaceutical composition comprises a conjugate of LLP2A and alendronate (“LLP2A-Ale”), the conjugate having the formula: I have TIFF2026042861000002.tif42140.

[0012] In some embodiments, the osteonecrosis is traumatic osteonecrosis. In some embodiments, the osteonecrosis is glucocorticoid-induced osteonecrosis or alcohol-induced osteonecrosis. In some embodiments, the at least one bone is a femur, hip, knee, shoulder, ankle, wrist, or jawbone.

[0013] In some embodiments, identifying a subject having at least one osteonecrotic lesion in at least one bone comprises magnetic resonance imaging of at least one bone. 2In some embodiments, the identifying step comprises detecting at least one osteonecrotic lesion having a size of at least 5 cm. 2 The method includes detecting at least one osteonecrotic lesion having a size of

[0014] In some embodiments, the method further comprises administering exogenous mesenchymal stem cells. In some embodiments, the pharmaceutical composition and the exogenous mesenchymal stem cells are administered simultaneously. In some embodiments, the pharmaceutical composition and the exogenous mesenchymal stem cells are administered sequentially.

[0015] In some embodiments, one or both of the pharmaceutical composition and exogenous mesenchymal stem cells are administered systemically.In some embodiments, one or both of the pharmaceutical composition and mesenchymal stem cells are administered locally (for example, locally to the site of at least one osteonecrotic lesion).In some embodiments, the site of at least one osteonecrotic lesion is in the femur, hip, knee, shoulder, ankle, wrist or jawbone.

[0016] In some embodiments, one or both of the pharmaceutical composition and mesenchymal stem cells are administered intravenously. In some embodiments, one or both of the pharmaceutical composition and mesenchymal stem cells are administered by injection. In some embodiments, one or both of the pharmaceutical composition and mesenchymal stem cells are administered in a series of doses separated by intervals of days or weeks.

[0017] In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is an adult. In some embodiments, the subject is a juvenile.

[0018] In another aspect, there is provided a use of a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug for the treatment of osteonecrosis, for increasing vascularity in osteonecrotic tissue, and / or for preventing or reducing cell death in osteonecrotic tissue. In some embodiments, the pharmaceutical composition comprises a conjugate of LLP2A and alendronate ("LLP2A-Ale"), the conjugate having the formula: I have TIFF2026042861000003.tif42140.

[0019] In some embodiments, the pharmaceutical composition further comprises exogenous mesenchymal stem cells.

[0020] In yet another aspect, there is provided a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug for the manufacture of a medicament for the treatment of osteonecrosis, for increasing vascularity in osteonecrotic tissue, and / or for preventing or reducing cell death in osteonecrotic tissue. In some embodiments, the pharmaceutical composition comprises a conjugate of LLP2A and alendronate ("LLP2A-Ale"), the conjugate having the formula: I have TIFF2026042861000004.tif42140.

[0021] In some embodiments, the pharmaceutical composition further comprises exogenous mesenchymal stem cells. [The present invention 1001] administering to a subject a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug. A method of treating osteonecrosis in a subject, comprising: [The present invention 1002] The pharmaceutical composition comprises a conjugate of LLP2A and alendronate (“LLP2A-Ale”), the conjugate having the formula: The method of the present invention 1001 having TIFF2026042861000005.tif42140. [The present invention 1003] The method of any one of claims 1001 to 1002, further comprising the step of identifying a subject having at least one osteonecrotic lesion in at least one bone prior to said administering step. [The present invention 1004] 1004. The method of claim 1003, wherein said at least one bone is a femur, hip, knee, shoulder, ankle, wrist, or jaw bone. [The present invention 1005] 10. The method of claim 1003 or 1004, wherein said identifying step comprises magnetic resonance imaging of said at least one bone. [The present invention 1006] The step of identifying comprises determining a distance of at least 3.5 cm 2 1006. The method of any of claims 1003 to 1005, comprising detecting at least one osteonecrotic lesion having a size of [The present invention 1007] The step of identifying comprises determining a distance of at least 5 cm 2 1006. The method of claim 1006, comprising detecting at least one osteonecrotic lesion having a size of [The present invention 1008] Any of the methods of claims 1003 to 1007, further comprising the step of measuring the size of said at least one osteonecrotic lesion after said administering step, and detecting a decrease in the size of said osteonecrotic lesion compared to the size of said osteonecrotic lesion before said administering step. [The present invention 1009] 1009. The method of any one of claims 1001 to 1008, wherein said osteonecrosis is traumatic osteonecrosis. [The present invention 1010] 1009. The method of any of claims 1001 to 1008, wherein said osteonecrosis is glucocorticoid-induced osteonecrosis or alcohol-induced osteonecrosis. [The present invention 1011] 1011. The method of any of claims 1001 to 1010, further comprising the step of administering exogenous mesenchymal stem cells. [The present invention 1012] 10. The method of claim 10, wherein said pharmaceutical composition and said exogenous mesenchymal stem cells are administered simultaneously. [The present invention 1013] 1012. The method of claim 1011, wherein said pharmaceutical composition and said exogenous mesenchymal stem cells are administered sequentially. [The present invention 1014] 1014. The method of any of claims 1001 to 1013, wherein said pharmaceutical composition is administered systemically. [The present invention 1015] 1014. The method of any of claims 1001 to 1013, wherein said pharmaceutical composition is administered topically. [The present invention 1016] 1015. The method of claim 1015, wherein said pharmaceutical composition is administered locally to the site of said at least one osteonecrotic lesion. [The present invention 1017] 1015. The method of any of claims 1001 to 1014, wherein said pharmaceutical composition is administered intravenously. [The present invention 1018] 1017. The method of any of claims 1001 to 1016, wherein said pharmaceutical composition is administered by injection. [The present invention 1019] administering to the subject a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug. 10. A method for increasing vascularity in osteonecrotic tissue, comprising: [The present invention 1020] The pharmaceutical composition comprises a conjugate of LLP2A and alendronate ("LLP2A-Ale"). wherein the conjugate comprises a compound of the formula: The method of the present invention 1019 having TIFF2026042861000006.tif42140. [The present invention 1021] The method of any one of claims 1019 to 1020, further comprising the step of identifying a subject having at least one osteonecrotic lesion in at least one bone prior to said administering step. [The present invention 1022] 1021. The method of claim 1021, wherein said at least one bone is a femur, hip, knee, shoulder, ankle, wrist, or jaw bone. [The present invention 1023] 1023. The method of claim 1021 or 1022, wherein said identifying step comprises magnetic resonance imaging of said at least one bone. [The present invention 1024] The step of identifying comprises determining a distance of at least 3.5 cm 2 1024. The method of any of claims 1021 to 1023, comprising detecting at least one osteonecrotic lesion having a size of [The present invention 1025] The step of identifying comprises determining a distance of at least 5 cm 2 1024. The method of claim 1024, comprising detecting at least one osteonecrotic lesion having a size of [The present invention 1026] 1026. The method of any one of claims 1019 to 1025, wherein the osteonecrosis is traumatic osteonecrosis. [The present invention 1027] 1026. The method of any of claims 1019 to 1025, wherein said osteonecrosis is glucocorticoid-induced osteonecrosis or alcohol-induced osteonecrosis. [The present invention 1028] 1028. The method of any of claims 1019 to 1027, further comprising the step of administering exogenous mesenchymal stem cells. [The present invention 1029] 1029. The method of claim 1028, wherein said pharmaceutical composition and said exogenous mesenchymal stem cells are administered simultaneously. [The present invention 1030] 1029. The method of claim 1028, wherein said pharmaceutical composition and said exogenous mesenchymal stem cells are administered sequentially. [The present invention 1031] 1031. The method of any of claims 1019 to 1030, wherein said pharmaceutical composition is administered systemically. [The present invention 1032] 1031. The method of any of claims 1019 to 1030, wherein said pharmaceutical composition is administered topically. [The present invention 1033] 1033. The method of claim 1032, wherein said pharmaceutical composition is administered locally to the site of said at least one osteonecrotic lesion. [The present invention 1034] 1032. The method of any of claims 1019 to 1031, wherein said pharmaceutical composition is administered intravenously. [This invention 1035] The method of any of claims 1019 to 1033, wherein said pharmaceutical composition is administered by injection. [The present invention 1036] administering to a subject a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug. 10. A method of preventing or reducing cell death in osteonecrotic tissue, comprising: [This invention 1037] The pharmaceutical composition comprises a conjugate of LLP2A and alendronate ("LLP2A-Ale"). wherein the conjugate comprises a compound of the formula: The method of the present invention 1036 having TIFF2026042861000007.tif42140. [The present invention 1038] The method of any one of claims 1036 to 1037, further comprising the step of identifying a subject having at least one osteonecrotic lesion in at least one bone prior to said administering step. [This invention 1039] 1038. The method of claim 1038, wherein said at least one bone is a femur, hip, knee, shoulder, ankle, wrist, or jaw bone. [The present invention 1040] 1039. The method of any one of claims 1038 to 1039, wherein said identifying step comprises magnetic resonance imaging of said at least one bone. [This invention 1041] The step of identifying comprises determining a distance of at least 3.5 cm 2 1041. The method of any of claims 1038 to 1040, comprising detecting at least one osteonecrotic lesion having a size of [The present invention 1042] The step of identifying comprises determining a distance of at least 5 cm 2 1041. The method of claim 1041, comprising detecting at least one osteonecrotic lesion having a size of [This invention 1043] 1043. The method of any one of claims 1036 to 1042, wherein the osteonecrosis is traumatic osteonecrosis. [This invention 1044] 1043. The method of any of claims 1036 to 1042, wherein said osteonecrosis is glucocorticoid-induced osteonecrosis or alcohol-induced osteonecrosis. [This invention 1045] 1045. The method of any one of claims 1036 to 1044, further comprising the step of administering exogenous mesenchymal stem cells. [The present invention 1046] 1046. The method of claim 1045, wherein said pharmaceutical composition and said exogenous mesenchymal stem cells are administered simultaneously. [This invention 1047] 1046. The method of claim 1045, wherein said pharmaceutical composition and said exogenous mesenchymal stem cells are administered sequentially. [This invention 1048] 1052. The method of any of claims 1036 to 1051, wherein said pharmaceutical composition is administered systemically. [This invention 1049] 1048. The method of any of claims 1036 to 1047, wherein said pharmaceutical composition is administered topically. [The present invention 1050] 1049. The method of claim 1049, wherein said pharmaceutical composition is administered locally to the site of said at least one osteonecrotic lesion. [This invention 1051] 1049. The method of any of claims 1036 to 1048, wherein said pharmaceutical composition is administered intravenously. [This invention 1052] The method of any of claims 1036 to 1050, wherein said pharmaceutical composition is administered by injection. [Brief explanation of the drawings]

