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Synthesis, Characterization and Biodistribution Studies of 125I-Radioiodinated di-PEGylated Bone Targeting Salmon Calcitonin Analogue in Healthy Rats

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Abstract

Purpose

The objective of this study was to prepare a bisphosphonate (BP) mediated bone targeting di-PEGylated salmon calcitonin analogue sCT-2(PEG-BP) as a novel bone targeting pharmaceutical.

Methods

HPLC was used for isolation of sCT-2(PEG-BP) from the reaction mixture, followed by determination of possible PEGylation sites by trypsin digestion. Stability of the compound over time, bone mineral affinity using hydroxyapatite, and biodistribution in normal rats after radiolabeling of sCT-2(PEG-BP) or control sCT with 125I was evaluated.

Results

PEGylated sCT analogues were synthesized, and sCT-2(PEG-BP) was isolated by HPLC and confirmed by MALDI-TOF and ICP-MS. MALDI-TOF analysis of trypsinized fragments suggested Cys1 (or Lys11) and Lys18 to be the two PEGylation sites. Bone mineral affinity test showed sCT-2(PEG-BP) or 125I-sCT-2(PEG-BP) exhibited significantly increased bone mineral affinity over sCT or 125I-sCT, respectively. sCT-2(PEG-BP) remained stable for at least 1 month. In vivo biodistribution study showed significantly increased bone retention and prolonged plasma circulation time for sCT-2(PEG-BP) compared to the control sCT.

Conclusion

Those results support sCT-2(PEG-BP) as a promising new drug candidate for the treatment of resorptive and/or maladaptive bone conditions, such as Osteoporosis, Osteoarthritis, Rheumatoid Arthritis, Paget’s disease and bone cancers.

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Abbreviations

%ID/g:

Percentage injected dose per gram of tissue

AMW:

Average Molecular Weight

BP:

Bisphosphonate

BSA:

Bovine Serum Albumin

DMSO:

Dimethyl Sulfoxide

DTT:

Dithiothreitol

GFR:

Glomerular Filtration Rate

HA:

Hydroxyapatite

HPLC:

High Performance Liquid Chromatography

ICP-MS:

Inductively Coupled Plasma Mass Spectrometry

MAL:

Maleimide

MALDI-ToF:

Matrix-Assisted Laser Desorption/Ionization Time of Flight

MW:

Molecular Weight

NHS:

N-Hydroxysuccinimide

RTLC:

Radio-Thin Layer Chromatography

sCT:

Salmon calcitonin

SD:

Standard Deviation

TCEP:

Tris(2-carboxyethyl)phosphine

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors would like to thank Dr. Fakhreddin (Mo) Jamali and his research group for access to HPLC and Savant Speedy Vac concentrator. This research was funded by the Alberta Osteoarthritis Team grant from Alberta Heritage Foundation for Medical Research (AHFMR).

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Authors and Affiliations

  1. Pharmaceutical Orthopaedic Research Laboratory Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada

    Yang Yang, Krishna H. Bhandari, Arash Panahifar & Michael R. Doschak

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  1. Yang Yang
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  2. Krishna H. Bhandari
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  3. Arash Panahifar
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  4. Michael R. Doschak
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Correspondence to Michael R. Doschak.

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Yang, Y., Bhandari, K.H., Panahifar, A. et al. Synthesis, Characterization and Biodistribution Studies of 125I-Radioiodinated di-PEGylated Bone Targeting Salmon Calcitonin Analogue in Healthy Rats. Pharm Res 31, 1146–1157 (2014). https://doi.org/10.1007/s11095-013-1237-7

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  • DOI: https://doi.org/10.1007/s11095-013-1237-7

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