BONE TREKThe “Next Generation”of Drugs for  Osteoporosis, Osteoarthritis &          Bone Cancer      Michael R. Doschak, M...
Outline of Talk • Bone Metabolism   – Bone Cells and Remodeling • Calcium Physiology   – Key Hormones Involved in Bone Hea...
Outline of Talk • Bone Metabolism   – Bone Cells and Remodeling • Calcium Physiology   – Key Hormones Involved in Bone Hea...
‘Bone’ is often   perceived asbeing ‘dead’ - butin reality, bone is   very dynamic and very much       ‘alive’- constant r...
•‘Bone’ is often                 perceived as               being ‘dead’ - but               in reality, bone is          ...
Bone: Matrix and Mineral• Matrix  – 90% Type I collagen  – Non-collagenous proteins  (growth / differentiation / mineraliz...
Adult Human Skeleton   – Comprised of 206 bones   – Classified primarily by      • Location: axial vs. appendicular      •...
- Trabecular bone        - compact boneremodels according to    - spongy bonelines of stress(mechanical loading)     •comp...
Cortical bone Compact (dense) bone Osteon - Concentric layers (lamellar) Central Haversian Canal      (blood vessels, ...
OsteocyteOsteocytes:- mature bone cells- lacunae- canaliculi
Osteocytes:           Osteoblasts:            Osteoprogenitors:- mature bone cells   - surface               - surface- la...
Osteocytes:           Osteoblasts:            Osteoprogenitors:   Osteoclasts:- mature bone cells   - surface             ...
Bone Remodeling (metabolism)• Resorption of existing bone• Formation of new “osteoid” bone matrix• MineralizationDynamic p...
Steps in Bone Remodeling
Outline of Talk • Bone Metabolism   – Bone Cells and Remodeling • Calcium Physiology   – Key Hormones Involved in Bone Hea...
Calcium Physiology – The Major Players   •   Calcium   •   Phosphate   •   Calcium Sensing Receptor   •   Parathyroid Horm...
Calcium• Ca/PO4 - the major mineral of bone• 99% of total body Ca is bone• 1% is extracellular  – Co-factor for enzymatic ...
Phosphate  • 85% of total body PO4 is bone-    resident  • 15% as organic form    – Part of biological molecules    – Nucl...
Parathyroid Hormone (PTH) • 84 aa peptide hormone that controls   minute to minute ionized [Ca] • Tight control of serum c...
Parathyroid Glands
Actions of Parathyroid Hormone (PTH)• Acts on kidney (within minutes)  – Increases renal calcium reabsorption  – Increases...
Factors that influence PTH secretion  • Low serum Ca++ - primary stimulus  • High serum PO4 – stimulates PTH    – Direct e...
Low serum            Parathyroid calcium              Gland
Parathyroidcalcium            Gland           PTH
Parathyroidcalcium            Gland           PTH
Parathyroidcalcium            Gland             PTH
phosphate          Parathyroidcalcium            Gland             PTH
phosphate          Parathyroidcalcium            Gland             PTH
phosphate          Parathyroidcalcium            Gland           PTH
phosphate          Parathyroidcalcium            Gland           PTH
phosphate          Parathyroidcalcium            Gland           PTH
phosphate          Parathyroidcalcium            Gland           PTH
phosphate          Parathyroidcalcium            Gland           PTH
phosphate            Parathyroidcalcium              Gland             PTH  Vitamin D (active)
phosphate            Parathyroidcalcium              Gland             PTH  Vitamin D (active)
phosphate            Parathyroidcalcium              Gland             PTH  Vitamin D (active)
phosphate            Parathyroid      ?calcium              Gland               PTH  Vitamin D (active)
phosphate            Parathyroidcalcium              Gland               PTH  Vitamin D (active)
Summary: Actions of PTH                       Parathyroid                          Gland            PTH                   ...
Calcitonin• Produced by Thyroid C cells• 32 aa polypeptide hormone• blocks osteoclast activity (Calcitonin Rc)• role in ca...
Vitamin D This term describes 2 molecules: • Ergocalciferol (Vitamin D2)   – Produced by UV irradiation of plant steroid e...
Vitamin D conversion to active form  • Vitamin D2 & D3, ingested or made in the    skin, metabolized in liver to    25-hyd...
1,25-[OH]2-VitD (calcitriol)   Promotes:   • Absorption of Calcium and PO4 from     small intestine   • Extracellular Calc...