[0022] [Figure 1] Stages of Osteonecrosis. For osteonecrosis of the humeral head, the stages of osteonecrosis are shown. First there is a loss of blood supply, followed by death of bone cells, and then the humerus collapses because the dead bone area cannot support the use of the joint. [Figure 2]Blood vessel density in femurs from glucocorticoid-treated mice. Four-month-old male mice were treated with glucocorticoids ("GC") or placebo for 56 days. On day 28, groups of glucocorticoid-treated mice were treated with LLP2A-Ale or PTH for an additional 28 days. Blood vessel density in femurs was measured after sacrifice by micro-CT according to published methods. *p<0.05 between GC and PL; GC+LLP2A-Ale was significantly different from all other groups; GC+PTH was significantly different from all other groups. [Figure 3] Blood vessel density in femurs from glucocorticoid-treated mice. Four-month-old male mice were treated with glucocorticoids or placebo for 56 days. On day 28, the glucocorticoid-treated mice were treated with LLP2A-Ale or PTH for an additional 28 days. On day 56, the animals received contrast medium and were then sacrificed. Their femurs were then decalcified and scanned by micro-CT. Compared to the placebo group, GC treatment reduced blood vessel density. GC + LLP2A-Ale increased blood vessel density compared to GC alone, while GC + PTH did not significantly change blood vessel density. [Figure 4] Serum VEGF-A levels in glucocorticoid-treated mice. Mice treated with placebo (PL), glucocorticoid (GC), glucocorticoid and 250 μg / kg LLP2A-Ale (GC+LLP2A-Ale), glucocorticoid and PTH (GC+PTH), or glucocorticoid, PTH, and 250 μg / kg LLP2A-Ale (GC+PTH+LLP2A-Ale) were measured for serum VEGF levels. GC treatment reduced serum VEGF levels, while GC+LLP2A-Ale restored them to the levels of placebo-treated animals. [Figure 5]Cortical bone osteocyte apoptosis. Mice were treated with PL for 56 days, GC alone for 56 days, or GC alone for 28 days, then GC plus one dose of LLP2A-Ale on day 28. Animals were sacrificed on day 56, and the percentage of osteocytes that were apoptotic was determined. GC treatment alone increased the number of apoptotic osteocytes on day 56, and GC+LLP2A-Ale prevented osteocyte apoptosis. *p<0.05 from all other groups on day 28 of the study. [Figure 6] Trabecular bone volume in glucocorticoid-treated mice. Mice were treated with glucocorticoids (GC) for 28 days and then with either LLP2A-Ale, PTH, or LLP2A-Ale plus PTH. Evaluation criteria were the change in trabecular bone volume measured in vivo in the distal femur from 28 to 56 days (left panel) and the change in trabecular bone volume in vertebral trabecular bone volume (right panel). Mice receiving GC treatment alone lost approximately 40% trabecular bone volume in the distal femur from 28 to 56 days, whereas treatment with GC and either LLP2A-Ale or PTH increased trabecular bone volume to baseline / sham levels. The combination of GC plus LLP2A-Ale plus PTH increased trabecular bone volume in the distal femur above the placebo (PL) group. In the vertebrae, the combination of GC+LLP2A-Ale+PTH restored cancellous bone mass to the level of the placebo (PL) group. *p<0.05 from all other groups on day 56, #p<0.05 vs. GC. [Figure 7]Prevalence of osteonecrosis in dexamethasone-treated mice. Mice were treated with dexamethasone (Dex), 4 mg / L in drinking water, or placebo (PL) (fresh water) for 90 days. On day 30, Dex-treated mice were re-randomized to Dex alone, Dex + 250 μg / kg LLP2A-Ale, Dex + 500 μg / kg LLP2A-Ale, and Dex + 750 μg / kg LLP2A-Ale. LLP2A-Ale treatment was given on days 30, 45, 60, and 75. All mice were euthanized on day 90. Right and left distal femurs were fixed in 10% neutral-buffered formalin and decalcified. Specimens were dehydrated, embedded in paraffin, cut into 5-micrometer sections, and stained with H&E. Sections were evaluated for osteonecrosis by bright-field microscopy using modified criteria reported by Yang et al., J. Orthop. Res., 27: 169-75 (2009), which define osteonecrosis by the presence of five features: (1) empty lacunae, (2) pyknotic osteocyte nuclei in the vacuolated lacunae and adjacent areas of bone marrow necrosis, (3) the presence of excess adipocytes in the bone marrow, (4) cartilage degradation, and (5) the presence of fibrin clots in blood vessels. Each GC-treated group and placebo group was evaluated for the presence of all five features of osteonecrosis or at least three of the five features of osteonecrosis. Fewer mice receiving LLP2A-Ale treatment developed three or more features of osteonecrosis compared with mice treated with Dex alone (Dex alone group: 14 / 16 (88%); Dex + LLP2A-Ale 250 μg / kg: 12 / 16 (75%); Dex + LLP2A-Ale 500 μg / kg: 9 / 16 (56%); and LLP2A-Ale 750 μg / kg: 11 / 16 (69%)). The number of mice exhibiting all five features of osteonecrosis per treatment group was also reduced in mice receiving LLP2A-Ale treatment compared with mice treated with Dex alone. [Figure 8A]Figure 8A-B. Vacuolated bone lacuna density in femurs of dexamethasone-treated mice. Mice treated with placebo (PL), dexamethasone alone (Dex), Dex + 250 μg / kg LLP2A-Ale, Dex + 500 μg / kg LLP2A-Ale, or Dex + 750 μg / kg LLP2A-Ale as described above in Figure 7 were evaluated for vacuolated bone lacuna density within the distal femoral epiphyseal region. Vacuolated bone lacuna density was calculated by determining the number of vacuolated bone lacunae in the entire distal femoral epiphyseal region divided by the total number of osteocytes within the same region for each specimen. In the Dex-only group, 75% of samples had 10% or more vacuolated bone lacunae, compared with 44–62% of samples in the LLP2A-Ale-treated group. Compared with Dex-only treatment, there were more samples treated with LLP2A-Ale 250 μg / kg, LLP2A-Ale 500 μg / kg, and LLP2A-Ale 750 μg / kg that had less than 10% vacuolated bone lacunae. [Figure 8B] See legend to Figure 8A. [Figure 9-1]Figures 9A-9F. Histological evaluation of femurs from mice treated with placebo or dexamethasone. Mice were treated with placebo (PL) or dexamethasone alone (Dex) as described above in Figure 7. All mice were euthanized on day 90. Right and left distal femurs were fixed in 10% neutral buffered formalin and decalcified. Specimens were dehydrated, embedded in paraffin, cut into 5-micrometer sections, and stained with H&E. Sections were scored by two experienced histologists, followed by consensus reading by two additional histologists. Figures 9A-9B: Representative proximal femoral epiphysis at 4x (A) and 20x (B) magnification in a placebo-treated mouse. (C) Representative proximal femoral epiphysis at 4x magnification in a mouse treated with dexamethasone (Dex) alone. Figures 9D, 9E, and 9F: Representative proximal femoral epiphysis at 20x magnification in a mouse treated with dexamethasone (Dex) alone. In dexamethasone-treated mice, the majority of the marrow cavity is occupied by fat (blue arrows in (D-F)), necrotic marrow deposits (green arrow in (E)), and trabecular bone, characterized by vacuolated bone lacunae (black arrows), pyknotic nuclei of osteocytes (yellow arrow in (D)), and surrounded by necrotic fat debris. [Figure 9-2] See description of Figure 9-1. [Figure 9-3] See description of Figure 9-1. [Figure 10A] Figure 10A-B. Fat volume and sinusoidal capillary volume in femurs from treated mice. Mice were treated with placebo (PL), dexamethasone alone (Dex), Dex + 250 μg / kg LLP2A-Ale, Dex + 500 μg / kg LLP2A-Ale, or Dex + 750 μg / kg LLP2A-Ale as described above in Figure 7. (A) Mice were evaluated for fat volume within the distal femoral epiphyseal region. Mice receiving LLP2A-Ale treatment had reduced fat volume compared to mice treated with Dex alone. (B) Mice were evaluated for sinusoidal capillaries in the bone marrow. Mice receiving LLP2A-Ale treatment had increased sinusoidal capillary volume compared to mice treated with Dex alone or placebo. *p<0.05 from Dex + LLP2A-Ale treatment at study day 90. [Figure 10B] See legend to Figure 10A. [Figure 11-1] Figure 11A-F. Histological evaluation of femurs from LLP2A-Ale-treated mice. Mice were treated with Dex + 250 μg / kg LLP2A-Ale as described above in Figure 7. All mice were euthanized on day 90. Right and left distal femurs were fixed in 10% neutral buffered formalin and decalcified. Specimens were dehydrated, embedded in paraffin, cut into 5-micrometer sections, and stained with H&E. Sections were scored by two experienced histologists, followed by consensus reading by two additional histologists. (A, D): Representative proximal femoral epiphysis at 4x magnification in a mouse treated with Dex + LLP2A-Ale. (B, C, E, F): Representative proximal femoral epiphysis at 20x magnification in a mouse treated with Dex + LLP2A-Ale. In LLP2A-Ale-treated mice, increased sinusoid formation was observed, protruding into the necrotic bone marrow (B) or occupying bone marrow without bone marrow necrosis (E–F). Some osteoblasts were observed on the trabecular surface and near the sinusoids (yellow arrows in (E)). [Figure 11-2] See description of Figure 11-1. [Figure 12] Reduced incidence of glucocorticoid-induced osteonecrosis in mice treated with recombinant LLP2A-Ale. Mice were randomized to receive placebo (PL) (fresh water), dexamethasone (Dex) 4 mg / L in drinking water, Dex + 250 μg / kg LLP2A-Ale, Dex + 500 μg / kg LLP2A-Ale, or Dex + 40 μg / kg hPTH(1-34) for a 30-day or 45-day treatment period. The incidence of Dex-induced osteonecrosis was 57% at 30 days and 73% at 45 days. Both LLP2A-Ale (both doses) and hPTH(1-34) treatment prevented glucocorticoid-induced osteonecrotic lesions. DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description of the Invention I. Introduction Osteonecrosis is a bone disease caused by a temporary or permanent loss of blood supply to the bone. Without an adequate blood supply, bone tissue dies, which can eventually lead to the collapse of the bone and surrounding joint.

