Martin John Calverley

20-epi-vitamin D3 analogues: a novel class of potent regulators of cell growth and immune responses.

The 20-epi-vitamin D3 analogues are a novel class of vitamin D3 derivatives, structurally related to 1 alpha,25-dihydroxycholecalciferol (1 alpha,25(OH)2D3). They are characterized by an altered stereochemistry at carbon 20 in the side-chain. In vitro, these new analogues were found to be considerably more potent as regulators of growth and differentiation in the human histiocytic lymphoma cell line U 937 than 1 alpha,25(OH)2D3, despite a practically unchanged calcemic activity in vivo. The most potent analogue, KH 1060, inhibited cell proliferation by 50% at 10(-12) M (14,000 times more active than 1 alpha,25(OH)2D3). At the same time, KH 1060 induced cell differentiation at concentrations as low as 10(-14)M. In addition, the 20-epi-vitamin D3 analogues were found to be very potent inhibitors of T-lymphocyte proliferation induced by interleukin-1 or alloantigen. In this respect, they were several orders of magnitude more active than the potent immunosuppressive agent cyclosporin A (CyA). KH 1060, the most potent analogue, inhibited interleukin-1-induced mouse thymocyte proliferation by 50% at 3 x 10(-16) M and allogeneic stimulation of mouse spleen lymphocytes at 5 x 10(15) M. These effects were considered to be mediated by inhibition of interleukin-2 release from activated T-lymphocytes. The new analogues are of potential interest in the prevention of graft rejection and in the treatment of psoriasis, cancer and auto-immune diseases.

Increased biological activity of 20-EPI-1,25-dihydroxyvitamin D3 is due to reduced catabolism and altered protein binding☆

The 20-epi series of vitamin D3 analogs has been shown to be made up of more potent inducers of cell differentiation than calcitriol in vitro. Using 20-epi-1 alpha,25-dihydroxyvitamin D3 (MC 1288), we attempted to rationalize this increased biological activity by examining several parameters including the binding affinity of the analog for the plasma binding globulin (DBP) and the target cell vitamin D receptor (VDR), as well as attempting to measure the rate at which MC 1288 is metabolized. MC 1288 was found to be metabolized 36 times more slowly than its epimer 1,25-dihydroxy vitamin D3 (1,25-(OH)2D3), forming several metabolites which were analogous to metabolites of 1,25-(OH)2D3 formed in the side chain oxidation pathway. Bovine thymus VDR bound MC 1288 with five times greater affinity than calcitriol, while rat plasma DBP did not bind MC 1288 even at a concentration of 50 microM, 5000 times the B50 of 25-OH-D3, the ligand used in the assay. Using a vitamin D-inducible growth hormone gene reporter system we were able to demonstrate that MC 1288 induces human growth hormone (hGH) activity 30-fold more efficiently than 1,25-(OH)2D3 in the presence of fetal calf serum (FCS), while the analog is only 10 times more efficient than 1,25-(OH)2D3 in the absence of FCS. We therefore conclude that MC 1288 is more biologically active than calcitriol in vitro due to a combination of factors: the increased VDR binding affinity, the decreased DBP binding affinity, and the decreased rate of metabolism. As with other analogs of vitamin D, the altered protein binding and decreased catabolism of MC 1288 may be important in pharmaceutical applications such as a topical treatment for psoriasis or skin cancer.

In vitro metabolism of Calcipotriol (MC 903), a vitamin D analogue

Calcipotriol (MC 903) is a novel vitamin D analogue which combines potent effects on cell proliferation and differentiation with a decreased activity on calcium metabolism. During preliminary pharmacokinetic studies in rats and pigs, a rapid metabolism of MC 903 was observed, and the presence of a metabolite (X) in plasma was disclosed by the HPLC method used for determination of MC 903. The present report describes the production of this metabolite by incubation of MC 903 with post-mitochondrial supernatant from rat liver, its isolation, and its identification by comparison of UV, NMR and MS properties with synthetic material

Metabolism of a cyclopropane-ring-containing analog of 1α-hydroxyvitamin D3 in a hepatocyte cell model: Identification of 24-oxidized metabolites

MC969 is an analog of the calcemic drug 1 alpha-hydroxyvitamin D3 (1 alpha-OH-D3) in which carbons 25,26, and 27 in the side chain are incorporated into a cyclopropane ring. Metabolites of MC 969 were generated in an in vitro human hepatocyte cell model, Hep 3B. The identity of the metabolites was established by comigration on HPLC with authentic standards, and by mass spectrometry of native and chemically modified metabolites. Unequivocal identification of the 24-keto- and the two epimeric 24-alcohol metabolites is provided. No 25-hydroxylated metabolites were detected. In competition studies, MC 969 was able to inhibit 25-hydroxylation of tritiated vitamin D3 more effectively than 1 alpha-OH-D3 itself, indicating that the vitamin D3-25-hydroxylase may be responsible for generation of one or more of the metabolites observed.