Childhood “Rickets”                      •ASBMR Bone Curriculum
Adult Osteomalacia  • Pseudofracture• Pathognomonic for    Osteomalacia      •ASBMR Bone Curriculum
Vitamin D Deficiency•   Lack of sun exposure•   Dietary deficiency•   Malabsorption pathology•   Liver or Renal disease (c...
Vitamin D Deficiency• Incidence reduced in North America due to  supplemented foods (milk, orange juice)• Still identified...
Vitamin D Deficiency   Produces Osteomalacia in stages   • Initially ↓Calcium absorption    Results in ↑ PTH (2ary Hyperp...
Outline of Talk • Bone Metabolism   – Bone Cells and Remodeling • Calcium Physiology   – Key Hormones Involved in Bone Hea...
Bone Health and Bone Disease• Excessive bone turnover associated with:  – Osteoporosis  – Rheumatoid Arthritis  – Paget’s ...
Therapeutic agents Four main types of therapeutics currently    indicated: • Selective estrogen receptor modulators    (SE...
Problems with Current Bone Therapeutics• Selective estrogen receptor modulators (SERMs) have a variety of  hormonal effect...
Next Generation Drugs –Targeted Drug DeliverySite-specific Drug Delivery:• Enhances therapeutic potency• Diminishes off-si...
Targeting Bone with BP-conjugates    This image cannot currently be displayed.  BISPHOSPHONATES (BPs)     – First used to ...
Targeting Bone with BP-conjugates    This image cannot currently be displayed.  BISPHOSPHONATES (BPs)     – High affinity ...
General Hypothesis The targeted delivery of Calcitonin to  hydroxyapatite bone surfaces will  increase drug interaction wi...
Radiolabeled  Bisphosphonate  Distribution  (Bone scan)    of Knee  OsteoArthritis (OA)Image from: Addison S. et al. Arthr...
Bone-Targeting Peptide Hormones:        BP-conjugated Calcitonin (BP-CT)                       and   BP-conjugated Parathy...
Salmon Calcitonin-Bisphosphonate Conjugation Scheme       1.   0.06 µMol Calcitonin in DMF + 0.3 µMol Sulfo-SMCC in DMF + ...
Scientific (BP-CT)• Chemical Coupling ofBisphosphonate (BP) moiety tosalmon Calcitonin (sCT)• Di-conjugate utilizes amineg...
Characterization of Salmon Calcitonin (sCT): MALDI-TOF                 x104Intens. [a.u.]                                 ...
Derivatization of sCT with crosslinker: MALDI-TOFIntens. [a.u.]                                                           ...
Conjugate Characterization: MALDI-TOF:                                                   sCT-crosslinker-bone targeting mo...
Hence we hypothesized targeting sCT to bone by         conjugation to Bisphosphonate (BP)                                 ...
Scientific (BP-PTH)•Chemical Coupling of Bisphosphonate (BP)moiety to synthetic human 1-34 PTH• Mono-conjugate utilizes Ly...
Mechanism: Intermittently administered PTH(in low doses) presents an anabolic effect on        bones; stimulates osteoblas...
Intens. [a.u.]                                     4119.990                                                               ...
•*           •*                     •*                          •* •*                                  •*   * SIG DIF P<0....
Calcitonin Receptor Binding                               and In Vitro Bioactivity            Intracellular cAMP stimulati...
Procedures• Six week old female OVX rats were injected  sub cu with 2.5 IU/kg/day sCT or equivalent  analogues• Analyzed u...
Micro-Computed TomographySkyscan 1076 “in vivo”Micro-CT (9 µm, 100kV)
Pharmacy Micro-CT Imaging and Pharmaceutical Research Labs
(Jones and Doschak, Arth Rheum, 2010)
UntreatedOVX rat            baseline     4 weeks   8 weeks     12 week       16 weekRisedronate  treated  OVX ratSTR (Prot...
Initial BP-CT Data – Rat Osteoporosis                                25Bone Volume / Tissue Volume %                      ...
How? Evidence of New Bone Formation in Rat OVX Model          [Dynamic Bone Labeling (light blue colour)]       Light blue...
Osteometabolix Pharmaceuticals Inc.         Bone-targeting biologics   The “Next Generation” of Bone Drugs                ...
Osteometabolix (OMX)•   Start-up company based on a new    bone-targeting drug delivery platform    developed at the Unive...
Summary            BP-conjugated Calcitonin and        BP-conjugated Parathyroid Hormone• We have developed a novel bone-t...
Summary 2:            BP-conjugated Calcitonin and       BP-conjugated Parathyroid Hormone• OMX has engaged local Alberta ...
