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WO2018162722A1 - Inhibiteurs de dpp-4 à utiliser dans le traitement de fractures osseuses - Google Patents

Inhibiteurs de dpp-4 à utiliser dans le traitement de fractures osseuses Download PDF

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Publication number
WO2018162722A1
WO2018162722A1 PCT/EP2018/055931 EP2018055931W WO2018162722A1 WO 2018162722 A1 WO2018162722 A1 WO 2018162722A1 EP 2018055931 W EP2018055931 W EP 2018055931W WO 2018162722 A1 WO2018162722 A1 WO 2018162722A1
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WIPO (PCT)
Prior art keywords
dpp
inhibitor
cells
subject
bone
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PCT/EP2018/055931
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English (en)
Inventor
Luís Miguel Rodrigues SARAIVA
Tim Julius Schulz
Thomas Hans AMBROSI
Original Assignee
Deutsches Institut Für Ernährungsforschung Potsdam-Rehbrücke
Sidra Medical and Research Center
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Publication of WO2018162722A1 publication Critical patent/WO2018162722A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth

Definitions

  • Bone breaks An average of 8 million bone breaks occurs in the United States each year. Most bone fractures heal without issues. However, about 3-10% of these breaks are slow to heal or do not heal at all with traditional methods. In particular, about 5-10% of all bone fractures also display non-union and/or delayed unions, which have a substantial economic impact and substantial effects on quality of life. Fracture healing is considered to be a complicated metabolic process that requires the interaction of many factors, including the recruitment of reparative cells and expression of corresponding genes. If these factors are inadequate or interrupted, healing is delayed or impaired, resulting in a nonunion of the bone. The causes of nonunions or delayed healings of fractures are usually unknown.
  • the present invention relates to a DPP-4 inhibitor for use in treating a bone fracture in a subject in need thereof.
  • the present invention relates to a DPP-4 inhibitor for use in preventing non-union of a bone fracture or preventing healing complications following bone fracture in a subject in need thereof.
  • the present invention relates to a DPP-4 inhibitor for use in promoting fracture healing in a subject in need thereof.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a DPP-4 inhibitor for use in treating a bone fracture in a subject in need thereof, wherein said pharmaceutical composition further comprises a pharmaceutically-acceptable diluent, excipient, or carrier, and wherein the DPP-4 inhibitor is present in an effective amount to treat or prevent bone fractures.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a DPP-4 inhibitor for use in preventing non-union of a bone fracture or in preventing healing complications following bone fracture in a subject in need thereof, wherein said pharmaceutical composition further comprises a pharmaceutically-acceptable diluent, excipient, or carrier, and wherein the DPP-4 inhibitor is present in an effective amount to treat or prevent bone fractures.
  • the bone fracture is a non-union bone fracture, a compound fracture or a fracture with delayed healing.
  • the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, is administered systemically.
  • the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the DPP-4 inhibitor is administered locally.
  • the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the DPP-4 inhibitor is administered to the site of fracture.
  • the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, is administered in one or more doses over a period of less than 6 months.
  • the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, is administered in one or more doses over a period of less than 3 months.
  • the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, is administered in one or more doses over a period of less than 1 month.
  • the present invention relates to the DPP-4 inhibitor for use according to the first, second or third aspect, or to the pharmaceutical composition for use according to the fourth or fifth aspect, wherein, prior to diagnosis of the bone fracture, the subject was not receiving a DPP-4 inhibitor.
  • the present invention relates to the DPP-4 inhibitor for use according to the first, second or third aspect, or to the pharmaceutical composition for use according to the fourth or fifth aspect, wherein the subject in need thereof was not, prior to the bone fracture, diagnosed with or treated for Type II diabetes.
  • the subject in need thereof is over age 65.
  • the subject in need thereof is under age 40.
  • the subject in need thereof has a BMI less than 25.
  • the subject in need thereof has a BMI of greater than or equal to 25 and less than 30.
  • the subject in need thereof has a BMI of equal to or greater than 30.
  • the present invention relates to the DPP-4 inhibitor for use according to the first, second or third aspect, or to the pharmaceutical composition for use according to the fourth or fifth aspect, wherein the DPP-4 inhibitor is administered in one or more doses, and wherein at least one of the doses is administered locally during surgery to set the fracture.
  • DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, wherein the subject in need thereof is a human subject.
  • the healing complications are osteoporosis related healing complications to bone fracture.
  • the DPP-4 inhibitor is selected from one or more of alogliptin, linagliptin, saxagliptin, sitagliptin, vildapliptin, gemigliptin, or teneligliptin.
  • the use further comprises administering metformin, in the same or a different formulation as the DPP-4 inhibitor.
  • the use further comprises administering one or more other therapeutic agent.
  • the present invention relates to a DPP-4 inhibitor for use in reducing the inhibitory effects of marrow adipose tissue (MAT) on fracture healing in a subject in need thereof.
  • MAT marrow adipose tissue
  • the present invention relates to a method of preparing cells for transplantation, comprising
  • OPCs osteogenic progenitor cells
  • mesenchymal stem cells OPCs
  • the present invention relates to a method of preparing cells for transplantation, comprising
  • OPCs osteogenic progenitor cells
  • the present invention relates to DPP-4 treated cells prepared by the method of the seventh or eights aspect for use in transplantation to a fracture in a subject in need thereof.
  • the present invention relates to a DPP-4 inhibitor for use in preventing loss of bone mineral density (BMD) in an astronaut or other individuals exposed to an altered gravity environment.
  • BMD bone mineral density
  • the astronaut is exposed to an altered gravity environment for greater than one week, and the DPP-4 inhibitor is administered to the astronaut prior to and/or during and/or after the exposure.
  • the present invention relates to a DPP-4 inhibitor for use in preventing loss of bone mineral density (BMD) in a subject in need thereof.
  • BMD bone mineral density
  • Figure 1A shows flow cytometric separation of CD45 CD31 " (upper and lower left quadrants in dot plot) cells by Seal -selection.
  • Figure IB shows Oil Red-0 (Adipogenesis), Alizarin Red S (Osteogenesis) and Alician Blue (Chondrogenesis) staining of Scal + and Seal " cells differentiated under corresponding conditions.
  • Figure 1C shows FACS analysis plot of CD45 " CD31 " Scal + cells separated by CD24 expression.
  • Figure ID shows Oil Red-0 (Adipogenesis), Alizarin Red S (Osteogenesis) and Alician Blue (Chondrogenesis) staining of CD45 " CD31 " Scal + CD24 + and CD45 CD31 " Scal + CD24 " cells differentiated under corresponding conditions.
  • Figure 2A shows FACS-analysis of viable cells from 2-month old male -eGFP reporter mice for expression of GFP followed by Seal and CD45/CD31 expression analysis within GFP + cells.
  • Figure 2B shows adipogenic (Oil Red O) and osteogenic (Alizarin Red S) differentiation assays of CD45 " CD31 " Scal + Pa + and CD45 " CD31 " Scal " Pa + populations.
  • Figure 5 shows flow cytometric dot plot analyses of bone resident CD45 " CD31 " Scal " Pa + cells (left panel) and bone resident CD45 " CD31 " Scal " Pa " cells separated into CD24 " and CD24 + cells by FACS.
  • Figure 6 shows FACS-analysis of CD45 CD31 " cells from 2-month old male Zfp423- eGFP reporter mice for expression of Seal and GFP.
  • Figure 9 upper panel shows quantification of osteogenic Scal " Pa+, multipotent Scal + Pa + CD24 + and adipogenic Scal + Pa + CD24 " cells in metaphysis or diaphysis of bones derived from -eGFP mice.
  • Figure 10 shows quantification of bone marrow- localized Pa-GFP+ cells associated to blood vessels with diameters smaller or larger than 10 ⁇ (upper panel).
  • Figure 12A shows transgene alleles of the rep AdlLuc reporter mouse strain:
  • the Zfp423- eGFP reporter mouse strain was crossed to a strain expressing Cre-recombinase under control of the Adiponectin promoter (Adipoq-Cre) and a constitutive Luciferase (Luc)-reporter where the Luc-encoding cDNA is suppressed by a loxP-flanked Stop-signal.
  • Adipoq-Cre Adipoq-Cre
  • Luc constitutive Luciferase
  • FIG. 12B shows transgene alleles of the rep tdTom reporter mouse strain: The Zfp423-eGFV reporter was crossed to an mTmG-reporter mouse strain without presence of a Cre-transgene. Thus, the cells maintained constitutive red fluorescence and can be detected by immunofluorescence for tdTomato or by in vivo imaging.
  • Figure 13 shows the FACS-gating strategy for the isolation of the four investigated cell populations (PreAd, APC, CD45 CD31 Scal + CD24 + , OPC) from both the rep AdiLuc reporter mouse strain and the rep tdTom reporter mouse strain for subsequent in vivo transplantation assays.
  • Figure 14 shows in vivo luciferase imaging (top panels) and macroscopic identification (arrows; middle panels) of transplants 8 weeks after sternal s.c. -injection of the indicated cell populations isolated from repAdiLuc mice. The lower panels show microscopy of corresponding Movat-Pentachrome stains (yellow: mineralized structure; blue: cartilage; purple: nuclei). Scale bars, 30 ⁇ .
  • Figure 15 shows FACS-analysis of transplants of initially CD45 CD31 Scal + CD24 + cells identified by tdTomato-expression giving rise to the CD45 ⁇ CD31 ⁇ Scal + CD24 ⁇ population within the transplant.
  • Figure 16 shows a summary of sternal transplantation experiments of the four investigated cell populations including numbers of transplanted animals and respective differentiation fates as determined by histological analysis and engraftment efficiency.
