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Home , CYP27A1, CYP2R1, CYP3A4

ANTICANCER RESEARCH 26: 2589-2596 (2006)

Hepatic and Inactivation of Clinically-relevant D Analogs and

GLENVILLE JONES1, VALARIE BYFORD1, SHELLY WEST1, SONOKO MASUDA1, GEORGE IBRAHIM1, MARTIN KAUFMANN1, JOYCE C KNUTSON2, STEPHEN STRUGNELL2 and RAJENDRA MEHTA3

1Department of Biochemistry, Queen’s University, Kingston, ON, Canada; 2Bone Care International, Madison, WI; 3IIT Research Institute, Chicago IL, U.S.A.

Abstract. Like most pharmaceutical agents, analogs the nature of the P450 (CYP) species are subject to hepatic by a variety of cytochrome involved in the 25- of D2 and D3 P450 (CYP)-based systems. Metabolism can involve activation has become much clearer, even if it has not been fully as well as inactivation of the vitamin D analog and one of the resolved (2). One mitochondrial CYP and four more successful families includes the 1·-hydroxyvitamin D microsomal CYPs have been implicated as potential prodrugs (1·-OH-D2,1·-OH-D3,1·-OH-D4,1·-OH-D5), that all vitamin D-25-hydroxylases, though it appears that require a step of activation. Some of these prodrugs are in use or CYP2R1 possesses many of the criteria to be identified clinical trial because they have a therapeutic advantage over as the physiologically-relevant (3), including a . However, the nature of the activation of these type of human associated molecules is poorly understood, particularly with regard to the with a mutant form of the CYP2R1 enzyme (4). These CYP isoform involved. Various transfected CYPs and hepatic cell criteria are worth repeating: lines combined with tandem LC-MS analysis were used to i) Kinetic experiments suggest that there are at least two investigate the metabolism of a spectrum of vitamin D analogs, isoforms in the mitochondrial and including 1·-OH-Ds and the topical analog, calcipotriol. In the (microsomal) compartments. case of the 1·-OH-Ds, evidence was found of multiple sites of ii) The mitochondrial form is a high capacity, low affinity side-chain hydroxylation consistent with the generation of more ‘pharmacologically-relevant’ enzyme while the microsomal than one active form. The potential involvement of CYP27A and form is a low capacity, high affinity “physiologically-relevant” other putative 25-hydroxylases in 1·-OH-D activation was also enzyme. shown, as well as the potential for CYP24 activation and iii) Together the two must explain the in vivo inactivation. In the case of calcipotriol, the respective roles of observations that 25-hydroxylase is weakly regulated and non-vitamin D-related CYPs and CYP24 in the catabolism of leads to side-chain hydroxylation of both vitamins D2 and D3. this anti-psoriatic drug were dissected out using cell lines with or There are claims that CYP27A1 has many of the without CYP24 expression, allowing us to demonstrate the properties ascribed to the mitochondrial enzyme (5), though potential contribution of CYP24 to "vitamin D resistance". The strangely it will 24-hydroxylate vitamin D2 but will not implications of hepatic metabolism in the context of other facets 25-hydroxylate it at micromolar concentrations. Of thought to play a role in the mechanism of action of anticancer the microsomal forms, only CYP2R1 meets all the criteria and antiproliferative vitamin D analogs are discussed. ascribed to the “physiologically-relevant” enzyme including the ability to 25-hydroxylate vitamin D2. Comparisons of The importance of the liver in the activation of vitamin putative microsomal 25-hydroxylases have been published D has long been recognized (1). Over the past decade, (6, 7), but no studies have demonstrated the high affinity nature of the enzyme. Few studies of the hepatic metabolism of vitamin D analogs have been published. The principal exceptions to Correspondence to: Glenville Jones, Department of Biochemistry, this statement are studies of the 1·-hydroxyvitamin D Queen’s University, Kingston, ON, Canada. e-mail: gj1@post. prodrugs including 1·-OH-D (One-alpha; Leo Pharma, queensu.ca 3 Denmark); 1·-OH-D2 (Hectorol; Bone Care International, Key Words: Vitamin D analogs and prodrugs, hepatic activation, Madison, WI, USA); 1·-OH-D4; 1·-OH-D5 (IIT, Chicago calcitriol, CYP. IL, USA) (8). These compounds have proven to be highly