3M Microneedle Technologyhollow Microstructured Transdermal System                  (hMTS)                          • 18 h...
Next Steps• Projected Costs for FY13-FY15   – $1M to bring compound to Investigational New Drug (IND)   – $1M for Phase I ...
Outline of Talk • Bone Metabolism   – Bone Cells and Remodeling • Calcium Physiology   – Key Hormones Involved in Bone Hea...
Evidence of New Bone Formation in Rat OVX Model       [Dynamic Bone Labeling (light blue colour)]    Light blue colour sho...
Quantitative Imaging Data(% Vertebral Bone Volume)            (Bhandari and Doschak; Wu and Doschak, ASBMR 2010)
Principle of K-Edge Subtraction (KES)imaging of Strontium in Bone
StrontianiteBelow Sr K-edge (16.020 keV)Above Sr K-edge (16.180 keV)Subtraction Image
Strontium Solution Standards(3-D Synchrotron KES micro-CT Imaging)
Dynamic Strontium Label in RemodelingRat Bone (3-D Synchrotron KES Render)
Dynamic Strontium Label in RemodelingRat Bone (3-D Synchrotron KES Render)
Acknowledgements• Pharmaceutical Orthopaedic Research Lab  (PORL) Staff & Students  – Dr. Krishna Bhandari (Senior Scienti...
Bone Trek: The Next Generations of Drugs for Osteoporosis, Osteoarthritis and Bone Cancer
Bone Trek: The Next Generations of Drugs for Osteoporosis, Osteoarthritis and Bone Cancer
Bone Trek: The Next Generations of Drugs for Osteoporosis, Osteoarthritis and Bone Cancer
Bone Trek: The Next Generations of Drugs for Osteoporosis, Osteoarthritis and Bone Cancer
Bone Trek: The Next Generations of Drugs for Osteoporosis, Osteoarthritis and Bone Cancer
Bone Trek: The Next Generations of Drugs for Osteoporosis, Osteoarthritis and Bone Cancer
Bone Trek: The Next Generations of Drugs for Osteoporosis, Osteoarthritis and Bone Cancer
Bone Trek: The Next Generations of Drugs for Osteoporosis, Osteoarthritis and Bone Cancer
Bone Trek: The Next Generations of Drugs for Osteoporosis, Osteoarthritis and Bone Cancer
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Bone Trek: The Next Generations of Drugs for Osteoporosis, Osteoarthritis and Bone Cancer

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Dr. Michael Doschak, Faculty of Pharmacy and Pharmaceutical Science, University of Alberta, presented January 22, 2013 at the University of Alberta Calgary Centre.

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Bone Trek: The Next Generations of Drugs for Osteoporosis, Osteoarthritis and Bone Cancer

  1. 1. BONE TREKThe “Next Generation”of Drugs for Osteoporosis, Osteoarthritis & Bone Cancer Michael R. Doschak, MSc, PhD Associate Professor Faculty of Pharmacy & Pharmaceutical Sciences University of Alberta
  2. 2. Outline of Talk • Bone Metabolism – Bone Cells and Remodeling • Calcium Physiology – Key Hormones Involved in Bone Health • Bone-Targeting Drug Delivery – Case study: Calcitonin delivery to Bone • Imaging Dynamic Bone Turnover – Synchrotron Imaging of Strontium Drugs
  3. 3. Outline of Talk • Bone Metabolism – Bone Cells and Remodeling • Calcium Physiology – Key Hormones Involved in Bone Health • Bone-Targeting Drug Delivery – Case study: Calcitonin delivery to Bone • Imaging Dynamic Bone Turnover – Synchrotron Imaging of Strontium Drugs
  4. 4. ‘Bone’ is often perceived asbeing ‘dead’ - butin reality, bone is very dynamic and very much ‘alive’- constant remodeling- maintains Calcium levels- balanced turnover: - acromegaly - osteoporosis
  5. 5. •‘Bone’ is often perceived as being ‘dead’ - but in reality, bone is very dynamic and very much ‘alive’•Normal Bone •Osteoporotic Bone - constant remodeling - maintains Calcium levels - balanced turnover: - acromegaly - osteoporosis