[0024] As described herein, it has been found that LLP2A-Ale conjugates can prevent the loss of blood supply to bone and increase bone vascularity in an animal model of glucocorticoid-induced osteonecrosis. LLP2A-Ale conjugates can also prevent bone cell death and increase new bone formation in an animal model of glucocorticoid-induced osteonecrosis.

[0025] Thus, in one aspect, a method for preventing and / or treating osteonecrosis is provided, comprising administering a pharmaceutical composition of a peptidomimetic ligand (e.g., LLP2A) conjugated to a bisphosphonate drug (e.g., alendronate). In another aspect, a method for increasing vascularity in osteoporotic tissue is provided, comprising administering a pharmaceutical composition of a peptidomimetic ligand (e.g., LLP2A) conjugated to a bisphosphonate drug (e.g., alendronate). In yet another aspect, a method for reducing or preventing cell death in osteonecrotic tissue is provided, comprising administering a pharmaceutical composition of a peptidomimetic ligand (e.g., LLP2A) conjugated to a bisphosphonate drug (e.g., alendronate). In some embodiments, the peptidomimetic ligand-bisphosphonate drug conjugate is administered in combination with mesenchymal stem cells and / or an anabolic agent such as PTH.

[0026] II. Definition Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular biology, and chemistry described below are those well known and commonly employed in the art.

[0027] As used herein, the terms "a" or "an," when used herein in connection with a substituted moiety group or "substituent," means at least one. For example, when a compound is substituted with "an" alkyl or aryl, the compound may be substituted with at least one alkyl and / or at least one aryl, where each alkyl and / or aryl may be different. In another example, when a compound is substituted with "a" substituent, the compound is substituted with at least one substituent, where each substituent may be different.

[0028] The description of the compounds of the present invention is limited by the principles of chemical bonding known to those skilled in the art. Thus, when a group can be substituted with one or more of several substitution moieties, such substitutions are selected to obey the principles of chemical bonding and to result in compounds that are not inherently unstable and / or that are known to those skilled in the art as potentially unstable under environmental conditions, such as aqueous, neutral, or physiological conditions.

[0029] As used herein, the terms "Ale" or "Alen" refer to alendronate.

[0030] As used herein, the term "peptide" refers to a compound composed of a single chain of D or L amino acids or a mixture of D and L amino acids linked by peptide bonds. Generally, peptides are about 2 to about 50 amino acids in length. Preferably, peptides are about 2 to about 25 amino acids in length, about 3 to 20 amino acids in length, or about 3 to 10 amino acids in length.

[0031] As used herein, the term "amino acid" refers to natural, unnatural, and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted one-letter codes.

[0032] The term "salt" as used herein refers to the acid salt and / or base salt of the compound used in the method of the present invention. Examples of pharmaceutically acceptable salts include mineral acid salts (such as hydrochloric acid, hydrobromic acid, and phosphoric acid), organic carboxylic acid salts (such as acetic acid, propionic acid, glutamic acid, and citric acid), organic sulfonic acid salts (such as methanesulfonic acid), and quaternary ammonium salts (such as methyl iodide and ethyl iodide). Pharmaceutically acceptable salts include salts formed with bases, i.e., cationic salts, for example, alkali and alkaline earth metal salts such as sodium, lithium, potassium, calcium, magnesium, and ammonium salts such as ammonium, trimethylammonium, diethylammonium, and tris(hydroxymethyl)methylammonium salt. It is understood that pharmaceutically acceptable salts are non-toxic. Additional information about suitable pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, which is incorporated herein by reference.

[0033] As used herein, the terms "pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to a substance that aids in the administration and absorption of an active agent by a subject. A "pharmaceutically acceptable excipient" refers to an excipient that can be included in the compositions described herein and that does not cause significant adverse toxicological effects in a subject. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, and coloring agents. Those skilled in the art will recognize that other pharmaceutical excipients can also be used.

[0034] As used herein, the term "hydrate" refers to a compound that is complexed with at least one water molecule. In some embodiments, the LLP2A-bisphosphonate compounds described herein can be complexed with 1 to 10 water molecules.

[0035] As used herein, the term "isomers" refers to compounds that have the same chemical formula but are structurally distinguishable.

[0036] As used herein, the term "mesenchymal stem cells" refers to pluripotent stem cells (i.e., cells that have the ability to differentiate into a subset of cell types) that can differentiate into a variety of cell types, including osteoblasts, chondrocytes, and adipocytes. Mesenchymal stem cells can be obtained from a variety of tissues, including, but not limited to, bone marrow tissue, adipose tissue, muscle tissue, birth tissue (e.g., amniotic membrane, amniotic fluid, or umbilical cord tissue), skin tissue, bone tissue, and dental tissue.

[0037] As used herein, the term "subject" refers to a living organism that has or is prone to a condition that can be treated by administering a pharmaceutical composition as provided herein (e.g., a living organism that has been diagnosed with, is suspected of having, or is at risk of having an osteonecrotic lesion). Non-limiting examples include humans, other mammals, and other non-mammalian animals.

[0038] As used herein, the term "therapeutically effective amount" refers to an amount of a pharmaceutical composition (e.g., a conjugate comprising an LLP2A peptidomimetic ligand and a bisphosphonate drug, and / or stem cells, as described herein) useful for treating or ameliorating a specified disease or condition, or for exhibiting a detectable therapeutic or inhibitory effect. The effect can be detected by any assay known in the art.

[0039] As used herein, the terms "treat," "treating," and "treatment" refer to any objective or subjective parameter, such as relief; remission; diminishing symptoms or making the injury, condition, or state more tolerable for the patient; slowing the rate of degeneration or decline; making the end point of degeneration less debilitating; and / or improving the patient's physical or mental well-being, and any other indication of success in treating or ameliorating an injury, condition, or state. Treatment or amelioration of symptoms may be based on objective or subjective parameters, including, but not limited to, the results of a physical examination and / or biological assay performed on a sample from the subject.

[0040] III. Compositions for the Treatment of Osteonecrosis In one aspect, the present invention relates to peptidomimetic ligands, e.g., LLP2A, conjugated to bisphosphonates such as alendronate, which can be used alone or in combination with mesenchymal stem cells and / or anabolic agents to prevent and / or treat osteonecrosis, to increase vascular density in osteonecrotic tissue, or to prevent or reduce cell death in osteonecrotic tissue.

[0041] LLP2A-bisphosphonate conjugates In some embodiments, the peptidomimetic ligand has the following structure: It is an LLP2A compound with TIFF2026042861000008.tif49128.

[0042] LLP2A is a high-affinity, high-specificity peptidomimetic ligand that binds to α4β1 integrin. See Liu et al., Biopolymers, 84:595-604(2006); Peng et al., Nature Chemical Biology 2:381-389(2006); DeNardo et al., J. Nucl. Med. 50:625-634(2009).

[0043] In some embodiments, the LLP2A peptidomimetic ligand is conjugated to a bisphosphonate.Bisphosphonates are a class of drugs that have two phosphonate groups and are used to treat osteoporosis and prevent bone loss.Bisphosphonates contain two phosphonate groups that are covalently linked to carbon.Bisphosphonates can be nitrogen-containing (e.g., etidronate) or nitrogen-free (e.g., alendronate). Bisphosphonate drugs suitable for use in conjugation to a peptidomimetic ligand include, but are not limited to, etidronate (Didronel®), clodronate (Bonefos®, Loron®), tiludronate (Skelid®), pamidronate (Aredia®), neridronate, olpadronate, alendronate (Fosamax®), ibandronate (Boniva®), risedronate (Actonel®), and zoledronate (Zometa®, Reclast®). One of skill in the art will appreciate that other bisphosphonates are useful in the present invention. In some embodiments, the bisphosphonate is alendronate.

[0044] In some embodiments, the pharmaceutical composition comprises a compound of Formula I: TIFF2026042861000009.tif55149 compounds (e.g., LLP2A-Ale).

[0045] In some embodiments, the pharmaceutical composition comprises a salt, hydrate, solvate, prodrug form, isomer, or metabolite of an LLP2A compound (e.g., a compound of Formula I) conjugated to a bisphosphonate drug.

[0046] Salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, superphosphate, phosphonate, isonicotinate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, metasulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoic acid)) salts. Other salts include salts with inorganic bases, including, but not limited to, alkali metal salts such as sodium, lithium, and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; aluminum salts; and ammonium salts such as ammonium, trimethylammonium, diethylammonium, and tris-(hydroxymethyl)-methylammonium salts. Other salts with organic bases include salts with diethylamine, diethanolamine, meglumine, and N,N'-dibenzylethylenediamine.

[0047] The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating from the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for purposes of the present invention.

[0048] In some embodiments, compounds as described herein can exist in unsolvated forms as well as solvated forms, including hydrated forms. Generally, solvated forms are equivalent to unsolvated forms and are included within the scope of the present invention. Certain compounds may exist in multiple crystalline or amorphous forms. Generally, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0049] In some embodiments, compounds as described herein possess asymmetric carbon atoms (optical centers) or double bonds; enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomers, and individual isomers, which may be defined in terms of absolute stereochemistry as (R)- or (S)-, or, for amino acids, (D)- or (L)-, are encompassed within the scope of the present invention. Compounds of the present invention do not include compounds known in the art to be too unstable to synthesize and / or isolate. The present invention is meant to include compounds in racemic or optically pure form. Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or separated using conventional techniques.

[0050] In some embodiments, the compounds are in prodrug form. Prodrugs of the compounds described herein are compounds that easily undergo chemical changes under physiological conditions to provide the compounds of the present invention. In addition, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

[0051] The compounds described herein can be synthesized by various methods known to those skilled in the art (see Comprehensive Organic Transformations by Richard C. Larock, 1989) or by an appropriate combination of commonly known synthetic methods. Techniques useful for synthesizing the compounds of the present invention are readily apparent and accessible to those skilled in the relevant art. As a non-limiting example, LLP2A-alendronate (LLP2A-Ale) can be prepared by the conjugate addition of the sulfhydryl group of LLP2A-Lys(D-Cys) to alendronate maleimide (Ale-Mal). The latter can be prepared in situ from alendronate and sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid (sulfo-SMCC). LLP2A-Lys(D-Cys) can be prepared by solid-phase synthesis from several commercially available starting materials and one characterized intermediate, 4-[(N'-2-methylphenyl)ureido]phenylacetic acid (UPA), which can also be prepared from commercially available starting materials. Methods for making LLP2A compounds conjugated to bisphosphonate drugs, including detailed descriptions of the synthesis of LLP2A-Ale, can be found, for example, in International Application Publication Nos. WO 2012 / 031228 and WO 2013 / 032527, the disclosures of which are incorporated herein in their entireties for all purposes.