  • Figure 17 shows a summary of the four investigated cell populations with phenotypic marker expression and differentiation potential performances during in vitro and in vivo experiments.
  • markers that are required to define and isolate the respective populations by flow cytometry are labeled in bold.
  • Figure 19 shows a western blot analysis of UCP1 protein with ⁇ -Actin as a loading control measured in differentiated CD45 " CD31 " Scal + populations isolated from inguinal white adipose tissue (iWAT), brown adipose tissue (BAT), bone or muscle after adipogenic differentiation in the presence of the browning agent rosiglitazone (Rosi).
  • Figure 22 shows hematoxylin and eosin (H&E) stains of femora from 2-month old or
  • Figure 23B shows quantification of multipotent CD45 " CD31 " Scal + CD24 + , adipogenic
  • CD45 CD31 Scal + CD24- (APC), and osteogenic CD45 CD31 Scal-CD24-Pa + (OPC) subpopulations in 2-month old and 25 -month old male mice fed SD (white bars) or high fat diet for 10 days (lOdHFD) (black bars) (n 9). Results are shown as mean ⁇ SEM (****p ⁇ 0.0001).
  • SD standard diet
  • LdHFD 1 day high fat diet
  • Figure 28 shows red fluorescence in tibiae (top panels) and ⁇ CT images (lower panels) of fracture calluses 14 days after fracture and intratibial injection of the indicated cell populations.
  • Figure 29 shows flow cytometric analysis of fracture calluses two weeks after surgery either injected with bone-derived Scal + Pa + cells or Scal " Pa + cells isolated from animals constitutively expressing GFP. Shown are viable cells previously gated for CD45 " CD31 " to show retention of cells after transplantation.
  • Figure 3 IB provides immuno florescence showing the contribution of transplanted multipotent CD45 CD31 Scal + CD24 + (upper panels) and osteogenic CD45 CD31 Scal Pa + (OPC, lower panels) cell populations to osteochondrogenic structures in the fractured tibiae that were not observed in adipogenic cell transplants (Red: tdTomato; Blue: DAPI; right panels indicate merge of immuno florescence and light microscopic images). Scale bar, 20 ⁇ .
  • Figure 31C provides immuno florescence showing the contribution of transplanted multipotent CD45 " CD31 " Scal + CD24 + (upper panels) and osteogenic CD45 CD3 rScal Pa + (OPC, lower panels) cell populations to endosteal bone linings in the fractured tibiae (Red: tdTomato; Blue: DAPI; dotted lines indicate areas of compact bone as seen in right-side panels of merged immunoflorescence and light microscopic images). Scale bar, 10 ⁇ .
  • Figure 3 ID provides immunoflorescence co-staining of tdTomato + cells (red fluorescence) with Osteocalcin to show an osteogenic differentiation fate of transplanted multipotent CD45 " CD31 " Scal + CD24 + (upper panels) osteogenic CD31 " CD45 “ Scal Pa + (lower panels) cell populations. No co-staining detected in adipogenesis-committed populations, e.g. APCs and preAds (not shown). Scale bar, 10 um.
  • Figure 3 IE provides immunoflorescence co-staining of tdTomato+ cells (red fluorescence) with Aggrecan to show a chondrogenic differentiation fate of transplanted multipotent CD45 CD31 Scal + CD24 + (upper panels) osteogenic CD45 CD31 Scal Pa + (lower panels) cell populations. No co-staining detected in adipogenesis-committed populations, e.g. APCs and preAds (not shown). Scale bar, 10 um.
  • Figure 32 shows characterization results of RNA-Seq samples with read counts (left panel) and the fraction of reads mapped to exons (right panel).
  • Figure 33A shows the Principal Component Analysis (PCA) of the RNA-seq samples.
  • Figure 33B shows the correlation scores of top 10 genes driving PCI and PC2 in Figure 33 A.
  • Figure 33C shows hierarchical clustering analyses of RNA-Seq data from all four cell populations.
  • Figure 34 shows a heat map of selected differentially expressed (DE) genes, divided by candidates reported in the literature (known, asterisks indicate no significant DE between individual groups) and novel markers, enriched in CD45 ⁇ CD31 ⁇ Scal + CD24 + cell populations.
  • DE differentially expressed
  • Figure 35 shows a heat map of selected differentially expressed (DE) genes, divided by candidates reported in the literature (known, asterisks indicate no significant DE between individual groups) and novel markers, enriched in OPC cell populations.
  • Figure 36 shows a heat map of selected differentially expressed (DE) genes, divided by candidates reported in the literature (known, asterisks indicate no significant DE between individual groups) and novel markers, enriched in APC cell populations.
  • DE differentially expressed
  • Figure 37 shows a heat map of selected differentially expressed (DE) genes, divided by candidates reported in the literature (known, asterisks indicate no significant DE between individual groups) and novel markers, enriched in preAd cell populations.
  • Figure 38 shows gene expression intensities of Dpp4 from RNA-Seq analysis. Mean ⁇ SEM; *p ⁇ 0.05, **p ⁇ 0.01, ****p ⁇ 0.0001.
  • FIG. 39 shows FACS analysis of DPP4/CD26 surface marker expression in
  • Figure 42A shows mRNA expression (as a percentage of control) of Runx2 and Osterix
  • Figure 43A shows Oil Red-0 staining of CD45 CD31 Scal + CD24 + and APCs either treated with PBS or Sitagliptin (100 ⁇ ) during adipogenic differentiation.
  • Figure 47A shows representative Movat Pentachrome stains of fracture calluses from control PBS-treated mice that received osteogenic (PBS/OPC) or adipogenic (PBS/APC) intratibial transplants and animals treated with Sitaglitpin for 1 week after fracture and receiving the same transplants of osteogenic (Sita/OPC) or adipogenic (Sita/APC) cells.
  • PBS/OPC osteogenic
  • PBS/APC adipogenic
  • Figure 47B shows quantification from histomorphometric analysis of fracture calluses from control PBS-treated mice that received osteogenic (PBS/OPC) or adipogenic (PBS/APC) intratibial transplants and animals treated with Sitaglitpin for 1 week after fracture and receiving the same transplants of osteogenic (Sita/OPC) or adipogenic (Sita/APC) cells.
  • Figure 48 shows FACS analysis of bone-resident MSCs, APCs, and OPCs from animals treated with Sitagliptin (Sita) or control (Ctrl) for 3 days at a dose of 10 mg/kg body weight, either by intraperitoneal injections (i.p.) or by oral gavage (per oral - p.o.). Results are shown as mean ⁇ SEM.
  • the disclosure provides dipeptidyl peptidase-4 (DPP-4) inhibitor agents (which may be referred to herein simply as “agents” or “DPP-4 inhibitors” or “gliptins”) for use in any of the methods or compositions described herein.
  • the agents are for use in treating a subject having a bone fracture.
  • the agent is a DPP-4 inhibitor.
  • the agent is for use in promoting fracture healing in a subject in need thereof (e.g., a subject having a fracture; a subject diagnosed with a fracture).
  • the agent is for use in decreasing or preventing complications following fracture, such as non-union.
  • the agent is for use in reducing inhibitory effects of marrow adipose tissue (MAT) on fracture healing in a subject in need thereof.
  • the agent is for use in preventing loss of bone density.
  • the disclosure provides methods of preparing cells for transplantation into a subject, wherein the cells have been treated with the agent in an amount effective to increase osteogenic gene expression and differentiation, e.g. osteogenic (or chondrogenic) bone cell formation. Such cells are suitable for transplantation into a subject having a bone fracture, such as to promote fracture healing or to prevent or decrease complications from bone fracture. Other uses and methods are described in further detail herein.
  • compositions comprising a DPP-4 inhibitor, and any such compositions may be used in any of the methods described herein.
  • formulations of a DPP-4 inhibitor to be applied systemically or topically/locally in or onto the fracture and any such compositions, such as to promote fracture healing or to prevent or decrease complications from bone fracture.
  • the term “has the ability” or “is capable of is meant that the recited agent, proteins or polypeptides will carry out the stated bioactivity under suitable conditions (e.g., physiological conditions or standard laboratory conditions).
  • suitable conditions e.g., physiological conditions or standard laboratory conditions.
  • the term “can” may be used to describe this ability (e.g., “can bind” or “binds” to a given sequence).
  • inhibitor when used to refer to any of the agents disclosed herein, mean that the agent is capable of blocking, reducing, attenuating and/or reversing activation of the protein targeted by the agent (e.g., DPP-4).
  • a DPP-4 inhibitor or antagonist is an agent that is capable of blocking, reducing, attenuating and or reversing DPP-4 serine exopeptidase activity.
  • the agent for use in any of the methods disclosed herein is a DPP-
  • the DPP-4 inhibitor is a small organic molecule. In some embodiments, the DPP-4 inhibitor is a polypeptide or peptide. In some embodiments, the DPP- 4 inhibitor is an antibody (e.g., an antibody that binds to and inhibits the activity of DPP-4). In some embodiments, the DPP-4 inhibitor is a polynucleotide.
  • the agent is isolated and/or purified.
  • Any of the agents described herein, including those provided in an isolated or purified form, may be provided as a composition, such as a composition comprising an agent formulated with one or more pharmaceutical and/or physiological acceptable carriers and/or excipients.
  • Any of the agents described herein, including compositions may be used in any of the methods described herein. Examples of particular pharmaceutical compositions formulated for preferred routes of delivery are provided herein.
  • the agents inhibit a biological activity of DPP-4. In some embodiments, the agent binds to DPP-4 and inhibits a biological activity of DPP-4. In some embodiments, the agent binds to another protein or agent and indirectly inhibits a biological activity of DPP-4. In some embodiments, the agent binds to a substrate of DPP-4 and prevents DPP-4 from interacting with that substrate. In some embodiments, the agent is an antibody or antigen-binding fragment that binds to DPP-4 or a DPP-4 substrate in a manner that prevents DPP-4 or the substrate from interacting with each other.