  6. 6. Bone: Matrix and Mineral• Matrix – 90% Type I collagen – Non-collagenous proteins (growth / differentiation / mineralization) – Bone cells (Osteoclast, Osteoblast, Osteocyte) and other cells (marrow, blood vessels, nerves)• Mineral – Calcium Phosphate – Hydroxyapatite Ca10(PO4)6(OH)2 – Deposited on (and within) matrix
  7. 7. Adult Human Skeleton – Comprised of 206 bones – Classified primarily by • Location: axial vs. appendicular • Shape: long, short, flat, irregular – Two structural forms of bone • Cortical (dense) • Trabecular (honeycomb-like) – Bones function as • sites of attachment • for protection • locomotion • haematopoietic function • mineral storage and homeostasis
  8. 8. - Trabecular bone - compact boneremodels according to - spongy bonelines of stress(mechanical loading) •compact bone: - formed (active)- Cortical bone is the - osteon (Haversianthickest system) - concentric lamellae - central canal - Volkmann canals - interstitial lamellae - circumferential lamellae •spongy bone: - missing? - trabeculae
  9. 9. Cortical bone Compact (dense) bone Osteon - Concentric layers (lamellar) Central Haversian Canal  (blood vessels, nerves, fluid) Volkmann canal (Transverse) Microscopic canaliculi  Channels linking osteocytes  (osteocyte cellular processes, extracellular fluid)
  10. 10. OsteocyteOsteocytes:- mature bone cells- lacunae- canaliculi
  11. 11. Osteocytes: Osteoblasts: Osteoprogenitors:- mature bone cells - surface - surface- lacunae - secrete bone matrix - daughter cells- canaliculi - osteoid - osteoblasts
  12. 12. Osteocytes: Osteoblasts: Osteoprogenitors: Osteoclasts:- mature bone cells - surface - surface - giant cells- lacunae - secrete bone matrix - daughter cells - multinucleated- canaliculi - osteoid - osteoblasts - erode bone
  13. 13. Bone Remodeling (metabolism)• Resorption of existing bone• Formation of new “osteoid” bone matrix• MineralizationDynamic process that allows• Bone renewal and microcrack repair• Liberation of bone calcium stores• Fracture healing
  14. 14. Steps in Bone Remodeling
  15. 15. Outline of Talk • Bone Metabolism – Bone Cells and Remodeling • Calcium Physiology – Key Hormones Involved in Bone Health • Bone-Targeting Drug Delivery – Case study: Calcitonin delivery to Bone • Imaging Dynamic Bone Turnover – Synchrotron Imaging of Strontium Drugs
  16. 16. Calcium Physiology – The Major Players • Calcium • Phosphate • Calcium Sensing Receptor • Parathyroid Hormone (PTH) • Vitamin D (pro-hormone!) • Kidneys / Small Bowel / Bone • Other hormones (calcitonin, estrogen)
  17. 17. Calcium• Ca/PO4 - the major mineral of bone• 99% of total body Ca is bone• 1% is extracellular – Co-factor for enzymatic rxns – Coagulation – Muscle contraction – Neurotransmitter release – Endocrine/exocrine secretion
  18. 18. Phosphate • 85% of total body PO4 is bone- resident • 15% as organic form – Part of biological molecules – Nucleic Acids, phospholipids, CHO, Enzymes, cofactors, ATP
  19. 19. Parathyroid Hormone (PTH) • 84 aa peptide hormone that controls minute to minute ionized [Ca] • Tight control of serum calcium (2.10 – 2.55 mmol/L) • Synthesized and secreted by “chief cells” of the parathyroid glands • Primary function to maintain (raise) serum calcium levels
  20. 20. Parathyroid Glands
  21. 21. Actions of Parathyroid Hormone (PTH)• Acts on kidney (within minutes) – Increases renal calcium reabsorption – Increases production of active Vitamin D (intestinal Ca++ absorption)• Increases bone resorption – Mobilizes calcium from bone by stimulating cellular matrix dissolution• Subsequently, PTH stimulates bone formation (as bone resorption-and-formation are coupled!!)