[0052] Mesenchymal stem cells In some embodiments, a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug (e.g., LLP2A-Ale) is co-administered with mesenchymal stem cells (MSCs) to treat osteonecrosis, increase vascular density in osteonecrotic tissue, or prevent or reduce cell death in osteonecrotic tissue.

[0053] The administered mesenchymal stem cells may be a homogeneous composition or a mixed cell population containing or enriched for MSCs. Suitable MSCs can be obtained or derived, for example, from bone marrow mononuclear cells collected from bone marrow aspirates. In some embodiments, a homogeneous mesenchymal stem cell composition can be obtained by culturing bone marrow-adherent or periosteal cells in an appropriate culture medium, and the mesenchymal stem cell composition can be obtained by culturing bone marrow-adherent or periosteal cells to obtain an expanded MSC population. MSCs can be identified by specific cell surface markers identified by unique monoclonal antibodies. Methods for obtaining cell populations enriched in mesenchymal stem cells are described, for example, in U.S. Patent No. 5,486,359, incorporated herein by reference. Sources of mesenchymal stem cells include, but are not limited to, bone marrow, muscle, fat, placental tissue, umbilical cord tissue, dental pulp, skin tissue, peripheral blood, and synovium. Mesenchymal stem cells (MSCs) can be purified using methods known in the art (see, e.g., Wakitani et al., 1995; Fukuda and Yuasa, 2006; Woodbury et al., 2000; Deng et al., 2001; Kim et al., 2006; Mareschi et al., 2006; Krampera et al., 2007).

[0054] Compositions enriched for MSCs (e.g., having greater than about 95%, usually greater than about 98% mesenchymal stem cells) can be achieved using techniques for isolating, purifying, and culturing and expanding mesenchymal stem cells known in the art. As a non-limiting example, isolated and cultured mesenchymal stem cells can comprise a single phenotypic population (e.g., at least about 95% or about 98% homogeneous) by flow cytometry analysis of expressed surface antigens. Desired cells in such compositions are identified as expressing one or more cell surface markers of the cell type (e.g., CD73 or CD105).

[0055] Mesenchymal stem cells can be derived from a wide variety of sources, including autologous, allogeneic, or xenogeneic.

[0056] In some embodiments, the mesenchymal stem cells are about 1 x 10 cells. 4 Approximately 1 x 10 cells / kg body weight 8 cells / kg body weight (e.g., approximately 1 x 10 cells) 4 cells / kg, approximately 1×10 cells 5 cells / kg, approximately 1×10 cells 6 cells / kg, approximately 1×10 cells 7 cells / kg, or approximately 1 x 10 cells 8 The amount of mesenchymal stem cells to be administered will depend on a variety of factors, including the age, weight, and sex of the patient.

[0057] Mesenchymal stem cells can be administered together with acceptable pharmaceutical carriers.For example, mesenchymal stem cells can be administered as a cell suspension in a pharmaceutically acceptable liquid medium or gel for injection or topical application.In one embodiment, the pharmaceutically acceptable liquid medium is saline solution.Saline solution can contain additional substances such as dimethyl sulfoxide (DMSO) and human serum albumin.

[0058] anabolic agent In some embodiments, a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug (e.g., LLP2A-Ale) is co-administered with an anabolic agent to treat osteonecrosis, increase vascular density in osteonecrotic tissue, or prevent or reduce cell death in osteonecrotic tissue.

[0059] As used herein, an anabolic agent is an agent that stimulates or promotes new bone formation. Anabolic agents suitable for use in combination with LLP2A-bisphosphonate conjugates include, but are not limited to, parathyroid hormone (PTH), growth hormone (GH), insulin-like growth factor-I (IGF-I), parathyroid hormone-related peptide (PTHrp), or anti-sclerostin antibody. In some embodiments, the anabolic agent is PTH or an analog thereof. In some embodiments, the anabolic agent is a recombinant form of PTH (e.g., PTH 1-34 (teriparatide)).

[0060] The anabolic agent may be administered in conjunction with an acceptable pharmaceutical carrier, as described herein. For example, in some embodiments, the anabolic agent may be formulated for administration by injection, or by oral, parenteral, or intranasal administration.

[0061] Formulation and Administration In some embodiments, the compositions described herein (e.g., compositions comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug (e.g., LLP2A-Ale), mesenchymal stem cells, and / or an anabolic agent) are provided as pharmaceutical compositions comprising a pharmaceutically acceptable carrier or excipient. Details of techniques for formulation and administration are fully described in the scientific and patent literature; see, for example, the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co, Easton PA.

[0062] Pharmaceutically acceptable carriers for preparing pharmaceutical compositions as described herein can be either solid or liquid.Solid preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.Solid carriers can be one or more substances, and can also function as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials.

[0063] Generally, the type of carrier is selected based on the mode of administration. For example, in some embodiments, oral preparations include tablets, pills, powders, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. In some embodiments, pharmaceutical compositions for oral administration or delivery by injection may take the form of a liquid (e.g., an elixir, syrup, solution, emulsion, or suspension). Liquid pharmaceutical compositions may contain, for example, one or more of the following: water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, a sterile diluent such as fixed oils, polyethylene glycol, glycerin, polypropylene glycol, or other solvents that can serve as a solvent or suspending medium; antibacterial agents; antioxidants; chelating agents; buffers such as acetate, citrate, or phosphate, and agents for adjusting tonicity, such as sodium chloride or glucose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. The use of physiological saline is preferred, and injectable pharmaceutical compositions are preferably sterile.

[0064] The compositions typically include a common pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, adjuvants, diluents, tissue penetration enhancers, solubilizers, etc. In some embodiments, the compositions contain about 0.01% to about 90% by weight of the conjugate, mesenchymal stem cells, and / or anabolic agent, e.g., about 0.1% to about 75%, about 0.1% to 50%, or about 0.1% to 10%, with the remainder consisting of suitable pharmaceutical carriers and / or excipients. Appropriate excipients can be tailored to the particular composition and route of administration by methods well known in the art, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra.

[0065] 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; low melting point waxes; cocoa butter; carbohydrates; sugars, including but not limited to, lactose, glucose, sucrose, mannitol, or sorbitol; starches, including but not limited to, starches derived from corn, wheat, rice, potato, or other plants; celluloses, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums, including acacia, tragacanth, and acacia; and proteins, including but not limited to, gelatin, collagen; microcrystalline cellulose, water, saline, syrup, ethylcellulose, and polyacrylic acids, e.g., Carbopol 941, Carbopol 980, Carbopol Carbopol such as 981; lubricants; mineral oil; wetting agents; emulsifying agents; suspending agents; preservatives such as methyl-, ethyl-, and propyl-hydroxybenzoates (i.e., parabens); pH adjusters such as inorganic and organic acids and bases; sweeteners; and flavoring agents; biodegradable polymer beads. If desired, disintegrating or solubilizing agents, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, alginatetone, or a salt thereof, sodium alginate, and the like, may be added.

[0066] Pharmaceutically acceptable carriers can include, for example, physiologically acceptable compounds that function to stabilize the compounds of the present invention or regulate their absorption, or other desired excipients.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.Those skilled in the art will understand that the selection of a pharmaceutically acceptable carrier containing a physiologically acceptable compound depends, for example, on the route of administration of the compound and the specific physiochemical characteristics of the compound.

[0067] Generally, such carriers should be non-toxic to recipients at the dosages and concentrations used.Optionally, the preparation of such compositions involves combining one or more compounds 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 dextrin, chelating agents such as EDTA, glutathione, and other stabilizers and excipients.Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary suitable diluents.

[0068] Dragee cores are provided with suitable coatings, such as concentrated sugar solutions, and may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol 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., dosage). Pharmaceutical preparations can also be used orally, using push-fit capsules made of, for example, gelatin, and sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain the compound mixed with a filler or binder, such as lactose or starch, a lubricant, such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the compound can be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol, with or without stabilizers.

[0069] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions. Liquid compositions can be prepared, for example, by dissolving or dispersing the conjugate and optionally one or more pharmaceutically acceptable adjuvants in a carrier such as aqueous saline (e.g., 0.9% w / v sodium chloride), aqueous dextrose, glycerol, and ethanol to form a solution or suspension for oral, topical, or intravenous administration. For parenteral injection, liquid preparations can be formulated in a solution in aqueous polyethylene glycol.

[0070] Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active ingredient in water with a viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and a dispersing or wetting agent, such as a natural phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long-chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a fatty acid and a partial ester derived from a hexitol (e.g., polyoxyethylene sorbitol monooleate), or a condensation product of ethylene oxide with a fatty acid and a partial ester derived from a hexitol anhydride (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 coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, aspartame, or saccharin. The formulations may also be adjusted for osmolarity.

[0071] Oily suspensions can be prepared by suspending the compounds described herein in vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, or mineral oils such as liquid paraffin; or mixtures thereof.Oily suspensions can contain thickening agents such as beeswax, hard paraffin, or cetyl alcohol.Sweeteners such as glycerol, sorbitol, or sucrose can be added to provide a palatable oral preparation.These preparations can be preserved by adding antioxidants such as ascorbic acid.For examples of injectable oily vehicles, see Minto, J. Pharmacol. Exp. Ther. 281:93-102, 1997.Pharmaceutical preparations can also be in the form of oil-in-water emulsions.The oily phase can be the above-mentioned vegetable oil or mineral oil, or mixtures thereof. Suitable emulsifying agents include natural gums such as gum acacia and gum tragacanth, natural phosphatides 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, such as polyoxyethylene sorbitan monooleate. Emulsions can also contain sweetening and flavoring agents, as can the formulation of syrups and elixirs. Such formulations can also contain a demulcent, preservative, or coloring agent.

[0072] Also comprise solid preparations that are intended to be converted into the liquid form preparations for oral administration immediately before use.Such liquid forms include solution, suspension and emulsion.These preparations may contain, in addition to active ingredient, coloring agent, flavoring agent, stabilizer, buffer, artificial and natural sweetener, dispersant, thickener and solubilizer etc.

[0073] For oral administration, the composition 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, talc, cellulose, glucose, gelatin, sucrose, magnesium carbonate, etc.

[0074] For topical administration, the composition of the present invention can be in the form of emulsion, lotion, gel, cream, jelly, solution, suspension, ointment, and transdermal patch.For inhalation delivery, the composition can be delivered as dry powder or in liquid form via nebulizer.For parenteral administration, the composition can be in the form of sterile injection solution and sterile packaged powder.Preferably, the injection solution is formulated at a pH of about 4.5 to about 7.5.