  • the agent is capable of binding to a DPP-4 protein, or fragment thereof, having an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 97% or 100% identical to the amino acid sequence of SEQ ID NO: 1 or 3, or a fragment thereof.
  • the agent is capable of binding to the caveolin-1 binding domain, the fibronectin binding domain and/or the ADA binding domain of DPP-4.
  • the agent is capable of binding to the amino acid residues corresponding to residues 201-211 and/or 603 of SEQ ID NO: 1.
  • the agent is capable of binding to residues GWSYG of SEQ ID NO: 1.
  • the agent is capable of binding to residues corresponding to Ser630, Asp708 and/or His740 of SEQ ID NO: 1. In some embodiments, the agent inhibits or prevents homodimerization of DPP-4. In some embodiments, the agent inhibits secretion of DPP-4, such as from cells expressing DPP-4 on their surface in or near a fracture site.
  • the agent inhibits the expression of DPP-4. In some embodiments, the agent inhibits transcription of the DPP4 gene. In some embodiments, the agent inhibits translation of the DPP4 mRNA transcript. In some embodiments, the agent is capable of binding to a polynucleotide having a nucleotide sequence that is at least 80%>, 85%, 90%, 92%, 95%, 97% or 100% identical to the nucleotide sequence of SEQ ID NO: 2 or 4, or a portion or complement thereof.
  • the agent is an antisense molecule, an RNAi molecule, an siRNA or a CRISPR-based therapeutic agent (e.g., a CRISPR/Cas9 complex) that inhibits DPP4 expression.
  • the agent inhibits the expression or activity of DPP-4 in a cell by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to DPP-4 expression or activity in the same or substantially the same cell in the same or substantially the same conditions in the absence of the agent.
  • biological activity By the terms “biological activity”, “bioactivity”, “bioactive” or “functional”, when used in the context of DPP-4, is meant the ability of the DPP-4 polypeptide to carry out one or more functions associated with wildtype DPP-4 polypeptides ⁇ e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 1), for example, serine exopeptidase activity.
  • wildtype DPP-4 polypeptides ⁇ e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 1), for example, serine exopeptidase activity.
  • biological activity Bioactivity
  • bioactivity biological activity
  • functional are used interchangeably herein.
  • any of the DPP-4 inhibitors disclosed herein is capable of inhibiting activity by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the DPP-4 activity as compared to DPP-4 activity under the same or substantially the same physiological conditions in the absence of the agent.
  • the DPP-4 inhibitor preserves the action of DPP-4 substrate molecules, e.g., glucagon- like peptide- 1, gastric inhibitory polypeptide, peptide histidine methionine, substance P, neuropeptide Y, CXCL12, and other molecules typically containing alanine or proline residues in the second aminoterminal position.
  • treatment with DPP-4 inhibitors prolongs the duration of action of DPP-4 peptide substrates and increases levels of their intact, undegraded forms.
  • any of the agents disclosed herein decreases the half-life (tm) of a DPP-4 polypeptide.
  • the half-life of the DPP-4 polypeptide is decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative to the half-life of the DPP-4 polypeptide in the absence of the agent.
  • the protein half- life is determined in vitro, such as in a buffered saline solution or in serum.
  • the protein half-life is an in vivo half life, such as the half-life of the DPP-4 in the serum or other bodily fluid of an animal.
  • a DPP-4 inhibitor is also intended to comprise active metabolites and prodrugs thereof, such as active metabolites and prodrugs of DPP-4 inhibitors.
  • a “metabolite” is an active derivative of a DPP-4 inhibitor produced when the DPP-4 inhibitor is metabolized.
  • a “prodrug” is a compound that is either metabolized to a DPP-4 inhibitor or is metabolized to the same metabolite(s) as a DPP-4 inhibitor.
  • the term "a DPP-4 inhibitor” is also intended to comprise pharmaceutical salts thereof.
  • DPP-4 inhibitors are known in the art.
  • representative DPP- 4 inhibitors are disclosed in WO 98/19998, DE19616 486 Al, WO 00/34241, WO 95/15309, WO 01/72290, WO01/52825, WO03/002553, WO 9310127, WO 99/61431, WO 9925719, WO 9938501, WO 9946272, WO 9967278, WO 9967279, WO 02053548, WO 02067918, WO 02066627, WO 02/068420, W0 02083128, WO 2004/037181, WO 0168603, EP1258480, WO 0181337, WO 02083109, WO 030003250, WO 03035067, WO 03/035057, US2003216450, WO 99/46272, WO 0197808, WO 03002553, WO 01/34594, WO 02051836, EP1245568, EP
  • the agent e.g., the DPP-4 inhibitor
  • the agent is or comprises alogliptin, sitagliptin, vildagliptin, saxagliptin, gemigliptin, anagliptin, teneligliptin, trelagliptin, omarigliptin, evogliptin, dutogliptin and/or linagliptin or derivatives or pharmaceutically acceptable salts thereof.
  • the agent is or comprises diprotin A, berberine and/or lupeol, or a derivative or pharmaceutically acceptable salt thereof.
  • the agent is or comprises any natural plant extract or bioactive compounds which inhibit DPP-4 activity, or a derivative or pharmaceutically acceptable salt thereof. Such agents may be provided as pharmaceutical compositions and, as noted above, as prodrugs.
  • the agent is combined (e.g., in the same or different formulation) with metformin.
  • the methods of the present disclosure include administering a DPP-4 inhibitor and metformin, either as a co-formulation or in separate formulations. When administered as separate formulations, the two may be administered at the same or different times and via the same or different routes of administration.
  • metformin is not used (e.g., the methods of the disclosure do not include administering metformin).
  • any of the methods disclosed herein may include treatment with a single DPP-4 inhibitor or with more than one DPP-4 inhibitor, such as two DPP-4 inhibitors that act via different mechanisms of action.
  • the two agents may be administered at the same or different times and via the same or different routes of administration.
  • the methods further include metformin.
  • any of the agents disclosed herein is conjugated to a heterologous agent.
  • the heterologous agents include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, and an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), which are particularly useful for isolation of the agents by affinity chromatography.
  • the agent is conjugated to a detectable moiety.
  • the moiety is a fluorescently labeled or radiolabeled detectable moiety.
  • Various delivery systems are known and can be used to administer any of the agents of the disclosure, such as any of the DPP-4 inhibitors of the disclosure, e.g., various formulations, encapsulation in liposomes, nanoparticles, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432).
  • Methods of introduction can be enteral or parenteral, including but not limited to, intraosseous, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, pulmonary, intranasal, intrathecal, intraocular, epidural, and oral routes.
  • parenteral introduction includes intramuscular, subcutaneous, intravenous, intravascular, and intrapericardial administration.
  • the agents are administered locally to the subject.
  • the agents are administered systemically to the subject.
  • the agents are administered directly to a bone fracture site, e.g., during a surgical procedure or when setting a compound fracture.
  • the agents are administered as a topical formulation (e.g., as a gel formulation) to the site of the bone fracture.
  • routes of administration are combined.
  • a DPP4-inhibitor can be administered locally, such as during a surgical procedure to set a bone.
  • Subsequent doses of agent over subsequent days, such as over 1-4 weeks, may be by systemic administration (e.g., oral, intravenous, subcutaneous, or intraperitoneal).
  • systemic administration e.g., oral, intravenous, subcutaneous, or intraperitoneal.
  • the same route of administration may be used throughout a multi-dose regimen, such as systemic administration over 1-4 weeks.
  • the agents are administered by means of a device implanted within the subject.
  • the implantable device is coated by a composite surface coating comprising any of the DPP-4 inhibitor agents disclosed herein.
  • the implantable device delivers drug to the site of bone fracture.
  • the implantable device releases DPP-4 inhibitor in a controlled fashion (e.g., a controlled release device).
  • the agents of the disclosure are administered in one or more doses over a period of less than 1 year, less than or equal to 9 months, less than or equal to 6 months, less than or equal to 3 months, less than or equal to 1 month, less than or equal to 3 weeks, less than or equal to 2 weeks, or less than or equal to 1 week.
  • the total treatment period e.g., 1, 3, 6 months, etc.
  • the subject is administered the agent on a dosing schedule.
  • the schedule may be daily, every other day, twice weekly, weekly, twice monthly or monthly.
  • each infusion is part of an overall treatment plan where a composition of the disclosure is administered according to a regular schedule (e.g., weekly, monthly, etc.).
  • the agents of the disclosure are prepared in a formulation/composition appropriate for a specific route of administration.
  • the composition and route of administration is chosen depending on the particular use of the technology. For example, a different composition and/or route of administration may be appropriate when using the compositions of the disclosure for research purposes, such as in vitro or in an animal model, versus when using for diagnostic or therapeutic purposes in human patients.
  • One of skill in the art can select the appropriate route of administration depending on the particular application of the technology.
  • compositions of the disclosure for use in the methods of the present disclosure can be determined by standard clinical techniques and may vary depending on the particular indication or use. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • Dosages may be determined by techniques known to those of skill in the art or as taught herein. Toxicity and therapeutic efficacy of any of the agents disclosed herein may be determined by standard pharmaceutical procedures in experimental animals.
  • agents of the disclosure are formulated with a pharmaceutically acceptable carrier.
  • the disclosure provides a composition comprising an agent of the disclosure formulated with one or more pharmaceutically acceptable carriers and/or excipients.
  • Such pharmaceutical compositions include, where applicable, pharmaceutically acceptable salts of a DPP-4 inhibitor.