  22. 22. Factors that influence PTH secretion • Low serum Ca++ - primary stimulus • High serum PO4 – stimulates PTH – Direct effect to increase PTH mRNA – Indirect effect by decr. Serum Ca • High Vitamin D (active form) – Direct effect on gland that decreases PTH synthesis
  23. 23. Low serum Parathyroid calcium Gland
  24. 24. Parathyroidcalcium Gland PTH
  25. 25. Parathyroidcalcium Gland PTH
  26. 26. Parathyroidcalcium Gland PTH
  27. 27. phosphate Parathyroidcalcium Gland PTH
  28. 28. phosphate Parathyroidcalcium Gland PTH
  29. 29. phosphate Parathyroidcalcium Gland PTH
  30. 30. phosphate Parathyroidcalcium Gland PTH
  31. 31. phosphate Parathyroidcalcium Gland PTH
  32. 32. phosphate Parathyroidcalcium Gland PTH
  33. 33. phosphate Parathyroidcalcium Gland PTH
  34. 34. phosphate Parathyroidcalcium Gland PTH Vitamin D (active)
  35. 35. phosphate Parathyroidcalcium Gland PTH Vitamin D (active)
  36. 36. phosphate Parathyroidcalcium Gland PTH Vitamin D (active)
  37. 37. phosphate Parathyroid ?calcium Gland PTH Vitamin D (active)
  38. 38. phosphate Parathyroidcalcium Gland PTH Vitamin D (active)
  39. 39. Summary: Actions of PTH Parathyroid Gland PTH PTH Synthesis Release Conserves of active Absorb Ca++ in Vitamin DCa++ from Kidneys Ca++ and PO4 Bone But Promotes In small intestine Phosphaturia Maintain (raise) Serum Ca++ Levels
  40. 40. Calcitonin• Produced by Thyroid C cells• 32 aa polypeptide hormone• blocks osteoclast activity (Calcitonin Rc)• role in calcium regulation (will restore resting plasma calcium levels)• Synthetic human and salmon• s.c., i.m. (Intranasally for Osteoporosis)Therapeutic dosage forms: – Salmon calcitonin [Miacalcin; Calcimar]
  41. 41. Vitamin D This term describes 2 molecules: • Ergocalciferol (Vitamin D2) – Produced by UV irradiation of plant steroid ergosterol – Often the major form of supplemental Vitamin D • Cholecalciferol (Vitamin D3) – Formed in skin (epidermis) under action of ultra-violet (UV) light on 7-dehydrocholesterol
  42. 42. Vitamin D conversion to active form • Vitamin D2 & D3, ingested or made in the skin, metabolized in liver to 25-hydroxyvitamin D (25-OH-VitD [calcidiol]), the major circulating form! • Further hydroxylation in kidney to form the highly biologically active: 1,25-Dihydroxyvitamin D (1,25-[OH]2-VitD [calcitriol])
  43. 43. 1,25-[OH]2-VitD (calcitriol) Promotes: • Absorption of Calcium and PO4 from small intestine • Extracellular Calcium homeostasis, directly & also indirectly through ↓PTH • Mineralization of the skeleton
  44. 44. Childhood “Rickets” •ASBMR Bone Curriculum
  45. 45. Adult Osteomalacia • Pseudofracture• Pathognomonic for Osteomalacia •ASBMR Bone Curriculum
  46. 46. Vitamin D Deficiency• Lack of sun exposure• Dietary deficiency• Malabsorption pathology• Liver or Renal disease (conversion & loss)• Increased liver metabolism (drug-induced)• Genetic causes: – Renal 1-α-hydroxylase deficiency (Vit D dependant rickets, VDDR) – Vit D receptor defect (Vit D “resistance”)
  47. 47. Vitamin D Deficiency• Incidence reduced in North America due to supplemented foods (milk, orange juice)• Still identified in young, elderly, new immigrants – Dietary insufficiency – Countries with little sun exposure – Sunscreen overuse – Traditional clothing that covers skin – Dark skin requires more sunlight exposure
  48. 48. Vitamin D Deficiency Produces Osteomalacia in stages • Initially ↓Calcium absorption Results in ↑ PTH (2ary Hyperparathyroidism) • PTH prevents hypocalcemia At expense of phosphaturia and osteopenia • Bone mineralization impaired But Osteoblasts still actively form osteoid Leads to “hungry bone” effect with treatment (hypocalcemia, despite supplementation)
  49. 49. Outline of Talk • Bone Metabolism – Bone Cells and Remodeling • Calcium Physiology – Key Hormones Involved in Bone Health • Bone-Targeting Drug Delivery – Case study: Calcitonin delivery to Bone • Imaging Dynamic Bone Turnover – Synchrotron Imaging of Strontium Drugs
  50. 50. Bone Health and Bone Disease• Excessive bone turnover associated with: – Osteoporosis – Rheumatoid Arthritis – Paget’s Disease – Bone cancers – Post-traumatic Osteoarthritis – Fracture pain (particularly vertebral bone fractures, secondary to Osteoporosis)
  51. 51. Therapeutic agents Four main types of therapeutics currently indicated: • Selective estrogen receptor modulators (SERM’s) • Potent Nitrogen Bisphosphonates (BP) • Calcitonin (CT) • Parathyroid Hormone (PTH)