[0075] The compositions of the present invention may also be provided in lyophilized form. Such compositions may contain a buffer, such as bicarbonate, for reconstitution before administration, or a buffer may be included in the lyophilized composition for reconstitution with water, etc. The lyophilized composition may further contain a suitable vasoconstrictor, such as epinephrine. The lyophilized composition may be provided in a syringe, optionally packaged in combination with a buffer for reconstitution, so that the reconstituted composition can be immediately administered to a patient.

[0076] In some embodiments, the pharmaceutical composition is a slow-release formulation. Slow-release embodiments include polymeric materials that are biodegradable and / or slowly dissolving. Such polymeric substances include polyvinylpyrrolidone, low and medium molecular weight hydroxypropyl cellulose and hydroxypropylmethylcellulose, cross-linked sodium carboxymethylcellulose, carboxymethyl starch, potassium methacrylate divinylbenzene copolymer, polyvinyl alcohol, starch, starch derivatives, microcrystalline cellulose, ethyl cellulose, methyl cellulose, and cellulose derivatives, beta-cyclodextrin, poly(methyl vinyl ether / maleic anhydride), glucan, scleroglucan, mannan, xanthan, alzinic acid and its derivatives, dextrin derivatives, glyceryl monostearate, semi-synthetic glycerides, glyceryl palmitostearate, glyceryl behenate, polyvinylpyrrolidone, gelatin, magnesium stearate, stearic acid, sodium stearate, talc, sodium benzoate, boric acid, and colloidal silica.

[0077] The slow-release agents of the present invention may also include adjuvants such as starch, pregelled starch, calcium phosphate, mannitol, lactose, sucrose, glucose, sorbitol, microcrystalline cellulose, gelatin, polyvinylpyrrolidone, methylcellulose, starch solution, ethylcellulose, gum arabic, gum tragacanth, magnesium stearate, stearic acid, colloidal silica, glyceryl monostearate, hydrogenated castor oil, waxes, and mono-, di-, and tri-substituted glycerides. Slow-release agents may also be prepared as generally described in WO 94 / 06416.

[0078] Pharmaceutical preparations are preferably in unit dosage form.The term "unit dosage form" refers to a physically separate unit suitable for use as a unit dose in human subjects and other mammals (e.g., dogs), each unit containing a predetermined amount of active substance calculated to produce a desired expression, tolerability, and / or therapeutic effect in association with suitable pharmaceutical excipients (e.g., ampule).In addition, more concentrated compositions can be prepared, and then more diluted unit dosage compositions can be made from them.Therefore, more concentrated compositions will substantially contain, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more of the amount of LLP2A-bisphosphonate conjugate, mesenchymal stem cell, and / or anabolic agent.In such a form, the preparation is subdivided into unit doses containing an appropriate amount of active ingredient.The unit dosage form can also be a packaged preparation, and the package contains individual amounts of preparations, such as tablets, capsules, and powders, which are divided into vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The composition can also contain other compatible therapeutic agents, if desired. Preferred pharmaceutical preparations can deliver the compound in a sustained-release formulation.

[0079] Methods for preparing such dosage forms are known to those skilled in the art (see, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra). The composition to be administered contains an amount of the LLP2A-bisphosphonate conjugate, mesenchymal stem cells, and / or anabolic agent in a pharmaceutically effective amount for alleviating the condition being treated when administered in accordance with the teachings of the present invention. In addition, pharmaceutically acceptable salts (e.g., acid addition salts) of the conjugates described herein are prepared and included in the composition using standard techniques known to those skilled in the art of organic chemical synthesis and described, for example, by J. March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th Ed. (New York: Wiley-Interscience, 1992).

[0080] In some embodiments, the LLP2A-bisphosphonate conjugate, mesenchymal stem cells, and / or anabolic agent are administered in a therapeutically effective amount or dose. Daily dose ranges of about 0.01 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 200 mg / kg, or about 1 mg / kg to about 100 mg / kg, or about 10 mg / kg to about 50 mg / kg can be used. However, dosages can vary depending on several factors, including the selected route of administration, the formulation of the composition, patient response, the severity of the condition, the subject's weight, and the judgment of the prescribing physician. Dosages can be increased or decreased over time as needed by an individual patient. In certain instances, patients are initially given a low dose, which is then increased to an effective dose tolerated by the patient. Determining an effective amount is well within the capabilities of those skilled in the art.

[0081] In some embodiments, co-administration of an LLP2A-bisphosphonate conjugate with mesenchymal stem cells and / or an anabolic agent enhances the therapeutic effect such that one or more of the LLP2A-bisphosphonate conjugate, mesenchymal stem cells, or anabolic agent can be administered in a reduced amount or at a reduced frequency compared to the amount and / or frequency required to induce a therapeutic effect when administered alone. For example, in some embodiments, one or more of the LLP2A-bisphosphonate conjugate, mesenchymal stem cells, and anabolic agent can be administered 10%, 20%, 30%, 40%, 50%, 60%, or 70% less frequently than when administered alone. In some embodiments, one or more of the LLP2A-bisphosphonate conjugate, mesenchymal stem cells, and anabolic agent can be administered in an amount that is about 10%, 20%, 30%, 40%, 50%, 60%, or 70% less than the amount required to induce a therapeutic effect when the LLP2A-bisphosphonate conjugate or mesenchymal stem cells are administered alone.

[0082] In carrying out the methods described herein, pharmaceutical compositions can be used alone or in combination with other therapeutic or diagnostic agents.When multiple compositions are used in a treatment protocol, the compositions can be administered separately, or the compositions can be administered together, such as in a mixture.When one or more compositions are administered separately, the timing and schedule of administration of each composition can be different.Other therapeutic or diagnostic agents can be administered simultaneously, separately, or at different times with the compounds of the present invention.

[0083] In some embodiments, co-administration of compositions (e.g., an LLP2A-bisphosphonate conjugate as described herein with one or more of mesenchymal stem cells and an anabolic agent) includes administration of one composition within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of the second composition. In some embodiments, the two compositions are administered simultaneously, nearly 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 can be achieved by co-formulation, e.g., preparing a single pharmaceutical composition containing both the LLP2A-Ale conjugate and the mesenchymal stem cells and / or the anabolic agent. In other embodiments, the compositions can be formulated separately.

[0084] The administration of the compound of the present invention, optionally with suitable pharmaceutical excipients, can be carried out by any acceptable mode of administration.Therefore, administration can be, for example, intravenous, topical, subcutaneous, transcutaneous, transdermal, intramuscular, oral, intraarticular, parenteral, intraarteriolar, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, intrarectal, intravaginal or by inhalation.In some embodiments, one or more of LLP2A-bisphosphonate conjugates, mesenchymal stem cells and anabolic agents are administered locally, for example, to bone (for example, femur, hip, knee, shoulder, ankle, wrist, jawbone) or to the osteonecrotic lesion site in bone (for example, femur, hip, knee, shoulder, ankle, wrist, jawbone). In some embodiments, one or more of the LLP2A-bisphosphonate conjugate, mesenchymal stem cell, and anabolic agent are administered systemically.In some embodiments, the compositions are administered by different routes.For example, in some embodiments, one composition (e.g., LLP2A-bisphosphonate conjugate) is administered locally, and another composition (e.g., mesenchymal stem cell or anabolic agent) is administered systemically.

[0085] IV. METHODS OF PREVENTING OR TREATING OSTEONECROSIS, INCREASING VASCULAR DENSITY IN OSTEONECROSIS TISSUE, AND REDUCING CELL DEATH IN OSTEONECROSIS TISSUE In one aspect, the compositions described herein (e.g., compositions comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug (e.g., LLP2A-Ale)) are used to prevent and / or treat osteonecrosis, increase vascularity, or reduce cell death in osteonecrotic tissue. In some embodiments, the method comprises: administering to a subject a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug. Includes.

[0086] In some embodiments, the method comprises: identifying a subject having or suspected of having osteonecrosis of at least one bone; and administering to a subject a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug. Includes.

[0087] In some embodiments, the method comprises administering a pharmaceutical composition comprising a conjugate of LLP2A and alendronate ("LLP2A-Ale"), as shown in Formula I above. In some embodiments, the method further comprises administering mesenchymal stem cells, e.g., simultaneously or sequentially with the conjugate. In some embodiments, the method further comprises administering one or more anabolic agents (e.g., teriparatide), e.g., simultaneously or sequentially with the conjugate.

[0088] In some embodiments, the subject is an adult or juvenile subject who has been identified as having osteonecrosis. In some embodiments, the subject is an adult or juvenile subject suspected of having osteonecrosis. In some embodiments, the subject is an adult. In some embodiments, the subject is a juvenile.

[0089] Osteonecrosis, also known as avascular necrosis or aseptic necrosis, is the death of bone cells in bone due to reduced blood flow. If left untreated, the death of bone cells in bone can lead to the collapse of the bone site, which in turn can lead to degenerative arthritis of the joints near the bone. Osteonecrosis most commonly affects the hips and joints, but can also affect the shoulders, wrists, hands, ankles, feet, and jaw.

[0090] Osteonecrosis can have various causes, including traumatic and non-traumatic causes. Typically, in traumatic osteonecrosis, severe trauma to bone disrupts the blood supply of bone. Non-traumatic osteonecrosis can be caused by certain drug therapies, such as corticosteroid drug therapy (e.g., prednisone, cortisone, dexamethasone, or methylprednisolone), especially when high doses of medication are administered over a long period of time; excessive alcohol consumption; or radiation therapy; or can occur as a result of disease or condition. For example, see Xie et al., 2015, Journal of Orthopaedic Translation, 3:58-70, which is incorporated herein by reference. In some embodiments, the subject has post-traumatic osteonecrosis (e.g., osteonecrosis occurring after fracture or dislocation of bone). In some embodiments, the subject has non-traumatic osteonecrosis. In some embodiments, the subject has steroid-induced osteonecrosis (e.g., high-dose steroid treatment-induced osteonecrosis or glucocorticoid-induced osteonecrosis), alcohol-induced osteonecrosis, or smoking-induced osteonecrosis. In some embodiments, the subject has osteonecrosis induced as a secondary cause of another disease or condition, including, but not limited to, Legg-Calvé-Perthes disease, Caison's disease, sickle cell disease, post-irradiation, chemotherapy, arterial disease, Gaucher's disease, lipid disorders, connective tissue diseases, pancreatitis, kidney disease, liver disease, or lupus. In some embodiments, the osteonecrosis is idiopathic osteonecrosis.