0250-7005/2006 $2.00+.40 2589 ANTICANCER RESEARCH 26: 2589-2596 (2006) successful in the treatment of defects of calcium homeostasis and hyperparathyroidism associated with chronic kidney disease (CKD). All of these 1·-hydroxylated analogs are presumed to be active by virtue of being metabolized in vivo to side-chain hydroxylated species analogous to 1·,25-(OH)2D3 in order to execute their therapeutic effects (8). The 1·-OH-D5 (IIT) has been shown to act as a chemopreventative agent in mammary carcinogenesis models and has been approved for NCI trials in patients (9). However, currently no metabolic studies of 1·-OH-D5 have been Figure 1. Enzymatic activation of 1·-hydroxylated vitamin D prodrugs by published and, thus, in vitro metabolic studies using liver liver enzymes. The putative species are indicated by the letter codes. All CYPs, except for the mitochondrial CYP27A1, are systems would seem to be justified. Furthermore, though in microsomal. vitro studies of the metabolism of 1·-OH-D3 and 1·-OH-D2 have been carried out and have suggested that a variety of mitochondrial and microsomal enzymes can hydroxylate these prodrugs (Figure 1) (5-7), there have been no studies Cell culture. HepG2 and V79-4 were obtained from ATCC to determine if the newer isoforms (e.g., CYP2R1) can (Manassas,VA, USA) and V79-hCYP24 cells have been described side-chain hydroxylate 1·-hydroxylated prodrugs at elsewhere (5, 12). Vectors containing mCYP2R1 and hCYP2R1 (13) were kindly provided by Drs. David W. Russell and David physiological substrate concentrations. Mangelsdorf (Department of Molecular Genetics, University of Calcipotriol (MC-903) is a vitamin D analog well-studied Texas Southwestern Medical Center Dallas, TX, USA). The V79-4 metabolically in vitro. Sorensen and colleagues (10) established cells (1.5x105 cells) were transiently transfected with 0.2 Ìg of that various liver preparations are able to convert the pCMV6-mCYP2R1 or pCMV6-hCYP2R1 in the presence of 10 Ìl 24-hydroxylated analog into unsaturated and saturated 24- lipofectamine and 10 Ìl Plus reagents (Invitrogen) in serum-free ketones and this work was extended by Masuda et al. (11), who DMEM for 3 h at 37ÆC. Cultured or transfected cells were cultured showed that this metabolic pathway was present in vitamin D- to confluence in FCS-containing medium and then incubated with vitamin D substrates, in the absence of FCS but containing 1% target cells and went further to give 23- and 24- hydroxylated bovine serum albumin, for the indicated time period. products as well as side-chain-cleaved metabolites. One question not answered in earlier work was the nature of the Vitamin D analysis. HPLC was carried out using Waters Alliance enzyme(s) responsible for the metabolism of calcipotriol. Since 2695 separations module, a photo-diode array detector and a calcipotriol is topically applied, the metabolism of the drug in Berthold on-line radioactivity detector, as described previously keratinocytes would seem particularly relevant. To date, since (12). In the 1·-OH-D5 metabolic studies, metabolites were the highly inducible CYP24 represents the only vitamin analyzed by LC-MS (Micromass Ultima) in ES+ scanning mode over a m/z range of 50-600, using a methanol/water-based gradient D-metabolizing CYP identified in vitamin D target cells it system on Zorbax SB-C18 at 200 Ìl/min over 30 min. Metabolites seems logical to believe that CYP24 is involved in calcipotriol of 1·-OH-D5 were identified on the basis of their characteristic metabolism, but this hypothesis remains untested and molecular ions, dehydration products and other adducts after unproven. With the availability of newer tools such as isolated comparison to the side-chain metabolites of 1·-OH-D3 produced CYPs, newly-synthesized 1·-hydroxylated vitamin Ds and the in the same cell line. powerful new LC-MS technology, the metabolic objectives of the current study were to explore: Results i) The ability of CYP2R1 in a cell model in situ to 3 3 metabolize a physiological concentration of [9,11- H]1·- Enzymatic properties of CYP2R1 towards [9,11- H]1·-OH-D2. OH-D2. Paramount in the proposed criteria to discriminate between ii) The liver-cell mediated metabolism of 1·-OH-D5 “physiologically-relevant” 25-hydroxylases and those CYP using newer LC-MS analytical procedures (11). enzymes that will simply perform this reaction in vitro under iii) The putative CYP24-mediated metabolism of super-physiological conditions of high substrate concentration, calcipotriol. is the experiment to explore if a putative 25-hydroxylase will 25-hydroxylate a vitamin D2 substrate at nanomolar Materials and Methods concentration. The availability of high specific activity radiolabelled [9,11-3H]1·-OH-D allowed us to test CYP2R1 Vitamin D analogs. Synthetic standard 1·-OH-D and 1·,25-(OH) D 2 3 2 3 for this property using transient transfection in a host V79 were provided by Leo Pharma (Ballerup, Denmark), 1·-OH-D2 and 3 cell devoid of basal activity. Figure 2 shows the results of [9,11- H]1·-OH-D2 (35 Ci/mmol) by Bone Care International and 1·-OH-D5 by IIT Research Institute (Chicago, IL, USA). experiments using either mouse or human enzyme

