Anne-Marie Kissmeyer

Calcipotriol (MC 903): Pharmacokinetics in rats and biological activities of metabolites: A comparative study with 1,25(OH)2D3

Calcipotriol (MC 903) is a novel analogue of the physiologically active metabolite of vitamin D3, 1 alpha,25-dihydroxycholecalciferol [1,25(OH)2D3]. MC 903 and 1,25(OH)2D3 have similar effects on cell proliferation and cell differentiation in vitro using the human histiocytic lymphoma cell line U 937, but in vivo MC903 has 100-200 times less effect on calcium metabolism. To elucidate this difference, the pharmacokinetic profiles after a single intravenous dose (50 micrograms/kg) of the two compounds to rats were compared. The area under the serum level/time curve (AUC) was more than 100 times higher for 1,25(OH)2D3 than for MC903 and the rate of clearance was more than 100 times higher for MC903 than for 1,25(OH)D3. Serum from MC903 or 1,25(OH)2D3 dosed rats (i.v. 10 micrograms/kg) was investigated for biological activities by incubation of U 937 cells with serum collected 0-24 hr after drug administration. Serum from MC903 dosed rats had an effect only when collected shortly after dosing, whereas serum from 1,25(OH)2D3 dosed rats had an effect when collected up to 4 hr after dosing. The biological effects on the U937 cells of the two major metabolites of MC903 (MC 1046 and MC 1080) were investigated. The metabolites had effects that were more than 100 times weaker than those of the parent compound. The effect of MC903 on proliferative disorders, its fast elimination and the formation of inactive metabolites makes MC903 suitable for topical treatment of psoriasis.

Sensitive analysis of 1α,25-dihydroxyvitamin D3 in biological fluids by liquid chromatography–tandem mass spectrometry

Abstract A liquid chromatographic–tandem mass spectrometric assay using 5% bovine serum albumin as the calibration matrix has been developed for the quantitative analysis of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] in biological fluids. The analyte was extracted from the matrix after protein precipitation using an automated solid-phase extraction procedure involving both a reversed-phase and normal-phase procedure on a single C18 cartridge. The analytical chromatography was performed using a Symmetry C8 50×2.1 mm, 3.5 μm column. The mobile phase was a linear gradient from 75 to 99% methanol with a constant concentration of 2 mM ammonium acetate. 1α,25(OH)2D3 and the internal standard [2H6]1α,25(OH)2D3 were detected by using MS–MS. The ion source was operated in the positive electrospray ionisation mode. The assay is specific, sensitive, and has a capacity of more than 100 samples per day, with a limit of quantitation of 20 pg ml−1 for a 1.0-ml sample aliquot. The assay has been used for the analysis of 1α,25(OH)2D3 in serum from rats and pigs simultaneously with the analysis of the vitamin D analog seocalcitol.

Determination of the vitamin D analog EB 1089 (seocalcitol) in human and pig serum using liquid chromatography–tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric assay in human and pig serum has been developed for quantitative analysis of EB 1089 (seocalcitol). EB 1089 is a novel vitamin D analog under development for the treatment of cancer. The analyte was extracted from serum after protein precipitation using an automated solid-phase extraction procedure involving both a reversed-phase and normal-phase procedure on a single C18 cartridge. The analytical chromatography was performed using a Symmetri C8 50x2.1 mm, 3.5 microm column. The mobile phase was a linear gradient from 75% to 99% methanol with a constant concentration of 2 mM ammonium acetate. EB 1089 and the internal standard [d6]-EB 1089 were detected by using MS-MS. The ion source was operated in the positive electrospray ionisation (ESI) mode. The assay is specific, sensitive, and has a capacity of more than 100 samples per day, with a limit of quantitation of 10 pg ml(-1) for a 1.0-ml sample aliquot. It is now used for routine analysis in connection with pharmacokinetic studies in humans and toxicokinetic studies in pigs.

UVB‐induced production of 1,25‐dihydroxyvitamin D3 and vitamin D activity in human keratinocytes pretreated with a sterol Δ7‐reductase inhibitor

The skin fulfills an important role in the vitamin D photo‐endocrine system. Epidermis is not only the site of vitamin D3 photoproduction. In addition, epidermal keratinocytes contain the vitamin D receptor (VDR) and possess 25‐hydroxylase and 1α‐hydroxylase activity indicating that all components of the vitamin D system are present. We investigated whether these components cooperate in inducing vitamin D activity upon treatment with physiological UVB doses. Upon irradiation, 24‐hydroxylase mRNA was induced in keratinocytes pretreated with a sterol Δ7‐reductase inhibitor (BM15766) whereby the 7‐dehydrocholesterol content increased by 300‐fold. Transfection experiments with a vitamin D response element containing construct confirmed VDR‐dependent gene activation. Furthermore, the UVB‐dependent induction of 24‐hydroxylase was blocked by the cytochrome‐P450 inhibitor ketoconazole. The 24‐hydroxylase inducing photoproduct was transferable to unirradiated keratinocytes by medium and cellular homogenates of UVB‐irradiated, BM15766‐pretreated cells and was identified as 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] by high‐performance liquid chromatography with tandem mass spectrometric detection. Addition of vitamin D binding protein blunted UVB‐induced 24‐hydroxylase suggesting the possibility of a paracrine or autocrine role for 1,25(OH)2D3. In conclusion, epidermal keratinocytes can produce vitamin D3, convert it to 1,25(OH)2D3 and respond to it upon UVB irradiation in the absence of exogenous 7‐dehydrocholesterol and therefore contain a unique and complete photo‐endocrine vitamin D system. J. Cell. Biochem. 98: 81–92, 2006.