[0091] Osteonecrosis is classified into stages that refer to the degree of disease progression. In some embodiments, the Ficat classification system can be used to determine the stage of the disease. Other classification systems, such as the University of Pennsylvania system, the Association Research Circulation Osseous (ARCO) system, and the Japanese Orthopaedic Association system, can also be used. See, for example, Jawad et al., Clin Orthop Related Res, 2012, 470:2636-2639, which is incorporated herein by reference. In the Ficat system, there are five stages of osteonecrosis (stages 0 to 4). Stages 0 to 2 are generally described as the "early" stages of osteonecrosis, while stages 3 and 4 are generally described as the "late" stages. In stage 0, osteonecrosis is pre-clinical and pre-radiographic (i.e., not detectable by radiography), although evidence of bone defects may be detected by bone marrow pressure testing or core biopsy. In stage 1, evidence of osteonecrotic lesions can be seen by MRI, mild osteopenia may be visible by X-ray, and bone scans may show increased radioactive uptake in bone sites, indicating damage to the bone (e.g., tumor, fracture, or infection). Clinical symptoms of pain in the joints may also be present in stage 1. In stage 2, evidence of lesions and abnormalities in bone tissue can be detected by MRI, mixed osteopenia, sclerosis, and / or subchondral cysts can be detected by X-ray, and increased radioactive uptake can be detected by bone scan; clinical symptoms include pain and stiffness in the joints. In stage 3, changes in bone contour can be detected (e.g., as a "crescent" shape in the femur), and final cortical bone collapse can be detected by X-ray or MRI. In stage 4, the final stage of the disease, bone collapse and joint space reduction become detectable.

[0092] Symptoms can be correlated with the stage of osteonecrosis using a combination of diagnostic tools, such as X-ray, MRI, bone scan (bone scintigraphy), bone biopsy, clinical features, and "bone function checkup." For example, bone marrow pressure can be measured in bone area, and changes in bone marrow pressure can be measured by performing stress tests on bone (for example, by injecting isotonic saline into the bone, waiting a predetermined time, and then measuring the pressure at a certain time point after injection). Bone scans can be performed by intravenously injecting radioactive materials such as technetium and imaging the bone. For example, see Liehn et al., European Journal of Orthopaedic Surgery & traumatology, 1997, 4:263-265, which is incorporated herein by reference. MRI (magnetic resonance imaging) is a highly sensitive method for detecting osteonecrosis and can show areas of low signal intensity typical of osteonecrosis. See, for example, Saini et al., Clinical Radiology, 2004, 59:1079-1093, which is incorporated herein by reference. Computed tomography (CT) scans can also be used to detect osteonecrosis.

[0093] In some embodiments, a subject is identified as having or suspected of having osteonecrosis by examining at least one bone or joint in the subject and classifying the subject according to one of the classification systems described herein (e.g., Ficat or ARCO). In some embodiments, a subject is identified as having or suspected of having osteonecrosis, where the subject does not have detectable osteonecrotic lesions, but bone abnormalities can be detected (e.g., changes in bone pressure). In some embodiments, the subject is classified as having stage 0 disease by the Ficat classification system.

[0094] In some embodiments, a subject is identified as having osteonecrosis if there is an abnormality in blood flow, blood pool, and / or osteoblast activity in at least one bone or joint. In some embodiments, the abnormality is detected by bone scintigraphy.

[0095] In some embodiments, a subject is identified as having osteonecrosis if at least one osteonecrotic lesion is detected in at least one bone. In some embodiments, the osteonecrotic lesion is detected by MRI. In some embodiments, a subject has at least one osteonecrotic lesion measuring at least 3.5 cm. 2 In some embodiments, a subject is identified as having at least one osteonecrotic lesion having a size of at least 5 cm 2 In some embodiments, the subject has at least one osteonecrotic lesion or abnormality in the bone as detected by MRI or bone scan, but does not exhibit loss of bone density as measured by X-ray.

[0096] In some embodiments, the subject is classified as having stage 1 disease according to the Ficat classification system. In some embodiments, the subject has at least one osteonecrotic lesion or abnormality in bone as detected by MRI or bone scan, and also exhibits bone density loss, osteopenia, sclerosis, and / or bone cysts as measured by X-ray. In some embodiments, the subject is classified as having stage 2 disease according to the Ficat classification system.

[0097] In some embodiments, the subject exhibits bone flattening, impending bone collapse, or bone collapse. In some embodiments, the subject has late-stage osteonecrosis (e.g., stage 3 or 4 according to the Ficat classification system).

[0098] In some embodiments, the LLP2A peptidomimetic ligand-bisphosphonate drug conjugate as described herein is used to treat osteonecrosis. In some embodiments, the treatment of osteonecrosis is measured by assessing whether the osteonecrotic bone or joint exhibits a decrease in one or more symptoms of osteonecrosis, such as a decrease in the number or size of osteonecrotic lesions, a decrease in the number or size of sclerotic lesions, a decrease in the amount of cell death in the bone, an increase in the amount of angiogenesis (e.g., vascular density) in the bone, or a decrease in bone marrow adipocyte volume. In some embodiments, after administering a pharmaceutical composition as described herein (e.g., a composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug (e.g., LLP2A-Ale)), the method further comprises examining the osteonecrotic bone or joint of the subject to identify changes in one or more symptoms of osteonecrosis. Thus, in some embodiments, the method of treating osteonecrosis comprises: identifying a subject having one or more symptoms of osteonecrosis in at least one bone: administering to a subject a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug; and examining at least one bone to identify a change in one or more symptoms of osteonecrosis compared to before the administering step. Includes.

[0099] In some embodiments, the method includes identifying a change in two or more symptoms of osteonecrosis (eg, two, three, four, or more symptoms of osteonecrosis).

[0100] As a non-limiting example, in some embodiments, the method comprises, before administering a pharmaceutical composition as described herein, determining the amount of osteonecrotic lesions in a bone or joint and / or measuring the size of one or more osteonecrotic lesions in a bone or joint; and after administering the pharmaceutical composition, the method further comprises determining the number of osteonecrotic lesions and / or measuring the size of one or more osteonecrotic lesions in a bone or joint, and comparing the size of the lesions before and after administering the pharmaceutical composition. In some embodiments, a decrease in the amount of osteonecrotic lesions and / or the size of the osteonecrotic lesions after administering compared to before administering indicates that osteonecrosis is being treated in the subject. In some embodiments, at least about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in the size of osteonecrotic lesions after the administering step compared to the size of the osteonecrotic lesions before the administering step indicates that the osteonecrosis has been treated. In some embodiments, at least about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in the number of osteonecrotic lesions in the bone or tissue after the administering step compared to the number of osteonecrotic lesions before the administering step indicates that the osteonecrosis has been treated.

[0101] In some embodiments, the LLP2A peptidomimetic ligand-bisphosphonate drug conjugates described herein are used to increase vascular density in osteonecrotic tissue. In some embodiments, the increased vascular density comprises an increased amount of angiogenesis in the bone. In some embodiments, the increased vascular density comprises an increased sinusoidal formation or sinusoidal red blood cell volume in the bone. In some embodiments, after administering a pharmaceutical composition as described herein (e.g., a composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug (e.g., LLP2A-Ale)), the method further comprises examining the subject's osteonecrotic bone or joint to identify changes in the amount of angiogenesis.

[0102] In some embodiments, the angiogenesis of osteonecrotic tissue is measured by superselective angiography (SSA), digital subtraction arteriography (DSA), Doppler-laser hemodynamic measurement, bone scintigraphy, magnetic resonance imaging (MRI, for example, dynamic MRI with contrast injection), and the method of measuring angiogenesis in osteonecrotic tissue is described in the art.For example, see Ehlinger et al., Orthopedics & traumatology: Surgery & Research, 2001, 97:79-88, which is incorporated herein by reference.In some embodiments, the angiogenesis of osteonecrotic tissue is measured by radionuclide bone scintigraphy.In some embodiments, the angiogenesis of osteonecrotic tissue is measured by MRI, for example, dynamic MRI. In some embodiments, an increase of at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the degree of vascularization in the osteonecrotic tissue (e.g., osteonecrotic bone) compared to the degree of vascularization before the administering step indicates that vascular density is increased in the osteonecrotic tissue. In some embodiments, an increase of at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in sinusoidal formation or sinusoidal red blood cell volume in osteonecrotic tissue (e.g., osteonecrotic bone) compared to the degree of sinusoidal formation or sinusoidal red blood cell volume before the administering step indicates that vascular density is increased in the osteonecrotic tissue.

[0103] In some embodiments, the LLP2A peptidomimetic ligand-bisphosphonate drug conjugate as described herein is used to reduce or prevent cell death in osteonecrotic tissue. In some embodiments, cell death in osteonecrotic tissue is measured by detecting the presence of vacuolated osteocyte lacunae in bone. Thus, in some embodiments, the prevention of cell death or the reduction in the rate of cell death in osteonecrotic tissue (e.g., bone) caused by the administration of a pharmaceutical composition as described herein (e.g., a composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug (e.g., LLP2A-Ale)) can be measured by comparing the amount of vacuolated osteocyte lacunae in osteonecrotic tissue before and after administration of the pharmaceutical composition. In some embodiments, vacuolated bone lacunae are detected by imaging (e.g., MRI or X-ray). In some embodiments, vacuolated bone lacunae are detected, for example, by histopathological examination of a bone biopsy sample from a subject. In some embodiments, stabilization of the amount of vacuolated bone lacunae in the osteonecrotic tissue before and after administration of the pharmaceutical composition (e.g., the number of vacuolated bone lacunae in the osteonecrotic tissue after administration of the pharmaceutical composition is substantially the same as the number of vacuolated bone lacunae in the osteonecrotic tissue before administration of the pharmaceutical composition) indicates prevention or reduction of cell death in the osteonecrotic tissue.

[0104] In some embodiments, the methods for treating osteonecrosis as described herein further include increasing bone remodeling (e.g., in conjunction with increasing vascularity and / or reducing cell death in osteonecrotic bones or joints). In some embodiments, the methods for treating osteonecrosis include increasing the number of osteoblasts present on the bone surface at the site where osteonecrosis is observed. Bone growth can be measured by various methods known to those skilled in the art. Methods for measuring bone growth include, but are not limited to, microCT, dual-energy X-ray absorption, ultrasound, QCT, SPA, DPA, DXR, SEXA, QUS, X-ray, or marker analysis, such as alizarin red S, serum osteocalcin, serum alkaline phosphatase, serum bone gamma-carboxyglutamic acid-containing protein (BGP), bone mineral density, serum calcium, serum phosphorus, tantalum markers, and serum IGF-1. In some embodiments, an increase in bone density in the osteonecrotic tissue (e.g., osteonecrotic bone) of at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the bone density before the administering step indicates increased bone remodeling in the osteonecrotic tissue. [Example]

[0105] V. Working Example The following examples are offered to illustrate, but not to limit, the claimed invention.