  • the disclosure provides for a pharmaceutical composition for use in treating a bone fracture in a subject in need thereof, comprising a DPP-4 inhibitor in admixture with a pharmaceutically-acceptable diluent, excipient, or carrier, wherein the DPP-4 inhibitor is present in an effective amount to treat or prevent bone fractures.
  • the disclosure provides for a pharmaceutical composition for use in preventing non-union of a bone fracture or for use in preventing healing complications following bone fracture in a subject in need thereof, comprising a DPP-4 inhibitor in admixture with a pharmaceutically-acceptable diluent, excipient, or carrier, wherein the DPP-4 inhibitor is present in an effective amount to treat or prevent bone fractures.
  • compositions of the disclosure can be administered alone or as a component of a pharmaceutical formulation (composition).
  • compositions of the disclosure may be formulated for administration in any convenient way for use in human or veterinary medicine.
  • Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • Formulations of the compositions of the disclosure include those suitable for oral, nasal, topical, parenteral, rectal, and/or intravaginal administration.
  • the disclosure provides for a composition for administration directly to a bone fracture, e.g., by a liquid formulation applied directly to the site of bone fracture ⁇ e.g., by a spray).
  • the disclosure provides for a gel-based formulation for direct or topical administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
  • methods of preparing these formulations or compositions include combining the therapeutic agent and a carrier and, optionally, one or more accessory ingredients.
  • the formulations can be prepared with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions suitable for parenteral administration may comprise one or more compositions of the disclosure in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers ⁇ e.g., HEPES buffer), bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers ⁇ e.g., HEPES buffer), bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium
  • the compositions of the disclosure are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • a solubilizing agent such as lidocaine to ease pain at the site of the injection.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • compositions of the disclosure are non-pyrogenic.
  • the compositions are substantially pyrogen free.
  • the formulations of the disclosure are pyrogen- free formulations which are substantially free of endotoxins and/or related pyrogenic substances.
  • Endotoxins include toxins that are confined inside a microorganism and are released only when the microorganisms are broken down or die.
  • Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, even low amounts of endotoxins must be removed from intravenously administered pharmaceutical drug solutions.
  • FDA Food & Drug Administration
  • EU endotoxin units
  • the endotoxin and pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.
  • compositions, methods and uses described herein specifically contemplates any combination of the features of compositions of the present disclosure (alone or in combination) with the features described for the various pharmaceutical compositions and route of administration described in this section.
  • compositions of the disclosure contemplates the use of any of the compositions of the disclosure (whether alone or in combination with any of the additional therapeutic treatments disclosed herein).
  • Compositions of the disclosure may be described based on any combination of structural and/or functional features provided herein.
  • the disclosure contemplates the combination of any step or steps of one method or use with any step or steps from another method or use.
  • These methods and uses involve administering to an individual in need thereof an effective amount of a compound of the disclosure (including as a pharmaceutical composition) appropriate for the particular disease or condition (e.g., a bone fracture).
  • these methods and uses involve delivering any of the agents disclosed herein to the cells of a subject in need thereof.
  • the disclosure provides uses in treating a bone fracture in a subject in need thereof. In some embodiments, the disclosure provides a use in preventing nonunion of a bone fracture in a subject in need thereof. In some embodiments, the disclosure provides a use in preventing healing complications following bone fracture in a subject in need thereof. In some embodiments, the disclosure provides a use in promoting fracture healing in a subject in need thereof. In some embodiments, the disclosure provides a use in reducing the inhibitory effects of marrow adipose tissue (MAT) on fracture healing in a subject in need thereof. In some embodiments, the disclosure provides a use in preventing loss of bone mineral density (BMD) in a subject (e.g. , an astronaut) exposed to an altered gravity environment.
  • BMD bone mineral density
  • any of the methods and uses disclosed herein comprises administering to the subject an effective amount of any of the DPP-4 inhibitors disclosed herein. In some embodiments, any of the methods and uses disclosed herein comprises administering an effective amount of any of the agents disclosed herein (alone or in combination with any of the additional therapeutic treatments disclosed herein), to a subject in need thereof according to a dosing regimen (e.g., a dose and dosing schedule) and/or dosing schedule.
  • a dosing regimen e.g., a dose and dosing schedule
  • a method or use may comprise administering a DPP-4 inhibitor to a subject in need thereof, such as a subject having a fracture, in one or more doses over a period of time (e.g., a single administration or multiple administrations over a period of less than or equal to 6, 3, 2, 1 month).
  • a period of time e.g., a single administration or multiple administrations over a period of less than or equal to 6, 3, 2, 1 month.
  • treatment generally mean obtaining a desired pharmacologic and/or physiologic effect, and may also be used to refer to improving, alleviating, and/or decreasing the severity of one or more symptoms of a condition being treated.
  • the effect may be prophylactic in terms of completely or partially delaying the onset or recurrence of a disease, condition, or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for a disease or condition and/or adverse effect attributable to the disease or condition.
  • Treatment covers any treatment of a disease or condition of a mammal, particularly a human, and includes: (a) preventing complications from a fracture or other bone related disease or condition, such as non-union; (b) preventing further complications of an existing disease or condition (e.g. preventing formation of non-union fractures in osteoporosis patients or preventing healing complications in osteoporosis patients having a fracture); (c) inhibiting the disease or condition (e.g., arresting its development or further progression); (d) relieving the disease or condition (e.g., causing regression of the disease or condition, providing improvement in one or more symptoms); promoting healing of a tissue (e.g. , bone) that was damaged or compromised as a result of the disease or condition (e.g. , a bone fracture) ; or (e) acceleration of the healing process or decrease of processes that inhibit the healing process .
  • a tissue e.g. , bone
  • the disease or condition e.g. , a bone fracture
  • the disclosure provides a use in treating a bone-related disease (e.g. , osteoporosis) or bone-related condition (e.g., a bone fracture) or preventing or decreasing complications associated with a bone fracture with any of the agents described herein (alone or in combination with any of the additional therapeutic treatments disclosed herein).
  • a bone-related disease e.g. , osteoporosis
  • bone-related condition e.g., a bone fracture
  • the subject is a human.
  • the disclosure provides a use in treating a therapeutically induced bone loss or bone-related disease such as those induced by medication (e.g. corticosteroid-induced bone loss) or a medical procedure (e.g. patients who have undergone a hysterectomy) or preventing or decreasing complications associated with a bone fracture with any of the agents described herein (alone or in combination with any of the additional therapeutic treatments disclosed herein).
  • the subject is human. Treating a bone-related disease (e.g., osteoporosis) or bone-related condition (e.g. , a bone fracture) in a subject refers to improving (e.g.
  • a bone -related disease e.g., osteoporosis
  • bone-related condition e.g., osteoporosis
  • a bone fracture) or secondary complications arising after bone injury includes any one or more of: improved or accelerated healing of a bone fracture, reduction in pain associated with a bone fracture (e.g., back pain), preventing height loss, improving posture, reducing frequency of bone fractures, or increasing bone mineral density (BMD) to prevent fractures and to decrease general proneness to bone fractures in subjects (for instance in patients with metabolic diseases such as diabetes and obesity, or in subjects affected by osteoporosis, or in subjects of advanced age alone, or subjects primarily affected by a combination of these disorders and advanced age).
  • BMD bone mineral density
  • the effects of administration of any of the agents disclosed herein may be determined by assessing the treated subject before and after treatment, and determining whether the treatment has any effect on the bone-related disease (e.g., osteoporosis) or bone-related condition (e.g., a bone fracture).
  • the bone-related disease e.g., osteoporosis
  • bone-related condition e.g., a bone fracture
  • Efficacy can be measured, for example, by: the ability of a bone to heal from a fracture; a reduction in pain experienced by the subject due to the bone fracture; assessing the time for a bone to heal from a fracture or the time for pain associated with a bone fracture to be alleviated; an improvement in posture or height loss. This can be assessed by means of instruments such as X-rays and/or bone scans, or by asking the patient about their degree of pain associated with the site of injury.
  • a “therapeutically effective amount” or “effective amount” of a composition is a predetermined amount calculated to achieve the desired result (e.g. , effective in promoting bone fracture healing).
  • the activity contemplated by the present methods and uses includes both medical therapeutic and/or prophylactic treatment, as appropriate.
  • the specific dose of an agent administered according to this invention to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the agent administered, the route of administration, and the condition being treated.
  • a therapeutically effective amount of agent of this invention is typically an amount such that when it is administered in a physiologically tolerable excipient composition, it is sufficient.
  • Therapeutically effective amounts may be administered according to a dosing schedule such that the amount of each dose is effective such that, in the aggregate, the combined doses achieve an end result. Each dose is still considered effective even if it adding to an overall therapeutic effect.
  • a "patient,” “subject” or “individual” are used interchangeably and refer to either a human or non-human animal.
  • the term includes mammals such as humans.
  • Bone-related disease e.g. , osteoporosis
  • bone-related condition e.g. , a bone fracture
  • the mammal is human.
  • the mammal is postnatal.
  • the mammal is pediatric.
  • the subject is at a life stage that is associated with a greater risk of developing a bone-related condition such as osteoporosis.
  • the mammal is adult.
  • the subject is a human that is at least 50 years in age, at least 60 years in age, at least 65 years in age, at least 70 years in age, at least 80 years of age, at least 90 years of age, at least 100 years of age, at least 1 10 years of age or at least 120 years of age.
  • the subject is a human subject under 40 years of age, under 35 years of age, under 30 years of age, or under 20 years of age.
  • the subject is a human subject that is still growing (e.g., whose growth plates have not yet fused).
  • the subject treated with any of the DPP-4 inhibitors disclosed herein is not a diabetic patient.
  • the subject in need thereof has Type I or Type II diabetes.