  52. 52. Problems with Current Bone Therapeutics• Selective estrogen receptor modulators (SERMs) have a variety of hormonal effects on the body and are associated with increased number of blood clots*• Nitrogen-containing Bisphosphonates (BP) are highly selective for bone but have been associated with osteonecrosis of the jaw and other severe side effects**• Native peptide hormones (e.g., Calcitonin, PTH) lack potency and have very short half-lives (~43 mins), which requires more frequent and supra-physiological dosing of the drug *SERMs for the treatment and prevention of breast cancer. Rev Endocr Metab Disord 2007 **Systematic review: bisphosphonates and osteonecrosis of the jaw. Ann Int Med 2006; Spontaneous femoral shaft fracture after long term bisphosphonate therapy
  53. 53. Next Generation Drugs –Targeted Drug DeliverySite-specific Drug Delivery:• Enhances therapeutic potency• Diminishes off-site drug side-effects• Can be achieved by – Identifying cellular metabolic process as a means of uptake – Synthesizing a “pro-drug” by chemical conjugation of the active ligand to a carrier that is uptaken or incorporated
  54. 54. Targeting Bone with BP-conjugates This image cannot currently be displayed. BISPHOSPHONATES (BPs) – First used to treat Paget’s disease in 1971 – Initially termed “di-phosphonates”) – Analogues of inorganic pyrophosphate – Non-hydrolyzable (stable P-C-P bonds)
  55. 55. Targeting Bone with BP-conjugates This image cannot currently be displayed. BISPHOSPHONATES (BPs) – High affinity for bone mineral – Adsorb to bone (large avail. surface area) – Uptake with osteoclast resorption, interfere with cell function – Many different bisphosphonates constructed, with differing affinity for hydroxyapatite
  56. 56. General Hypothesis The targeted delivery of Calcitonin to hydroxyapatite bone surfaces will increase drug interaction with bone cell Calcitonin-receptors, and will preserve bone mass in Osteoporosis
  57. 57. Radiolabeled Bisphosphonate Distribution (Bone scan) of Knee OsteoArthritis (OA)Image from: Addison S. et al. Arthritis Rheum. 2009 Nov;60(11):3366-73.
  58. 58. Bone-Targeting Peptide Hormones: BP-conjugated Calcitonin (BP-CT) and BP-conjugated Parathyroid Hormone (BP-PTH)• Derivatize peptide hormones with bone-specific bisphosphonate (BP) moieties• Our lead compounds BP-Calcitonin and BP-PTH significantly increase bone specificity of CT or PTH• Bone targeting lowers dose levels (and dosing frequency) and decreases side effects relative to the competing therapeutics in the market
  59. 59. Salmon Calcitonin-Bisphosphonate Conjugation Scheme 1. 0.06 µMol Calcitonin in DMF + 0.3 µMol Sulfo-SMCC in DMF + 0.1 % TEA…………..Reacted at room temperature for 1 hr. 2. 3 µMol Thiol-BP in 10 mM PBS pH 6.7……..Reacted for 2 hr at room temperature then overnight at 4°C (Bhandari and Doschak, Int J Pharmaceut, 2010)
  60. 60. Scientific (BP-CT)• Chemical Coupling ofBisphosphonate (BP) moiety tosalmon Calcitonin (sCT)• Di-conjugate utilizes aminegroups of Lysine11 and Lysine18• Bone-targeting deliverysystem for sCT whilst retainingbioactivity•PEGylated formulations ofBP-CT have also beendeveloped to increasedterminal half-life and decreasedosing interval • sCT-Tri (SMCC-BP)
  61. 61. Characterization of Salmon Calcitonin (sCT): MALDI-TOF x104Intens. [a.u.] 3414.808 3430.449 3436.956 1.25 1.00 0.75 0.50 3654.569 3636.524 1830.709 1321.509 1578.592 2069.884 2112.864 2301.009 2816.187 0.25 Salmon Calcitonin: Peak at 3430.449 0.00 000 1500 2000 2500 3000 3500 4000 4500 5000 5500 m/z
  62. 62. Derivatization of sCT with crosslinker: MALDI-TOFIntens. [a.u.] 3872.108 3868.030 6000 5000 Salmon Calcitonin: 4000 Peak at 3430.449 is lost (i.e., converted) 3000 Mono-substituted crosslinker: Peak at 3648.999 4088.172 2000 3648.999 1935.555 3690.977 Di-substituted 4313.169 2045.553 2046.077 1000 crosslinker: Peak at 3868.03 0 000 1500 2000 2500 3000 3500 4000 4500 5000 5500 m/z Tri-substituted crosslinker: Peak at 4088.172
  63. 63. Conjugate Characterization: MALDI-TOF: sCT-crosslinker-bone targeting moiety (Proof-of-principle compound)Intens. [a.u.] 4113.367 5000 4000 Salmon Calcitonin: Peak at 3430.449 is absent (i.e., conjugates are stable) 3000 sCT-Mono substituted bone targeting conjugate: 3650.261 2000 Peak at 3770.219 4453.437 2096.811 3991.436 1404.516 2212.872 2057.734 2126.989 1267.510 3814.270 1170.493 2244.846 1890.881 1897.616 4319.479 3770.219 1630.678 1766.711 1827.173 1000 sCT-Di substituted bone targeting conjugate: 0 Peak at 4113.367 000 1500 2000 2500 3000 3500 4000 4500 5000 5500 m/z sCT-Tri substituted bone targeting conjugate: Peak at 4453.437
  64. 64. Hence we hypothesized targeting sCT to bone by conjugation to Bisphosphonate (BP) Osteoclast CTR CTR CTR sCT sCT sCT Localization & retention of sCT to bone BP BP BP Bone resorption site [sCT] at its site of BP binds to antiresorptive action hydroxyapatite Improved Bone bioavailability & efficacy of sCT(Pierce, 1987; Kasugai, 2000; Yokogawa, 2001; Orme, 1994; Fujisaki, 1997).
