Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • 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
• • 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
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/10—Drugs for disorders of the endocrine system of the posterior pituitary hormones, e.g. oxytocin, ADH
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

• the present invention concerns peptides, pharmaceutical formulations containing the same, and methods of use thereof in controlling weight, treating bone disorders and improving bone marrow reconstitution.
• IGFBP-2 Insulin-like growth factor binding protein-2
• IGFBP-2 is a 36,000 dalton protein that is a member of the IGFBP family.
• IGFBP-2 There are six forms of high affinity IGF binding proteins. In addition to binding the insulin-like growth factors I and II and acting as transport proteins, these proteins have been shown to have some actions that are independent of their ability to bind to IGFs.
• IGFBP-2 is the second most abundant binding protein in serum. It circulates in concentrations in humans that vary between 100-600 ng/ml. Protein concentrations are high during fetal life and at birth and fall progressively during childhood and adolescence. There is a slight rise, an approximately 25% increase that occurs between 60-80 years. Serum concentrations of IGFBP-2 are regulated by hormones and nutrients.
• a first aspect of the invention is a compound comprising, consisting of or consisting essentially of the IGFBP-2 fragment KHHLGLEEPKKLR, KHHLGLEEPKK, HLGLEEPKKLR, analogs thereof, prodrug of any thereof, or pharmaceutically acceptable salts of any thereof (all hereinafter sometimes referred to as "active compounds” or “active agents”).
• a second aspect of the invention is a composition comprising an active compound as described herein, which can be in combination with a pharmaceutically acceptable carrier, and optionally including at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. etc.) additional active agent such as a bone resorption inhibitor or a weight control agent.
• a further aspect of the invention is a method for inducing deposition and maturation of bone in a subject having a compromised bone condition, comprising administering the subject an active compound in an amount effective to induce deposition and maturation of bone in the subject.
• the method may further comprise concurrently administering a bone resorption inhibitor to the subject in an effective amount.
• An additional aspect of the invention is a method for enhancing bone formation and inhibiting bone resorption simultaneously in a subject in need thereof, comprising administering to the subject an active compound in an amount effective to enhance bone formation and inhibit bone resorption simultaneously in said subject.
• a further aspect of the invention is a method for controlling body weight in a subject in need thereof, comprising administering the subject an active compound as described herein in an amount effective to control body weight.
• the method may further comprise concurrently administering the subject an additional weight control agent.
• Another aspect of the invention is a method for improving bone marrow reconstitution in a subject in need thereof, comprising administering to said subject an active compound of this invention in an amount effective to improve bone marrow reconstitution.
• a further aspect of the invention is the use of an active compound as described herein for carrying out a method as described herein, and/or for the preparation of a medicament for carrying out a method as described herein.
• FIG. 1 The heparin-binding domain of IGFBP2 enhances osteoblastogenesis in vitro, a. Von Kossa staining was performed using tibia from 8 weeks old Igfbp2 + + and Ig ⁇ p2 ⁇ ' male mouse, b. Calvarial osteoblasts (COBs) were harvested from day 7 old male Igfbp2 + + and Igfbp2 ⁇ ' male mouse, and treated with osteogenic media in the presence of 2.5% FCS. At day 7, alkaline phosphatase (ALP) staining was performed, c and d.
• ALP alkaline phosphatase
• BMSCs Bone marrow stromal cells
• PEG-HBD pegylated heparin-binding domain peptide
• FIG. 1 The heparin binding domain of IGFBP2 enhances periosteal expansion of metacarpals ex vivo.
• Metacarpals were collected from day 1 male mouse, and treated with osteogenic media with IGFBP2 (200 ng/ml) or the heparin-binding domain peptide (HBD- peptide) (2 ⁇ g/ml) in the presence or absence of IGF-I (20 ng/ml) for 10 days. Bones were labeled with calcein (500 ng/ml) and calcein incorporated area was visualized (a) and longitudinal length of calcein incorporated area was quantified (b).
• FIG. 3 The heparin binding domain of IGFBP5 does not show any effect on in vitro osteoblastogenesis or periosteal expansion of metacarpals ex vivo.
• a Primary calvarial osteoblasts were collected from male Ig ⁇ p2 ⁇ ⁇ male mouse and treated with osteogenic media with or without the heparin-binding domain of IGFBP-5 (HBD-BP5) (2 ⁇ g/ml). Osteoblastogenesis was evaluated by Alizarin Red staining, b and c. Metacarpals were collected from day 1 male mouse, and treated with osteogenic media with or without HBD-BP5 (2 ⁇ g/ml) for 10 days.
• FIG. 4 The heparin-binding domain of IGFBP2 partially rescues osteopenic phenotype of Igfbp2 " mice.
• Ig ⁇ p2 " mice were treated with PBS or pegylated heparin- binding domain (PEG-HBD) from 6 to 9 weeks of age.
• a. MicroCT image of distal femur
• b. Parameters by three-point bending study were shown
• d Osteoblast number/bone perimeter
• ObS/BS osteoblast surface/bone surface
• ES/BS erosion surface/bone surface
• g and h. Igfbp2 ⁇ l ⁇ COBs were treated with or without PEG-HBD for 2 hours (h).
• I ⁇ p2 ⁇ ' ⁇ BMSCs were cultured with osteogenic media with PBS or PEG-HBD for 2 weeks (i).
• FIG. 5 The heparin-binding domain of IGFBP2 suppresses PTEN expression
• a and b, Igft>p2 ⁇ ' calvarial osteoblasts (COBs) were isolated, and treated with PBS, IGFBP-2 or pegylated heparin-binding domain peptide (PEG-HBD) in MEM ⁇ containing 0.1% BSA overnight.
• PEG-HBD pegylated heparin-binding domain peptide
• the heparin-binding domain of IGFBP-2 stimulates cytosolic accumulation and Ser552 phosphorylation of ⁇ -catenin by IGF-I.
• a MC3T3-E1 cells were serum-starved over-night and treated with IGF-I at the indicated concentration for 6 hours.
• Expression of ⁇ -catenin and ⁇ -actin was analyzed by Western blot analysis with membrane and cytosolic fraction
• b, Igfbp2 ⁇ / ⁇ calvarial osteoblasts (COBs) were serum- starved over-night with PBS or pegylated heparin-binding domain peptide (PEG-HBD) and then treated with IGF-I (100 ng/ml) for 6 hours.
• PEG-HBD pegylated heparin-binding domain peptide
• c MC3T3-E1 cells were serum-starved over-night and treated with IGF-I (100 ng/ml) for 15 min.
• Expression of pSer552- ⁇ -catenin, ⁇ -catenin, pSer473-Akt, Akt and ⁇ -actin was analyzed by Western blot analysis with whole cell lysates.
• Igfbp2 ⁇ l ⁇ COBs were serum-starved overnight with PBS or PEG-HBD and then treated with IGF-I (100 ng/ml) for 15 min.
• Control peptide for heparin-binding domain peptide does not have any anabolic effects on in vitro osteoblastogenesis.
• Igfbp2 ⁇ / ⁇ calvarial osteoblasts COBs were cultured with osteogenic media with PBS, control peptide (2 ⁇ g/ml) or heparin binding domain peptide (HBD-peptide) (2 ⁇ g/ml). Osteoblastogenesis was evaluated by Alizarin Red staining.
• Control peptide for heparin binding domain peptide does not have any anabolic effects on metacarpals ex vivo.
• Metacarpals were collected from day 1 male Ig ⁇ p2 + + mouse, and cultured in DMEM (1 g/L of glucose) containing ImM ⁇ - glycerophosphate and 50 ⁇ g/ml ascorbic acid. Either PBS or control peptide (2 ⁇ g/ml) was added to the media.
• bones were treated with 500 ng/ml of calcein for two hours. Calcein incorporated area was visualized (b) and longitudinal length of incorporated area was quantified (c).
• Control peptide for heparin-binding domain peptide does not suppress PTEN expression in Ig ⁇ p2 +/+ calvarial osteoblasts.
• Ig ⁇ p2 +/+ calvarial osteoblasts were treated with PBS, control peptide, or heparin-binding domain peptide (HBD-peptide) overnight, and whole cell lysate was collected.
• Statistical significance was analyzed by ANOVA followed by Bonferroni post-hoc test. a ; p ⁇ 0.01 vs. PBS, b ; p ⁇ 0.01 vs. control peptide.
• IGFBP-2 increased cell proliferation of osteoblasts and bone marrow stromal cells as compared to full length (FL) IGFBP-2 (BP2) and control peptide and as seen by increased alkaline phosphatase and alizarin red staining, respectively.
• FIG 11. The HBD of IGBP-2 rescued the low bone mass phenotype of Igfbp2 ⁇ / ⁇ mice in vivo, as shown by ⁇ CT analysis.
• Figure 12. HBD reversed the increased lymphoid and myeloid hematopoietic phenotype of Ig ⁇ p2 ⁇ / ⁇ mice.
• the present invention is based on the discovery that peptides of IGFBP-2, (e.g., the heparin binding domain (HBD)) can be employed in methods of treating bone disorders, methods of weight control and methods of improving bone marrow reconstitution.
• peptides of IGFBP-2 e.g., the heparin binding domain (HBD)
• HBD heparin binding domain
• the present invention provides a method for enhancing bone formation (i.e., increasing the amount of new bone that is laid down) and inhibiting bone resorption (i.e., reducing the amount of bone that is dissolved) simultaneously in a subject in need thereof, comprising administering to the subject an active compound of this invention in an amount effective to enhance bone formation and inhibit bone resorption simultaneously in said subject.
• Nonlimiting examples of subjects for whom such treatment would be indicated and/or beneficial include women (e.g., postmenopausal; premenopausal) with osteoporosis or low bone mass, men with osteoporosis or low bone mass, subjects with a healing fracture, subjects undergoing prolonged immobilization, subjects who have been or are immobilized for a prolonged period, subjects likely to undergo or experience prolonged immobilization, subjects with estrogen deficiency, etc., as would be known in the art.
• a method for inducing deposition and maturation of bone in a subject in need thereof comprising administering to the subject an active compound of this invention in an amount effective to induce deposition and maturation of bone in the subject.
• the methods of this invention may further comprise concurrently administering a bone resorption inhibitor to the subject in an effective amount.
• a compromised bone condition is at a targeted site of the subject.
• the site may be an intervertebral space, a facet joint, a site of a bone fracture, bones of the mouth, chin and jaw, or an implant site.
• a subject undergoing chemotherapy with or without radiation would benefit from more rapid restoration of cells in the bone marrow in order to prevent opportunistic infections.
• a subject of these methods can also be a subject having or suspected of having a hematologic disorder (e.g., aplastic anemia; myelodysplasia) that depletes bone marrow cells.
• a hematologic disorder e.g., aplastic anemia; myelodysplasia
• Such an improvement or enhancement or increase in bone marrow reconstitution is in comparison to a subject to whom the active compound has not been administered.
• the methods of this invention can be employed in methods of ex vivo expansion of stem cells, carried out according to protocols known in the art.
• the present invention provides a method of expanding stem cells ex vivo, comprising contacting the compound of this invention with stem cells from a subject, wherein said stem cells are maintained under conditions whereby they are reintroduced into the subject.
• the stem cells are obtained from a subject, e.g., a human, e.g., from peripheral blood, umbilical cord blood, or bone marrow, and the stem cells are contacted with the compound of this invention outside the body of the subject.
• Ex vivo embodiments include obtaining stem cells from a subject and culturing the cells for a period of time prior to use (e.g. , for transplantation).
• the cells are delivered to a subject, e.g., the same subject from which the cells were isolated (autologous donation) or a different subject (non-autologous (e.g., syngeneic or allogeneic) donation).
• Nonlimiting examples of a subject for whom these methods would be indicated or beneficial include a subject having or who has had chemotherapy, a subject having or who has had radiation, a subject having aplastic anemia, a subject having myelodysplasia, and any combination thereof.
• Administration of the compound or composition of this invention may be by any suitable route, including intrathecal injection, subcutaneous, cutaneous, oral, intravenous, intraperitoneal, intramuscular injection, in an implant, in a matrix, in a gel, or any combination thereof.
• a bone condition that can be treated according methods of this invention may be one or more of broken bones, bone defects, bone transplant, bone grafts, bone cancer, joint replacements, joint repair, fusion, facet repair, bone degeneration, dental implants and repair, bone marrow deficits and other conditions associated with bone and boney tissue.
• Bone defects may be a gap, deformation and/or a nonunion fracture in a bone.
• Bone degeneration may be due to osteopenia or osteoporosis (e.g. the patient is afflicted with geriatric or senile osteoporosis, with post-menopausal osteoporosis, etc.), or due to dwarfism.
• Joint replacements that may be treated include vertebral, knee, hip, tarsal, phalangeal, elbow, ankle and/or other articulating joints or replacements thereof.
• Joint repairs include, but are not limited to, vertebral, knee, hip, tarsal, phalangeal, elbow, ankle, and sacroiliac joint repairs.