  • the subject was not, prior to the bone fracture, diagosed with or treated for Type II diabetes.
  • the subject was not receiving a DPP-4 inhibitor prior to the diagnosis with a bone fracture.
  • the subject has not previously received a treatment for diabetes.
  • the subject has been administered metformin. In some embodiments, the subject has not been and/or is not being administered metformin.
  • the subject has a body mass index (BMI) less than 50, less than 45, less than 40, less than 39, less than 38, less than 37, less than 36, less than 35, less than 34, less than 33, less than 32, less than 31 , less than 30, less than 29, less than 28, less than 27, less than 26 or less than 25.
  • BMI body mass index
  • the subject has a BMI of greater than or equal to 25 and less than 30.
  • the subject has a BMI of equal to or greater than 30.
  • the subject has a BMI of 18.5- 24.
  • the subject has a BMI of less than 18.5.
  • the subject to be treated with any of the DPP-4 inhibitors disclosed herein is a subject having increased marrow adipose tissue (MAT) as compared to a healthy control subject.
  • MAT marrow adipose tissue
  • the subject in need thereof has, at the time of treatment of or immediately prior to the fracture, an Ale (hemoglobin Ale) level below 5.7%. In some embodiments, the subject in need thereof has, at the time of treatment of or immediately prior to the fracture, an Ale level of 5.7-6.4%. In some embodiments, the subject in need thereof has, at the time of treatment of or immediately prior to the fracture, an Ale level greater than 6.5%.
  • the subject treated with any of the DPP-4 inhibitors disclosed herein is a subject to be exposed to, currently exposed to, or previously exposed to altered gravity environment. In some embodiments, the subject is exposed to an altered gravity environment for at least 3 days, one week, two weeks, one month, two months, three months, four months, five months, six months, nine months, and/or one year. In some embodiments, the subject is administered the DPP-4 inhibitor prior to and/or during the exposure. In some embodiments, the subject is an astronaut.
  • the agents of the present disclosure may be used to treat a bone- related disease.
  • the bone-related disease is selected from the group consisting of osteoporosis, osteogenesis imperfect, primary bone cancer, cancer that has metastasized to bone, rickets, osteomalacia, renal osteodystrophy, and/or Paget's Disease.
  • the osteoporosis is primary osteoporosis.
  • the primary osteoporosis is idiopathic primary osteoporosis or age-related osteoporosis.
  • the osteoporosis is secondary osteoporosis.
  • the osteoporosis is caused by idiopathic hyper-calcinuria, cystic fibrosis, glucocorticoid treatment, cyclosporine A treatment, and/or tacromilus treatment.
  • the bone related disease occurs in post-meopausal women, patients who have undergone hysterectomy, patients who are undergoing or have undergone long-term administration of corticosteroids, patients suffereing from Cushing's syndrome, patients who display one or more symptoms of the frailty syndrome, patients with cachexia, patients with conditions associated with wasting of body tissues, or patients who have gonadal dysgenesis.
  • the disclosure provides any of the foregoing methods or uses comprising administering any of the agents of the disclosure.
  • the subject is suffering from more than one of the diseases disclosed herein.
  • the subject is suffering from a bone -related condition, such as a fracture of a bone.
  • the subject is suffering from a fracture in more than one bone.
  • the bone is a flat, long, short, irregular or sesamoid bone.
  • the bone is a long bone (e.g., a femur, humerus, tibia, metacarpal, metatarsal and/or phalange).
  • the bone is a short bone (e.g., a carpal or tarsal).
  • the bone is a flat bone (e.g., a scapula, sternum, cranium, os coxae, pelvis and/or rib).
  • the bone is an irregular bone (e.g., vertebrae, sacrum and/or mandible).
  • the bone is a sesamoid bone (e.g., knee cap and/or pisiform).
  • the fracture is a compound fracture.
  • the disclosure provides for a use in treating a bone fracture.
  • the fracture is a non-union fracture, a compound fracture, a fracture with delayed healing, a stable fracture, a displaced fracture, a non-displaced fracture, an open fracture, a closed fracture, a Greenstick fracture, a complete fracture, an incomplete fracture, a transverse fracture, an oblique fracture, a comminuted fracture, a buckled fracture, a pathologic fracture, and/or a stress fracture.
  • the bone fracture is a non-union bone fracture, a compound fracture or a fracture with delayed healing.
  • non-union bone fracture is meant to relate to a permanent failure of healing following a broken bone unless intervention (such as surgery) is performed.
  • a "fracture with delayed healing” is meant to relate to a failure to reach bony union by 6 months post-injury. This also includes fractures that are taking longer than expected to heal (ie. distal radial fractures).
  • the fracture is the result of a disease or condition such as osteoporosis.
  • the fracture is the result of a trauma to the fractured bone.
  • the fracture is the result of overuse of the bone (e.g. , as the result of repetitive motion, such as in an athlete).
  • the disclosure provides for a use in inducing differentiation of a multipotent stem cell or a progenitor cell into a cell of the bone lineage (e.g., into an osteoblast or an osteocyte) in a subject in need thereof. In some embodiments, the disclosure provides for a use in inducing differentiation of a multipotent stem cell or a progenitor cell into a cell of the cartilage lineage (e.g., into a chondrocyte) in a subject in need thereof.
  • the multipotent stem cell or its progeny, the adipogenic progenitor cell is a cell that expresses high levels of genes associated with adipocytic lineage (e.g., CD34, EBF2, VIM, PPARA and/or DPP4).
  • the multipotent stem cell or its progeny, the osteochondrogenic progenitor cell is a cell that expresses high levels of genes associated with osteogenic lineage (e.g., AlpI, Dmpl, Collal/2) or is a cell that expresses high levels of genes associated with chondrogenic lineage (e.g., Acan, Col2al, Sox9).
  • the multipotent stem cell expresses elevated levels of GREM1 or other markers such as Cxcll2, Kitl/Scf, Vcam-1, Lepr.
  • the multipotent stem cell is a CD45 CD31 " Scal + CD24 + multipotent stem cell.
  • the adipogenic progenitor cell (APC) is a CD45 ⁇ CD31 ⁇ Scal + CD24 ⁇ cell.
  • the osteogenic progenitor cell (OPC) is a CD45 ⁇ CD31 ⁇ Scal ⁇ Pa + cell.
  • treatment of the multipotent stem cell with any of the DPP-4 inhibitors disclosed herein enhances osteogenic gene expression.
  • treatment of the multipotent stem cell with any of the DPP-4 inhibitors disclosed herein enhances chondrogenic gene expression.
  • the subject is a mammal. In some embodiments, the subject is a human.
  • the disclosure provides for a method of preparing cells for transplantation into a subject having a bone-related disease ⁇ e.g., osteoporosis) or bone-related condition ⁇ e.g., a bone fracture).
  • the method comprises providing a cell culture comprising osteogenic progenitor cells and/or mesenchymal stem cells, contacting the cells with any of the DPP-4 inhibitors disclosed herein in an amount effective to increase osteogenic or chondrogenic gene expression thereby generating a culture comprising DPP-4 treated cells.
  • the thus generated DPP-4 treated cells from the culture are transplanted to a fracture in a subject in need thereof.
  • the cells for transplantation are multipotent stem cells.
  • the multipotent stem cell is a cell that expresses high levels of genes associated with adipocytic lineage ⁇ e.g., CD34, EBF2, VIM, PPARA and/or DPP 4). In some embodiments, the multipotent stem cell is a CD45-CD31 - Scal+CD24+ multipotent stem cell.
  • the multipotent stem cell or its progeny, the osteochondrogenic progenitor cell is a cell that expresses high levels of genes associated with osteogenic lineage ⁇ e.g., AlpI, Dmpl, Collal/2) or is a cell that expresses high levels of genes associated with chondrogenic lineage ⁇ e.g., Acan, Col2al, Sox9).
  • the OPC is a cell whose surface marker configuration is CD45 " CD31 " Scal " Pa + .
  • treatment of the multipotent stem cell with any of the DPP-4 inhibitors disclosed herein enhances osteogenic gene expression and/or osteogenic lineage commitment of the initially multipotent cell.
  • any of the agents described herein may be administered in combination with any of the additional therapeutic treatments described herein.
  • the additional therapeutic treatment is reduction ⁇ e.g. , closed reduction) and or the use of medical devices (e.g., casts, pins, plates, screws, rods or glue) to hold the fracture in place.
  • the additional therapeutic treatments may include treatment with one or more compounds selected from the group consisting of: an anti-infective agent, a pain and/or inflammation reliever (e.g., acetominophen, ibuprofen), a growth factor (e.g., bone morphogenic proteins (BMPs), TGF-beta, insulin-like growth factor (IGF), fibroblast growth factor (FGF), FGF-2, platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF)), a hormone (e.g., parathyroid hormone (PTH) or growth hormone (GH)) and a soluble receptor (e.g., an ActRIIA receptor).
  • a pain and/or inflammation reliever e.g., acetominophen, ibuprofen
  • a growth factor e.g., bone morphogenic proteins (BMPs), TGF-beta, insulin-like growth factor (IGF), fibroblast growth factor (FGF), FGF-2
  • the BMP is selected from the group consisting of: OP-1 , OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP- 10, BMP-1 1 , BMP- 15, BMP- 16, DPP, Vgl , Vgr-1 , 60A protein, GDF-1 , GDF- 2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-1 1 , GDF-12, NODAL, UNIVIN, SCREW, ADMP, NEURAL, and amino acid sequence variants thereof.