Synthesis and biological evaluation of MC 1357, a new 20-epi-23-oxa-1α,25-dihydroxy-vitamin D3 analogue with potent non-classical effects

Abstract The synthesis of the 20-epi-23-oxa-24a,24b-dihomo analogue (MC 1357) of 1α,25-dihydroxy-vitamin vitamin D3 is described. In classical receptor binding and in vivo calcemic effect assays MC 1357 is slightly less potent than the natural hormone. However, in its in vitro effects on cancer cell differentiation and proliferation and on the mixed lymphocyte reaction it is 2 to 3 orders of magnitude more potent.

Polyclonal antibodies to EB1089 (seocalcitol), an analog of 1α, 25-dihydroxyvitamin D3

Abstract A hapten derivative of EB1089 [1(R),3(S),25-trihydroxy-26,27-dimethyl-9,10-seco-24-homocholesta-5(Z),7(E),10(19),22(E),24(E)-pentaene], a side-chain analog of 1α,25-dihydroxyvitamin D 3 , was synthesized for raising antibodies with a high specificity for EB1089. The A-ring moiety of EB1089 was replaced in the hapten by a linker for conjugation to a protein. Three polyclonal antibodies were obtained by immunizing rabbits with a BSA-conjugate of the hapten. The antibodies were characterized for titer, avidity and specificity using an enzyme immunoassay with covalently bound EB1089. The three antibodies had similar binding profiles and were highly selective for EB1089 and its metabolites over the naturally occurring vitamin D metabolites. Cross-reactivities with 25-hydroxyvitamin D 3 , the most abundant vitamin D metabolite in serum, were in the range 0.01–0.2% relative to EB1089.

Polar metabolites of dihydrotachysterol3 in the rat : comparison with in vitro metabolites of 1α,25-dihydroxydihydrotachysterol3

The metabolism of 25-hydroxydihydrotachysterol3 (25-OH-DHT3) to more polar metabolites was investigated in vivo in the rat and compared with the in vitro metabolism of 1 alpha,25-dihydroxy-DHT3 (1 alpha,25-(OH)2DHT3) in the osteosarcoma cell line UMR 106. Rats were given 2 mg of DHT3 in divided doses at 0 and 6 hr. Plasma was collected 24 hr after the initial dose, extracted, separated, and polar metabolites purified by HPLC. A number of polar metabolites were formed in vivo with mass spectrometric characteristics which suggested that they were derived from a previously isolated metabolite of 25-OH-DHT3, T3/H. Of these, four were isolated and identified as 24-oxo-T3/H, 24-hydroxy-T3/H, 26-hydroxy-T3/H and the 26,23-lactone of T3/H. In view of the identification of T3/H as a mixture of 1 alpha- and 1 beta-hydroxylated 25-OH-DHT3, osteosarcoma cells (UMR 106) were incubated with chemically synthesized 1 alpha,25-(OH)2DHT3 in an attempt to determine from which component of the T3/H mixture these metabolites were derived. Again, more polar metabolites were formed and five of these were isolated by lipid extraction, purified by HPLC and identified as 24-oxo-1 alpha,25-(OH)2DHT3, 1 alpha,23,25-(OH)3DHT3, 24-oxo-1 alpha,23,25-(OH)3DHT3, 1 alpha,24,25-(OH)3DHT3 and 1 alpha,25,26-(OH)3DHT3. Three of the in vitro metabolites were similar to those found in rat plasma but only two of these metabolites were available in sufficient amounts to allow comparison. The chromatographic characteristics, using HPLC and gas chromatography, of these two pairs of metabolites (24-oxo and 24-hydroxy) were examined and it was demonstrated that they were not the same. It is therefore suggested that the polar metabolites formed in vivo are in fact metabolites of the T3/Hb component (1 beta,25-(OH)2DHT3) rather than the T3/Ha component (1 alpha,25-(OH)2DHT3). Supporting evidence for this suggestion was obtained when a small quantity of 1 beta,25-(OH)2DHT3, obtained from chemically synthesized 1 beta-OH-DHT3 by incubation with Hep 3B cells, was further incubated in the osteosarcoma UMR 106 system. Preliminary studies indicated that the putative 24-oxo and 24-hydroxy metabolites formed from 1 beta,25-(OH)2DHT3 had chromatographic and mass spectral properties almost indistinguishable from those of corresponding metabolites of T3/H formed in vivo. All the metabolites formed in vivo and in vitro are components of two metabolic pathways described previously for 25-hydroxyvitamin D3 and also for 25-OH-DHT3.

1α,24S-dihydroxy-26,27-cyclo-22-yne-vitamin D3: the side chain triple bond analogue of MC 903 (Calcipotriol)

Abstract The side chain propargylic alcohol function (established stereoselectivity via S-Alpine-BoraneR reduction of ynone 8 and correlated with MC 903) in the title compound 1 replaces the metabolically labile allylic alcohol function of MC 903, a selective analogue of the vitamin D hormone used for treating psoriasis. 1 exhibits reduced in vitro activity but still shows selectively much lower in vivo calcemic effects.