• a further aspect of the invention is a method for controlling body weight in a subject in need thereof, comprising administering the subject an active compound as described herein in an amount effective to control body weight.
• the method may further comprise concurrently administering the subject an additional weight control agent.
• the subject is obese.
• the compound is administered in an amount effective to reduce weight gain or induce weight loss (or particularly fat loss) in the subject.
• the compound is administered in an amount effective to reduce the body mass index of the subject.
• the subject has a body mass index of 25 or 30 kg/m 2 or above.
• the administering is continued for a period of at least 16 or 24 weeks, and/or until the individual has achieved at least 5% weight loss or the individual's body mass index is reduced to less than 25 kg/m 2 .
• the active compound is administered in an amount effective to inhibit fat cell differentiation (e.g., inhibiting fat cell precursor differentiation into mature adipocytes) in the subject.
• Some embodiments further comprise concurrently administering the subject an additional weight control agent.
• the therapeutically effective dosage of any specific active compound of this invention will vary from compound to compound, and patient to patient, and will depend, among other things, upon the effect or result to be achieved, the condition of the patient and the route of delivery. In some embodiments, a dosage from about 0.001 (i.e., 1 ug/kg), 0.05, 0.1, 0,2, 0.3.
• Subjects as used herein are generally human subjects and includes, but is not limited to, post-menopausal subjects.
• the subjects may be male or female and may be of any race or ethnicity, including, but not limited to, Caucasian, African-American, African, Asian, Hispanic, Indian, etc.
• the subjects may be of any age, including newborn, neonate, infant, child, adolescent, adult, and geriatric.
• the subjects are postmenopausal female subjects, or subjects afflicted with senile osteoporosis.
• Subjects may also include animal subjects, particularly mammalian subjects such as canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g.
• Treating refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the disease, etc.
• “Pharmaceutically acceptable” as used herein means that the compound or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.
• Concurrently administering or “concurrently administer” as used herein means that the two or more compounds or compositions are administered closely enough in time to produce a combined effect (that is, concurrently may be simultaneously, or it may be two or more events occurring within a short time period before or after each other, e.g., sequentially). Simultaneous concurrent administration may be carried out by mixing the compounds prior to administration, or by administering the compounds at the same point in time but at different anatomic sites and/or by using different routes of administration.
• “Bone resorption inhibitor” as used herein may be any suitable bone resorption inhibitor, including but not limited to a bisphosphonate, a selective estrogen receptor modulator, calcitonin, a vitamin D analog, and a calcium salt.
• Weight control agent includes any additional weight control active agent, including but not limited to appetite suppressants such as sibutramine, phentermine, diethylpropion, phendimetrazine, etc.; lipase inhibitors such as orilstat; antidepressants such as bupropion; anti-seizure agents such as topiramate, zonisamide, and metformin, etc.
• appetite suppressants such as sibutramine, phentermine, diethylpropion, phendimetrazine, etc.
• lipase inhibitors such as orilstat
• antidepressants such as bupropion
• anti-seizure agents such as topiramate, zonisamide, and metformin, etc.
• Capping group includes, but is not limited to, acetyl, benzoyl, formyl, trifluoroacetyl, benzyloxycarbonyl, tert-butyloxycarbonyl, biphenylylisopropyloxycarbonyl, triphenylmethyl, o-nitrobenzenesulfenyl, and diphenylphosphinyl.
• the capping groups may consist of such groups as R 10 CO-, R 10 -O-CO-, R 10 -PO-, R 10 -SO 2 - and arylalkyl-; where R 10 is selected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl, and arylalkyl.
• Linking group or “linker” as used herein includes non-amino acid linking groups such as are known in the art (see, e.g., US Patent Nos. 7,468,418; 7402,652; and 7,351,797) or variations thereof that will be apparent to those skilled in the art.
• Alkyl refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms.
• Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n- heptyl, n-octyl, n-nonyl, n-decyl, and the like.
• Lower alkyl as used herein, is a subset of alkyl and refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms.
• Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, and the like.
• Alkenyl refers to a straight or branched chain hydrocarbon containing from 2 to 10 carbons and containing at least one carbon-carbon double bond formed by the removal of two hydrogens.
• alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5- hexenyl, 2-heptenyl, 2-methyl-l-heptenyl, 3-decenyl and the like.
• “Lower alkenyl” as used herein, is a subset of alkenyl and refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms.
• Alkynyl refers to a straight or branched chain hydrocarbon group containing from 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond.
• Representative examples of alkynyl include, but are not limited, to acetylenyl, 1-propynyl, 2- propynyl, 3-butynyl, 2-pentynyl, 1-butynyl and the like.
• “Lower alkynyl” as used herein, is a subset of alkyl and refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms.
• alkyl, alkenyl, and alkynyl groups of the invention can be substituted or unsubstituted and are either unless otherwise specified.
• alkenyl or alkynyl groups of the invention can be substituted with 1, 2, 3, 4, or 5 or more substituents independently selected from alkenyl, alkenyloxy, alkoxy, alkoxyalkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy, alkylsulfmyl, alkylsulfonyl, alkylthio, alkynyl, aryl, azido, arylalkoxy, arylalkyl, aryloxy, carboxy, cyano, formyl, halogen, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, mercapto, nitro, sulfamyl, sulfo, sulfonate,
• Aryl refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused ring system having one or more aromatic rings.
• Representative examples of aryl include, azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like.
• aryl groups of this invention can be substituted with 1, 2, 3, 4, or 5 or more substituents independently selected from alkenyl, alkenyloxy, alkoxy, alkoxyalkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy, alkylsulfmyl, alkylsulfonyl, alkylthio, alkynyl, aryl, azido, arylalkoxy, arylalkyl, aryloxy, carboxy, cyano, formyl, halogen, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, mercapto, nitro, sulfamyl, sulfo, sulfonate,
• Arylalkyl refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkenyl group, as defined herein.
• Representative examples of arylalkenyl include, but are not limited to, 2-phenylethenyl, 3-phenylpropen-2- yl, 2-naphth-2-ylethenyl, and the like, which may be substituted or unsubstituted as noted above.
• Polyalkylene oxides as used herein are known (see, e.g., US Patent No, 7,462,687) and include poly(ethylene glycol) or "PEG". Additional examples may contain hetero atoms such as S or N, and are typically linear polyalkylene oxides such as: 0-(CH 2 CH 2 O) x -, -O-
• “Analog” as used herein is a peptide that has the physiological activity of the parent compound thereof, and that includes one or more (e.g., two, three, four, five or six or more) amino acids different from the amino acid sequence of a naturally occurring parent peptide. Such an analog preferably has at least about 70% of the physiological activity of the parent peptide. Such different amino acids may be additions, substitutions, deletions, or combinations thereof, including addition of non-natural side-chain groups and backbone links. Modifications of peptides to produce analogs thereof are known. See, e.g., US Patent
• Active compounds of the present invention are, in general, compounds (from amino or N terminus to carboxy or C terminus) of Formula I:
• R 1 is present or absent and when present is a positively charged (at pH 6.0) amino acid, which can be an amino acid selected from the group consisting of K, R and H;
• R 2 is present or absent and when present is a positively charged (at pH 6.0) amino acid, which can be an amino acid selected from the group consisting of K, R and H;
• R is any amino acid and in some embodiments is a positively charged (at pH 6.0) amino acid, which can be an amino acid selected from the group consisting of K, R and H;
• R 4 is any amino acid and in some embodiments is an amino acid with a nonpolar side chain, which can be an amino acid selected from the group consisting of L, I or V;
• R 5 is any amino acid and in some embodiments is G;
• R is any amino acid and in some embodiments is an amino acid with a nonpolar side chain, which can be an amino acid selected from the group consisting of L, I and V;
• R 7 is any amino acid and in some embodiments is an amino acid selected from the group consisting of E and N;
• R 8 is any amino acid and in some embodiments is an amino acid selected from the group consisting of E and N;
• R 9 is any amino acid and in some embodiments is P;
• R is a positively charged (at pH 6.0) amino acid, which can be an amino acid selected from the group consisting of K, R and H;
• R 11 is a positively charged (at pH 6.0) amino acid, which can be an amino acid selected from the group consisting of K, R and H;
• R 12 is present or absent and when present is any amino acid and in some embodiments is an amino acid with a nonpolar side chain, which can be an amino acid selected from the group consisting of L, I or V;
• R 13 is present or absent and when present is a positively charged (at pH 6.0) amino acid, which can be an amino acid selected from the group consisting of K, R and H;
• Y' is present or absent and when present is a linking group
• X' is present or absent and when present is a capping group, a polyalkylene oxide (e.g., PEG), or a peptide consisting of from 2 to 6 or 10 additional amino acids, which peptide is optionally terminated by a capping group or polyalkylene oxide.
• a pharmaceutically acceptable salt of the compound of Formula I is also provided herein.
• Amino acids in peptides of the present invention may be in the D or L configuration: e.g., all D; all L; some D and some L in any combination.
• X is preferably a polyalkylene oxide, or a peptide consisting of from 2 to 6 or 10 additional amino acids, which peptide is terminated by polyalkylene oxide.
• Nonlimiting examples of an active agent of this invention include KHHLGLEEPKKLR (SEQ ID NO: 1), KHHLGLEEPKK (SEQ ID NO:2), HLGLEEPKKLR (SEQ ID NO: 3), analogs thereof, prodrug of any thereof, or pharmaceutically acceptable salts of any thereof
• Additional examples of compounds of the present invention include all of the foregoing compounds, where the first one or two N-terminal amino acids are deleted. Additional examples of compounds of the present invention include all of the foregoing compounds, with one, two or three additional C-terminal amino acids of any type coupled thereto.
• Additional examples of compounds of the present invention include all of the foregoing compounds, with a 10,000 to 30,000 molecular weight of poly(ethylene glycol) (or "PEG”) moiety coupled to either the N or C terminus thereof.
• PEG poly(ethylene glycol)
• Polyalkylene glycol means straight or branched polyalkylene glycol polymers including, but not limited to, polyethylene glycol (PEG), polypropylene glycol (PPG), and polybutylene glycol (PBG), as well as co-polymers of PEG, PPG and PBG in any combination, and includes the monoalkylether of the polyalkylene glycol.
• the polyalkylene glycol in the compositions of this invention can be, but is not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and any combination thereof.
• the polyalkylene glycol of the composition is polyethylene glycol or "PEG.”
• PEG subunit refers to a single polyethylene glycol unit, i.e., - (CH 2 CH 2 O)-.
• the polyalkylene glycol e.g., PEG
• PEG polyalkylene glycol
• “Monodispersed” is used to describe a mixture of compounds wherein about 100 percent of the compounds in the mixture have the same molecular weight.
• Substantially monodispersed is used to describe a mixture of compounds wherein at least about 95 percent of the compounds in the mixture have the same molecular weight. “Purely monodispersed” is used to describe a mixture of compounds wherein about
• a purely monodispersed mixture is a monodispersed mixture, but a monodispersed mixture is not necessarily a purely monodispersed mixture.
• substantially purely monodispersed is used to describe a mixture of compounds wherein at least about 95 percent of the compounds in the mixture have the same molecular weight and have the same molecular structure.
• a substantially purely monodispersed mixture is a substantially monodispersed mixture, but a substantially monodispersed mixture is not necessarily a substantially purely monodispersed mixture.
• the active compounds described above may be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (9 U Ed. 1995).
• the active compound (including the physiologically acceptable salts thereof) is typically admixed with, inter alia, an acceptable carrier.
• the carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient.
• the carrier may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation, for example, a tablet, which may contain from 0.01 or 0.5% to 95% or 99% by weight of the active compound.
• One or more active compounds may be incorporated in the formulations of the invention, which may be prepared by any of the well known techniques of pharmacy comprising admixing the components, optionally including one or more accessory ingredients.
• compositions of the invention include those suitable for oral, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), topical (i.e., both skin and mucosal surfaces, including airway surfaces) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active compound which is being used.
• Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion.
• Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and a suitable carrier (which may contain one or more accessory ingredients as noted above).
• the formulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture.
• a tablet may be prepared by compressing or molding a powder or granules containing the active compound, optionally with one or more accessory ingredients.
• Compressed tablets may be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s).
• Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.
• Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the active compound in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.
• Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the active compound(s), which preparations are preferably isotonic with the blood of the intended recipient. These preparations may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient.
• Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents.
• the formulations may be presented in unrt ⁇ dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use.
• sterile liquid carrier for example, saline or water-for-injection immediately prior to use.
• Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
• an injectable, stable, sterile composition comprising an active compound(s), or a salt thereof, in a unit dosage form in a sealed container.
• the compound or salt is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject.
• the unit dosage form typically comprises from about 10 mg to about 10 grams of the compound or salt.
• a sufficient amount of emulsifying agent which is ' physiologically acceptable may be employed in sufficient quantity to emulsify the compound or salt in an aqueous carrier.