  • the additional therapeutic treatment is a compound for treating osteoporosis, such as a bisphosphonate (e.g., Alendronate (Fosamax), Risedronate (Actonel), Ibandronate (Boniva) and Zoledronic acid (Reclast)), a hormone (e.g., raloxifene (Evista)), a RANKL inhibitor (e.g., Denosumab (Prolia)) and/or a synthetic hormone (e.g., a synthetic parathyroid hormone such as Teriparatide (Forteo)).
  • a bisphosphonate e.g., Alendronate (Fosamax), Risedronate (Actonel), Ibandronate (Boniva) and Zoledronic acid (Reclast)
  • a hormone e.g., raloxifene (Evista)
  • a RANKL inhibitor e.g., Denosumab (Prolia)
  • Administration "in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
  • any of the agents described herein are administered to a subject in combination with an additional therapeutic treatment, wherein the subject has a bone-related disease (e.g., osteoporosis) or bone-related condition (e.g., a bone fracture).
  • the additional therapeutic treatment is a physical therapy, massage therapy, electrical and electromagnetic stimulation, ultrasound, extracorporeal shock waves and/or rest.
  • the combined administration includes coadministration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.
  • the additional therapeutic treatment and the agent are administered consecutively.
  • the additional therapeutic treatment is administered concurrently with the agent.
  • the additional therapeutic treatment is administered prior to the administration of the agent.
  • combined therapy results in a cumulative therapeutic effect.
  • combined therapy results in a synergistic therapeutic effect.
  • the therapeutically effective amount of each of: a) the DPP-4 inhibitor, and/or b) the additional therapeutic treatment is less than that required to achieve a therapeutic effect when one or both agents is administered as a monotherapy.
  • compositions of the disclosure have numerous uses.
  • the DPP-4 inhibitor agents disclosed herein are useful for studying effects on different cells and tissues in vitro and/or in vivo.
  • the agents are useful as imaging agents, such as for ex vivo or in vivo diagnostic applications.
  • agents conjugated to a radioactive moiety are useful for ex vivo or in vivo imaging studies.
  • the disclosure provides for a method of inducing differentiation of a multipotent stem cell into a cell of the bone lineage (e.g., into an osteoblast).
  • the multipotent stem cell is a cell that expresses high levels of genes associated with adipocytic lineage (e.g. , CD34, EBF2, VIM, PPARA and/or DPP4).
  • the multipotent stem cell is a CD45-CD31-Scal+CD24+ multipotent stem cell.
  • treatment of the multipotent stem cell with any of the DPP-4 inhibitors disclosed herein enhances osteogenic gene expression and/or osteogenic cell differentiation and/or osteogenic lineage commitment of the multipotent cell.
  • treatment of the multipotent stem cell with any of the DPP-4 inhibitors disclosed herein enhances chondrogenic gene expression and chondrogenic cell differentiation.
  • the multipotent cell is in vitro.
  • the multipotent cell is in a subject.
  • the subject is a mammal.
  • the subject is a human.
  • the agents of the disclosure are suitable for identifying binding partners and/or tissue distribution for the agents delivered and for evaluating localization and trafficking. Similarly, effects of the agent on gene expression or DPP-4 half- life may be studied in vivo or in vitro.
  • Suitable animal models of bone-related diseases or conditions are known in the art. Suitable animals for studying the effects of any the DPP-4 inhibitors disclosed herein include mice, rats, dogs, and primates. Representative animal models are described in Histing et al, 2011, Bone, 49(4):591-9. An example of a model is also discussed in the Example section below. In this model, anesthetized mice are injected with 1.5 x 10 4 cells in a 50 % matrigel suspension through the proximal articular surface of the tibia. A steel pin (diameter 0.35 mm) is inserted into the medullary cavity for stabilization and a fracture was induced with scissors 0.5 cm distal from the knee. The effects of any of the DPP-4 inhibitors disclosed herein on the healing of the bone-related diseases or conditions may be assessed in these models.
  • CD45 CD31 non-hematopoietic, non-endothelial cells
  • Sca Stem cell antigen
  • Pa Platelet-derived growth factor-a
  • a population of unilaterally committed adipogenic progenitors was isolated that was CD45 CD3 l " Scal + CD24 " and a population that displayed tri- lineage differentiation potentials and was CD45 " CD31 " Scal + CD24 + ( Figures 1C and ID).
  • Colony forming potentials (CFU-F) and in vitro recovery rates were highest in fibroblastic cells expressing Seal and/or Pa and were highest in the CD45 CD3 l " Scal + Pa + CD24 + subset ( Figures 3 and 4).
  • CFU-F potential was enriched in the osteogenic CD45 CD3 rScal " Pa + population but absent in CD45 " CD31 " Scal " Pa " cells.
  • the transcription factor zinc finger protein (Zfp)-423 labels adipogenic cells in white adipose tissue (WAT).
  • WAT white adipose tissue
  • Zfp423-eGFV + (Zfp423 + ) cells occurred as a small subpopulation of less than 1% within the CD45 " CD31 " Sca population while all adipogenic CD45 CD3 l " Scal + cells were GFP " ( Figure 6). This small subset of adipogenic cells was likely not initially detected due to a dilution effect within the strongly osteochondrogenic cell fraction.
  • CD45 CD3 Scal ⁇ Zfp423 + cells maintained GFP-expression before and after differentiation into adipocytes.
  • all CD45 CD3 l " Scal + cells uniformly acquired GFP-expression only during differentiation, a process that correlated with a concomitant loss of Seal -expression ( Figures 7A, 7B, and 8).
  • multipotent cells and osteogenic progenitors were more abundant in the metaphysis compared to the diaphysis whereas adipogenic progenitors were evenly distributed. Further analyses of Pa, Seal and CD24 expression localized osteogenic progenitors to the endosteum. The majority of multipotent and adipogenic progenitors cells resided in a non-endosteal localization within 40 ⁇ of the bone surface and revealed a perivascular association of all non-endosteal Pa + cells to blood vessels of less than 10 ⁇ in diameter ( Figures 9 and 10).
  • a tri-potent, perivascular population with stem cell-like characteristics (CD45 ⁇ CD31 ⁇ Scal + CD24 + ), two functionally and anatomically distinct progenitor populations that are fate-committed towards either the osteochondrogenic (CD45 " CD31 Scal Pa + ; also referred to as OPC) or adipogenic (CD45 CD31 Scal + CD24-; also referred to as APC) lineages, and a more mature CD45 CD3 rScal " Zfp423 + adipocyte precursor stage (also referred to as preAd) cell population.
  • a triple transgenic mouse strain was generated carrying alleles of the Zfp423-eGFF reporter, Adiponectin (Adipoq)-Cre, and a luciferase reporter within the Rosa26-locus that is only expressed after Cre -mediated recombination, e.g. in mature adipocytes (rep AdlLuc , Figure 12 A) and a second strain with constitutive red fluorescence (mTmG reporter allele) crossed to the Zfp423-eGFF reporter (rep tdTom , Figure 12B).
  • RNA-Sequencing was used to further characterize the molecular identity of all four populations ( Figure 32). Principal component and hierarchical clustering analyses clearly supported the distinct nature of each population, providing a second line of evidence for the lineage restriction of adipogenic commitment of the closely related APC and pre Ad populations ( Figure 33 A, Figure 33B, and Figure 33C). Differential expression (DE) analysis produced several sets of known and potential new candidate genes to define each population ( Figures 34-37). For instance, canonical stem cell markers (e.g. Nog, Illrn, Myc) were enriched in the CD45-CD31- Scal+CD24+ multipotent stem cell population ( Figure 34).
  • canonical stem cell markers e.g. Nog, Illrn, Myc
  • signals known to regulate HSC quiescence and maintenance showed highest expression in this population, along with the highest, but not exclusive, expression level of LepR that was also expressed in the other cells types.
  • the OPC population expressed the classical osteogenic (e.g. AlpI, Dmpl, Collal/2) and chondrogenic markers (e.g. Acan, Col2al, Sox9), as well as previously described skeletal stem cell markers (Itga5, CD200) at elevated levels ( Figure 35).
  • the adipogenic populations expressed high levels of markers that have been linked to the adipocytic lineage (i.e.
  • RNA-Seq analysis confirmed the cellular characteristics of the four populations and establishes the CD45-CD31-Scal+CD24+ multipotent stem cell population as a population expressing elevated levels of Cxcll2 and Lepr that are important regulators of HSCs and osteogenesis.
  • Dpp4 Dipeptidyl peptidase-4
  • Figure 38 Consistent with the RNA-Seq data, CD26 (the membrane bound form of DPP4), was enriched on the surface of adipogenic cell populations, and only CD45-CD31-Scal+CD24+ and APCs, but not OPCs, released DPP4 into the medium after adipogenic differentiation ( Figures 39 and 40).
  • Sitagliptin Sita or control (Ctrl) were administered to the animals daily at a dose of 10 mg/kg body weight, either by intraperitoneal injections (i.p.) or by oral gavage (per oral - p.o.). For each condition, 3 animals were used.
  • Flow cytrometric analysis of bone-resident mesenchymal stromal cells (MSCs), APCs, and OPCs was conducted as described below. Per oral administration and intraperitoneal injection of Sitagliptin showed equal effects on the distribution of these cell poulations (Figure 48).
  • mice All procedures were approved by the ethics committee for animal welfare of the State Office of Environment, Health, and Consumer Protection (State of Brandenburg, Germany). Animals were housed in a controlled environment (20 ⁇ 2 °C, 12 hour/12 hour light/dark cycle), maintained on a standard diet (SD) (Ssniff, Soest, Germany), or fed a high fat diet (HFD) (45% energy from fat, D12451, Research Diets, New Brunswick, NJ,USA) for 1 or 10 days. Male mice were used for all experiments at the indicated ages, where applicable.