Metabolism of the vitamin D3 analogue EB1089 alters receptor complex formation and reduces promoter selectivity

1 1α,25‐dihydroxyvitamin3 (VD) is a nuclear hormone that has important cell regulatory functions but also a strong calcemic effect. EB1089 is a potent antiproliferative VD analogue, which has a modified side chain resulting in increased metabolic stability and a selective functional profile. Since EB1089 is considered for potential systemic application, it will be investigated to what extent its recently identified metabolites (hydroxylated at positions C26 and C26a) contribute to biological profile of the VD analogue. 2 Limited protease digestion analysis demonstrated that EB1089 is able to stabilize the high affinity ligand binding conformation of the VDR, starting at concentrations of 0.1 nM and affecting up to 80% of all receptor molecules. The metabolites EB1445 and EB1470 showed to be 100 fold less potent than EB1089, whereas the remaining three metabolites (EB1435, EB1436 and EB1446) showed a clearly reduced ability to stabilize the high affinity ligand binding conformation. Interestingly, at pharmacological concentrations all EB1089 metabolites stabilized a second, apparently lower affinity conformation to a much higher extent than EB1089. 3 In reporter gene assays all metabolites showed lower potency than EB1089. Moreover, the preference of EB1089 for activation of VDR binding to sites formed by inverted palindromic arrangements spaced by nine nucleotide (IP9‐type VD response elements) appeared to be reduced (with EB1445 and EB1470) or completely lost (with EB1435, EB1436 and EB1446). The ranking of EB1089 and its metabolites that was obtained by limited protease digestion and reporter gene assays was confirmed by an analysis of their antiproliferative effect in breast cancer cells. 4 The potency and selectivity of the EB1089 metabolites in mediating gene regulatory effects was found to be drastically reduced in comparison to the parent compound suggesting that the contribution of the metabolites to the biological effect of EB1089 is minor. However, the compounds showed to be interesting tools for understanding the selective biological profile of EB1089.

The tissue-specific distribution of 3H-seocalcitol (EB 1089) and 3H-calcitriol in rats

Seocalcitol (EB 1089) is under development for the treatment of hepato-cellular carcinoma (HCC). The tissue distribution of 3H-seocalcitol was investigated in comparison to 3H-calcitriol in rats. Quantitative whole-body autoradiography was used to quantify the tissue distribution. The greatest difference in distribution between the two compounds was observed in the bloodstream. For most tissues the ratio seocalcitol/calcitriol varied between 0.2 and 3.1. The concentration of radioactivity in the liver was almost the same for the two compounds. For seocalcitol the concentration in the liver was 10 times higher than in serum. Assuming that the liver/serum concentration ratio is the same in rats and humans, the concentration of seocalcitol in the human liver is expected to be higher than the concentration resulting in more than 50% inhibition of cancer cell proliferation, and thus pharmacologically effective in HCC. It is questionable whether calcitriol would be present in the human liver in sufficient concentrations to be effective for the treatment of HCC, as the antiproliferative activity of calcitriol is generally more than 10-fold lower compared to that of seocalcitol and as calcitriol can only be administered at a dose that is ca. three-fold lower than the dose of seocalcitol.

Metabolism of 1|[alpha]|,25(OH)2-20-epi-D3 Into a Stable, Biologically Active, Intermediary Metabolite. |[dagger]| 418

1α,25(OH)2-20-epi-D3, a synthetic analog of 1α,25(OH)2D3, differs from the natural hormone, by a simple modification in the stereochemistry of the methyl group at carbon-20 of the side chain. Previous studies have shown that the activity of the analog in inhibiting proliferation and inducing differentiation of leukemic cells is several fold greater than that of 1α,25(OH)2D3. At present, the various mechanisms responsible for the enhanced biological activities of the analog have not been clearly elucidated. In one study it is shown that the 20-epi analog induces a conformation of the vitamin D receptor(VDR) that is distinct from that induced by 1α,25(OH)2D3. The modified conformation of the VDR enhances dimerization of VDR with the retinoid X receptor resulting in increased transactivation activity. It is now established that the enhanced biological activities of synthetic analogs of 1α,25(OH)2D3 also depend on their stability and the rate at which they are metabolized. Therefore, in our present study we compared the metabolism of 1α,25(OH)2-20-epi-D3 with that of 1α,25(OH)2D3 in the isolated perfused rat kidney. The metabolism studies reveal that there is a partial block in the further metabolism of the analog, resulting in accumulation of the intermediary metabolite, 1α,25(OH)2-20-epi-24-oxo-D3 and its precursor, 1α,24,25(OH)3-20-epi-D3. We determined that 1α,25(OH)2-20-epi-24-oxo-D3 also induces a conformation of the VDR similar to that induced by the parent analog. Furthermore, this intermediary metabolite is nearly as potent as the parent analog at inducing transactivation of a gene construct containing the human osteocalcin vitamin D-responsive element linked to the thymidine kinase promoter and growth hormone reporter gene. Thus, we conclude that 1α,25(OH)2-20-epi-D3 is metabolically stable and the stability is at the level of its intermediary metabolites, which in turn contribute significantly to the overall biological activity of the analog.