• emulsifying agent is phosphatidyl choline.
• Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
• Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
• Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6):318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound. Suitable formulations comprise citrate or bis ⁇ tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2M active ingredient.
• the present invention provides liposomal formulations of the compounds disclosed herein and salts thereof.
• the technology for forming liposomal suspensions is well known in the art.
• the compound or salt thereof is an aqueous-soluble salt, using conventional liposome technology, the same may be incorporated into lipid vesicles. In such an instance, due to the water solubility of the compound or salt, the compound or salt will be substantially entrained within the hydrophilic center or core of the liposomes.
• the lipid layer employed may be of any conventional composition and may either contain cholesterol or may be cholesterol-free.
• the salt When the compound or salt of interest is water-insoluble, again employing conventional liposome formation technology, the salt may be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome. In either instance, the liposomes which are produced may be reduced in size, as through the use of standard sonication and homogenization techniques.
• the liposomal formulations containing the compounds disclosed herein or salts thereof may be lyophilized to produce a lyophilizate which may be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.
• compositions may be prepared from the water-insoluble compounds disclosed herein, or salts thereof, such as aqueous base emulsions.
• the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of the compound or salt thereof.
• Particularly useful emulsifying agents include phosphatidyl cholines, and lecithin.
• the pharmaceutical compositions may contain other additives, such as pH-adjusting additives.
• useful pH-adjusting agents include acids, such as hydrochloric acid, bases or buffers, such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate.
• the compositions may contain microbial preservatives.
• Useful microbial preservatives include methylparaben, propylparaben, and benzyl alcohol. The microbial preservative is typically employed when the formulation is placed in a vial designed for multidose use.
• the pharmaceutical compositions of the present invention may be lyophilized using techniques well known in the art.
• KHHLGLEEPKKLR (SEQ ID NO: 1).
• amino acid sequence of an 11 amino acid peptide of the present invention (a fragment of SEQ ID NO: 1 that retains biological activity) is as follows:
• KHHLGLEEPKK (SEQ ID NO: 2).
• the amino acid sequence of an 11 amino acid peptide of the present invention (a fragment of SEQ ID NO: 1 that retains biological activity) is as follows:
• Variations on the foregoing sequences that are biologically active include substitutions that can be made for the K or H in positions 1, 2, and 3 (positions referring to SEQ ID NO: 1) with arginine or lysine, and which retain activity. Similarly, substitutions can be made for L at position 4 and 6 with I or V. Substitutions can be made at positions 7 or 8, for either E, with N. Substitutions can be made at positions 10 and 11 with either H or R. Substitutions can be made at position 12 with L for I or V, and at position 13 for R with K or H. The rationale for picking this particular region of the IGFBP-2 protein was primarily based on certain factors.
• the first is its charge density; that is, the peptide contains 6 basic residues in 13 positions and 2 acidic residues. This gives it an extremely high charge density.
• the IGFBP family contains 6 members that have a high degree of sequence conservation at the N terminus and C terminus of the proteins. However, the mid region of each protein shows sequence divergence.
• This 13 amino acid sequence is completely unique for IGFBP-2 and is not contained in any of the other 6 IGF binding proteins. The importance of this is that knockout of IGFBP-2 in mice gives a unique phenotype: low bone turnover and high fat mass. Knockout of any of the other 5 IGF binding proteins does not give this phenotype. Therefore, it was hypothesized that there was a region within IGBP-2 that accounted for these effects.
• the 13 amino acid peptide of SEQ ID NO: 1 described herein was prepared by solid phase synthesis, purified by HPLC, and then assessed for purity using mass spectrometry.
• Polyethylene glycol) (“PEG”) was purchased from JenKem Technology USA (2033 W. McDermott Dr., Suite 320 #188, Allen, TX 75013-4675). 10.3 mg of the peptide was dissolved in 1.0 ml of 5OmM sodium phosphate pH 6.5. 406 mg of methoxyPEG propinaldehyde molecular weight 20 IcDa was dissolved in 4 ml of 5OmM sodium phosphate.
• the compound was also analyzed by immunoblotting using an antibody that had been specifically prepared against the 13 amino acid sequence.
• the electrophoretic mobility of the Coumassie stained protein and the immunoblotted protein were identical, indicating that the immunoreactive peptide had been pegylated.
• fat cell data were obtained by using the cell line 3T3L1, which is used to study fat cell differentiation. These cells are grown as fat cell precursors. These cells are maintained in Hams Fl 2 with fetal calf serum and 15mM glutamine. To stimulate them to enter the fat cell differentiation pathway, isomethylbutylxanine, dexamethasone and insulin are added using concentrations of 0.5mM IBMX 16.7nM insulin and 0.IuM dexamethasone. In some cases 100 ng/ml IGF-I was added instead of insulin. Three days after the addition, differentiation into adipocytes is apparent and is detected by oil red O staining.
• 60 ml of solution is mixed with 40 ml of water and allowed to sit at room temperature for an hour and then filtered.
• the media is aspirated from the adipocyte cultures and cells are fixed in 1% formaldehyde.
• Formaldehyde is aspirated and the oil red O solution is added in an amount necessary to cover the cells, which are then left at room temperature for one hour.
• the stain is then removed and the plates are washed with distilled water and dried. Typically 60-70% of the cells stain positive after 3 days.
• the cells were then plated in DMEM F12 containing 10% fetal calf serum and then centrifuged at 700 x g to separate the stromal vascular cells from the adipocytes, which float. The supernatant was discarded and the cell pellet which contained the stromal vascular cells was resuspended in DMEM F12 with 10% FCS and plated at a density of 15,000 cells/cm 2 , The medium was aspirated 24 hr later and fresh medium was replaced every 3 days until the cells reached 80-90% confluence.
• the method for osteoclast differentiation is as follows: Balb/c mice in which the IGFBP-2 gene had been deleted ⁇ see V. DeMambro et al, Endocrinology, 149(5): 2051-2061 (2007)) were anesthetized and sacrificed using cervical dislocation. The femurs from each mouse were dissected free of soft tissue and the bone marrow aspirated by needle puncture. Cells were removed sterile and resuspended in DMEM with 10% fetal calf serum. The cells were centrifuged at 700 x g and the cell pellet resuspended in medium and plated in 24 well plates at a concentration of 5000 cells/cm 2 .
• the plates that are used are low adherence plates that have no additives to facilitate cell adherence. Under these conditions macrophages will adhere while other cells will not.
• the media is supplemented with mCSF 20 ng/ml. After 3 days the cells are removed from the plate and the adherent macrophages are exposed to fresh media containing 10% FBS and mCSF. This is repeated again after 3 days and then after an additional 3 days cells are trypsinized and replated in fresh plates at 6 x 10 4 cells/cm. At this time RANK ligand, 50 ng/ml, is added, although in some experiments concentrations as low as 10 ng/ml were utilized.
• IGFBP-2 was added at concentrations between 50 ng/ml and 500 mcg/ml.
• the peptide was added at 20 or 200 ng/ml. The results showed that the cells would not differentiate into osteoclasts without addition of the peptide or native IGFBP-2. Additionally, in the presence of the peptide, the serum concentration could be lowered to as low as 0.5% and osteoclast differentiation was still detected. Additionally, if 2% serum was utilized, mCSF could be deleted from the medium and RANK ligand lowered to as low at 30 ng/ml. However it was not possible to remove IGFBP-2. That is, cultures maintained without IGFBP-2 or the peptide did not differentiate into osteoclasts.
• the cells are also derived from the same bone marrow aspirates. However in some experiments they were derived from calvaria obtained from newborn animals. Cells are plated in 6 well plates at 20 x 10 cells/well in alpha MEM with 10% fetal bovine serum. Media was changed on day 1 and 3. At day 3 the cells received differentiation medium, which is alphaMEM containing 10% fetal calf serum, 8mM glycerol phosphate and 50 ug/ml ascorbic acid . Media subsequently is changed every 2 days until termination of the experiment, generally at 18 days. In some experiments fetal calf serum concentration was lowered to either 5% or 2%.
• IGFBP-2 200 ng/ml was added to some cultures or the IGFBP-2 peptide at concentrations between 2 ng/ml and 2 mcg/ml.
• the cells were fixed with 4% paraformaldehyde and then stained for alkaline phosphatase. The cells were then counterstained with von kossa stain to detect mineralization.
• the pegylated 13 amino acid peptide was compared to the nonpegylated peptide and found to have equal biologic activity.
• This system uses an ex vivo explant system, therefore it is a transition between cultured cells and in vivo experiments.
• the bone forming tissue is removed directly from a mouse and then grown in organ culture for several days.
• the test additives can be added directly to the medium at any concentration in order to test for biological activity.
• the method is as follows: mouse metatarsal bones were isolated from newborn mice between days 0 and 3 after birth using the dissecting microscope. The middle three metatarsals were used for all experiments. Three metatarsals were incubated per well in a 24 well tissue culture dish in DMEM containing 0.5% bovine serum, 0.5M ascorbic acid, ImM beta glycerol phosphate at 37°C.
• the medium was replaced one day later and the metatarsals are cultured for up to 10 days. Growth was measured by quantitative increase in size, both length and width of the bones using an inverted phase microscope with a built in micrometer scaling bar for measurements. Mineralization was assessed by the addition of calcein (500 ng/ml) to the medium for 2 hrs prior to tissue fixation. Fluorescent images were captured using a digital camera attached to an inverted microscope and quantifying calcein fluorescence under the appropriate weight length stimulus. Bone and cartilage histology were measured by fixing the bones in 4% PFA overnight and storing at 70% ethanol, followed by parafilm embedding sectioning and staining. Sections were stained with H&E, Alizaran red or alkaline phosphatase.
• IGFBP-2 -/- mice have reduced bone mineral density, reduced cortical and trabecular thickness and reduced bone turnover, both formation and resorption.
• a pegylated preparation was prepared in order to extend halflife. This preparation was then injected into the mice. Mice between the ages of 16 and 20 weeks were utilized. These mice are known to have a major bone phenotype.
• the peptide was injected at a dose of 50 meg of peptide plus conjugate, that is the total molecular weight of PEG plus the peptide, which is approximately 23,000 molecular weight was injected intraperitoneally five days per week for three weeks. Six mice were injected in each group.
• the major measure of bone density is the bone volume over total volume as calculated by microCT analysis. This measures the amount of total bone within a given space. Control animals receive PBS injections.
• the BV/TV values were as follows: For the normal mice, the value of the volume occupied by bone was 0.22 (22%); for the mice with the IGFBP-2 gene deletion, the value was 0.12; and for mice that received the peptide, it was 0.18. Therefore, injection of the peptide rescued the bone phenotype to within 70% of normal even though these mice had gone the first 6 weeks of life without exposure to this protein. Measurement of the cortical thickness also showed that it was substantially increased by administration of the peptide. Analysis of the bones biochemically showed that PTEN was downregulated. Therefore it appears that the peptide is fully capable of rescuing a bone phenotype in a whole animal model.
• EXAMPLE 8 The heparin binding domain of IGFBP2 is anabolic to the skeleton and works through a mechanism that is independent of IGF-I binding
• mice Generation of the original mixed background strain, B6;l29-Ig ⁇ p2 ⁇ tmlJep>, which are refer to Ig ⁇ p2 " mice, has been described previously 10 ' 52 .
• the original mice were backcrossed onto C57BL/6J background for at least 10 generations.
• AU in vivo and ex vivo experimental studies were performed using male mice. AU the animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Maine Medical Center Research Institute. Mice had free access to water and diet for the duration of the study. Colony environmental conditions included 14:10 hour light: dark cycles.
• Cell culture. Neonatal calvarial osteoblasts were collected from 7-10 day old mice as previously described 53 .
• calvariae were digested 5 times with coUagenase P and Trypsin.
• Cells released from digests 2 through 5 were collected as primary calvarial osteoblasts, and maintained in DMEM supplemented with 10% FCS and non-essential amino acids.
• Osteoblastogenesis of primary calvarial osteoblasts was carried out by treating cells with 4mM ⁇ -glycerophosphate and 50 ⁇ g/ml ascorbic acid in ⁇ MEM.
• Bone marrow stromal cells were harvested from femurs and tibias of 8 weeks old mice as described previously 10 .
• Osteoblastogenesis of BMSCs was induced by the treatment with osteogenic media consisting of ⁇ MEM containing 10% FCS, 8mM ⁇ -glycerophosphate, and 50 ⁇ g/ml ascorbic acid.
• Metacarpal culture Metacarpals were isolated from Igfbp2 ⁇ / ⁇ mice at 1-day old and incubated in DMEM containing 0.5% BSA, 50 ⁇ g/ml ascorbic acid and ImM ⁇ -glycerol phosphate for 10 days 54 . Stimulants were added to culture media from day 1. At day 10, metacarpals were incubated in media containing calcein (500 ng/ml) for 2 hours for staining of calcium deposition, and images were obtained. Then, bones were fixed with 4% PFA, embedded in paraffin, and processed for Alcian blue and von Kossa double staining.