2590 Jones et al: Hepatic Activation and Inactivation of Clinically-relevant Vitamin D Analogs and Prodrugs

Figure 2. Transfection studies of (A) human CYP2R1 and (B) mouse CYP2R1. Vectors containing the CYPs under investigation were transiently 3 transfected in V79-4 host cells and then incubated with 10 nM [9,11- H]1·-OH-D2 for 24 h. HPLC of the extracts using an on-line radioactivity detector gave the traces shown. Standard 1·,25-(OH)2D2 co-migrated with the radioactive peak in each case. Note that the slight shifts in the retention times of both substrate and product in panels (A) and (B) were due to day-to-day variability in column conditions. NTCC=non-transfected cell control; DCC=dead cell control.

3 incubated with 10 nM [9,11- H]1·-OH-D2 for 24 h. In the metabolites of 1·-OH-D5 ran more polar than the parent chromatogram traces depicted, no products were formed in molecule but less polar than 1·,25-(OH)2D3. On LC-MS control extracts (parental V79 cells or dead-cell controls) (Figure 3), these metabolites all showed spectra consistent with while, after the expression of mouse or human CYP2R1, an them being mono-hydroxylated products with MH+ ions of m/z additional peak appeared running between 12-13 min in 445 (MWt=444; MWt of parent 1·-OH-D5=428). In each trace and co-migrating exactly with 1·,25-(OH)2D2. comparison, the LC-MS of 1·,25-(OH)2D3 derived from 1·- This peak was absent when CYP27A1 was transfected into OH-D3 is also contrasted with its parent molecule in Figure the same cells (data not shown). 3. Though the mono-hydroxylated products of 1·-OH-D5 are likely to be hydroxylated in the side-chain, LC-MS/MS Metabolites of 1·-OH-D5 from HepG2 cells evaluated using analysis, and possibly other technologies are required to LC-MS-based analytical procedures. When 1·-OH-D5 (10 ÌM) pinpoint the exact site of this hydroxylation. These studies is incubated with HepG2 cells for 24-48 h a number of putative are on-going, but the current experiments have established hydroxylated products are produced which elute that hepatoma cells are able to metabolize the 1·-OH-D5 chromatographically more slowly than the substrate and close substrate to hydroxylated products, albeit less efficiently to 1·,25-(OH)2D3 on straight-phase HPLC. Compared with than 1·-OH-D3. control incubations using the same cells and 1·-OH-D3 (10 ÌM), where there is a demonstrable production of 1·,25- Metabolism of calcipotriol(MC-903) by V79-hCYP24 cells. (OH)2D3 and 1·,26-(OH)2D3, the yield of metabolites from Calcipotriol metabolism can be readily followed in both 1·-OH-D5 was disappointingly low, suggesting that the rate of hepatoma and keratinocyte cell models, suggesting that this activation in the liver was much less efficient for 1·-OH-D5 cyclopropane ring-containing analog is subject to the same than for 1·-OH-D3. On reverse-phase HPLC, hydroxylated type of modification at the C-24 position that is observed

