In vitro metabolism of 19-nor-1alpha, 25-(OH)2D2 in cultured cell lines: inducible synthesis of lipid- and water-soluble metabolites.

The active vitamin D analog, 19-nor-1alpha,25-dihydroxyvitamin D2 (19-nor-1alpha,25-(OH)2D2), has a similar structure to the natural vitamin D hormone, 1a,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3), but lacks the C10-19 methylene group and possesses an ergosterol/ vitamin D2 rather than a cholesterol/vitamin D3 side chain. We have used this analog to investigate whether any of these structural features has any effect upon the type and rate of in vitro metabolism observed. Using a vitamin D-target cell, the human keratinocyte, HPK1A-ras, we observed formation of a number of metabolites, three of which were purified by extensive HPLC and conclusively identified by a combination of GC-MS and chemical derivatization as 19-nor-1alpha,24,25-(OH) 3D2, 19-nor-1alpha,24,25,26-(OH) 4D2, and 19-nor-1alpha,24,25,28-(OH)4,D2. The first metabolite is probably a product of the vitamin D-inducible cytochrome P450, P450cc24 (CYP24), while the latter two metabolites are likely to be further metabolic products of 19-nor-1alpha,24,25-(OH)3D2. These hydroxylated metabolites resemble those identified by other workers as products of the metabolism of 1alpha,25-(OH)2D2 in the perfused rat kidney. It therefore appears from the similar metabolic fate of 19-nor-1alpha,25-(OH)2D2 and 1alpha,25-(OH)2D2 that the lack of the C10-19 methylene group has little effect upon the nature of the lipid-soluble metabolic products and the rate of formation of these products seems to be comparable to that of products of 1alpha,25-(OH)2D3 in vitamin D-target cells. We also found extensive metabolism of 19-nor-1alpha,25(OH)2D2 to water-soluble metabolites in HPK1A-ras, metabolites which remain unidentified at this time. When we incubated 19-nor-1alpha,25-(OH)2D2 with the liver cell line HepG2, we obtained only 19-nor-1alpha,24,25-(OH)3D2. We conclude that 19-nor-1alpha,25-(OH)2D2 is efficiently metabolized by both vitamin D-target cells and liver cells.

Metabolism of a 20-methyl substituted series of vitamin D analogs by cultured human cells: apparent reduction of 23-hydroxylation of the side chain by the 20-methyl group.

We describe here for the first time the effect of introducing a 20-methyl group on the side-chain metabolism of the vitamin D molecule. Using a series of 20-methyl-derivatives of 1alpha,25-(OH)2D3 incubated with two different cultured human cell lines, HPK1A-ras and HepG2, previously shown to metabolize vitamin D compounds, we obtained a series of metabolic products that were identified by comparison to chemically synthesized standards on HPLC and GC-MS. 24-Hydroxylated-, 24-oxo-hydroxylated-, and 24-oxo-23-hydroxylated products of 20-methyl-1alpha,25-(OH)2D3 were observed, but the efficiency of 23-hydroxylation was low as compared with that of the natural hormone and, in contrast to 1alpha,25-(OH)2D3, no truncated 23-alcohol was formed from the 20-methyl analog. These data, taken together with results from other analogs with changes in the vicinity of the C17-C20 positions, lead us to speculate that such changes must alter the accessibility of the C-23 position to the cytochrome P450 involved. Using the HepG2 cell line, we found evidence that the 24S-hydroxylated product of 20-methyl-1alpha,25-(OH)2D3 predominates, implying that the liver cytochrome involved in metabolism is a different isoform. Studies with a more metabolically resistant analog of the series, 20-methyl-Delta(23)-1alpha,25-(OH)2D3, gave the expected block in 23- and 24-hydroxylation, and evidence of an alternative pathway, namely 26-hydroxylation. 20-Methyl-Delta(23)-1alpha,25-(OH)2D3 was also more potent in biological assays, and the metabolic studies reported here help us to suggest explanations for this increased potency. We conclude that the 20-methyl series of vitamin D analogs offers new perspectives into vitamin D analog action, as well as insights into the substrate preferences of the cytochrome(s) P450 involved in vitamin D catabolism.

