METABOLISM OF THE VITAMIN-D ANALOG EB1089 BY CULTURED HUMAN-CELLS - REDIRECTION OF HYDROXYLATION SITE TO DISTAL CARBONS OF THE SIDE-CHAIN

oxy-hepta-1'(E),3'(E)-dien-1'-yl)-9,10-secopregna- 5(Z),7(E),10(19)-tr iene (EB1089) is a novel synthetic analog of 1 alpha,25-dihydroxyvitam in D [1,25-(OH)(2)D-3] with potential for use in the treatment of hype rproliferative disorders. It has an altered side-chain structure compa red to 1,25-(OH)(2)D-3, featuring 26,27 dimethyl groups, insertion of an extra carbon atom (24a) at C-24, and two double bonds at C-22,23 an d C-24,24a. In vitro metabolism of EB1089 was studied in a human kerat inocyte cell model, HPK1A-ras, previously shown to metabolize 1,25-(OH )(2)D-3. 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 insufficie nt quantities to identify them as 26-hydroxy EB1089 (major product) an d 26a-hydroxy EB1089 (minor product), based on mass spectral analysis and cochromatography with synthetic standards. Similar metabolites wer e generated in vivo and using a liver postmitochondrial fraction in vi tro (Kissmeyer et al., companion paper). Studies with the human hepato ma Hep G2 gave rise to 2 isomers of 26-hydroxy EB1089. Studies using k etoconazole, a general cytochrome P450 inhibitor, implicated cytochrom e P450s in the formation of the EB1089 metabolites. COS-1 transfection cell experiments using vectors containing CYP27 and CYP24 suggest tha t these cytochrome P450s are probably not involved in 26- or 26a-hydro xylation of EB1089. Other experiments that examined the HPK1A-ras meta bolism of related analogs containing only a single side-chain double b ond: -en-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (MC1473; doubl e bond at C-22,23) and -en-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-tri ene (MC1611; double bond at C-24,24a) revealed that the former compoun d was subject to 24-hydroxylation and the latter compound was mainly 2 3-hydroxylated. Metabolism experiments involving EB1089, MC1473, and M C1611 in competition with [1 beta-H-3]1,25-(OH)(2)D-3 in HPK1A-ras con firmed that CYP24 is probably not involved in the metabolism of EB1089 whereas, in the case of MC1473 and MC1611, it does appear to carry ou t side-chain hydroxylation. Our interpretation is that the conjugated double bond system in the side-chain of EB1089 is responsible for dire cting the target cell hydroxylation to the distal positions, C-26 and C-26a. We conclude that EB1089 is slowly metabolized via unique in vit ro metabolic pathways, and that these features may explain the relativ e stability of EB1089 compared to other analogs in vivo. (C) 1997 Else vier Science Inc.