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Bone Targeting Salmon Calcitonin Analogues as Drug Delivery Systems for Bone Disease

  • Author / Creator
    Bhandari, Krishna H
  • The objective of this thesis was to design bone targeting salmon calcitonin (sCT) analogues as drug delivery systems for bone diseases. Non-PEGylated salmon calcitonin-bisphosphonate (sCT-BP) and PEGylated salmon calcitonin-bisphosphonate (sCT-PEG-BP) were investigated. sCT-BP conjugates were synthesized by initially reacting sCT with sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) in dimethylformamide (DMF) in presence of triethylamine (TEA) at room temperature. Thiolated bisphosphonate (thiol-BP) was then reacted with sCT-SMCC intermediate to generate sCT-BP conjugates. To synthesize sCT-PEG-BP, sCT was reacted with maleimide-PEG-N-hydroxy succinimidyl carboxy methyl ester (NHS-PEG-MAL) to generate sCT-PEG-MAL which was then reacted with thiol-BP to generate sCT-PEG-BP. Conjugates were characterized by tris-tricine SDS-PAGE. The number of BP per sCT molecule was determined by phosphate and sCT assays and the effect of PEG-BP or BP on sCT secondary structure was examined by Circular Dichroism (CD). sCT analogues were evaluated for in vitro bone mineral affinity and specificity using a binding assay for bone hydroxyapatite and several other calcium salts. Successful conjugation of sCT with BP and the effect of such conjugation on antibody binding ability of sCT was indirectly determined using ELISA. Conjugates were incubated with bone mineral in bone mineral coated osteologic plates. Presence of BP resulted into the binding of these conjugates on the bone mineral surface of these plates. sCT on such bound conjugates was then reacted with anti-sCT antibody. Finally, the intensity of the developed colour was measured. In vitro cell viability was evaluated in osteoclast (OC) precursor RAW 264.7 cells and sCT bioactivity and CTR binding potential was evaluated by in vitro intracellular cAMP stimulation assay in human T47D breast cancer cells. Similarly, in vivo activity was evaluated by determining the plasma levels of calcium after s. c. administration in normal, osteoporotic (OP) and osteoarthritic (OA) rats. Finally, efficacy in bone diseases like OP and adjuvant arthritis (AA) was studied using in- vivo micro-CT and a dose escalation study was performed in OP rats. Our results showed that the chemical coupling of BP or PEG-BP to sCT has a tendency to alter its secondary structure from a less stable and readily aggregation prone random coil orientation into a more stable and desirable alpha helical form without altering its receptor and antibody binding specificity. Bone targeting sCT analogues exhibited significantly greater affinity and specificity for bone mineral over unmodified sCT, retained strong sCT bioactivity and CTR binding affinity. They were non-toxic to bone marrow cells and exhibited comparable hypocalcaemic effect to that of sCT in normal rats. They decreased plasma calcium in OP and OA rats. Compared to marketed unmodified sCT, these analogues showed significantly improved efficacy in terms of preserving bone volume, BMD and trabecular micro-architecture in OP and AA rats. Bisphosphonate-mediated bone targeting of PEGylated or non-PEGylated sCT represents a new class of targeted antiresorptive compounds that have not previously been attempted. These bone targeting sCT analogues hold the promise of facilitating the delivery of sCT preferentially to skeletal bony tissues, thereby increasing its local concentration to bone surfaces, whilst maintaining sCT bioactivity and increasing efficacy.

  • Subjects / Keywords
  • Graduation date
    2012-11
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3ST3D
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Faculty of Pharmacy and Pharmaceutical Sciences
  • Specialization
    • Pharmaceutical Sciences
  • Supervisor / co-supervisor and their department(s)
    • Doschak, Michael R (Faculty of Pharmacy & Pharmaceutical Sciences)
  • Examining committee members and their departments
    • Velázquez, Carlos A (Faculty of Pharmacy & Pharmaceutical Sciences)
    • Barralet, Jake (Faculty of Dentistry, McGill University, Montreal Canada)
    • Mercer, Johns (Department of Oncology)
    • Löbenberg, Raimar (Faculty of Pharmacy & Pharmaceutical Sciences)
    • Klotz , Lars-Oliver (Faculty of Pharmacy & Pharmaceutical Sciences)