(P11-015-23) Evaluating the Absorption of Various Forms of Water-Solubilized 3-Hydroxy-Cholecalciferol (Vitamin D3) Using In Vitro Models

Objectives: It is estimated that 70% of the global population > 50 are insufficient/deficient in vitamin D3. We estimate that 20% of this population are unable to achieve repletion even with prolonged supplementation of D3 or D2. Our objective was to develop 3-hydroxy-cholecalciferol (3HC) variants that self-emulsify resulting in increased absorption when compared to unmodified D3.

Methods: Molecular Synthesis: To a solution of 3HC in DCM, succinic anhydride was added in the presence of anhydrous pyridine and DMAP to generate an ester bond on the D3 hydroxyl group. The D3-succinic acid conjugate was further subjected to a coupling reaction with polyethyleneglycol-1000 (DPGS) or b-propyl-cyclodextrin (DCDS), in the presence of EDC and DMAP. After purification methods, the pure white solid was isolated and prepared for testing in Caco-2 cells.

In vitro assessment: Caco-2 cells were grown for 21 d in transwell assay chambers. Test compounds were dissolved in media to a concentration of 50µM. Apical media was replaced with 1mL of media containing the test compound. Lower chamber media was replaced with PBS. The assay was allowed to proceed for 6 hours at which point PBS was removed from the lower chamber. A liquid:liquid extraction using 5 excess volumes of hexane was performed. The resulting eluants were evaporated under vacuum centrifugation. The residue was reconstituted in EtOH for HPLC detection.

Analytical Procedure: The analysis was performed on a Shimadzu 1020 equipped with a variable wavelength detector set at 265 nm. The isocratic system was on a 15-cm long C18 column with 5 um particle size. The mobile phase was methanol: ethanol (17:3- v:v) with flow rate of 0.80 mL/min. Prior to sample injection, the mobile phase was run for 20 min to equilibrate the chromatography condition. Standards and samples were dissolved in ethanol and injection volume was 100 uL.

Results: Caco-2 series 1: 3HC 9.53+/-10.107 ppm, 3HC-Succinate 18.5+/-0.82 ppm, DPGS 103.16+/-3.359 ppm, Fold Change 1: 1.94: 10.83.

Caco-2 series 2: 3HC 29.7+/-5.99 ppm, DCDS prep 1 90.43+/-5.99 ppm, DCDS prep 2 109.83+/-14.45 ppm, DCDS prep 3 108.84+/-10.81 ppm, Fold Change 1: 3.04: 3.7: 3.66.

Conclusions: Water solubilized D3 variants resulted in increased absorption as measured in the Caco-2 model. Next steps include testing in an animal model for calcium homeostasis and immunomodulatory effects.

Funding Sources: None