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Background <p>Salmon calcitonin (sCT) is a therapeutic hormone for treating postmenopausal osteoporosis, hypercalcemia, and Paget’s disease. Studies indicate that the absorption of orally taken sCT is negligible (&lt; 0.1% bioavailability).</p> Objective <p>This research work aimed to synthesize, characterize, and evaluate a bioactive trypsin-resistant derivative of sCT for improving its oral delivery.</p> Methods <p>Structural analysis of free sCT and sCT bound to its receptor enabled the identification of Lys<sup>11</sup> and Lys<sup>18</sup> residues for potential biotinylation without disrupting the binding ability. Lys<sup>11,18</sup>-di-bio–sCT was prepared using biotin-N-hydroxy succinimide ester and was isolated using RP-HPLC. The product was characterized using LC–MS and NMR, and positional substitution was verified using the Lys-C enzymatic digestion. Further, the derivative was digested with trypsin to evaluate its proteolytic resistance. To evaluate translational relevance, we conducted in-vitro biocompatibility testing (MTT), stability studies in simulated intestinal fluid, and transepithelial uptake across Caco-2 monolayers. Bioactivity was examined using ELISA in human T47D cells.</p> Results <p>The Lys<sup>11,18</sup>-di-biotinylated derivative was cytocompatible at concentrations applied in transport assays, exhibited greater stability under intestinally relevant conditions (with additional protection afforded by citric acid), and showed improved transepithelial transport compared with unmodified sCT. ELISA confirmed that the derivative retained bioactivity comparable to native sCT while exhibiting greater trypsin resistance. Alongside biotinylation, we also explored the co-incubation of the sCT derivative with citric acid. This resulted in a local decrease in reaction pH, effectively reducing the trypsin activity due to acidic inhibition of proteolysis.</p> Conclusion <p> This dual strategy of chemical modification and citric acid co-administration effectively enhances the oral bioavailability of sCT, potentially improving its therapeutic efficacy.</p>

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Resisting Enzymatic Degradation: Synthesis, Characterization, and In-Vitro Bioassay of Vitamin-Conjugated Salmon Calcitonin Analogue

  • 期刊论文

  • 发表日期：

    2025年10月10日

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  • Aaditi Belnekar,
  • Triveni Manchekar,
  • Shubham Vishwakarma,
  • Ameya Parkar,
  • Deepa Chaturvedi,
  • Aswani Kancherla,
  • Prajakta Dandekar Jain,
  • Ratnesh Jain

摘要

Background

Salmon calcitonin (sCT) is a therapeutic hormone for treating postmenopausal osteoporosis, hypercalcemia, and Paget’s disease. Studies indicate that the absorption of orally taken sCT is negligible (< 0.1% bioavailability).

Objective

This research work aimed to synthesize, characterize, and evaluate a bioactive trypsin-resistant derivative of sCT for improving its oral delivery.

Methods

Structural analysis of free sCT and sCT bound to its receptor enabled the identification of Lys11 and Lys18 residues for potential biotinylation without disrupting the binding ability. Lys11,18-di-bio–sCT was prepared using biotin-N-hydroxy succinimide ester and was isolated using RP-HPLC. The product was characterized using LC–MS and NMR, and positional substitution was verified using the Lys-C enzymatic digestion. Further, the derivative was digested with trypsin to evaluate its proteolytic resistance. To evaluate translational relevance, we conducted in-vitro biocompatibility testing (MTT), stability studies in simulated intestinal fluid, and transepithelial uptake across Caco-2 monolayers. Bioactivity was examined using ELISA in human T47D cells.

Results

The Lys11,18-di-biotinylated derivative was cytocompatible at concentrations applied in transport assays, exhibited greater stability under intestinally relevant conditions (with additional protection afforded by citric acid), and showed improved transepithelial transport compared with unmodified sCT. ELISA confirmed that the derivative retained bioactivity comparable to native sCT while exhibiting greater trypsin resistance. Alongside biotinylation, we also explored the co-incubation of the sCT derivative with citric acid. This resulted in a local decrease in reaction pH, effectively reducing the trypsin activity due to acidic inhibition of proteolysis.

Conclusion

This dual strategy of chemical modification and citric acid co-administration effectively enhances the oral bioavailability of sCT, potentially improving its therapeutic efficacy.