  • SD standard diet
  • HFD high fat diet
  • mice strains were obtained from The Jackson Laboratory: C57BL/6J, B6(Cg)-Tyr c 2J /J (B6-albino), B6.Cg-Tg(Gt(ROSA)26Sor- EGFP)IlAble/J, B6.129S4-Pdgfra tml l(EGFp)So 7J ( ⁇ -eGFP reporter), B6;FVB-Tg(Zfp423- EGFP)7Brsp/J (Zfp423-eGFP reporter), B6.129(Cg)-Gt ⁇ O&4 2 ⁇ or im ⁇ cra - irfromflto - £G ⁇ "°/J (mTmG-reporter), FVBA29S6(B6)-Gt(ROSA)26So ⁇ Jml(Luc)Kael /J (Rosa26-Luciferase reporter), B6;FVB-Tg(Adipoq-
  • Zfp423-eGFP reporter mice were either intercrossed with mTmG- reporter mice (rep tdTom ), or to AdipoQ-Cre mice and a lox-Stop-lox reporter strain expressing luciferase after Cre-mediated removal of the fioxed Stop-cassette from the Rosa26-locus (rep AdlLuc ).
  • Freshly sorted primary murine cells were used throughout this study and isolated by FACS and cultured as previously described (Schulz et al, 2011, Proc. Natl. Acad. Sci. U. S. A. 108, 143-148 and Steenhuis et al, 2008, Calcif. Tissue Int. 82, 44-56).
  • a complex medium of 60% DMEM low glucose (Invitrogen) and 40% MCDB201 (Sigma) was supplemented with 100 U/ml penicillin and 1,000 U/mL streptomycin (Invitrogen).
  • 2% FBS, l x insulin-transferrin-selenium (ITS) mix, l x linoleic acid conjugated to BSA, 1 nM dexamethasone, and 0.1 mM L-ascorbic acid 2-phosphate (all from Sigma) were added.
  • growth factors were added to the medium: 10 ng/ml epidermal growth factor (PeproTech), 10 ng/ml leukemia inhibitory factor (MerckMillipore), 10 ng/ml platelet-derived growth factor BB (PeproTech), and 5 ng/ml basic fibroblast growth factor (bFGF; Sigma- Aldrich).
  • the bFGF was added daily throughout the culture period except where stated otherwise.
  • For adipogenic differentiation cells were induced for 48 hours after three days of expansion, followed by a differentiation period of 5 days.
  • induction medium growth medium without growth factors
  • human insulin Gibcose IGF
  • indomethacin 1 ⁇ dexamethasone
  • 0.5 ⁇ isobutylmethylxanthine 1 nM 3,3',5- triiodo-L-thyronine (T3) (all from Sigma-Aldrich)
  • T3 3,3',5- triiodo-L-thyronine
  • Oil Red O staining was performed by fixing cells with 4% Histofix for 15 minutes at room temperature.
  • Oil Red O working solution For the preparation of Oil Red O working solution, a 0.5% stock solution in isopropanol was diluted with distilled water at a ratio of 3:2. The working solution was filtered and applied to fixed cells for at least one hour at room temperature. Cells were washed four times with tap water before evaluation. For quantification, Oil Red O was extracted by adding a defined volume of isopropanol and absorbance was read in a micro-plate reader (Synergy HI , BioTek) at 510 nm.
  • osteogenic medium DMEM low glucose (Invitrogen)
  • FBS fetal bovine serum
  • Dexamethasone 0.2 mM L-ascorbic acid 2-phosphate, lOmM ⁇ -glycerophosphate, and 50 ng/ml L-thyroxine
  • Cells were then formalin- fixed and stained with 2% Alizarin Red S (Roth) in distilled water.
  • Wells were washed twice with PBS and once with distilled water. De-staining was conducted to quantitatively determine mineralization by adding a 10% cetylpyridinium chloride solution. Absorbance was measured in a micro-plate reader (Synergy HI, BioTek) at 570 nm.
  • a micromass culture was used for the chondrogenesis assay.
  • a 5 ⁇ droplet of cell suspension (appr. 1.5 x 10 7 cells/ml) was pipetted in the center of a well (48-well plate).
  • warm chondrogenic media (DMEMhigh (Invitrogen)) with 10% FBS, 100 nM Dexamethasone, 1 ⁇ L-ascorbic acid-2- phosphate, lOx ITS mix, and 10 ng/ml Transforming growth factor ⁇ ) was added.
  • Cell media was changed every other day. After 21 days, cells were fixed and stained with 1% Alcian-Blue staining (Sigma) for 30 minutes at room temperature. Cells were rinsed three times with 0.1 M HC1. To neutralize acidity, a washing step with dH20 was conducted before microscopic analysis.
  • DPP4 in vitro experiments cell populations were differentiated with adipogenic or osteogenic assays as described above.
  • Mouse recombinant DPP4 250 ng/ml; R&D Systems
  • the DPP-4 inhibitor Sitagliptin 100 ⁇ ; biomol
  • DPP4 secretion into cell culture media was determined by ELISA (ThermoFisher). Either supernatant of freshly isolated tibia explants maintained in culture media for 24 hours or supernatant from cell populations following 10 days of adipogenic differentiation were used.
  • CFU-F assay was conducted as follows: Freshly isolated cell populations were seeded in expansion media at 500 cells per 6-well plate. Medium was changed every other day. At day 10, cells were fixed and stained with Crystal Violet (Sigma). Colonies consisting of more than 20 cells were counted as CFU. At least 6 independent assays were performed per cell population. For total recovery rate experiments, cell populations were seeded as described for the CFU-F assay. Analysis of fixed and crystal violet stained cell populations was conducted on day 7, 11, and 15 by quantification of total cell invasion area of well-plate surface using ImageJ software.
  • Flow cytometry & cell sorting Flow cytometry and cell sorting were performed on a FACS Aria III cell sorter (BD Biosciences) and analyzed using Flow Jo software (Tree Star). Soft-tissue free bones (tibia/femur) were crushed with bone scissors and incubated for 1 hour in a shaking water bath at 37 °C in 10 ml of 20% FBS/PBS containing 0.5% type-2 collagenase (CellSystems). The suspension was filtered through a 70 ⁇ mesh to remove bone fragments and centrifuged at 1200 rpm for 5 minutes at 4 °C.
  • the pellet was re-suspended in ACK (Ammonium-Chloride -Potassium) lysing buffer to eliminate red blood cells and centrifuged again at 1200 rpm for 5 minutes at 4 °C.
  • the pellet was re-suspended in 100 ⁇ sorting buffer (2% FBS/PBS) and stained with antibodies for at least 30 minutes at 4 °C.
  • the applied FACS antibodies can be found in the Key Resources Table. Living cells were gated for lack of PI (propidium iodide; 1 : 1,000 diluted stock solution: 1 ⁇ g/mL in water) fluorescence and accumulation of Calcein (1 : 1,000 dilution; stock of 1 mg in 215 DMSO).
  • Single-cell clonal assays For the co-culture approach, a feeder layer of CD45 CD31 " Pa + cells was isolated from long bones of 8-week old male C57BL/6J mice and seeded in 100 ⁇ of expansion medium at 750 cells per well of a 96-well plate. On the next day, a single CD45 " CD3 Scal + CD24 + tdTomato + cell freshly isolated from 8-week old male Rosa26-mTmG mice was FACS-sorted into each well. Cells were expanded for 10 days to sub-confiuency with media changes every other day. After 10 days, clonal expansion of a single cell was verified by fluorescence microscopy.
  • Wells containing a readily detectable single colony of tdTomato + cells were trypsinized, washed, and collected in 100 ⁇ sorting medium.
  • Five to ten cells (per condition) of each clone were directly FACS-sorted onto freshly prepared 96-well plate feeder layers of expanded CD45 " CD31 " Pa + cells for adipogenic and osteogenic differentiation protocols, or onto a micromass culture for chondrogenic differentiation.
  • clones were analyzed for their differentiation capacity by immunocytochemistry.
  • a tdTomato positive clone was considered adipogenic if it co-stained with Perilipin, osteogenic if it co-stained with Osteocalcin, and chondrogenic if it co-stained with Aggrecan.
  • a single CD45 CD31 " Scal + CD24 + tdTomato + cell freshly isolated from 8-weeks old male C57BL/6J mice, was FACS-sorted into a well of a 96-well plate without feeder cells. Single cells were expanded for 10 days with media changes every other day. After 10 days, clones giving rise to colonies were re-seeded in a new 96-well plate and expanded until sub-confluency.
  • bone marrow regions of 0.05 mm 2 from bone sections were selected on fluorescence images.
  • fixated cells in well plates were permeabilized with 0.1% Triton X-100 solution and blocked with 3%) BSA in PBS.
  • Antibodies were used as listed in the resources table.
  • nuclear staining specimens were treated with DAPI. Sections and cells were analyzed using a Keyence BZ-9000 (Biorevo) fluorescence microscope (for up to two fluorescences) or a Zeiss confocal laser scanning microscope (LSM) 700 (for three fluorescences).
  • Sternal transplantation Sorted cell populations from luciferase-expressing rep or tdTomato-expressing r ep tdTom mice were subcutaneously injected at 1.5 x 10 4 cells in a 50% matrigel suspension into the sternal area of B6-albino mice. Eight weeks after transplantation, engrafted tissues were excised, fixed, and histologically analyzed. Mice injected with cells from rep Luc animals were additionally subjected to Luciferase imaging with an IVIS imaging system (Perkin Elmer) before sacrifice. To this end, animals were intraperitoneally injected with luciferin (150 mg/kg) and subsequently anesthetized. After 12 to 18 minutes, the animals were imaged. Image analysis was performed with Living Image 4.4 software (Xenogen).