• RNA was prepared using RNeasy Mini Kit (Qiagen) and treated with DNase (Qiagen).
• cDNA was generated using a random hexamer and reverse transcriptase (Superscript III, Invitrogen) according to the manufacturer's instructions.
• Quantification of mRNA expression was carried out using an iQ SYBR Green Supermix in a iQ5 thermal cycler and detection system (Bio-Rad). 18S rRNA or Gapdh was used as an internal standard control gene for all quantification.
• the lysates were frozen at -8O 0 C for 1 h and thawed at room temperature. After three cycles, they were centrifuged at 13,000 x g for 25 min. The pellet was suspended with membrane protein isolation buffer (2OmM Tris-HCl; 15OmM NaCl; ImM EDTA; ImM EGTA; and 1% Triton X-100, pH 7.5) containing protease and phosphatase inhibitors. Protein concentration was assessed by BCA protein assay kit (Thermo Scientific) and equal amounts of sample were separated by SDS-PAGE and transferred electrophoretically to nitrocellulose membranes. Membranes were blocked in 5% BSA in Tris-buffered saline.
• the membranes were immunoblotted with anti- PTEN (Cell signaling, #9559), anti-Akt (Cell signaling, #9272), anti-pSer473-Akt (Cell signaling, #9271), anti- ⁇ -catenin (BD Transduction Laboratories, 610153), anti-pSer552- ⁇ - catenin (Cell signaling, #9566P), or anti- ⁇ -actin (Santa Cruz, sc-47778), and developed with horseradish peroxidase-coupled anti mouse or rabbit IgG antibodies, followed by enhancement with SuperSignal West Dura Extended Duration Substrate Antibodies (Pierce Chemical Co.).
• HBD-peptide The synthetic peptide containing the mouse homologue of the heparin binding domain (AA246-249) and 9 additional amino acids of mouse IGFBP2, 237 KHLSLEEPKKLRP 250 (referred to as HBD-peptide), and control peptide for HBD-peptide (AALSLEEP AALAP) were synthesized by the Protein Chemistry Core Facility at the University of North Carolina at Chapel Hill.
• the synthetic peptide for heparin-binding domain of IGFBP5 201 RKGFYKRKQCKPSRGKRK 218 ) was generated in the same way. Purity and sequence confirmation were determined by mass spectrometry.
• the IGFBP-2 HBP-peptide was pegylated as follows: 10 mg of peptide was mixed with 380 ug of methoxy PEG maleimide (20000 kDa)(l:3 molar ratio peptide to PEG) (Jenkem Biotechnology) in 4.0 ml of 0.05M NaH 2 PO 41 pH 7.0. Following an overnight incubation at 4 0 C, cysteine was added to a final concentration of 17mM to block unreacted sites. To remove the nonpegylated peptide and cysteine the mixture was desalted using a Zebra Desalt spin column (Thermo Scientific) following the manufacturer's instructions. Pegylation was verified by SDS-PAGE analysis with Coomassie staining.
• Ig ⁇ p2 ⁇ / ⁇ mice In vivo treatment of Ig ⁇ p2 ⁇ / ⁇ mice with pegylated heparin-binding peptide (PEG- HBD-peptide). Igfbp2 +/+ and I ⁇ pl 1' mice were administered with PBS or PEG-HBD- peptide (50 ⁇ g/day) intraperitoneally 5 times per week from 6 to 9 weeks of age. Dual- energy x-ray absorptiometry was performed before the initiation of treatment. Mice were injected with 20 mg/kg calcein intraperitoneally 7 days and 2 days before sample collection.
• PEG- HBD-peptide pegylated heparin-binding peptide
• Dual-energy x-ray absorptiometry DXA
• Dual-energy x-ray absorptiometry DXA for whole body and femoral areal bone mineral density (aBMD) and body composition exclusive of the head were performed using the PIXImus (GE-Lunar) 10 .
• the PIXImus was calibrated daily with a mouse phantom provided by the manufacturer.
• MicroCT Microarchitecture of distal trabecular bone and midshaft cortical bone was analyzed with femurs and vertebrae (L5) by MicroCT (MicroCT40) 10 . Approximately 100 slices were measured just proximal to the distal growth plate, with an isotropic pixel size of 12 ⁇ m and slice thickness of 12 ⁇ m. Midshaft cortical bone thickness was analysis in a similar fashion using approximately 18 slices obtained at the exact midpoint of the femur.
• Bone Histomorphometry In vivo histomorphometry differences were analyzed between Ig ⁇ p2 ⁇ / ⁇ mice treated with PBS or PEG-HBD-peptide at 9 weeks of age, Mice were injected with 20 mg/kg calcein intraperitoneally 7 days and 2 days before sample collection. Femurs were analyzed as described previously 10 .
• Femoral Biomechanics Femoral Biomechanical properties were compared by three- point bending between Igfbp2 " mice treated with PBS or PEG-HBD-peptide at 9 weeks of age. Load was applied at a constant rate (0.05 mm/sec) until failure. Failure load (newtons), bending stiffness (newtons per millimeter), and work-to-failure (newton-millimeters) from the load-displacement curve were measured and the apparent elastic modulus (megapascal) and ultimate strength (gigapascal) were measured using the relevant midfemoral cross- sectional geometry measured from ⁇ CT.
• IGFBP-2 The heparin-binding domain of IGFBP-2 stimulates in vitro osteoblastogenesis. It has been reported previously that Igfbp2 ⁇ ' mice exhibited an osteopenic phenotype with low bone formation and impaired osteoblastogenesis in bone marrow stromal cells (BMSCs) from Ig ⁇ p2 ⁇ / ⁇ mice compared to wild-type cells (Fig. Ia) 10 . Consistent with this, Igfbp2- deficient calvarial osteoblasts (COBs) exhibited impaired alkaline phosphatase (ALP) activity during osteoblastogenesis compared to wild-type cells (Fig. Ib).
• BMSCs bone marrow stromal cells
• COBs Igfbp2- deficient calvarial osteoblasts
• ALP alkaline phosphatase
• HBD-peptide heparin-binding domain of IGFBP-2
• pegylation of the HBD- peptide was performed (PEG-HBD).
• Ig ⁇ p2 ⁇ / ⁇ COBs and BMSCs were cultured in osteogenic media (i.e., ascorbic acid and ⁇ -glycerol phosphate) in the presence of the PEG-HBD.
• PEG- HBD strongly enhanced osteogenesis of COBs and BMSCs as measured by alkaline phosphatase and Alizarin Red staining (Fig. Ic). Consistent with this, ALP, R ⁇ nx2 and osteocalcin (OC) expression were enhanced in BMSCs treated with PEG-HBD compared to control treated cells (Fig. Id).
• the heparin-binding domain of IGFBP-2 stimulates periosteal expansion of metacarpals ex vivo.
• metacarpals from 1 day old mouse were collected and incubated with osteogenic media in the absence of fetal calf serum and analyzed for the effects of HBD-peptide on skeletal growth. Bone accrual was evaluated by the longitudinal length of calcein incorporated area as well as width and length of metacarpals. The control peptide had no effect on calcein incorporated area, and length and width of metacarpals was comparable to the metacarpals treated with PBS. Hence PBS was used as the subsequent control for further studies (Fig. 8). As shown in Fig.
• IGFBP-2 and HBD-peptide significantly enhanced calcein incorporated area of metacarpals (Fig. 2a and b).
• IGF-I also stimulated expansion of calcein incorporated area to a similar extent as IGFBP-2 and HBD-peptide (Fig. 2a and b).
• the metacarpals displayed further enhancement of calcein incorporated area.
• IGFBP-2 and HBD-peptide also increased the width of metacarpals, implying that IGFBP-2 has an important role in periosteal expansion (Fig. 2c and d).
• the heparin-binding domain of IGFBP-5 does not have an anabolic effect on the skeleton in vitro and ex vivo. Because other IGFBPs possess HBD regions as well as IGFBP-2, analyses were carried out to determine whether the osteogenic effect of the HBD peptide is specific for this region in IGFBP-2 or whether the HBD domain in other forms of IGFBPs also had this effect. For this purpose a peptide was prepared that contained the HBD region in IGFBP-5 (HBD-BP5) and its effect on osteoblastogenesis was determined. Ig ⁇ p2- deficient COBs were cultured in the presence of HBD-BP5 and it was determined that HBD- BP5 did not show any enhancement of mineralization compared to PBS-treated cell (Fig.
• Igfbp2 mice in vivo by improving skeletal micro-architecture. Based on these in vitro and ex vivo observations studies were conducted to determine if the HBD-peptide could rescue the osteopenic phenotype of Ig ⁇ p2 ⁇ / ⁇ mice. For this purpose, Ig ⁇ p2 ⁇ l ⁇ mice were treated with PBS or PEG-HBD. Based on the half-life analysis of PEG-HBD (data not shown), 50 ⁇ g of PEG-HBD were administered 5 times per week for 3 weeks in mice from 6 to 9 weeks of age.
• aBMD bone mineral density
• aBMC bone mineral content
• BW body weight
• microCT analysis revealed an increase in trabecular bone volume both in the distal femur and L5 vertebrae of Igfbp2 " mice treated with PEG-HBD vs PBS-treated Ig ⁇ p2 " mice. This was accompanied by increased trabecular thickness (Fig. 4a, Table 1, Table 5). Cortical thickness at the midshaft of the femur was not affected by administration of PEG-HBD, but total area analyzed at the level of the femur midshaft was increased in Ig ⁇ p2 ⁇ ' ⁇ mice treated with PEG- HBD compared to PBS-treated Ig ⁇ p2 ⁇ ' ⁇ mice.
• the heparin-binding domain of IGFBP -2 suppresses PTEN expression and stimulates IGF-I/Akt signaling.
• IGFBP-2 heparin-binding domain of IGFBP-2
• PTEN expression has beef shown to be enhanced in Igfbp2 deficient osteoblasts 10 .
• IGFBP-2 negatively regulates PTEN expression and enhances IGF-I signaling in osteoblasts.
• First analyses were carried out to determine whether IGFBP-2 suppressed PTEN expression using primary Igfbp2 ' COBs. It was found that full-length IGFBP-2 markedly suppressed PTEN expression in these cells (Fig. 5a).
• IGFBP-2 enhances IGF-I stimulated/ Akt activation by suppressing PTEN expression in part through the HBD.
• the heparin-binding domain of IGFBP-2 stimulates IGF-I induced cytosolic ⁇ - catenin accumulation and Ser552 phosphorylation of ⁇ -catenin.
• IGF-I reduced membrane-bound ⁇ -catenin expression and enhanced cytosolic accumulation of ⁇ -catenin in MC3T3-E1 cells (Fig. 6a).
• cytosolic accumulation of ⁇ -catenin in response to IGF-I was enhanced in Igfbp2 ⁇ l ⁇ COBs treated with PEG-HBD compared to PBS- treated control cells (Fig. 6b). This suggests that the HBD-peptide is involved in IGF-I/ ⁇ - catenin signaling possibly by suppressing PTEN expression.
• Serine 552 (Ser552) phosphorylation of ⁇ -catenin, which can be induced by Akt has been shown to be important for dissociation of ⁇ -catenin from membrane and re-localization to cytoplasm and nucleus 30 .
• IGF-I stimulated ⁇ -catenin phosphorylation on Ser552 was enhanced in cells treated with PEG-HBD. Because activation of ⁇ -catenin is important for bone formation, the osteogenic effect of the HBD-peptide might be mediated by IGF-I ⁇ -catenin signaling in osteoblasts.
• IGFBP-2 may be critical for the anabolic actions of IGFBP-2 on bone.
• the determination that the HBD suppresses PTEN expression in osteoblasts provides one mechanism by which IGFBP-2 could regulate cell fate.
• sorting the IGF-I dependent and independent activities of intact IGFBP-2 is challenging.
• IGF-I signaling is a prerequisite for skeletal acquisition and modulation of this signaling pathway results in alterations in skeletal homeostasis 17 ' 34"40 . Therefore, an anabolic effect of the HBD on the skeleton could in part be mediated by enhancement of IGF-I-induced activation of the PI3K/Akt pathway as a result of PTEN suppression.
• HBD rescued the low osteoblast number in Igfbp2 ⁇ / ⁇ mice
• osteoclast number was further diminished by administration of HBD. This observation is consistent with the observed increase in bone mass but suggests that bone turnover is not accelerated by HBD treatment. Indeed, mineral apposition rate and bone formation rate showed minimal changes in Ig ⁇ p2 ⁇ ' mice treated with HBD.
• IGF-I signaling has been shown to directly stimulate osteoclastogenesis, and prior data suggest that physiological levels of IGFBP-2 in the skeletal microenvironment are required for osteoclastogenesis 41 .
