Gino J. Sasso

Isolation and identification of 1α-hydroxy-3-epi-vitamin D3, a potent suppressor of parathyroid hormone secretion

Since our original demonstration of the metabolism of 1α,25(OH)2D3 into 1α,25(OH)2‐3‐epi‐D3 in human keratinocytes, there have been several reports indicating that epimerization of the 3 hydroxyl group of vitamin D compounds is a common metabolic process. Recent studies reported the metabolism of 25OHD3 and 24(R),25(OH)2D3 into their respective C‐3 epimers, indicating that the presence of 1α hydroxyl group is not necessary for the 3‐epimerization of vitamin D compounds. To determine whether the presence of a 25 hydroxyl group is required for 3‐epimerization of vitamin D compounds, we investigated the metabolism of 1αOHD3, a non‐25 hydroxylated vitamin D compound, in rat osteosarcoma cells (ROS 17/2.8). We noted metabolism of 1αOHD3 into a less polar metabolite which was unequivocally identified as 1αOH‐3‐epi‐D3 using the techniques of HPLC, GC/MS, and 1H‐NMR analysis. We also identified 1αOH‐3‐epi‐D3 as a circulating metabolite in rats treated with pharmacological concentrations of 1αOHD3. Thus, these results indicated that the presence of a 25 hydroxyl group is not required for 3‐epimerization of vitamin D compounds. Furthermore, the results from the same studies also provided evidence to indicate that 1αOH‐3‐epi‐D3, like 1αOHD3, is hydroxylated at C‐25. We then evaluated the biological activities of 1αOH‐3‐epi‐D3. Treatment of normal rats every other day for 7 days with 2.5 nmol/kg of 1αOH‐3‐epi‐D3 did not raise serum calcium, while the same dose of 1αOHD3 increased serum calcium by 3.39 ± 0.52 mg/dl. Interestingly, in the same rats which received 1αOH‐3‐epi‐D3 we also noted a reduction in circulating PTH levels by 65 ± 7%. This ability of 1αOH‐3‐epi‐D3 to suppress PTH levels in normal rats without altering serum calcium was further tested in rats with reduced renal function. The results indicated that the ED50 of 1αOH‐3‐epi‐D3 for suppression of PTH was only slightly higher than that of 1α,25(OH)2D3, but that the threshold dose of the development of hypercalcemia (total serum Ca > 10.5 mg/dl) was nearly 80 times higher. These findings indicate that 1αOH‐3‐epi‐D3 is a highly selective vitamin D analog with tremendous potential for treatment of secondary hyperparathyroidism in chronic renal failure patients.

Synthesis of (+)-negamycin from D-glucose

Abstract The broad spectrum antibiotic (+)-negamycin (1) was prepared from 1,2-O-isopropylidene- d -glucose ( 3 ) in nine steps.

Metabolism of selective 20-epi-vitamin D3 analogs in rat osteosarcoma UMR-106 cells: Isolation and identification of four novel C-1 fatty acid esters of 1α,25-dihydroxy-16-ene-20-epi-vitamin D3

&NA; Analogs of 1&agr;,25‐dihydroxyvitamin D3 (S1) with 20‐epi modification (20‐epi analogs) possess unique biological properties. We previously reported that 1&agr;,25‐dihydroxy‐20‐epi‐vitamin D3 (S2), the basic 20‐epi analog is metabolized into less polar metabolites (LPMs) in rat osteosarcoma cells (UMR‐106) but not in a perfused rat kidney. Furthermore, we also noted that only selective 20‐epi analogs are metabolized into LPMs. For example, 1&agr;,25‐dihydroxy‐16‐ene‐20‐epi‐vitamin D3 (S4), but not 1&agr;,25‐dihydroxy‐16‐ene‐23‐yne‐20‐epi‐vitamin D3 (S5) is metabolized into LPMs. In spite of these novel findings, the unequivocal identification of LPMs has not been achieved to date. We report here on a thorough investigation of the metabolism of S4 in UMR‐106 cells and isolated two major LPMs produced directly from the substrate S4 itself and two minor LPMs produced from 3‐epi‐S4, a metabolite of S4 produced through C‐3 epimerization pathway. Using GC/MS, ESI‐MS and 1H NMR analysis, we identified all the four LPMs of S4 as 25‐hydroxy‐16‐ene‐20‐epi‐vitamin D3‐1‐stearate and 25‐hydroxy‐16‐ene‐20‐epi‐vitamin D3‐1‐oleate and their respective C‐3 epimers. We report here for the first time the elucidation of a novel pathway of metabolism in UMR‐106 cells in which both 1&agr;,25(OH)2‐16‐ene‐20‐epi‐D3 and 1&agr;,25(OH)2‐16‐ene‐20‐epi‐3‐epi‐D3 undergo C‐1 esterification into stearic and oleic acid esters. Graphical abstract Figure. No caption available. HighlightsA novel pathway of 20‐epi‐3‐epi analogs of vitamin D3 in UMR‐106 cells.C‐1 esterification of both 16‐ene‐20‐epi‐D3 and 16‐ene‐20‐epi‐3‐epi‐D3.GC–MS, ESI‐MS and NMR confirm esterification into stearic and oleic acid esters.The modifications may alter the biological activity of synthetic VD3 analogs.