  • mice were i.p. -injected with a single dose of 100 mg BrdU/kg (Sigma Aldrich) diluted in sterile PBS. Mice receiving a SD or HFD for ten days were given BrdU via drinking water at a concentration of 0.5 mg/ml. Drinking water was refreshed every other day.
  • BrdU cell proliferation in vivo assay For 24 hour experiments, mice were i.p. -injected with a single dose of 100 mg BrdU/kg (Sigma Aldrich) diluted in sterile PBS. Mice receiving a SD or HFD for ten days were given BrdU via drinking water at a concentration of 0.5 mg/ml. Drinking water was refreshed every other day.
  • For single-cell immunostaining was approximately 2 x 10 3 cells/mouse of each population of interest were double-sorted on glass cover slips pre-coated with a 5 ⁇ drop of DMEM(low). Coverslips were incubated for 30 minutes, allowing cells to attach.
  • Fracture model Mice were given an analgetic (MediGel, ClearFbO) starting two days prior to surgery. Anesthetized mice were injected with 1.5 x 10 4 cells in a 50% matrigel suspension through the proximal articular surface of the tibia. A steel pin (diameter 0.35 mm) was inserted into the medullary cavity for stabilization and a fracture was induced with scissors 0.5 cm distal from the knee. At the indicated time point after fracture induction, tibiae were harvested for analyses. After removal of the pin from extracted tibiae, ⁇ CT analysis was conducted with LaTheta LCT-200 (Hitachi-Aloka) using manufacturer's pre-defined parameters for isolated bone measurements.
  • LaTheta LCT-200 Hitachi-Aloka
  • tibiae were fixed and decalcified followed by paraffin embedding and sectioning at 3 ⁇ per slice. Samples were stained using SafraninO/Fast green and Movat Pentachrome. ImageJ software was used for computer-assisted histomorphometric analysis of fracture calluses. Six representative sections of each callus were analyzed for bone, fibrous, and cartilaginous tissue areas in a blinded manner.
  • mice received a daily dose of PBS, Diprotin A (5 mg/kg body weight; Sigma) or Sitagliptin (10 mg/kg body weight; biomol) i.p. for 9 consecutive days.
  • PBS diprotin A
  • Sitagliptin 10 mg/kg body weight; biomol
  • RNA extraction, reverse transcription and cDNA pre-amplification, Nextera XT libraries and R A-sequencing of the cell populations was done as previously described.
  • Pa + cells were FACS-sorted from bones of 4 mice (the 3 biological replicates were done on 3 different days), collected in a 1.5 ml Eppendorf tube containing 50 ⁇ RLT Plus Buffer (Qiagen) supplemented with 1% 2-Mercaptoethanol, immediately frozen in dry-ice and kept at -80 °C.
  • RNA-seq data processing and analysis Sequencing data were aligned to the Mus musculus genome (Ensembl version 38.82) using GSNAP (version 2014-10-07) with default parameters. HTseq-count was used to count the number of reads mapped to each gene (default options). Almost all libraries showed good quality, with sizes ranging between 2-3.5xl0 7 read counts and a fraction of reads mapped to exons greater than 75%. One library yielded less than 300 reads and was excluded from downstream analysis. The data was normalized for sequencing depth using size factors. The union of the top 1,000 genes expressed in each library was selected, which resulted in a list of 2,120 genes.
  • RNA-seq data was statistically analyzed using the R-statistical package and Paleontological Statistics (PAST, version 3.10, http://folk.uio.no/ohammer/past/, accessed December 2015).
  • PAST paleontological Statistics
  • gene expression was compared between all investigated cell populations.
  • a p-value of ⁇ 0.05 was used as a cut-off for differentially expressed genes.
  • Heat-maps contain representative top-regulated genes, which were further divided by known cell type specific functions as previously described in the literature and unknown novel marker genes.
  • RNA isolation and gene expression analysis were conducted using standard methods as described before (Schulz et al, 2011, Proc. Natl. Acad. Sci. U. S. A. 108, 143-148) using column-based RNA-isolation, reserve transcription for cDNA synthesis, and SYBR green-based detection during quantified real-time PCR. Primer sequences were used as noted in the Key Resources Table.
  • RNA-seq data generated in this study was deposited at the European Nucleotide Archive (http://www.ebi.ac.uk/ena) under secondary sample accession number (ID code) ERP013883.
  • the invention relates to a method of treating a bone fracture in a subject in need thereof, comprising administering an effective amount of a DPP-4 inhibitor to the subject.
  • the invention relates to a menthod of preventing non-union of a bone fracture or of preventing healing complications following bone fracture in a subject in need thereof, comprising administering an effective amount of a DPP-4 inhibitor to the subject.
  • the invention relates to a method of promoting fracture healing in a subject in need thereof, comprising administering an effective amount of a DPP-4 inhibitor to the subject.
  • the bone fracture is a non-union bone fracture, a compound fracture or a delayed fracture healing.
  • the DPP-4 inhibitor is administered systemically. In a further embodiment, the DPP-4 inhibitor is administered locally. In a further embodiment, the DPP-4 inhibitor is administered to the site of fracture. In a further embodiment the DPP-4 inhibitor is administered in one or more doses over a period of less than 6 months. In a further embodiment, the DPP-4 inhibitor is administered in one or more doses over a period of less than 3 months. In a further embodiment, the DPP-4 inhibitor is administered in one or more doses over a period of less than 1 month.
  • the subject prior to diagnosis of the bone fracture, was not receiving a DPP-4 inhibitor.
  • the subject in need thereof was not, prior to the bone fracture, diagnosed with or treated for Type II diabetes.
  • the subject in need thereof is over age 65.
  • the subject in need thereof is under age 40.
  • the subject in need thereof has a BMI less than 25.
  • the subject in need thereof has a BMI of greater than or equal to 25 and less than 30.
  • the subject in need thereof has a BMI of equal to or greater than 30.
  • the DPP-4 inhibitor is administered in one or more doses, and wherein at least one of the doses is administered locally during surgery to set the fracture.
  • the subject in need thereof is a human subject.
  • the healing complications are osteoporosis related healing complications to bone fracture.
  • the DPP-4 inhibitor is selected from one or more of alogliptin, linagliptin, saxagliptin, sitagliptin, vildapliptin, gemigliptin, or teneligliptin.
  • the method further comprises administering metformin, in the same or a different formulation as the DPP-4 inhibitor.
  • the method further comprises administering one or more other therapeutic agent.
  • the invention relates to a method for reducing the inhibitory effects of marrow adipose tissue (MAT) on fracture healing in a subject in need thereof, comprising administering an effective amount of a DPP-4 inhibitor to the subject.
  • MAT marrow adipose tissue
  • the invention in a further aspect relates to a method of preparing cells for transplantation, comprising providing a cell culture comprising osteogenic progenitor cells (OPCs) and/or mesenchymal stem cells, contacting the cells with a DPP-4 inhibitor in an amount effective to increase osteogenic gene expression, or osteo-/ chondrogenic cell differentiation, or osteogenic lineage commitment, thereby generating a culture comprising DPP-4 treated cells, and transplanting cells from the culture to a fracture in a subject in need thereof.
  • OPCs osteogenic progenitor cells
  • DPP-4 inhibitor in an amount effective to increase osteogenic gene expression, or osteo-/ chondrogenic cell differentiation, or osteogenic lineage commitment
  • the invention in a further aspect relates to a method of preparing cells for transplantation, comprising providing a cell culture comprising CD45 " CD31 " Scal + CD24 + multipotent cells, treating the cell culture to promote lineage commitment and/or differentiation into osteogenic progenitor cells (OPCs) and/or mature cells of the osteogenic lineage/bone tissues, sorting the cells to isolate or enrich for OPCs, and transplanting the OPCs to a fracture in a subject in need thereof.
  • OPCs osteogenic progenitor cells
  • the invention in a further aspect relates to a method for preventing lose of bone mineral density (BMD) in an astronaut or other individuals exposed to an altered gravity environment, said method comprising administering to the astronaut or individual an effective amount of a DPP-4 inhibitor.
  • BMD bone mineral density
  • the astronaut is exposed to an altered gravity environment for greater than one week, and the DPP-4 inhibitor is administered prior to and/or during and/or after the exposure.
  • the invention relates to a method of preventing loss of bone mineral density (BMD) in a subject in need thereof, comprising administering to the subject an effective amount of a DPP-4 inhibitor.
  • BMD bone mineral density

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Abstract

La présente invention concerne des inhibiteurs de DPP-4 à utiliser pour traiter une fracture osseuse, pour prévenir l'absence de consolidation d'une fracture osseuse ou pour prévenir des complications de cicatrisation à la suite d'une fracture osseuse, pour réduire des effets inhibiteurs de tissus adipeux de moelle (MAT) sur la cicatrisation d'une fracture, ou pour prévenir la perte de densité minérale osseuse (BMD) chez un patient nécessitant un tel traitement. L'invention concerne également des méthodes de préparation de cellules en vue d'une greffe qui consistent à fournir une culture cellulaire comprenant des cellules progénitrices ostéogéniques (OPC) et/ou des cellules souches mésenchymateuses, et à mettre en contact les cellules avec un inhibiteur de DPP-4. L'invention concerne en outre des méthodes de préparation de cellules en vue d'une greffe qui consistent à fournir une culture cellulaire comprenant des cellules multipotentes CD45-CD31- Sca1+ CD24+, et à traiter la culture cellulaire pour favoriser l'implication et/ou la différenciation de lignée en cellules progénitrices ostéogéniques (OPC) et/ou en cellules matures des tissus osseux/de lignée ostéogéniques.
PCT/EP2018/055931 2017-03-09 2018-03-09 Inhibiteurs de dpp-4 à utiliser dans le traitement de fractures osseuses WO2018162722A1 (fr)

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