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Figure 3. LC-MS of specific isolated metabolic products of 1·-OH-D3 and 1·-OH-D5 produced by HepG2 cells along with their respective substrates. MS1 spectra were produced in electrospray positive (ES+) mode using a Micromass Quattro Ultima triple-quadrapole instrument in MS1 mode. Metabolites were generated by incubating 1·-OH-D3 or 1·-OH-D5 with HepG2 cells and the products isolated using preparative LC on Zorbax-SIL. Mass spectra were performed on-line using a reverse-phase LC step. ES+ spectra showed the usual MH+ ions, their dehydration products and various adducts of the metabolites. Asterisks on the 1·-OH-D5 molecule are putative hydroxylation sites targeted by hepatic CYPs.

in broken cell systems and in vivo and described previously although this was not observed when the experiments were 3 (10, 11). The studies performed here represent the first performed with [1‚- H]1·,25-(OH)2D3. In any event, the attempt to use an individual CYP, expressed in a stable ability of V79-4 to generate metabolites was extremely manner in a Chinese hamster lung V79 host cell, to study weak. In contrast, V79-hCYP24 cells showed a robust its potential involvement in the metabolism of calcipotriol. production of a full spectrum of calcipotriol metabolites, In Figure 4, it is important to note that the host cell showed observed previously in liver and keratinocyte models (10, virtually undetectable metabolism without the introduction 11). While it was believed possible that CYP24 might be of the hCYP24 (Panel A). It is possible that the trace responsible for the production of C-24 ketone Peak 3, the amount of Peak 3 in the chromatogram was incorrectly subsequent 23-hydroxylated products (Peak 4; actually a identified as the metabolite simply co-migrates with the mixture of 4a and 4b) and side-chain cleaved products, saturated ketone of MC903 depicted in the inset of Figure there was no firm evidence to prove this. Though the data 4A. This possibility is rendered more plausible considering suggest CYP24 may be responsible for the saturation of the that there was no evidence of the formation of its ¢22-23 in the side-chain of calcipotriol, these studies cannot precursor, the unsaturated ketone (Peak 2 of Figure 4A). exclude the fact that another enzyme is responsible and is Another trivial explanation is that there was a minor unmasked by the production of the unsaturated ketone amount of native, inducible CYP24 in the host V79 cells, (Peak 2 of Figure 4B) by CYP24.

2592 Jones et al: Hepatic Activation and Inactivation of Clinically-relevant Vitamin D Analogs and Prodrugs

Figure 4. Lipidomic studies of the metabolites of calcipotriol produced by (A) V79-4 cells and (B)V79-hCYP24 over a 48-h incubation period. Cells were incubated with calcipotriol (10 ÌM) for incubation times from 0-48 h and the lipid extracts run on HPLC using a photo-diode array detector. Chromatograms show A265nm traces but putative hCYP24-generated metabolite peaks were checked for their vitamin D chromophore. Only peaks marked #1-4 in the traces possessed the characteristic vitamin D chromophore. Calcipotriol was Peak 1 running at 13.5-14 min and the shoulder just preceding at 13 min was pre-vitamin D. The structures of putative metabolites marked Peaks 2, 3 and 4 are shown in Panel (A).