• IGFBP-2 acts to maintain IGF-I concentrations within the pericellular niche but that the HBD, which lacks the IGF-I binding domain, may play a distinct role by inhibiting bone resorption when supraphysiologic concentrations are administered. Since the HBD does not directly suppress osteoclast differentiation in vitro, the underlying mechanism whereby the HBD decreases bone resorption is likely indirect and due to decreased Rankl expression in osteoblasts. Expression of Rankl is regulated by numerous factors including the Wnt/ ⁇ -catenin signaling pathway 42 . Glass et al. reported that mice expressing the stabilized form of ⁇ -catenin exhibit increased trabecular bone volume with a reduction in osteoclast number primarily caused by an increased Opgl Rankl ratio 43 .
• HBD enhances ⁇ -catenin stability that is induced by IGF-I
• the switch of mesenchymal cells towards the osteogenic lineage might explain the increased osteoblasts and reduced adipocytes in bone marrow from Ig ⁇ p2 ⁇ / ⁇ mice treated with PEG-HBD.
• IGFBP insulin-like growth factor-binding protein
• IGF insulin-like growth factor
• Insulin-like growth factor binding protein 2 is a growth inhibitory protein conserved in zebrafish. Proc Natl Acad Sci U 5 ⁇ 96, 15274-9 (1999).
• Insulin-like growth factor 1 regulates the location, stability, and transcriptional activity of beta-catenin.
• IGFBP-2 Insulin-like growth factor-binding protein-2
• Integrin-mediated action of insulin-like growth factor binding protein-2 in tumor cells JMo/ Endocrinol 32, 859-68 (2004). 50. Wang, G.K., Hu, L., Fuller, G.N. & Zhang, W. An interaction between insulin-like growth factor-binding protein 2 (IGFBP2) and integrin alpha5 is essential for IGFBP2-induced cell mobility. J Biol Chem 281, 14085-91 (2006).
• IGFBP2 insulin-like growth factor-binding protein 2
• IGFBP-2 inhibits fat accumulation. These data include studies in which IGFBP-2 was overexpressed in mice; that is transgenic mice that produced excessive IGFBP-2 were created. These animals had decreased fat mass. Importantly when they were fed a high fat diet which normally induces glucose intolerance, they maintained normal glucose tolerance compared to control mice that were fed the same high fat diet and developed hyperglycemia. This suggests that IGFBP-2 protects against insulin resistance. In contrast the mice in which the IGFBP-2 gene was deleted had the opposite phenotype; that is they were significantly fatter than control mice. After 24 weeks the IGFBP-2 knockout mice weighed 37 ⁇ 1.5 gm whereas control animals weighed 32 ⁇ 1.3 gm. After 52 weeks this difference was maintained.
• mice had 3.82 ⁇ 0.67 grams and the knockout mice administered the heparin binding peptide had 3.67 ⁇ 0.36 grams. Again these are highly significant differences. Glucose tolerance tests were also performed in these animals and showed that the knockout animals have impaired glucose tolerance and this was significantly improved with administration of the peptide and not significantly different from control mice.
• HBD IGFBP-2 heparin binding domain
• B220+ B cell progenitor cells in the spleens of BP2-/- M compared to WT-M but no difference in the percentages of all other mature WBC populations. Bone marrow transplants into lethally irradiated WT and BP2-/- male recipients were next performed.
• IGFBP2 is one of 6 IGF binding proteins that shuttles IGFs to their respective receptors. PTH has been shown to induce IGFBP2 synthesis in calvarial osteoblasts and global deletion of BP2 results in small spleens, very low bone formation, impaired bone resorption and increased marrow adiposity. In vitro, bone marrow from bp2-/- mice show impaired recruitment of both osteoblasts and osteoclasts. BP2 null males also have a hematopoietic phenotype in which increased percentages of lymphoid and myeloid subpopulations are found in both the peripheral blood and spleen.
• BP2 serves a critical role as a proliferative factor in the bone marrow niche.
• RNA from purified mouse mesenchymal stem cells Stem Cell Technologies
• BMT bone marrow transplants
• Donor engraftment was not different between strains as shown by greater than 70% donor cells present in the peripheral blood of recipient mice from both strains.
• BP2 which is synthesized by MSCs and inducible by PTH, is a potent regulator of hematopoiesis and may specifically control stem cell fate in an IGF binding independent manner within the marrow niche.
• Niche regulation by IGFBP2 may be therapeutically important to clinical bone marrow transplantation which is currently the only treatment option for many hematologic and solid tissue malignancies.
• Hematopoietic stem cell transplantation is used primarily in the treatment of hematopoietic cancers (leukemias and lymphomas), following high dose chemotherapy for non hematopoietic malignancies. It is also used for diseases involving genetic or acquired bone marrow failure, such as aplastic anemia and autoimmune diseases. Positive transplant outcomes are dependent upon (1) retrieval of sufficient numbers of hematopoietic stem cells (HSCs) from donor bone marrow, cord blood or peripheral blood and (2) successful homing of HSCs to the recipient's bone marrow 1 .
• HSCs hematopoietic stem cells
• IGFBP-2 has been shown to be a necessary factor for the in vitro proliferation of both human and murine hematopoietic stem cells 2 ' 3 .
• HSCs bone marrow hematopoietic stem cells
• Propagation of HSCs prior to transplant increases the likelihood of successful engraftment and re-establishment of steady state hematopoiesis 4 .
• IGFBP-2 has been shown to be a potent stimulus for the in vitro proliferation of mouse and human hematopoietic stem cells ' .
• the mechanism responsible for this effect is unknown.
• IGFBP-2 Insulin-Like Growth Factor Binding Protein 2
• IGFBP 1-6 Insulin-Like Growth Factor Binding Protein 2
• IGFBP-2 is one of six IGF binding proteins (IGFBP 1-6) that regulates the bioavailability of the proliferative growth factors IGF-I and IGF-II.
• IGFBP-2 can act independently of IGF, but the mechanisms underlying these actions have yet to be investigated.
• HBD heparin binding domain
• This peptide is studied for its effect on HSC proliferation, migration, and bone marrow homing with the ultimate goal of defining the usefulness of the HBD as an adjuvant for HSC transplantation therapies. Data in Support of Proposed Investigation. In the context of bone formation, the IGF independent actions of IGFBP-2 were investigated using a synthesized peptide of the HBD contained in the linker region of IGFBP-2. This peptide is distinct from the other IGF binding proteins.
• the HBD peptide rescued the low bone mineralization phenotype of Igfbp2-/- bone marrow stromal cells and calvarial osteoblasts (Fig 10) and stimulated periosteal expansion and growth in ex vivo explants of Ig ⁇ p2-/- metacarpals.
• the HBD peptide rescued the low bone mass phenotype of Igfbp2-/- mice, as seen by increased osteoblast number, restored bone mass, and improved bone strength (Fig 11).
• IGFBP-2 In addition to its actions on mesenchymal cells, IGFBP-2 has been shown to be a necessary factor for the in vitro proliferation of murine and human hematopoietic stem cells. Global deletion of IGFBP-2 results in altered hematopoiesis such that the peripheral blood and spleen of Ig ⁇ p2-/- mice contain significantly increased B lymphocytes and myeloid lineage cells. In vivo administration of the HBD reduced the number of differentiated lymphoid and myeloid cells to the baseline percentages of PBS injected controls (Fig 12). Furthermore, in competitive repopulation bone marrow transplants, Ig ⁇ p2-/- donor cells failed to efficiently repopulate the bone marrow of same strain recipients (Fig 13).
• HBD may be an important component of IGFBP-2 signaling to enhance cell proliferation and suppress differentiation in HSCs.
• the studies described herein are to determine if the growth promoting effects of IGFBP-2 on HSCs are mediated through the HBD and are independent of the actions of IGF-I.
• HBD heparin binding domain peptide
• IGFBP-2 acts to increase the proliferation of HSCs, but the mechanism underlying this effect is unknown. IGFBP-2 has been shown to act independently of IGF-I signaling via the RGD and HBD domains. Using a synthesized HBD peptide unique to IGFBP-2, the ability of the HBD to increase the proliferation of human and murine HSCs and maintain the HSCs in an undifferentiated state will be tested. To delineate IGFBP-2 independent effects, the HBD will be tested in vitro on mouse HSCs collected from both Ig ⁇ p2-/- and Ig ⁇ p2+/+ mice.
• the Igfbp2-/- mouse was created by Dr. John Pintar in the 1990s and then backcrossed to B6 by Dr. Ten ⁇ Wood in the early 2000s. These mice were obtained from Dr. Wood and they continued to be backcrossed to C57BL6 at The Jackson Laboratory.
• the HBD peptide and scrambled control peptide are provided by David Clemmons, MD, at UNC Chapel Hill. To extend the short half-life, the HBD has been pegylated. In vitro experiments have demonstrated that HBD and the pegylated HBD are identical in their ability to stimulate osteoblast proliferation and differentiation.
• HSCs isolated from cord blood will be purchased from Stem Cell Technologies and cultured in Methocult stem cell media, according to the manufacturer's instructions.
• Murine bone marrow stromal cells (BMSC) will be recovered from the femurs of Ig ⁇ p2-/ ⁇ and Ig ⁇ p2+/+ mice.
• BMSC will be stained with fluorescently conjugated antibodies and HSCs isolated using the FACS Aria cell sorter.
• HSCs will be defined as Lin “ CD34 " Sea- 1 + Kit + Flk-2 " . Twenty HSCs from each strain will be cultured in StemSpan serum free media (Stem Cell Technologies) supplemented with heparin, stem cell factor, thrombopoietin, and FGF-I, as previously reported 3 .
• HBD full length IGFBP-2, or scrambled control peptide will be added to cell cultures at predetermined concentrations and for optimized exposure time.
• cell proliferation for test specimens and controls will be quantified by the uptake of H 3 Thymidine as counts of radioactivity per minute using the L56500 Liquid Scintillation Counter. Results will be expressed as the average of triplicate tests.
• Cells will also be analyzed by flow cytometry to determine percentages of short term (ST) and long term (LT) hematopoietic stem cells as defined by CD34 + Sca-1 + Thyl.l +/1 ° c-kit + lin " CD135 " Slamfl/CD150 + CDl Ib 10 and CD34 " Sca-1 + Thyl.l +/1 ° c-kit + lin " CD135 " Slamfl/CD150 +J respectively.
• ST short term
• LT long term
• percentages of differentiated cells will also be determined by flow cytometry using antibodies to lineage specific markers including Terl 19 (erythrocytes) LY6G (Gr-I, myeloid), CD3 (T cell), CD19 (B cell) and CDl Ib (Mac-1, macrophage). Percentages will be based on collection of 20,000 events using the FACSCalibur flow cytometer. Cell culture experiments will be repeated three times. Analysis of variance (ANOVA) will be used to assess differences across groups. The confidence interval will be set at 95% (p ⁇ 0.05).
• IGF-I is a growth promoter for both normal and malignant cells. Elevated serum levels of IGFBP-2 are recognized as a poor prognostic indicator in recurrent metastatic disease and in childhood acute myelogenous leukemia following hematopoietic stem cell transplantation 10 ' 11 . It is, therefore, important to determine if the effects of IGFBP-2 on HSC expansion in vitro are independent of IGF signaling and whether there is synergy between IGF and IGFBP-2 with respect to cell signaling. Design and Methods: HSCs will be cultured from murine bone marrow stromal cells, as described herein.
• IGF signaling will be inhibited by blocking the IGF receptor using the alpha IR-3 antibody in murine HSC cultures.
• the alpha IR-3 antibody used in these experiments has been published as effectively blocking IGF signaling in a variety of cell types, including murine hematopoietic stem cells 12 .
• successful blocking of the IGF receptor will be assessed by adding IGF 1 to cell cultures and measuring cell proliferation by H 3 Thymidine uptake.
• HBD, scrambled peptide and full length IGFBP-2 will be added to HSC cultures, and cell proliferation quantified by H3 Thymidine uptake. Data will be analyzed by ANOVA as described herein.
• HBD has been shown to increase osteoblast proliferation by decreasing PTEN expression and increasing AKT phosphorylation.
• the mechanisms by which IGFBP-2 increases HSC proliferation have not been determined.
• Osteoblasts and hematopoietic cells reside together in close proximity in the borie marrow, and many cell signaling pathways are common to both the bone and hematopoietic niches.
• gene expression profiling will be performed on HSC cell cultures with and without the HBD, scrambled peptide, and full length IGFBP-2. Through PCR gene arrays, the contribution of cell proliferation and/or inhibition of apoptosis to HSC expansion will be determined,
• HSCs will be exposed to HBD, full length IGFBP-2, and a scrambled peptide, as described herein.
• RNA will be extracted from cultured HSCs on day 10 of culture.
• cDNA will be prepared using the RT2 First Strand Kit (SABiosciences, Frederick, MD), according to the manufacturer's directions. A small aliquot (i.e., 25 ul) of the experimental cocktail consisting of cDNA, Syber Green RT2 qPCR Master Mix (SABiosciences) will be added to each well of the PCR array in a 96 well plate format (SABiosciences).
