In Vitro Metabolism of the Vitamin D Analog, 22-Oxacalcitriol, Using Cultured Osteosarcoma, Hepatoma, and Keratinocyte Cell Lines

Abstract

1 has potent cell differentiating/anti-proliferative activities in addition to its role in calcium homeostasis (1). This has led researchers in both universities and the pharmaceutical industry to search for so-called "noncalcemic" vitamin D analogs with accentuated differentiating/anti-proliferative properties and reduced ability to cause hypercalcemia (2, 3). 22-Oxacalcitriol (OCT) 2 was an early analog developed for this purpose and contains a 22-oxygen atom that replaces the 22-carbon of calcitriol. OCT binds to the chicken vitamin D receptor (VDR) with an approximately 8 times lower affinity than 1␣,25-(OH) 2 D 3 (4). However, OCT is 10 times more effective than 1␣,25-(OH) 2 D 3 in suppressing cell growth and inducing differentiation of the mouse myelocytic leukemic cell, WEHI-3, in vitro (5). OCT also possesses an enhanced in vivo immunomodulatory potency in mice that is 50 times higher than that of 1␣,25-(OH) 2 D 3 (6). In contrast, OCT has reduced calcemic activity in vivo ( 1 ⁄50-1 ⁄100 that of 1␣, 25-(OH) 2 D 3 ), both in terms of mobilizing calcium from bone and in stimulating intestinal calcium transport in vitamin D-deficient and normal rats (7, 8). The mechanisms responsible for these differences in biological activity remain unclear, but factors such as cellular uptake and intracellular metabolism could contribute to these differences.OCT binds poorly to the vitamin D binding protein (DBP) and is transported in the plasma bound by lipoproteins (chylomicrons and low density lipoprotein) in vivo (9), and this leads to an unusual distribution pattern with a degree of concentration of the vitamin D analog in parathyroid tissue (10). In contrast, little is known about OCT metabolism except that it appears to be excreted as a glucuronide conjugate in the bile, possibly a derivative of a truncated version of OCT (10). Furthermore, there have been suggestions that a metabolite with a truncated side chain may be formed in bovine parathyroid cell cultures in vitro (11), although details of this have yet to be published.In these studies we set out to provide convincing physicochemical identification for metabolites generated in a variety of cultured cell models, namely the human hepatoma lines HepG2 and Hep3B (12), the rat osteosarcoma cell line UMR-106 (13), and the human keratinocyte cell lines HPK1A and HPK1A-ras (14, 15). Because these cell models mimic vitamin D metabolism found in vivo, we expected to observe the same metabolites found and in some cases tentatively identified by others (10, 11). In addition, our objective was to study the rate of OCT metabolism in various cell lines in order to gauge the involvement of vitamin D-inducible catabolic pathways as compared with more general metabolizing systems. Our results support the concept that OCT is subject to extensive metabolism in a variety of tissues that leads to side chain truncated forms excreted in the bile. * This work was partially supported by grants from the Medical Research Council of Canada (to G. J.). The costs of publication of this a...

Effects of ‘Non-Calcaemic’ Vitamin D Analogues on 24-Hydroxylase Expression in MG-63 Osteoblast-Like Cells

“…At this concentration we did not find accumulation of more polar metabolites as we have seen in many previous studies which employed higher (10 µ M ) concentrations [30, 31]. The recovery of metabolites during the extraction procedure was >90% for all samples (data not shown).…”

“…The base peak at m/z 131 is indicative of an intact C25-hydroxyl group and absent if either C26 or C27 has occurred, and our previous studies have shown that the m/z 131 peak is more intense, relative to the other ion species, when 24-hydroxylation has occurred. Although there was insufficient material to perform further chemical characterisation the spectrum mirrors precisely that of authentic 24-hydroxy-OCT as we have previously published [30]. …”

“…Other distinctive ions were seen at m/z 459, m/z 369 and m/z 279 which represent sequential losses of silanol (HOTMSi) groups following side-chain cleavage between C20/C22. These ions are characteristic of the OCT substrate and its metabolites [30]. The base peak at m/z 131 is indicative of an intact C25-hydroxyl group and absent if either C26 or C27 has occurred, and our previous studies have shown that the m/z 131 peak is more intense, relative to the other ion species, when 24-hydroxylation has occurred.…”

Intestinal and Hepatic CYP3A4 Catalyze Hydroxylation of 1α,25-Dihydroxyvitamin D3: Implications for Drug-Induced Osteomalacia

“…Upon LC/MS analysis (data not shown), we observed the disappearance of M2, but not M1 or M3 (both 23-hydroxylated) or the substrate, 1,25(OH) 2 D 3 . In the second experiment, purified M2 was esterified with n-butylboronic acid, which produces a cyclic boronate from a molecule that possesses vicinal hydroxyl groups (Masuda et al, 1996) and was then derivatized with BSTFA plus 1% TMCS. Its mass spectrum after GC/MS (Fig.…”

C-3 Epimerization of Vitamin D3 Metabolites and Further Metabolism of C-3 Epimers

“…Coexpression plasmids for CYP24, adrenodoxin, and adrenodoxin reductase were reported previously (pKSNdl-CYP24) (25). Recombinant Escherichia coli cells transfected with the above plasmids (JM109/ pKSNdl-CYP27B1 or JM109/pKSNdl-CYP24) were grown in TB medium (26) 70,000) in 3.5 mM barbiturate buffer (pH 8.6) containing 0.13 M NaCl and 0.1% ovalbumin was measured following the addition of the compounds as described previously (16,20,27).…”