[0106] Example 1 Use of LLP2A-Ale in a 56-day mouse model of glucocorticoid-induced osteonecrosis Osteonecrosis is a disease characterized by reduced blood flow to bones, especially in joints. The reduced blood supply leads to bone death and breakdown. Figure 1 illustrates the stages of glucocorticoid-induced osteonecrosis. Osteonecrosis is classified into stages (I to V) that refer to how advanced the disease is. In traumatic osteonecrosis, the reduced blood supply first leads to the death of bone cells, which then leads to bone collapse because the area of ​​dead bone cannot support bone use (Figure 1, humerus).

[0107] The efficacy of LLP2A-Ale was evaluated in a mouse model of glucocorticoid-induced osteonecrosis. Four-month-old male mice were treated with glucocorticoids for 56 days. On day 28, groups of mice were treated with either LLP2A-Ale or PTH for an additional 28 days. Vascular density in the femurs was measured after sacrifice by micro-CT according to published methods. As shown in Figure 2, the vascular density in the femurs of mice treated with glucocorticoids and LLP2A-Ale was significantly higher than that of negative control (glucocorticoid-only) or glucocorticoid- and PTH-treated mice. Vascular density in the femurs of glucocorticoid-treated mice treated with LLP2A-Ale or PTH was also assessed by administering contrast agent to the mice, sacrificing them, decalcifying the femurs, and scanning the femurs by micro-CT. As shown in Figure 3, glucocorticoid treatment reduced vascular density. Treatment of glucocorticoid-treated mice with LLP2A-Ale increased vascular density compared with glucocorticoid alone, whereas glucocorticoid-treated mice additionally treated with PTH did not significantly alter vascular density compared with glucocorticoid treatment alone. These data suggest that glucocorticoid-induced osteonecrosis is partially attributable to reduced vascularity, and that LLP2A-Ale increases vascular density in osteonecrotic tissue. Without being bound by theory, it is believed that LLP2A-Ale promotes increased vascularity by recruiting MSCs to bone undergoing turnover due to osteonecrosis, stimulating new blood vessel formation, and that this blood vessel formation is required to stimulate new bone formation in osteonecrotic bone.

[0108] Increased angiogenesis can also be assessed by marker analysis. For example, VEGF is a pro-angiogenic factor that stimulates new blood vessel formation. As shown in Figure 4, VEGF serum levels were measured on day 56 from animals in a treatment study that received glucocorticoid treatment alone on days 1–28, followed by glucocorticoid or placebo alone, or glucocorticoid plus LLP2A-Ale, or glucocorticoid plus PTH, or glucocorticoid plus PTH plus LLP2A-Ale. PTH was given at 20 μg / kg 5× weekly, and LLP2A-Ale was given at 500 μg / kg on day 28 only. Glucocorticoid treatment reduced serum VEGF levels as measured by ELISA in duplicate, while glucocorticoid plus LLP2A-Ale restored serum VEGF levels to those of placebo-treated animals.

[0109] Bone cells die from loss of blood supply and also die from glucocorticoid treatment. As shown in Figure 5, we counted apoptotic bone cells in cortical bone slices from the glucocorticoid treatment study described above, in which animals were treated with PL for 56 days, GC alone for 56 days, or GC alone for 28 days, followed by GC plus one dose of LLP2A-Ale on day 28. The animals were sacrificed on day 56, the bones were demineralized, and a TUNEL assay was then performed to determine the number of apoptotic bone cells. Glucocorticoid treatment alone increased the number of apoptotic bone cells on day 56. Glucocorticoid plus LLP2A-Ale treatment prevented osteocyte apoptosis. Because osteonecrosis involves apoptotic bone cells, this data suggests that LLP2A-Ale can treat osteonecrosis of bone.

[0110] Treatment with LLP2A-Ale also remodels bone in a mouse model of glucocorticoid-induced osteonecrosis. As shown in Figure 6, mice were treated with glucocorticoids for 28 days and then with either LLP2A-Ale, PTH, or LLP2A-Ale + PTH. Changes in cancellous bone volume during the treatment period were measured for the distal femur and vertebrae. Treatment with LLP2A-Ale increased bone volume in both the distal femur and vertebrae compared with glucocorticoid-treated mice. Regarding distal femur cancellous bone volume, treatment with LLP2A-Ale increased distal femur cancellous bone volume relative to baseline / sham levels.

[0111] Example 2 Use of LLP2A-Ale in a 90-day mouse model of glucocorticoid-induced osteonecrosis This study was conducted to determine whether LLP2A-Ale could reverse or treat established traumatic osteonecrosis in mice.

[0112] material and method: Animals and experimental methods The efficacy of LLP2A-Ale was evaluated in a 90-day study using a mouse model of glucocorticoid-induced osteonecrosis. Seven-week-old male mice (BALB / c (n=80, Jackson Laboratories, USA) were housed in a clean, ventilated animal room maintained at 20°C with a 12-h light / dark cycle. All animals were handled in accordance with USDA animal care guidelines approved by the UC Davis Committee on Animal Research. Mice were provided with water and standard commercial rodent chow (22 / 5 Rodent Diet; Teklad, Madison, WI) ad libitum. Animals were weight-randomized to either placebo (fresh water) or glucocorticoid only (4 mg / L dexamethasone (Dex) in drinking water) and treated for 90 days. Then, on day 30, Dex-treated mice were treated with Dex alone, Dex + 250 μg / kg LLP2A-Ale, Dex + 500 μg / kg LLP2A-Ale, and Dex + 750 μg / kg All mice were re-randomized to LLP2A-Ale. LLP2A-Ale treatment was given on days 30, 45, 60, and 75. In addition, 0.1 mg / kg methotrexate was administered twice weekly by subcutaneous injection on days 1-42 in all groups receiving glucocorticoids.

[0113] All mice were euthanized on day 90. The body weight of all test animals was evaluated weekly. If a test animal had a 10% weight loss compared to the previous week, the dose of dexamethasone was reduced to 2 mg / L until weight gain was observed. Seven and two days before euthanasia, all animals were injected with calcein (30 mg / kg) and alizarin red (20 mg / kg).

[0114] Histological analysis Both right and left distal femurs were fixed in 10% neutral buffered formalin, shaken for 3 days, and then decalcified in 5% EDTA until the bones were fully decalcified.The specimens were then treated with increasing concentrations of ethanol and embedded in paraffin.Tissue sections were cut into 5-micrometer sections, stained with hematoxylin and eosin, and evaluated for the presence of osteonecrosis under a bright-field light microscope at 20x magnification.The experimental method is also described in Mohan et al., Calif Tissue Int (2016) doi:10.1007 / s00223-016-0195-6, the entire contents of which are incorporated herein by reference for all purposes. Osteonecrosis was detected using a modified standard reported by Yang et al. (A mouse model for glucocorticoid-induced osteonecrosis: Effect of a steroid holiday. J Orthop Res. 2009;27:169-75), which defines osteonecrosis as requiring all of the following changes: (1) vacuolated bone lacunae, (2) condensed osteocyte nuclei in the vacuolated bone lacunae and adjacent bone marrow necrotic areas, (3) the presence of excessive adipocytes in the bone marrow, (4) cartilage degradation, and (5) the presence of fibrin clots in blood vessels. Additionally, cancellous bone volume, fat volume, sinusoidal capillary volume, and osteoblast surface in the distal femoral epiphysis were measured. Vacuolated bone lacuna density was calculated for each specimen by the ratio of vacuolated bone lacunae to total osteocytes within the entire distal femoral epiphysis region. Each section was scored by two experienced histologists, followed by a consensus reading by two additional experienced histologists.

[0115] result: body weight The body weight of BALB / c mice in the placebo group had a weight increase over the 90-day experimental period that was significantly different from baseline weight (p<0.05), but this was not observed in the other groups. The body weight of all dexamethasone (Dex)-treated animals was significantly lower compared to placebo from day 7 to day 90 (p<0.05).

[0116] Prevalence of osteonecrosis Evidence of osteonecrosis was present throughout the distal femoral epiphysis in Dex-treated mice. As shown in Figure 7, the Dex-only treatment group exhibited at least three or all five characteristics of osteonecrosis (as determined using modified criteria reported by Yang et al.), a significantly higher number of subjects than any of the Dex + LLP2A-Ale treatment groups. The number of subjects with three or more characteristics of osteonecrosis was: PL (0%); Dex alone group 14 / 16 (88%); Dex + LLP2A-Ale 250 μg / kg 12 / 16 (75%); Dex + LLP2A-Ale 500 μg / kg 9 / 16 (56%); and LLP2A-Ale 750 μg / kg 11 / 16 (69%). In the Dex-only group, 75% of samples had 10% or more vacuolated bone lacunae, compared with 44–62% of samples in the LLP2A-Ale-treated group (Fig. 8A–B). Also, as shown in Fig. 8A–B, more samples in the LLP2A-Ale-treated group exhibited less than 10% vacuolated bone lacunae compared with the Dex-treated group.

[0117] Histological evaluation of dexamethasone-treated mice As shown in Figures 9A and 9B, histological sections of the epiphysis from placebo-treated mice showed no evidence of osteonecrosis, and a few bone marrow adipocytes and sinusoidal capillaries were observed in the bone marrow. Histological sections of the epiphysis from Dex-treated mice (Figures 9C-9F) showed large vacuolated bone lacunae (Figures 9D and 9F, black arrows) and necrotic bone trabeculae characterized by pyknotic nuclei of osteocytes. Some dexamethasone-treated mice exhibited loss of articular cartilage (Figure 9D, yellow arrow) surrounded by necrotic bone marrow (Figure 9E, green arrow) and adipose tissue (Figures 9D, 9E, and 9F, blue arrows). These data indicate that glucocorticoid-induced osteonecrosis is observed in this mouse model over the 90-day study.

[0118] Repair of osteonecrotic lesions in LLP2A-Ale-treated mice As shown in Figure 10A, all three doses of LLP2A-Ale treatment reduced fat volume compared to that of Dex-treated mice, indicating evidence of osteonecrotic lesion repair. As shown in Figure 10B, all three doses of LLP2A-Ale treatment increased sinusoidal volume compared to placebo or Dex treatment alone. Increasing LLP2A-Ale treatment above 250 μg / kg resulted in a slight decrease in sinusoidal volume. These data suggest that LLP2A-Ale treatment can repair osteonecrotic lesions by increasing angiogenesis.