Discussion CYP2R1 is mutated in a patient with a form of hereditary rickets (4), and the distribution and substrate specificity The availability of expression systems which contain specific, studies produced by Russell’s group (13) and others (7) cloned cytochrome 450s has opened up the possibility of suggest that it must be the ‘physiologically-relevant’ dissecting out the role of individual enzymes in the microsomal form of the 25-hydroxylase. It remains to be metabolism of vitamin D and its analogs. Nowhere is this seen if others can demonstrate the relevance of CYP2J3 and truer than with the subset of CYPs found in the liver and CYP3A4 to vitamin D physiology, while for the believed to be primarily involved in activation of the parent mitochondrial form, CYP27A, the most generous view of its molecule and its prodrug analogs, but the approach can also role in vitamin D homeostasis is that it represents a be used for target cell CYPs involved in the metabolism of “pharmacologically-relevant” 25-hydroxylase enzyme. calcitriol analogs. Here the usefulness of the single CYP Though 1·-OH-D2 (Hectorol) is marketed for treatment of approach was demonstrated by showing that CYP2R1 was patients with secondary hyperparathyroidism associated with capable of the 25-hydroxylation of 1·-OH-D2 at nanomolar CKD, this analog has potential in the treatment of concentrations and that CYP24 was responsible for some (if hyperproliferative conditions. Indeed, one of its activated not all) of the steps of calcipotriol metabolism. Though, the metabolites, 1·,24-(OH)2D2, formed by the action of approaches used entirely different substrate concentrations CYP27A1 and CYP3A4, has been used to regulate the and, therefore, different modes of detection, the results growth of prostate cancer cells in vivo and in vitro (14). The were equally definitive. finding that there are also several enzymes in the The finding that CYP2R1 will 25-hydroxylate [9,11- microsomal fraction that will activate 1·-OH-D2 to 1·,25- 3 H]1·-OH-D2 at nanomolar concentrations makes this (OH)2D2, and that CYP2R1 is one of them, is valuable new isoform unique and, taken together with the assertion that information which opens up the possibility that the prodrug