• PCR amplification will be performed using the Bio-Rad iQ5 thermocycler at the following settings: one cycle for 10 minutes at 95 0 C, followed by 40 cycles of 15 seconds each at 95 0 C and 40 cycles of one minute each at 60 0 C. Baseline and threshold values will be determined manually.
• the ⁇ Ct (delta threshold change) will be calculated for each gene using the SABiosciences PCR Array Data Analysis Web Portal (http://www.SABiosciences.com).
• a fold change greater than 1 between the experimental ⁇ Ct (delta threshold change) and the control ⁇ Ct will be considered up-regulation of the gene.
• a fold change less than 1 will be considered down-regulation of the gene.
• Ct values above 35 will be considered negative.
• PCR arrays will be performed for apoptosis, hematopoietic stem cells and hematopoiesis, and the PDK-Akt, Wnt, MAP Kinase and Insulin signaling pathways. Investigation of the effect of the HBD of IGFBP-2 on bone marrow transplantation in vivo.
• HSCs While it is important that HSCs remain undifferentiated prior to transplant, successful transplantation is dependent upon a sufficient number of HSCs homing to the bone marrow and differentiating first into progenitor cells and eventually into mature and functional erythrocytes and white blood cells.
• the ability of the HSCs expanded in vitro by the HBD peptide to repopulate the bone marrow and to maintain steady state hematopoiesis when transplanted into lethally irradiated recipient mice will be tested.
• Design and Methods Murine HSCs collected from Ig ⁇ p2+/+ mice and expanded in vitro by the HBD peptide will be transplanted into Igfbp2+/+ mice.
• HSCs from human cord blood also expanded by the HBD peptide will be transplanted into non-obese, diabetic, severe combined immunodeficient (NOD/SCID) mice, a strain commonly used for transplantation studies of human bone marrow cells.
• Recipient mice will receive a lethal dose ( 10 Gy for IGFBP2+/+) or sublethal dose (3.5 Gy for NOD/SCID) of irradiation by exposure to a Cesium source using the JX Shepherd 143-45 Irradiator in two sessions and with 4 hours between sessions.
• Human and murine HSCs will be expanded in culture for 10 days, counted, and resuspended in PBS at a concentration of 10 X 10 6 cells/mL.
• donor HSCs 30 uL of donor HSCs will be transplanted into the retro orbitus of recipient mice.
• peripheral blood of recipient mice will be tested for successful bone marrow engraftment by determining the percentage of LY5.2+ murine donor cells and CD45/71+ human donor cells by flow cytometry in Igfbp2+/+ and NOD/SCID mice respectively. The presence of >80% donor cells will be indicative of successful engraftment.
• Recipient mice will be sacrificed at 16 weeks post transplant.
• Percentages of the major blood cell populations will be determined in peripheral blood by flow cytometry using conjugated antibodies to Ter 119 (erythrocytes), LY6G (Gr-I, myeloid), CD3 (T cell), CD 19 (B cell), and CDl Ib (Mac-1, macrophage).
• Ter 119 erythrocytes
• LY6G Gr-I, myeloid
• CD3 T cell
• CD 19 B cell
• CDl Ib Mac-1, macrophage
• the LY5.1/LY5.2 gene system will be used in competitive experiments to repopulate the bone marrow and distinguish donor engrafted bone marrow cells from recipient endogenous bone marrow cells.
• the B6.SJL-Ptprc a pepc b /BoyJ mouse strain (LY5.1 positive) will be used as the source of competitor bone marrow donor cells. All recipient mice will be LY5.2 positive.
• Donor and recipient mice will be male and 8 weeks of age at the time of transplant. To obtain donor bone marrow cells, mice will be sacrificed, and the femurs will be recovered aseptically.
• Bone marrow cells will be collected, washed, and resuspended in phosphate buffered saline (PBS) at a concentration of 0.3 X 10 cells per 30 uL injection volume.
• PBS phosphate buffered saline
• LY5.1+ donor cells are mixed with LY5.2+ (Igfbp2-/- or Igfbp2+/+) donor cells at 1 :0 (100% LY5.1 donor cells, positive control), 1 :3 and 1 :6 ratios.
• the HBD peptide, scrambled peptide, or full length IGFBP2 will be added to donor cell preparations at a concentration optimized in previous in vivo experiments (50 ug/30 uL).
• Recipient mice will be irradiated lethally and transplanted, as described herein.
• percentages of LY5.1 positive and LY5.2 positive engrafted donor cells will be determined in peripheral blood by flow cytometry.
• Transplant recipients will be sacrificed at 16 weeks post transplant, and white blood cell populations analyzed by flow cytometry.
• HSCs present in the bone marrow will be quantified and sorted using the FACSAria cell sorter. Sorted cells will be cultured and the number of colony forming units determined in a standard colony forming cell culture assay (Stem Cell Technologies).
• functionality of transplanted HSCs will be assessed by a second set of transplants, which will be performed as described herein.
• ANOVA will be used to assess statistical significance between group means of test (Ig ⁇ p2-/-) and control (Igfbp2+/+) recipients.
• the confidence interval will be set at ⁇ 0.05.
• PEG-HBD pegylated heparin-binding domain Values are expressed as the mean ⁇ SEM. a; p ⁇ 0.05 vs IGFBP2 7" with PBS b; p ⁇ 0.01 vs IGFBP2 +/+ with PBS c; p ⁇ 0.05 vs IGFBP2 "7” with PBS d; p ⁇ 0.01 vj IGFBP2 "A with PBS
• IGFBP-2 -/- 8 24.9 ⁇ 1.3 3.67 ⁇ 0.36
• PEG-HBD pegylated heparin-binding domain BMD
• bone mineral density BMC bone mineral content BW
• PEG-HBD pegylated heparin-binding domain BMD
• bone mineral density BMC bone mineral content BW
• PEG-HBD pegylated heparin-binding domain Values are expressed as the mean ⁇ SEM. a; p ⁇ 0.05 vs IGFBP2 " ' " with PBS
• PEG-HBD pegylated heparin-binding domain, OS; osteoid surface, BS; bone surface, ObS; osteoblast surface, Nob; osteoblast number, BPm; bone perimeter, ES; erosion surface, OcS; osteoclast surface, Noc; osteoclast number, MAR; mineral apposition rate, BFR; bone formation rate Values are expressed as the mean ⁇ SEM a; p ⁇ 0.05 vs IGFBP2 " ' " with PBS
• HHHLGLEEPICK HHVGLEEPICK
• RHHVGLEEPICIC ICHHIGIEEPKK
• HRRVGIEEPICIC HRRVGIEEPICIC
• KRHLGLEEPKK HICHVGLEEPICIC
• RKHIGIEEPICIC KICHLGVEEPICK
• HHKLGLEEPICIC RHKVGLEEPKK; KHKIGIEEPKK; HRHLGVEEPKIC;
• KKKLGLEEPKK HHICVGLEEPICIC; RHICIGIEEPKK; KHKLGVEEPKK;
• HHPVIGLEEPKK HHPVIGLEEPKK; RHRLGIEEPICK; ICHRVGIEEPKK; HRICIGVEEPKK;
• RRRIGLEEPICIC ICRRLGIEEPKK; HKRVGIEEPKK; RICRIGVEEPKK; HKRIGVEEPKK; KRRLGLNEPKK; RRRVGLNEPICIC; HRRIGINEPICK;
• KRRIGVEEPKIC RRRLGLNEPKK; HRRVGLNEPKK; KHHVGINEPKK;
• KHHVGVEEPKK RHHIGLNEPICK; HHHLGINEPKK; KICHVGINEPICK;
• HHHVGVEEPKK HHHVGVEEPKK
• KKHIGLNEPKK RIGLNEPKK
• RICHLGINEPICK HKHVGINEPKK
• KRHVGVEEPKK RRHIGLNEPKK
• HRHLGINEPICIC KHKVGINEPKK
• KHICVGVEEPKIC RHICIGLNEPICIC; HHICLGINEPKIC; ICICKVGINEPKK;
• HHICVGVEEPICIC KICKIGLNEPICIC; RICKLGINEPICIC; HICKVGINEPKIC;
• KKKVGVEEPICIC RKKIGLNEPKK; HICICLGPNEPKK; KRKVGINEPKK;
• HKKVGVEEPICIC KRKIGLNEPKK; RRICLGINEPKK; HRICVGINEPKK;
• KRKVGVEEPKK RRICIGLNEPKIC; HRICLGINEPKK; KHRVGINEPKK;
• HRICVGVEEPICK KHRIGLNEPKK; RHRLGINEPKK; HHRVGINEPKIC;
• RHRVGVEEPICK HHRIGLNEPKIC; ICICRLGINEPKK; RICRVGINEPKK;
• HHRVGVEEPICK HHRVGVEEPICK; KKRIGLNEPKK; RICRLGINEPKK; HKRVGINEPICIC;
• ICICRVGVEEPKIC RKRIGLNEPKK; HKRLGINEPKIC; ICRRVGINEPKK;