[0119] Histological evaluation of LLP2A-Ale-treated mice As shown in Figures 11A to 11F, histological sections of the epiphysis from LLP2A-Ale-treated mice showed increased sinusoid formation, with the sinusoids either protruding into the necrotic bone marrow (Figure 11B) or occupying bone marrow in areas where bone marrow necrosis was not observed (Figures 11E and 11F). In Figure 11E, several osteoblasts were observed on the trabecular surface and near the sinusoids (yellow arrows). These data suggest that LLP2A-Ale treatment after osteonecrosis, compared with dexamethasone alone, appeared to reduce the number of vacuolated bone lacunae / pyknotic nuclei and adipocyte density in the bone marrow, increased sinusoidal erythrocyte volume, and maintained bone marrow cellularity. In addition, LLP2A-Ale treatment appeared to reduce the amount of fibrin in the bone marrow and increased the number of osteoblasts present on the bone surface at epiphyseal sites where osteonecrosis was present, suggesting its ability to stimulate new bone formation.

[0120] Example 3 Use of LLP2A-Ale and hPTH(1-34) to prevent glucocorticoid-induced osteonecrosis and loss of vascularity in a mouse model This study was conducted to determine whether administration of LLP2A-Ale or hPTH(1-34) for 30 or 45 days could prevent glucocorticoid-induced osteonecrosis and preserve vascularity within the distal femur in mice.

[0121] material and method: Animals and experimental methods The efficacy of LLP2A-Ale and hPTH(1-34) was evaluated in a mouse model of glucocorticoid-induced osteonecrosis over 30 and 45-day studies. Seven-week-old male BALB / c mice were randomized to receive placebo (fresh water), glucocorticoid only (4 mg / L dexamethasone (Dex) in drinking water), Dex + 250 μg / kg LLP2A-Ale, Dex + 500 μg / kg LLP2A-Ale, or Dex + 40 μg / kg hPTH (administered five times weekly) for 30 or 45 days. Mice were sacrificed on days 30 and 45. Study endpoints included histological evidence of osteonecrosis, bone mass, and vascularity (CD31 and endomucin expression) in the distal femur. The study was performed as described in Example 2. Differences between groups were determined using a nonparametric Kruskal-Wallis test.

[0122] result: Prevalence of osteonecrosis Evidence of osteonecrosis was present throughout the distal femur epiphysis in Dex-treated mice. As shown in Figure 12, the Dex-only treatment group had a significantly higher percentage of osteonecrosis incidence than mice in the LLP2A-Ale or hPTH treatment groups. The incidence of glucocorticoid-induced osteonecrosis was 57% at 30 days and 73% at 45 days with Dex alone, and both LLP2A-Ale and hPTH(1-34) treatment prevented the concomitant GC-induced osteonecrotic lesions.

[0123] Histological evaluation Histological sections of the epiphysis from Dex-treated mice showed significant decreases in cancellous bone volume and trabecular thickness compared to placebo at 30 and 45 days (p<0.05). Trabecular number was significantly reduced at 45 days (p<0.05). In contrast, both LLP2A-Ale and hPTH(1-34)-treated animals had higher bone volume and lower trabecular spacing compared to the Dex group at 30 and 45 days (p<0.05). In addition, Dex treatment reduced the staining intensity of both CD31 and endomucin expression at 30 and 45 days compared to LLP2A-Ale, hPTH(1-34), or placebo treatment (data not shown). These data indicate that glucocorticoid-induced osteonecrosis is observed in this mouse model throughout the 30 and 45 days of the study.

[0124] conclusion Dex treatment induced cancellous bone loss and osteonecrotic lesions. Both LLP2A-Ale and hPTH treatment reduced the incidence and severity of osteonecrosis and maintained vascular integrity of the distal femur.

[0125] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will understand that certain changes and modifications may be practiced within the scope of the appended claims. Additionally, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

Claims

1. administering to the subject a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug. A method of treating osteonecrosis in a subject, comprising:

2. The pharmaceutical composition comprises a conjugate of LLP2A and alendronate (“LLP2A-Ale”), the conjugate having the formula:

2. The method of claim 1, comprising:

3. 3. The method of claim 1 or 2, further comprising, prior to said administering step, identifying a subject having at least one osteonecrotic lesion in at least one bone.

4. 4. The method of claim 3, wherein the at least one bone is a femur, hip, knee, shoulder, ankle, wrist, or jaw bone.

5. 5. The method of claim 3 or 4, wherein the identifying step comprises magnetic resonance imaging of the at least one bone.

6. The step of identifying comprises determining a distance of at least 3.5 cm 2 6. The method of claim 3, comprising detecting at least one osteonecrotic lesion having a size of:

7. The step of identifying comprises determining a distance of at least 5 cm 2 7. The method of claim 6, comprising detecting at least one osteonecrotic lesion having a size of:

8. 8. The method of claim 3, further comprising measuring the size of the at least one osteonecrotic lesion after the administering step and detecting a decrease in the size of the osteonecrotic lesion compared to the size of the osteonecrotic lesion before the administering step.

9. 9. The method of any one of claims 1 to 8, wherein the osteonecrosis is traumatic osteonecrosis.

10. 9. The method of any one of claims 1 to 8, wherein the osteonecrosis is glucocorticoid-induced osteonecrosis or alcohol-induced osteonecrosis.

11. The method of any one of claims 1 to 10, further comprising administering exogenous mesenchymal stem cells.

12. The method of claim 11 , wherein the pharmaceutical composition and the exogenous mesenchymal stem cells are administered simultaneously.

13. The method of claim 11 , wherein the pharmaceutical composition and the exogenous mesenchymal stem cells are administered sequentially.

14. 14. The method of any one of claims 1 to 13, wherein the pharmaceutical composition is administered systemically.

15. 14. The method of any one of claims 1 to 13, wherein the pharmaceutical composition is administered topically.

16. 16. The method of claim 15, wherein the pharmaceutical composition is administered locally to the site of the at least one osteonecrotic lesion.

17. 15. The method of any one of claims 1 to 14, wherein the pharmaceutical composition is administered intravenously.

18. 17. The method of any one of claims 1 to 16, wherein the pharmaceutical composition is administered by injection.

19. administering to a subject a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug.

10. A method for increasing vascularity in osteonecrotic tissue, comprising:

20. The pharmaceutical composition comprises a conjugate of LLP2A and alendronate ("LLP2A-Ale"). wherein the conjugate comprises a compound of the formula:

20. The method of claim 19, comprising:

21. 21. The method of claim 19 or 20, further comprising, prior to said administering step, identifying the subject as having at least one osteonecrotic lesion in at least one bone.

22. 22. The method of claim 21, wherein the at least one bone is a femur, hip, knee, shoulder, ankle, wrist, or jaw bone.

23. 23. The method of claim 21 or 22, wherein the identifying step comprises magnetic resonance imaging of the at least one bone.

24. The step of identifying comprises determining a distance of at least 3.5 cm 2 24. The method of any one of claims 21 to 23, comprising detecting at least one osteonecrotic lesion having a size of

25. The step of identifying comprises determining a distance of at least 5 cm 2 25. The method of claim 24, comprising detecting at least one osteonecrotic lesion having a size of

26. 26. The method of any one of claims 19 to 25, wherein the osteonecrosis is traumatic osteonecrosis.

27. 26. The method of any one of claims 19 to 25, wherein the osteonecrosis is glucocorticoid-induced osteonecrosis or alcohol-induced osteonecrosis.

28. 28. The method of any one of claims 19 to 27, further comprising administering exogenous mesenchymal stem cells.

29. 29. The method of claim 28, wherein the pharmaceutical composition and the exogenous mesenchymal stem cells are administered simultaneously.

30. 29. The method of claim 28, wherein the pharmaceutical composition and the exogenous mesenchymal stem cells are administered sequentially.

31. 31. The method of any one of claims 19 to 30, wherein the pharmaceutical composition is administered systemically.

32. 31. The method of any one of claims 19 to 30, wherein the pharmaceutical composition is administered topically.

33. 33. The method of claim 32, wherein the pharmaceutical composition is administered locally to the site of the at least one osteonecrotic lesion.

34. 32. The method of any one of claims 19 to 31, wherein the pharmaceutical composition is administered intravenously.

35. 34. The method of any one of claims 19 to 33, wherein the pharmaceutical composition is administered by injection.

36. administering to a subject a pharmaceutical composition comprising a conjugate of an LLP2A peptidomimetic ligand and a bisphosphonate drug.

10. A method of preventing or reducing cell death in osteonecrotic tissue, comprising:

37. The pharmaceutical composition comprises a conjugate of LLP2A and alendronate ("LLP2A-Ale"). wherein the conjugate comprises a compound of the formula:

37. The method of claim 36, comprising:

38. 38. The method of claim 36 or 37, further comprising, prior to said administering step, identifying the subject as having at least one osteonecrotic lesion in at least one bone.

39. 39. The method of claim 38, wherein the at least one bone is a femur, hip, knee, shoulder, ankle, wrist, or jaw bone.

40. 40. The method of claim 38 or 39, wherein the identifying step comprises magnetic resonance imaging of the at least one bone.

41. The step of identifying comprises determining a distance of at least 3.5 cm 2 41. The method of any one of claims 38 to 40, comprising detecting at least one osteonecrotic lesion having a size of

42. The step of identifying comprises determining a distance of at least 5 cm 2 42. The method of claim 41, comprising detecting at least one osteonecrotic lesion having a size of

43. 43. The method of any one of claims 36 to 42, wherein the osteonecrosis is traumatic osteonecrosis.

44. 43. The method of any one of claims 36 to 42, wherein the osteonecrosis is glucocorticoid-induced osteonecrosis or alcohol-induced osteonecrosis.

45. 45. The method of any one of claims 36 to 44, further comprising administering exogenous mesenchymal stem cells.

46. 46. ​​The method of claim 45, wherein the pharmaceutical composition and the exogenous mesenchymal stem cells are administered simultaneously.

47. 46. ​​The method of claim 45, wherein the pharmaceutical composition and the exogenous mesenchymal stem cells are administered sequentially.

48. 52. The method of any one of claims 36 to 51, wherein the pharmaceutical composition is administered systemically.

49. 48. The method of any one of claims 36 to 47, wherein the pharmaceutical composition is administered topically.

50. 50. The method of claim 49, wherein the pharmaceutical composition is administered locally to the site of the at least one osteonecrotic lesion.

51. 49. The method of any one of claims 36 to 48, wherein the pharmaceutical composition is administered intravenously.

52. 51. The method of any one of claims 36 to 50, wherein the pharmaceutical composition is administered by injection.

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