Defects in embryonic hindbrain development and fetal resorption resulting from vitamin A deficiency in the rat are prevented by feeding pharmacological levels of all-trans-retinoic acid.

Vitamin A is required for reproduction and normal embryonic development. We have determined that all-trans-retinoic acid (atRA) can support development of the mammalian embryo to parturition in vitamin A-deficient (VAD) rats. At embryonic day (E) 0.5, VAD dams were fed purified diets containing either 12 micrograms of atRA per g of diet (230 micrograms per rat per day) or 250 micrograms of atRA per g of diet (4.5 mg per rat per day) or were fed the purified diet supplemented with a source of retinol (100 units of retinyl palmitate per day). An additional group was fed both 250 micrograms of atRA per g of diet in combination with retinyl palmitate. Embryonic survival to E12.5 was similar for all groups. However, embryonic development in the group fed 12 micrograms of atRA per g of diet was grossly abnormal. The most notable defects were in the region of the hindbrain, which included a loss of posterior cranial nerves (IX, X, XI, and XII) and postotic pharyngeal arches as well as the presence of ectopic otic vesicles and a swollen anterior cardinal vein. All embryonic abnormalities at E12.5 were prevented by feeding pharmacological amounts of atRA (250 micrograms/g diet) or by supplementation with retinyl palmitate. Embryos from VAD dams receiving 12 micrograms of atRA per g of diet were resorbed by E18.5, whereas those in the group fed 250 micrograms of atRA per g of diet survived to parturition but died shortly thereafter. Equivalent results were obtained by using commercial grade atRA or atRA that had been purified to eliminate any potential contamination by neutral retinoids, such as retinol. Thus, 250 micrograms of atRA per g of diet fed to VAD dams (approximately 4.5 mg per rat per day) can prevent the death of embryos at midgestation and prevents the early embryonic abnormalities that arise when VAD dams are fed insufficient amounts of atRA.

Metabolism of the vitamin D analog EB 1089: identification of in vivo and in vitro liver metabolites and their biological activities.

1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3'(E)-dien -1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB 1089) is a novel analog of the vitamin D hormone, calcitriol that has been modified in the side-chain resulting in an increased metabolic stability relative to other side-chain modified analogs (e.g. calcipotriol and 22-oxacalcitriol). To further investigate the metabolism of EB 1089, we set out to study this metabolism both in the rat in vivo as well as in the postmitochondrial liver fractions from rat, man, and minipig in vitro. The same pattern of metabolism was observed in all biological systems employed, both in vivo and in vitro, namely 26- and 26a-hydroxylation of EB 1089. The same metabolites were produced using cultured cell systems (Shankar et al., see this issue). All the possible isomers of 26- and 26a-hydroxy EB 1089 were synthesised and these were compared to biologically generated material using HPLC, NMR, and GC-MS techniques. The predominant natural isomer observed in vitro and in vivo in rats as well as in vitro in humans was identified to be (25S),26R-hydroxy EB 1089. The biological activities of the EB 1089 metabolites on cell growth regulation were 10- to 100-fold lower than that of EB 1089. The effects of the metabolites on calcium metabolism in vivo were comparable to the effect of EB 1089; however, these effects were reduced for the major metabolite in rat and man and for the isomers of 26a-hydroxy EB 1089. We conclude that EB 1089 is metabolised by a different route of side-chain metabolism than calcitriol and that this may explain its relative metabolic stability in pharmacokinetic experiments in vivo compared to that of other vitamin D analogs.

Metabolism of the vitamin D analog EB1089 by cultured human cells: redirection of hydroxylation site to distal carbons of the side-chain.