2593 ANTICANCER RESEARCH 26: 2589-2596 (2006) can be activated and have growth inhibitory activity in extra- D analyst, it shows a disappointing inability to distinguish hepatic sites, since CYP2R1 is known to be more widely between isomers of vitamin D. In this respect, it is markedly distributed than in the liver alone (13). inferior to its more laborious but more structurally- The other finding that CYP24 was involved in calcipotriol informative sister technique of GC-MS (19). In the case metabolism is novel and relevant to those using calcipotriol such as here, where no synthetic hydroxylated derivatives of for the treatment of psoriasis. Clinical reviews of the use of 1·-OH-D5 are currently available to be compared with topical calcipotriol report that approximately 70% of mild unknown metabolites, the analyst is unable to assess LC to moderate psoriasis patients respond positively to retention times and has no definitive fragments to base treatment (15). Whether these patients develop a resistance identification upon. Consequently, the identification is to calcipotriol or whether there are other mechanisms to equivocal. Nevertheless, the number (three) of distinct explain the lack of responsiveness to calcipotriol in 30% of metabolite peaks in the extracts of liver-derived cells is less patients remains unclear. It is well known that the CYP24 than the number (seven) of possible hydroxylation sites in is highly inducible by vitamin D (16), so it is the side-chain of 1·-OH-D5 (See Figure 3, lower left panel), interesting to speculate that tissues exposed to such agents our work suggests that the enzymes involved generated a build up a resistance to the actions of the vitamin D analogs finite number of isomers. Based upon our experience with by CYP24 induction. Others have produced human tumor liver cells and 1·-OH-D3, we would expect one of these staging data to support the idea that CYP24 is a potential products to be 1·,25-(OH)2D5, though 1·,26-(OH)2D5 or tumor suppressor gene which presumably acts by lowering 1·,29-(OH)2D5 are other possibilities (20, 21). the 1·,25-(OH)2D3 levels in cancer cells (17). Thus, the Overall, the studies reported here demonstrate the demonstration that CYP24 is involved in the catabolism of versatility of the CYP complement of the liver to metabolize calcipotriol would suggest that patients who exhibit elevated any synthetic vitamin D analog made by pharmaceutical CYP24 in responding tissues might show short-term chemists and indicates the complexity of the task of making improvement or fail to respond altogether. A test of this effective anticancer vitamin D drugs that cause growth argument might be the use of a CYP24 inhibitor combined arrest in cancer cells before they are inactivated by CYPs. with a vitamin D in “resistant” or non-responsive patients. Such a strategy is not dissimilar to that currently Acknowledgements in advanced clinical trials by Cytochroma Inc. (Markham, Canada). The finding that CYP24 might be responsible for This work was supported by an operating grant MT-9475 to Glenville the saturation of the ¢22-23 in the side-chain of calcipotriol Jones from the Canadian Institutes of Health Research. Martin Kaufmann is supported by an Ontario Graduate Scholarship. is reminiscent of our work (18) using 1·,24-(OH)2D2 to produce saturated, side-chain-cleaved metabolites when incubated with the CYP24-containing keratinocyte HPK1A- References ras. The success of our studies suggests that V79-CYP24 cell 1 DeLuca HF: Chapter 1: "Historical Perspective". In: Vitamin D, assays (with the V79 control cells) could be used to Second Edition. Feldman D, Pike W and Glorieux F (eds.). determine the potential role of CYP24 in the catabolism of Academic Press, San Diego, CA, pp. 3-14, 2005. all calcitriol analogs in a routine manner, along with the 2 Prosser DE and Jones G: Enzymes involved in the activation usual battery of hepatic CYP assays commonly employed and inactivation of vitamin D. Trends Biochem Sci 29: 664-673, during pharmacology testing 2004. 3 Jones G, Strugnell S and DeLuca HF: Current understanding The study of 1·-OH-D5 by HepG2 cells provided the first positive evidence that the addition of the C24-C29 ethyl of the molecular actions of vitamin D. Physiolog Rev 78: 1193- group in the side-chain of 1·-OH-D does not block hepatic 1231, 1998. 5 4 Cheng JB, Levine MA, Bell NH, Mangelsdorf DJ and Russell cell activation of the prodrug. Though the efficiency of DW M: Genetic evidence that the human CYP2R1 enzyme is a 1·-OH-D5 modification was certainly reduced over that key vitamin D 25-hydroxylase. Proc Natl Acad Sci USA 101: observed in the control cultures with 1·-OH-D3, 7711-7715, 2004. measurable amounts of hydroxylated products were still 5 Guo YD, Strugnell S, Back DW and Jones G: Substrate formed. To date, these products have only been partially specificity of the liver mitochondrial cytochrome P-450, CYP- 27, towards vitamin D and its analogs. Proc Natl Acad Sci USA identified as mono-hydroxylated metabolites of 1·-OH-D5. A more definitive identification is impossible because of the 90: 8668-8672, 1993. 6 Gupta RP, Hollis BW, Patel SB, Patrick KS and Bell NH: lack of useful fragments in the MS1 mode. Further CYP3A4 is a human microsomal vitamin D 25-hydroxylase. J information should be available after the same compounds Bone Miner Res 19: 680-688, 2004. have been subjected to the MS2 mode of LC-MS/MS. It 7 Shinkyo R, Sakaki T, Kamakura M, Ohta M and Inouye K: should be noted that, while LC-MS/MS is a valuable and Metabolism of vitamin D by human microsomal CYP2R1. convenient addition to the armamentarium of the vitamin Biochem Biophys Res Commun 324: 451-457, 2004.

