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The Vitamin D Analog, KH1060, Is Rapidly Degraded Both in Vivo and in Vitro via Several Pathways: Principal Metabolites Generated Retain Significant Biological Activity¹

Departments of Biochemistry (F.J.D., G.J.) and Medicine (G.J.), Queen’s University, Kingston, Ontario, Canada K7L 3N6; Molecular Endocrinology Group (G.R.W.), Department of Medicine, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom W12 0NN; Leo Pharmaceutical Products (A.-M.K., J.L.N., E.B., M.J.C.), DK-2750 Ballerup, Denmark; and the Department of Clinical Biochemistry, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, University of London (H.L.J.M.), London, United Kingdom E1 2AD

Address all correspondence and requests for reprints to: Dr. Glenville Jones, Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vitamin D analogs are valuable drugs with established and potential uses in hyperproliferative disorders. Lexacalcitol (KH1060) is over 100 times more active than 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃], as judged by in vitro antiproliferative and cell differentiating assays. The underlying biochemical reasons for the increased biological activity of KH1060 are unknown, but are thought to include 1) metabolic considerations in addition to explanations based upon 2) enhanced stability of KH1060-liganded transcriptional complexes. In this study we explored the in vivo and in vitro metabolism of KH1060. We established by physicochemical techniques the existence of multiple side-chain hydroxylated metabolites of KH1060, including 24-, 24a-, 26-, and 26a-hydroxylated derivatives as well as side-chain truncated forms. KH1060 metabolism could be blocked by the cytochrome P450 inhibitor, ketoconazole. KH1060 was not an effective competitor of C24 oxidation of 1,25-(OH)₂D₃. Certain hydroxylated metabolites of KH1060 retained significant biological activity in vitamin D-dependent reporter gene systems (chloramphenicol acetyltransferase). Likewise, those metabolites accumulating in the target cell culture models in metabolism studies, particularly 24a-hydroxy-KH1060 and 26-hydroxy-KH1060, retained biological activities superior to those of 1,25-(OH)₂D₃ in native gene expression systems in vitamin D target cells (osteopontin and P450cc24). We conclude that KH1060 is rapidly metabolized by a variety of cytochrome P450-mediated enzyme systems to products, many of which retain significant biological activity in vitamin D-dependent assay systems. These results provide an explanation for the considerable biological activity advantage displayed by KH1060 compared with 1,25-(OH)₂D₃ in various in vitro assay systems.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
OVER THE past 15 yr, hundreds of vitamin D analogs have been synthesized in the hope of harnessing the cell differentiating/anticell proliferative properties of the hormone, 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] without its significant calcemic action (1). Some success has been realized in this pursuit, as judged by the widespread use of the antipsoriatic vitamin D analogs, calcipotriol and 1,24-(OH)₂D₃, and the continued development of several other analogs for psoriasis, several types of cancer, and hyperparathyroidism (2). Vitamin D analogs under advanced clinical testing include 22-oxa-calcitriol (OCT), 19-nor-1,25-(OH)₂D₂, and EB1089 (3, 4).

Perhaps the most promising analog recognized to date during routine in vitro screening is the Leo Pharmaceuticals drug, lexacalcitol, otherwise known by the Leo code, KH1060 (5) (Fig. 1). KH1060 is the most potent analog developed to date and is at least 100-fold more effective in cell proliferation assays than 1,25-(OH)₂D₃ while being only 1.3-fold more active in calcemic assays (6, 7, 8, 9, 10). In particular, KH1060 has been promoted as a potential immunosuppressive agent (reviewed in Ref.11), as it helps to prolong skin allograft survival in mice (12), suppresses renal allograft rejection in rats, and prevents type I diabetes in NOD (nonobese diabetic) mice.

View larger version (12K): [in this window] [in a new window]   Figure 1. Structures of KH1060 and 1,25-(OH)2D3.

	Materials and Methods

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Materials
The following cell lines and complementary DNAs (cDNAs) were acquired from the individuals listed below: human keratinocytes, HPK1A-ras (18), Dr. R. Kremer (McGill University, Quebec, Canada) and Dr. J. Rhim (NIH, Bethesda, MD); rat osteosarcoma, UMR-106 (19), T. J. Martin (St. Vincent’s Institute of Medical Research, Australia); human hepatoma, HepG2 (20), and the human colon cancer cell line Caco-2 (21), American Type Culture Collection (Rockville, MD); rat osteosarcoma ROS 25/1 and ROS 17/2.8 (22) and cDNA for rat osteopontin (23), Dr. G. A. Rodan (Merck Pharmaceuticals, West Point, PA); cDNA for human P450cc24 (24), Dr. H. F. DeLuca (University of Wisconsin, Madison, WI); and cDNA for rat 18S ribosomal RNA (rRNA) (25), Dr. Y.-L. Chan (University of Chicago, Chicago, IL). Ketoconazole, sodium metaperiodate, and sodium borohydride were purchased from Sigma (St. Louis, MO). All other chemicals and solvents were of analytical or HPLC grade and were purchased from a variety of commercial sources depending upon local suppliers.