• RICRVGVEEPKIC HKRIGLNEPICIC; KRRLGINEPICIC; RRRVGINEPKK;
• KRRVGVEEPKIC RRRIGLNEPICIC
• HRRLGINEPKIC KHHLGVNEPKK
• RRRVGVEEPICK HRRIGLNEPICIC; KHHIGIENPICIC; RHHLGVNEPKK;
• ICHHLGLNEPICK RHHVGLNEPKK; HHHIGINEPKIC; ICICHLGVNEPKK;
• HICHLGLNEPKK HICHLGLNEPKK
• KRHVGLNEPICIC KRHVGLNEPICIC
• RRHIGINEPICIC HRHLGVNEPICIC
• KRHLGLNEPICIC RRHVGLNEPKK
• HRHIGINEPICK KHICLGVNEPKIC
• HHICLGLNEPICK KICICVGLNEPKK; RKKIGINEPKK; HKKLGVNEPKK;
• RICKLGLNEPICIC HKKVGLNEPKK; ICRKIGINEPKK; RRICLGVNEPICIC;
• HRICLGLNEPICK KHRVGLNEPKK; RHRIGINEPICK; HHRLGVNEPICK;
• HHRLGLNEPICK ICICRVGLNEPKIC; RICRIGINEPICK; HKRLGVNEPKK;
• HKRLGLNEPICK KRRVGLNEPICIC; RRRIGINEPICIC; HRRLGVNEPICIC; KHHIGVNEPKK; RHHLGLENPKK; HHHVGLENPKK; ICICHIGIENPICK;
• HHHIGVNEPKIC KKHLGLENPICIC; RICHVGLENPKK; HKHIGIENPKK;
• KHKIGVNEPICIC RHICLGLENPICIC; HHKVGLENPICK; KKKIGIENPKK;
• KRICIGVNEPICIC RRKLGLENPKK; HRKVGLENPKK; KHRIGIENPKK;
• KHRIGVNEPKK RHRLGLENPKK; HHRVGLENPKK; KKRIGIENPKK;
• HHRIGVNEPICK KKRLGLENPICIC
• RKRVGLENPKK HICRIGIENPICK
• KKRIGVNEPICK RICRLGLENPICIC
• HKRVGLENPICIC HKRIGIENPICK
• RRRIGVNEPKIC RRRIGVNEPKIC
• HRRLGLENPICIC ICHHLGIENPKK
• RHHVGIENPKIC RRRIGVNEPKIC
• KHHVGVNEPICIC RHHIGLENPKK
• HHHLGIENPICIC HHHLGIENPICIC
• KKHVGIENPKK KKHVGIENPKK
• HHHVGVNEPICK KICHIGLENPKIC; RICHLGIENPICK; HICHVGIENPICK;
• KICHVGVNEPKIC RICHIGLENPKK
• HKHLGIENPKK HKHLGIENPKK
• KRHVGIENPKK KRHVGIENPKK
• HKHVGVNEPKK KRHIGLENPKK
• RRHLGIENPICIC HRHVGIENPKK
• KRHVGVNEPICK RRHIGLENPKIC
• HRHLGIENPICIC ICHICVGIENPICIC
• KHKVGVNEPKK RHICIGLENPICK; HHICLGIENPICIC; ICICICVGIENPICIC;
• HHKVGVNEPICK KKKIGLENPKK; RICKLGIENPICIC; HKKVGIENPKK;
• HKKVGVNEPKK KRICIGLENPICIC; RRICLGIENPKK; HRKVGIENPICIC;
• HRICVGVNEPICK HRICVGVNEPICK; ICHRIGLENPICIC; RHRLGIENPKK; HHRVGIENPKK;
• KHRVGVNEPICIC RHRIGLENPICK; HHRLGIENPICIC; ICKRVGIENPICIC;
• HHRVGVNEPKK HHRVGVNEPKK; ICKRIGLENPICK; RICRLGIENPICIC; HKRVGIENPICK;
• KKRVGVNEPKK RKRIGLENPICK; HICRLGIENPKK; KRRVGIENPKK;
• RICRVGVNEPKK HKRIGLENPKIC; KRRLGIENPICK; RRRVGIENPKK;
• KRRVGVNEPKK RRRIGLENPICIC
• HRRLGIENPKIC KHHLGVENPICIC
• RRRVGVNEPICIC HRRIGLENPKK; ICHHIGIENPKK; RHHLGVENPICIC;
• ICHHLGLENPKK RHHVGLENPKK; HHHIGIENPICK; KKHLGVENPKK; RKHLGVENPKK; HICHVGVENPKIC; KRHIGLNNPKK; RRHLGINNPKIC;
• HKHLGVENPKK KRHVGVENPICK; RRHIGLNNPICIC; HRHLGINNPKK;
• KRHLGVENPKK RRHVGVENPKK
• HRHIGLNNPICK KHICLGINNPKK
• RRHLGVENPKK RRHLGVENPKK; HRHVGVENPKIC; KHKIGLNNPKK; RHKLGINNPKK;
• ICHKLGVENPKK ICHKLGVENPKK; RHICVGVENPICK; HHKIGLNNPKK; KKKLGINNPKK;
• KKKLGVENPKK HKKIGLNNPKK
• KRICLGINNPKK HKKIGLNNPKK
• HKKLGVENPKK HKKLGVENPKK; KRKVGVENPKK; RRKIGLNNPKIC; HRICLGINNPICIC;
• KRICLGVENPKK RRKVGVEEPKK
• HRICIGLNNPICIC ICHRLGINNPICIC
• HRICLGVENPKK HRICLGVENPKK; KHRVGVENPICIC; RHRIGLNNPKK; HHRLGINNPKIC;
• HHRLGVENPKK HHRLGVENPKK; KKRVGVENPKK; RICRIGLNNPKK; HICRLGINNPKK;
• HKRLGVENPKK HKRLGVENPKK
• KRRVGVENPICK RRRIGLNNPICIC
• HRRLGINNPICIC HRRLGINNPICIC
• RRRLGVENPICK HRRVGVENPKIC
• KHHVGLNNPKK RHHIGINNPKK
• HRRLGVENPKK ICHHLGLNNPICIC; RHHVGLNNPICIC; HHHIGINNPKK;
• KHHIGVENPICK RHHLGLNNPKK; HHHVGLNNPKK; KKHIGINNPKK;
• HHHIGVENPICK HHHIGVENPICK
• KKHLGLNNPKK RICHVGLNNPICIC
• HICHIGINNPKIC HHHIGVENPICK
• KKHIGVENPKK RICHLGLNNPKK; HKHVGLNNPICK; KRHIGINNPKK;
• HKHIGVENPICIC KRHLGLNNPKK; RRHVGLNNPICK; HRHIGINNPKK;
• KRHIGVENPKK RRHLGLNNPKK
• HRHVGLNNPICK ICHKIGINNPKK
• KHKIGVENPICK RHICLGLNNPKK; HHKVGLNNPKK; KICKIGINNPKK;
• KKKIGVENPKK RKKLGLNNPICK; HICKVGLNNPKIC; ICRKIGINNPKK;
• ICRICIGVENPKK RRKLGLNNPKK; HRICVGLNNPKIC; ICHRIGINNPKK;
• ICHRIGVENPICIC RHRLGLNNPKK; HHRVGLNNPKK; KKRIGINNPICIC;
• RHRIGVENPICK HHRLGLNNPKK; KKRVGLNNPKK; RICRIGINNPKK;
• HHRIGVENPICK ICICRLGLNNPKIC; RKRVGLNNPKK; HKRIGINNPKK;
• KKRIGVENPKK RKRLGLNNPKK; HICRVGLNNPICIC; KRRIGINNPKIC;
• RICRIGVENPKK HKRLGLNNPKK; KRRVGLNNPKK; RRRIGINNPKK;
• HKRIGVENPICIC ICRRLGLNNPICK; RRRVGLNNPKK; HRRIGINNPICIC;
• RRRIGVENPKIC HRRLGLNNPKK; ICHHLGINNPKK; RHHVGINNPKK;
• KHHVGVENPKK RHHIGLNNPKK; HHHLGINNPKK; KKHVGINNPKK;
• HHHVGVENPKIC ICKHIGLNNPICK; RICHLGINNPICK; HICHVGINNPKK;
• RKHVGVENPKK HICHIGLNNPICIC; ICRHLGINNPKK; RRHVGINNPICIC; HRHVGINNPKK; KHKIGVNNPKK; RHKLGLEEPICR; HHICVGLEEPICR;
• HHICVGINNPKK ICICKIGVNNPKK; RKKLGLEEPICR; HKKVGLEEPKR;
• KKRVGINNPICK RICRIGVNNPICK
• HKRLGLEEPKR HKRLGLEEPKR
• ICRRVGLEEPKR ICRRVGLEEPKR
• KHHLGVNNPICIC RHHVGVNNPICK; HHHIGLEEPKR; KKHLGIEEPKR;
• HHHLGVNNPKK KKHVGVNNPKIC
• RICHIGLEEPICR HICHLGIEEPKR
• KKHLGVNNPKK RICHVGVNNPKK; HICHIGLEEPICR; KRHLGIEEPKR;
• KRHLGVNNPICIC RRHVGVNNPKIC
• HRHIGLEEPICR KHICLGIEEPKR
• KHKLGVNNPKK RHICVGVNNPICIC
• HHKIGLEEPKR KICICLGIEEPKR
• KKKLGVNNPICK HKKIGLEEPKR
• KRICLGIEEPKR KKKLGVNNPICK
• RKKVGVNNPKK HKKIGLEEPKR
• KRICLGIEEPKR KKKLGVNNPICK
• RKKVGVNNPKK HKKIGLEEPKR
• KRICLGIEEPKR KKKLGVNNPICK
• RKKVGVNNPKK HKKIGLEEPKR
• KRICLGIEEPKR KKKLGIEEPKR
• HKKLGVNNPICIC KRKVGVNNPKIC
• RRKIGLEEPKR HRICLGIEEPICR
• KRKLGVNNPICIC RRICVGVNNPICIC; HRKIGLEEPKR; KHRLGIEEPKR;
• HRICLGVNNPKIC KHRVGVNNPICK; RHRIGLEEPICR; HHRLGIEEPICR;
• HHRLGVNNPICK KKRVGVNNPKK
• RICRIGLEEPKR HICRLGIEEPKR
• KKRLGVNNPKK RKRVGVNNPKK; HICRIGLEEPKR; KRRLGIEEPKR;
• RKRLGVNNPICK HKRVGVNNPICIC
• ICRRIGLEEPKR RRRLGIEEPKR
• HICRLGVNNPKIC KRRVGVNNPKIC; RRRIGLEEPKR; HRRLGIEEPICR;
• HRRLGVNNPICK ICHHLGLEEPKR; RHHVGLEEPKR; HHHIGIEEPKR;
• ICHHIGVNNPICIC RHHLGLEEPKR; HHHVGLEEPKR; KICHIGIEEPKR;
• HHHIGVNNPKK HHHIGVNNPKK
• KKHLGLEEPKR KKHLGLEEPKR
• RKHVGLEEPKR HKHIGIEEPKR
• KKHIGVNNPKK RICHLGLEEPICR; HICHVGLEEPKR; ICRHIGIEEPICR;
• ICRKIGIEEPKR RRKLGVEEPKR; HRKVGVEEPKR; ICHRIGLNEPKR;
• KHRIGIEEPKR RHRLGVEEPKR
• HHRVGVEEPICR KICRIGLNEPICR
• HHRJGIEEPKR HHRJGIEEPKR; ICKRLGVEEPKR; RICRVGVEEPKR; HKRIGLNEPKR;
• KRRIGIEEPKR RRRLGVEEPKR
• HRRVGVEEPKR KHHVGLNEPKR
• KHHVGIEEPICR RHHIGVEEPICR
• HHHLGLNEPKR HHHLGLNEPKR
• KICHVGLNEPICR KHHVGIEEPICR
• HHHVGIEEPKR HHHVGIEEPKR
• ICKHIGVEEPKR ICKHIGVEEPKR
• RICHLGLNEPKR HICHVGLNEPKR
• HICHVGLNEPKR HHHVGIEEPKR
• HKHVGIEEPKR ICRHIGVEEPKR
• RRHLGLNEPICR RRHLGLNEPICR
• HRHVGLNEPKR HRHVGLNEPKR
• KRHVGIEEPKR KRHVGIEEPKR
• RRHIGVEEPKR HRHLGLNEPICR
• KHKVGLNEPICR KRHVGIEEPKR
• HRHLGLNEPICR KHKVGLNEPICR
• KHICVGIEEPKR KHICVGIEEPKR; RHICIGVEEPKR; HHKLGLNEPKR; ICKICVGLNEPKR;
• HHKVGIEEPKR HHKVGIEEPKR
• KKKIGVEEPKR RICICLGLNEPKR
• HICKVGLNEPKR HICKVGLNEPKR
• KKKVGIEEPKR RICKIGVEEPKR; HKKLGLNEPKR; KRICVGLNEPICR;
• HICKVGIEEPKR KRKIGVEEPICR; RRICLGLNEPICR; HRICVGLNEPKR;
• ICRKVGIEEPICR RRKIGVEEPKR; HRICLGLNEPKR; KHRVGLNEPKR;
• HPJCVGIEEPKR KHRIGVEEPICR; RHRLGLNEPKR; HHRVGLNEPKR;
• KHRVGIEEPICR KHRIGVEEPKR; HHRLGLNEPICR; KKRVGLNEPICR;
• HHRVGIEEPICR HHRVGIEEPICR; ICICRIGVEEPKR; RKRLGLNEPKR; HKRVGLNEPICR;
• KKRVGIEEPKR RICRIGVEEPKR
• HICRLGLNEPICR HICRLGLNEPICR
• KRRVGLNEPKR KRRVGLNEPKR
• RKRVGIEEPKR HKRIGVEEPKR; KRRLGLNEPKR; RRRVGLNEPKR;
• HKRVGIEEPKR KRRIGVEEPICR
• RRRLGLNEPKR RRRLGLNEPKR
• HRRVGLNEPKR HRRVGLNEPKR
• ICRRVGIEEPKR RRRIGVEEPICR; HRRLGLNEPICR; ICHHLGINEPKR;
• HHHLGVEEPKR ICICHVGVEEPKR
• RKHIGLNEPKR HICHLGINEPICR
• RICHLGVEEPKR HICHVGVEEPKR; ICRHIGLNEPICR; RRHLGINEPICR;
• HKHLGVEEPKR KRHVGVEEPICR
• RRHIGLNEPKR RRHIGLNEPKR
• HRHLGINEPKR HRHLGINEPKR
• KRHLGVEEPKR RRHVGVEEPICR
• HRHIGLNEPKR ICHKLGINEPKR
• KHICLGVEEPICR KHICLGVEEPICR
• RHICVGVEEPKR HHICIGLNEPICR
• KICICLGINEPKR KHICLGVEEPICR
• RHICVGVEEPKR HHICIGLNEPICR
• KICICLGINEPKR KHICLGVEEPICR
• RHICVGVEEPKR HHICIGLNEPICR
• KICICLGINEPKR KHICLGVEEPICR