1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3' (E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB1089) is a novel synthetic analog of 1 alpha,25-dihydroxyvitamin D [1,25-(OH)2D3] with potential for use in the treatment of hyperproliferative disorders. It has an altered side-chain structure compared to 1,25-(OH)2D3, featuring 26,27 dimethyl groups, insertion of an extra carbon atom (24a) at C-24, and two double bonds at C-22,23 and C-24,24a. In vitro metabolism of EB1089 was studied in a human keratinocyte cell model, HPK1A-ras, previously shown to metabolize 1,25-(OH)2D3. Four metabolites were formed, all of which possessed the same UV chromophore as EB1089, indicating the retention of the side-chain conjugated double bond system. Two metabolites were present in sufficient quantities to identify them as 26-hydroxy EB1089 (major product) and 26a-hydroxy EB1089 (minor product), based on mass spectral analysis and cochromatography with synthetic standards. Similar metabolites were generated in vivo and using a liver postmitochondrial fraction in vitro (Kissmeyer et al., companion paper). Studies with the human hepatoma Hep G2 gave rise to 2 isomers of 26-hydroxy EB1089. Studies using ketoconazole, a general cytochrome P450 inhibitor, implicated cytochrome P450s in the formation of the EB1089 metabolites. COS-1 transfection cell experiments using vectors containing CYP27 and CYP24 suggest that these cytochrome P450s are probably not involved in 26- or 26a-hydroxylation of EB1089. Other experiments that examined the HPK1A-ras metabolism of related analogs containing only a single side-chain double bond: 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1' (E)-en-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (MC1473; double bond at C-22,23) and 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-3'(E)-en-1'-yl)-9, 10-secopregna-5(Z),7(E),10(19)-triene (MC1611; double bond at C-24,24a) revealed that the former compound was subject to 24-hydroxylation and the latter compound was mainly 23-hydroxylated. Metabolism experiments involving EB1089, MC1473, and MC1611 in competition with [1 beta-3H]1,25-(OH)2D3 in HPK1A-ras confirmed that CYP24 is probably not involved in the metabolism of EB1089 whereas, in the case of MC1473 and MC1611, it does appear to carry out side-chain hydroxylation. Our interpretation is that the conjugated double bond system in the side-chain of EB1089 is responsible for directing the target cell hydroxylation to the distal positions, C-26 and C-26a. We conclude that EB1089 is slowly metabolized via unique in vitro metabolic pathways, and that these features may explain the relative stability of EB1089 compared to other analogs in vivo.

Role of neutral metabolites in microbial conversion of 3beta-acetoxy-19-hydroxycholest-5-ene into estrone.

Biotransformation of 3beta-acetoxy-19-hydroxycholest-5-ene (19-HCA, 6 g) by Moraxella sp. was studied. Estrone (712 mg) was the major metabolite formed. Minor metabolites identified were 5alpha-androst-1-en-19-ol-3,17-dione (33 mg), androst-4-en-19-ol-3,17-dione (58 mg), androst-4-en-9alpha,19-diol-3,17-dione (12 mg), and androstan-19-ol-3,17-dione (1 mg). Acidic metabolites were not formed. Time course experiments on the fermentation of 19-HCA indicated that androst-4-en-19-ol-3,17-dione was the major metabolite formed during the early stages of incubation. However, with continuing fermentation its level dropped, with a concomitant increase in estrone. Fermentation of 19-HCA in the presence of specific inhibitors or performing the fermentation for a shorter period (48 h) did not result in the formation of acidic metabolites. Resting-cell experiments carried out with 19-HCA (200 mg) in the presence of alpha,alpha'-bipyridyl led to the isolation of three additional metabolites, viz., cholestan-19-ol-3-one (2 mg), cholest-4-en-19-ol-3-one (10 mg), and cholest-5-en-3beta,19-diol (12 mg). Similar results were also obtained when n-propanol was used instead of alpha,alpha'-bipyridyl. Resting cells grown on 19-HCA readily converted both 5alpha-androst-1-en-19-ol-3,17-dione and androst-4-en-19-ol-3,17-dione into estrone. Partially purified 1,2-dehydrogenase from steroid-induced Moraxella cells transformed androst-4-en-19-ol-3,17-dione into estrone and formaldehyde in the presence of phenazine methosulfate, an artificial electron acceptor. These results suggest that the degradation of the hydrocarbon side chain of 19-HCA does not proceed via C(22) phenolic acid intermediates and complete removal of the C(17) side chain takes place prior to the aromatization of the A ring in estrone. The mode of degradation of the sterol side chain appears to be through the fission of the C(17)-C(20) bond. On the basis of these observations, a new pathway for the formation of estrone from 19-HCA in Moraxella sp. has been proposed.