  65. 65. Scientific (BP-PTH)•Chemical Coupling of Bisphosphonate (BP)moiety to synthetic human 1-34 PTH• Mono-conjugate utilizes Lysine13 aminegroup
  66. 66. Mechanism: Intermittently administered PTH(in low doses) presents an anabolic effect on bones; stimulates osteoblasts Localization of •PTH PTH to bone •BP after single dose PTH-mediated BP binds to Hydroxyapatite osteoblast activity surfaces Improved Bone bioavailability & efficacy of PTH therapy
  67. 67. Intens. [a.u.] 4119.990 Intens. [a.u.] 4119.990 6000 6000 4000 8195.185 8107.682 8019.068 7973.798 8063.079 8153.478 8239.548 8283.341 7929.963 8327.843 7886.641 8371.998 3970.689 4015.333 8416.423 7844.018 3926.007 7753.375 3882.898 7799.245 8504.527 3795.031 4000 2000 3750.510 0 8107.682 4000 5000 6000 7000 8000 9000 m/z 3882.898 2000 0 000 4000 6000 8000 10000 12000 14000 16000 18000 m/z Qualitative MALDI-ToF mass spectrum of PTH-PEG-BP after HPLC purification. PTH MW 4119 & PTH-PEG-BP (mono-substituted species) MW 8107; a, 0 to 20,000 Da scan of sample; b, Zoom-in view of mass spectra.
  68. 68. •* •* •* •* •* •* * SIG DIF P<0.05 PEGylated Bone-targeting Calcitonin shows significantly reduced kidney accumulation,and increased retention in bone after 24 hours “in vivo”
  69. 69. Calcitonin Receptor Binding and In Vitro Bioactivity Intracellular cAMP stimulation in human T47D cells. 120 100cAMP (% maximal) 80 s CT 60 s CT-SMCC s CT-SMCC-BP 40 20 0 0 20 40 60 80 100 Dose equivalent to [sCT] (nM)Ability to trigger calcitonin receptor was retained by sCT-SMCC and sCT-BP
  70. 70. Procedures• Six week old female OVX rats were injected sub cu with 2.5 IU/kg/day sCT or equivalent analogues• Analyzed using “in vivo” micro-CT (0, 4, 8, 12, 16 wks) to measure bone mass (% bone volume) and volumetric bone mineral density (BMD, after calibration against known HA “phantoms”)
  71. 71. Micro-Computed TomographySkyscan 1076 “in vivo”Micro-CT (9 µm, 100kV)
  72. 72. Pharmacy Micro-CT Imaging and Pharmaceutical Research Labs
  73. 73. (Jones and Doschak, Arth Rheum, 2010)
  74. 74. UntreatedOVX rat baseline 4 weeks 8 weeks 12 week 16 weekRisedronate treated OVX ratSTR (Protos) treated OVX rat baseline 4 weeks 8 weeks 12 week 16 week RIS+STR treated OVX rat (Doschak et al, ISCD, 2008)
  75. 75. Initial BP-CT Data – Rat Osteoporosis 25Bone Volume / Tissue Volume % 20 Baseline 4 week 15 * * 8 week 12 week 10 5 0 OVX untreated OVX + Calcimar OVX + BP-CT(v2) * BP-CT significantly outperforms regular Calcitonin “in vivo”
  76. 76. How? Evidence of New Bone Formation in Rat OVX Model [Dynamic Bone Labeling (light blue colour)] Light blue colour shows new BP-CONJUGATE RISEDRONATE bone formation DOSED DOSED
  77. 77. Osteometabolix Pharmaceuticals Inc. Bone-targeting biologics The “Next Generation” of Bone Drugs •79
  78. 78. Osteometabolix (OMX)• Start-up company based on a new bone-targeting drug delivery platform developed at the University of Alberta, Canada• OMX has expertise in development of bone- targeting peptide hormone therapeutics, and expertise in high-resolution Micro-CT bone imaging• OMX’s Two Lead Products are transformational drug compounds that offer the promise of safer and more effective treatments for Bone disease