• RHICVGVEEPKR HHICIGLNEPICR
• KICICLGINEPKR KHICLGINEPKR
• HHICLGVEEPICR KKKVGVEEPKR; RKKIGLNEPKR; HKKLGINEPKR;
• KKRLGINEPKR RJCRVGINEPKR; HICRIGVNEPICR; KRRLGLENPICR;
• RKRLGINEPKR HICRVGINEPKR; ICRRIGVNEPICR; RRRLGLENPKR;
• HKRLGINEPKR ICRRVGINEPICR
• RRRIGVNEPKR RRRIGVNEPKR
• HRRLGLENPICR HRRLGLENPICR
• RRRLGINEPICR HRRVGINEPKR; KHHVGVNEPKR; RHHIGLENPICR;
• HRRLGINEPICR ICHHLGVNEPICR; RHHVGVNEPICR; HHHIGLENPKR;
• KHHIGIENPKR RHHLGVNEPKR; HHHVGVNEPKR; KKHIGLENPKR;
• HHHIGINEPKR ICICHLGVNEPKR; RICHVGVNEPICR; HKHIGLENPKR;
• KKHIGINEPKR RICHLGVNEPICR; HICHVGVNEPICR; ICRHIGLENPICR;
• HKHIGINEPICR ICRHLGVNEPICR; RRHVGVNEPICR; HRHIGLENPKR;
• KRHIGINEPKR RRHLGVNEPICR
• HRHVGVNEPKR ICHKIGLENPKR
• RRHIGINEPICR HRHLGVNEPICR; ICHKVGVNEPICR; RHICIGLENPKR;
• KHKIGINEPICR RHICLGVNEPKR; HHICVGVNEPKR; ICICICIGLENPKR;
• HHICIGINEPKR KKKLGVNEPKR; RKKVGVNEPICR; HKKIGLENPKR;
• KICICIGINEPICR RICKLGVNEPKR; HKKVGVNEPKR; ICRKIGLENPKR;
• RKICIGINEPKR HICICLGVNEPICR; ICRKVGVNEPICR; RRKIGLENPKR;
• HKKIGINEPICR KRKLGVNEPKR; RRICVGVNEPKR; HRKIGLENPICR;
• KRKIGINEPICR RRKLGVNEPICR
• HRKVGVNEPKR KHRIGLENPKR
• ICHRIGINEPICR RHRLGVNEPKR; HHRVGVNEPKR; KKRIGLENPKR;
• HHRIGINEPICR HHRIGINEPICR
• KKRLGVNEPKR RIGLENPKR
• RICRVGVNEPICR HICRIGLENPKR
• ICICRIGINEPICR RKRLGVNEPKR; HICRVGVNEPICR; KRRIGLENPKR;
• RICRIGINEPICR HKRLGVNEPKR; KRRVGVNEPKR; RRRIGLENPKR;
• HKRIGINEPICR ICRRLGVNEPKR; RRRVGVNEPKR; HRRIGLENPICR;
• KRRIGINEPKR RRRLGVNEPKR
• HRRVGVNEPICR ICHHVGLENPKR
• RRRIGINEPKR HRRLGVNEPICR
• KHHLGLENPKR RHHVGLENPICR
• ICHHVGINEPKR RHHIGVNEPICR; HHHLGLENPICR; ICICHVGLENPKR;
• HHHVGINEPICR KICHIGVNEPICR; RICHLGLENPICR; HICHVGLENPKR;
• ICICHVGINEPKR RICHIGVNEPICR; HICHLGLENPKR; KRHVGLENPICR;
• RKHVGINEPKR HICHIGVNEPKR; ICRHLGLENPICR; RRHVGLENPKR;
• HKHVGINEPICR KRHIGVNEPKR; RRHLGLENPICR; HRHVGLENPICR;
• ICRHVGINEPKR RRHIGVNEPKR; HRHLGLENPICR; ICHICVGLENPKR;
• HRHVGINEPICR ICHKIGVNEPKR; RHKLGLENPKR; HHICVGLENPKR;
• ICHICVGINEPKR RHICIGVNEPKR; HHKLGLENPICR; KICKVGLENPICR;
• HHKVGINEPKR KKKIGVNEPKR; RICICLGLENPICR; HKKVGLENPICR;
• ICICJCVGINEPICR RJCKIGVNEPKR; HICICLGLENPKR; ICRICVGLENPKR;
• RKKVGINEPKR HICKIGVNEPKR
• ICRICLGLENPKR RRKVGLENPKR
• HKKVGINEPKR ICRKIGVNEPKR; RRICLGLENPICR; HRICVGLENPICR;
• ICRKVGINEPKR RRKIGVNEPICR; HRJCLGLENPICR; ICHRVGLENPICR;
• RRICVGINEPKR HRICIGVNEPKR; KHRLGLENPICR; RHRVGLENPKR; HHRVGLENPKR; KICRIGIENPICR; RICRLGVENPICR; HICRVGVENPKR;
• KKRVGLENPKR RICRIGIENPKR; HICRLGVENPKR; KRRVGVENPICR;
• RICRVGLENPICR HKRIGIENPKR; ICRRLGVENPKR; RRRVGVENPICR;
• HKRVGLENPICR ICRRIGIENPKR; RRRLGVENPICR; HRRVGVENPKR;
• KRRVGLENPKR RRRIGIENPKR; HRRLGVENPICR; ICHHLGLNNPKR;
• RRRVGLENPKR HRRIGIENPICR; ICHHIGVENPICR; RHHLGLNNPICR;
• KHHLGIENPKR RHHVGIENPICR; HHHIGVENPKR; KICHLGLNNPKR;
• HHHLGIENPICR KICHVGIENPKR; RICHIGVENPKR; HICHLGLNNPKR;
• KKHLGIENPKR RICHVGIENPKR; HKHIGVENPICR; KRHLGLNNPKR;
• RICHLGIENPKR HICHVGIENPICR; KRHIGVENPKR; RRHLGLNNPICR;
• HICHLGIENPKR KRHVGIENPICR; RRHIGVENPKR; HRHLGLNNPICR;
• KHKLGIENPICR KHKLGIENPICR
• RHKVGIENPKR HHICIGVENPKR
• KKKLGLNNPKR KHKLGIENPICR
• RHKVGIENPKR HHICIGVENPKR
• KKKLGLNNPKR KHKLGIENPICR
• RHKVGIENPKR HHICIGVENPKR
• KKKLGLNNPKR KHKLGIENPICR
• RHKVGIENPKR HHICIGVENPKR
• KKKLGLNNPKR KKKLGLNNPKR
• HHICLGIENPICR HHICLGIENPICR
• ICICKVGIENPICR RKICIGVENPKR
• HICICLGLNNPKR HHICLGIENPICR
• KICKLGIENPICR KICKLGIENPICR
• RICKVGIENPICR HKKIGVENPKR
• KRKLGLNNPICR KICKLGIENPICR
• HKKLGIENPKR HKKLGIENPKR
• KRICVGIENPKR RRICIGVENPICR
• HRICLGLNNPKR HKKLGIENPKR
• KRICVGIENPKR RRICIGVENPICR
• HRICLGLNNPKR HKKLGIENPKR
• KRICVGIENPKR RRICIGVENPICR
• HRICLGLNNPKR HKKLGIENPKR
• KRICVGIENPKR RRICIGVENPICR
• HRICLGLNNPKR HKKLGIENPKR
• RRICIGVENPICR HRICLGLNNPKR
• KRICLGIENPICR RRKVGIENPKR; HRICIGVENPKR; ICHRLGLNNPKR;
• HRICLGIENPICR HRICLGIENPICR; ICHRVGIENPICR; RHRIGVENPKR; HHRLGLNNPICR;
• HHRLGIENPICR KICRVGIENPKR; RICRIGVENPICR; HKRLGLNNPKR;
• ICICRLGIENPKR ICICRLGIENPKR
• RICRVGIENPKR HICRIGVENPKR
• ICRRLGLNNPICR ICRRLGLNNPICR
• RKRLGIENPICR HICRVGIENPKR; KRRIGVENPKR; RRRLGLNNPICR;
• HICRLGIENPKR HICRLGIENPKR; ICRRVGIENPKR; RRRIGVENPKR; HRRLGLNNPKR;
• KRRLGIENPICR RRRVGIENPKR; HRRIGVENPKR; KHHIGLNNPKR;
• KHHIGIENPKR RHHLGVENPKR; HHHVGVENPICR; KKHIGLNNPKR;
• HHHIGIENPICR HHHIGIENPICR
• KKHLGVENPKR RKHVGVENPKR
• HKHIGLNNPKR HHHIGIENPICR
• KKHLGVENPKR RKHVGVENPKR
• HKHIGLNNPKR HHHIGIENPICR
• KKHLGVENPKR RKHVGVENPKR
• HKHIGLNNPKR HKHIGLNNPKR
• ICICHIGIENPKR RICHLGVENPKR; HICHVGVENPICR; KRHIGLNNPKR;
• RICHIGIENPICR HKHLGVENPKR; KRHVGVENPKR; RRHIGLNNPICR;
• KRHIGIENPKR RRHLGVENPICR
• HRHVGVENPKR KHKIGLNNPKR
• ICHKIGIENPKR RHKLGVENPKR; HHKVGVENPKR; KKICIGLNNPKR;
• HHICIGIENPKR KKICLGVENPKR; RKKVGVENPKR; HKKIGLNNPKR;
• KKKIGIENPKR RICKLGVENPICR
• HKKVGVENPKR ICRICIGLNNPICR
• HICKIGIENPKR KRICLGVENPICR; RRICVGVEEPICR; HRKIGLNNPKR;
• KRICIGIENPKR RRKLGVENPKR; HRICVGVENPICR; ICHRIGLNNPKR;
• RRICIGIENPICR HRICLGVENPKR; ICHRVGVENPKR; RHRIGLNNPKR;
• KHRIGIENPKR RHRLGVENPICR
• HHRVGVENPKR HHRVGVENPKR
• ICICRIGLNNPKR ICICRIGLNNPKR
• HHRIGIENPICR ICICRLGVENPICR
• RICRVGVENPICR HICRIGLNNPKR
• KRRIGLNNPKR RRRLGMNPICR
• HRRVGINNPKR KHHVGVNNPKR
• RRRIGLNNPKR RRRIGLNNPKR; HRRLGINNPICR; ICHHLGVNNPKR; RHHVGVNNPKR;
• KHHVGLNNPKR RHHIGINNPICR; HHHLGVNNPICR; KKHVGVNNPICR;
• HHHVGLNNPKR HHHVGLNNPKR; KKHIGINNPKR; RICHLGVNNPKR; HICHVGVNNPKR;
• KKHVGLNNPKR RICHIGINNPKR
• HKHLGVNNPKR RICHIGINNPKR
• ICRHVGVNNPKR ICRHVGVNNPKR
• HKHVGLNNPKR ICRHIGINNPKR; RRHLGVNNPICR; HRHVGVNNPKR;
• KHKVGLNNPKR RHKIGINNPKR; HHKLGVNNPKR; KKKVGVNNPKR;
• HHJCVGLNNPKR KKKIGINNPICR; RICICLGVNNPKR; HICKVGVNNPKR;
• KICKVGLNNPKR RICKIGINNPICR; HICKLGVNNPKR; KRKVGVNNPICR;
• HKKVGLNNPKR ICRKIGINNPKR; RRICLGVNNPKR; HRICVGVNNPICR;
• KRKVGLNNPKR RRICIGINNPKR; HRICLGVNNPKR; ICHRVGVNNPKR;
• HRKVGLNNPKR ICHRIGINNPICR; RHRLGVNNPICR; HHRVGVNNPKR;
• HHRVGLNNPICR HHRVGLNNPICR; ICKRIGINNPKR; RICRLGVNNPKR; HKRVGVNNPKR;
• KKRVGLNNPKR RKRIGINNPKR; HICRLGVNNPICR; ICRRVGVNNPKR;
• RICRVGLNNPKR HICRIGINNPKR; KRRLGVNNPKR; RRRVGVNNPKR;
• HKRVGLNNPKR KRRIGINNPKR; RRRLGVNNPICR; HRRVGVNNPKR;
• KHHLGINNPKR RHHVGINNPICR; HHHIGVNNPICR; ICKHLGLEEPICH;
• HHHLGINNPICR HHHLGINNPICR
• KKHVGINNPKR RKHIGVNNPKR
• HICHLGLEEPKH HICHLGLEEPKH
• KKICLGINNPICR RKICVGINNPICR; HICKIGVNNPICR; KRICLGLEEPKH;
• HICKLGINNPICR HICKLGINNPICR
• KRICVGINNPICR RRICIGVNNPICR
• HRKLGLEEPKH HRKLGLEEPKH
• KRICLGINNPKR RRKVGINNPKR
• HRKIGVNNPKR KHRLGLEEPKH
• KKHIGLEEPKH RICHLGIEEPICH; HICHVGIEEPICH; KRHIGVEEPKH;
• KKHVGLEEPKH RICHIGIEEPKH; HICHLGVEEPICH; KRHVGVEEPKH;
• HICHVGLEEPICH ICRHIGIEEPICH; RRHLGVEEPKH; HRHVGVEEPICH;
• KKKLGLNEPKH RICKVGLNEPICH; HICICIGINEPICH; KRKLGVNEPKH;
• HHHVGLNEPKH HHHVGLNEPKH; KICHIGINEPICH; RKHLGVNEPKH; HKHVGVNEPICH;
• HHKVGVNEPKH HHKVGVNEPKH; ICICKIGLENPKH; RKKLGIENPKH; HICICVGIENPKH;
• KICKVGVNEPKH RICICIGLENPICH; HKICLGIENPICH; KRKVGIENPKH;
• HKKVGVNEPKH ICRICIGLENPKH; RRICLGIENPICH; HRICVGIENPICH;
• KRKVGVNEPICH RRICIGLENPICH; HRICLGIENPKH; ICHRVGIENPICH;
• HRICVGVNEPICH ICHRIGLENPICH; RHRLGIENPKH; HHRVGIENPICH;
• ICHRVGVNEPKH RHRIGLENPKH; HHRLGIENPICH; KKRVGIENPKH;
• HHRVGVNEPKH HHRVGVNEPKH; ICICRIGLENPICH; RICRLGIENPICH; HICRVGIENPICH;
• KKRVGVNEPKH RICRIGLENPICH; HICRLGIENPICH; KRRVGIENPKH;
• HHHLGLENPKH KICHVGLENPICH; RICHIGIENPICH; HKHLGVENPKH;

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• 2010
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