2594 Jones et al: Hepatic Activation and Inactivation of Clinically-relevant Vitamin D Analogs and Prodrugs

8 Jones G: Chapter 81: "Analog Metabolism". In: Vitamin D, 17 Hodgson G, Hager JH, Volik S, Hariono S, Wernick M, Moore Second Edition. Feldman D, Pike W and Glorieux F (eds.). D, Nowak N, Albertson DG, Pinkel D, Collins C, Hanahan D Academic Press, San Diego CA, pp. 1423-1448, 2005. and Gray JW: Genome scanning with array CGH delineates 9 Mehta RG, Hussain EA, Mehta RR and Das Gupta TK: regional alterations in mouse islet carcinomas. Nat Genet 29: Chemoprevention of mammary carcinogenesis by 1alpha- 459-464, 2001. hydroxyvitamin D5, a synthetic analog of Vitamin D. Mutat Res 18 Jones, G, Byford V, Kremer R, Makin HLJ, Rice RH, 523-524: 253-264, 2003. deGraffenreid LA, Knutson JC and Bishop CA: Anti- 10 Sorensen H, Binderup L, Calverley MJ, Hoffmeyer L and proliferative activity and target cell catabolism of the vitamin D Andersen NR: In vitro metabolism of calcipotriol (MC 903), a analog, 1·,24(S)-dihydroxyvitamin D2 in normal and vitamin D analogue. Biochem Pharmacol 39: 391-393, 1990. immortalized human epidermal cells. Biochem Pharmacol 52: 11 Masuda S, Strugnell S, Calverley M, Makin HLJ, Kremer R and 133-140, 1996. Jones G: In vitro metabolism of the anti-psoriatic vitamin D 19 Jones G and Makin HLJ: Chapter 2: Vitamin D. In: Modern analog, calcipotriol, in two cultured human keratinocyte Chromatographic Analysis of the Vitamins, Third Edition. models. J Biol Chem 269: 4794-4803, 1994. VanBocxlaer J, Lambert W, De Leenheer A and Marcel 12 Masuda S, Kaufmann M, Byford V, Gao M, St-Arnaud R, Dekker (eds.). New York, pp. 75-141, 2000. Arabian A, Makin HLJ, Knutson JC, Strugnell S and Jones G: 20 Dilworth FJ, Williams GR, Kissmeyer A-M, Løgsted-Nielsen J, Insights into vitamin D metabolism using CYP24 over- Binderup E, Calverley MJ, Makin HLJ and Jones G: The expression and knockout systems in conjunction with liquid vitamin D analog, KH1060 is rapidly degraded both in vivo and chromatography/mass spectrometry (LC/MS). J in vitro via several pathways: principal metabolites generated Biochem Molecular Biol 89-90: 149-153, 2004. retain significant biological activity. Endocrinology 138: 5485- 13 Cheng JB, Motola DL, Mangelsdorf DJ and Russell DW: De- 5496, 1997. orphanization of cytochrome P450 2R1: a microsomal vitamin 21 Shankar VN, Dilworth FJ, Makin HLJ, Schroeder NJ, Trafford D 25-hydroxylase. J Biol Chem 278: 38084-38093, 2003. DAJ, Kissmeyer A-M, Calverley MJ, Binderup E and Jones G: 14 Bauer JA, Thompson TA, Church DR, Ariazi EA and Wilding Metabolism of the vitamin D analog EB1089 by cultured human G: Growth inhibition and differentiation in human prostate cells: redirection of hydroxylation site to distal carbons of the carcinoma cells induced by the vitamin D analog 1·,24- side chain. Biochem Pharmacol 53: 783-793, 1997. dihydroxyvitamin D2. Prostate 55: 159-167, 2003. 15 Kragballe K: Treatment of psoriasis by the topical application of the novel analogue calcipotriol (MC 903) Arch Dermatol 125: 1647-1652, 1989. 16 Chibout S-D, de Brugerolle de Fraissinette A, Hartmann N, Picarles V, Grenet O, Cordier A, Molloy S and Medina J: Vitamin D3 24-hydroxylase mRNA expression in the skin of calcipotriol-treated psoriatic patients correlates with clinical Received December 29, 2005 efficacy. Archives Dermatolog Res Issue 295: 269-271, 2003. Accepted January 9, 2006

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