  79. 79. Summary BP-conjugated Calcitonin and BP-conjugated Parathyroid Hormone• We have developed a novel bone-targeting drug platform and several lead products (BP-CT and BP-PTH) that link peptide hormones with bone-specific BP molecules• Preclinical studies show significantly increased bone specificity• BP-conjugation lowers dose levels and decreases the side effects relative to unmodified competing therapeutics in the market• Patented bone-targeting methodology •81
  80. 80. Summary 2: BP-conjugated Calcitonin and BP-conjugated Parathyroid Hormone• OMX has engaged local Alberta government innovation entities (AITF), matching funds, and in-kind research contributions which are continuing to move our drug program towards Investigational New Drug (IND) status• Total Budget = $494,200 with AITF contributing $248,700. Approved Dec 2012. Expected completion is January 2014• The Joint Development Initiative (JDI) has 2 parts:  chemical synthesis, scale-up and purification = $93k (Duration: 4 months)  Pre-clinical toxicology studies and supporting analytical work = $401,200 (Duration: 1 year) •82
  81. 81. 3M Microneedle Technologyhollow Microstructured Transdermal System (hMTS) • 18 hollow microneedles / cm2 • 900 µm tall (0.9 mm) • Needles allow for fluid flow from device into skin • Wear time 3 to 40 mins
  82. 82. Next Steps• Projected Costs for FY13-FY15 – $1M to bring compound to Investigational New Drug (IND) – $1M for Phase I Clinical Trial (administered through the Alberta Osteoarthritis Team – Doschak is a key PI) – http://www.oarthritis.com/• Projected Revenue – $494K from provincial innovation entities (Approved Dec 2012) – (Alberta Innovates – Technology Futures Joint Development Initiative) – $300K from federal government Grants (Industry partnered)• Required – $1.2M from Pharma Partner (or Shareholder Round of Investment) – (Current discussions with Merck, Grunenthal, Asahi Kasei)
  83. 83. Outline of Talk • Bone Metabolism – Bone Cells and Remodeling • Calcium Physiology – Key Hormones Involved in Bone Health • Bone-Targeting Drug Delivery – Case study: Calcitonin delivery to Bone • Imaging Dynamic Bone Turnover – Synchrotron Imaging of Strontium Drugs
  84. 84. Evidence of New Bone Formation in Rat OVX Model [Dynamic Bone Labeling (light blue colour)] Light blue colour shows new BP-CONJUGATE RISEDRONATE bone formation DOSED DOSED
  85. 85. Quantitative Imaging Data(% Vertebral Bone Volume) (Bhandari and Doschak; Wu and Doschak, ASBMR 2010)
  86. 86. Principle of K-Edge Subtraction (KES)imaging of Strontium in Bone
  87. 87. StrontianiteBelow Sr K-edge (16.020 keV)Above Sr K-edge (16.180 keV)Subtraction Image
  88. 88. Strontium Solution Standards(3-D Synchrotron KES micro-CT Imaging)
  89. 89. Dynamic Strontium Label in RemodelingRat Bone (3-D Synchrotron KES Render)
  90. 90. Dynamic Strontium Label in RemodelingRat Bone (3-D Synchrotron KES Render)
  91. 91. Acknowledgements• Pharmaceutical Orthopaedic Research Lab (PORL) Staff & Students – Dr. Krishna Bhandari (Senior Scientist, Pharmasciences) – Dr. Yang Yang (Postdoctoral Fellow) – Madhuri Newa (PhD/ Pharmaceutical Sciences) – Arash Panahifar (PhD/ Pharmaceutical Sciences) – Kathy Tang (PhD/ Pharmaceutical Sciences) – Yuchin Wu (PhD/Biomedical Engineering) – Imran Khan (Micro-CT Imaging Technician)

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