Vitamin D analogs
1(S),3(R)-Dihydroxy-20(R)-(4'-hydroxy-4'ethyl-1'-hexyloxy)-9,10-secopregna-5(Z),7(E),10(19)-triene (KH1060) (5), 24a(S)-hydroxy (OH)-KH1060 (HS501) (26), 23,24,24a,25,26,26a,27,27a-octanor-20-oxo-KH1060 (EB1161), 23,24,24a,25,26,26a,27,27a-octanor-20-OH-KH1060 (EB1163), and 1,25-(OH)₂D₃ were synthesized by the Department of Chemical Research, Leo Pharmaceutical Products (Ballerup, Denmark).

Generation of metabolites of KH1060 using cultured human cell lines
Metabolites of KH1060 were generated using cultured human cell lines as previously described (27, 28). The human cell lines HPK1A-ras and HepG2 were maintained in 150-mm plates using DMEM and Earle’s culture medium, respectively [modified MEM, 10% FCS, penicillin G (100 µg/ml), fungizone (300 ng/ml), and gentamicin (5 µg/ml)]. Near confluence, cells were treated with 10 nM 1,25-(OH)₂D₃ (HPK1A-ras) or 10 µM all-trans-retinoic acid (HepG2) for 18 h to induce transcription of metabolic enzymes. The culture medium was then removed from induced cells, and replaced with incubation medium [modified MEM, 1% BSA, penicillin G (100 µg/ml), fungizone (300 ng/ml), gentamicin (5 µg/ml), and DPPD (100 µM)]. Cells were incubated for 48–72 h in the presence of vehicle (0.1% ethanol) or 10 µM KH1060.

Purification of metabolites from cultured cells
Lipids were extracted from both cells and medium as previously described (27) and separated on a modular HPLC system consisting of a model 590 pump, a U6K manual injector, a model 440 fixed wavelength detector (254 nm), and a model 990 photodiode array detector (Waters Scientific, Milford, MA). Separation of metabolites was achieved using a Zorbax-SIL column (3 µm; 0.62 x 8 cm) eluted with hexane-isopropanol-methanol (HIM; 91:7:2) at a flow rate of 1 ml/min. Metabolites were identified based on the characteristic vitamin D₃ chromophore (_max = 265, _min = 228, and _max/_min = 1.75).

Crude peaks obtained on the Zorbax-SIL column were further purified using a Zorbax-CN (6 µm; 0.46 x 25 cm) column and a HIM (91:7:2) solvent system at a flow rate of 1.5 ml/min. HPLC was repeated for several rounds until metabolites had been purified to homogeneity.

Chemical modification and gas chromatography-mass spectrometry (GC-MS) analysis of metabolites generated in cultured cells
Purified metabolites were subjected to chemical modification using sodium metaperiodate or sodium borohydride as previously described (27, 28) before analysis on HPLC using Zorbax-SIL. Metabolites were derivatized to pertrimethylsilyl ethers for GC-MS as previously described (27).

Cytochrome P450-specific inhibition of KH1060 metabolism using ketoconazole
Cells were cultured and induced as described above and then treated with incubation medium containing vehicle alone (0.05 M HCl) or ketoconazole at concentrations ranging from 100 nM to 10 µM. After 15 min, 10 µM KH1060 was added to cells and allowed to incubate for 48 h. In the case of HPK1A-ras cells, additional doses of ketoconazole were added at 12-h intervals to maintain suppression of cytochrome P450 activity. The viability of HPK1A-ras cells treated with ketoconazole was confirmed using a hemocytometer by measuring exclusion of a 0.2% trypan blue solution. Metabolites were extracted from the medium and analyzed by HPLC as described above.

In vivo metabolism of KH1060
For investigation of the time-course profile, groups of six male or six female mice (30 g) were given KH1060 iv at a dose of 0.3 mg/kg. Pooled blood samples were taken from different groups 0, 5, 10, 20, 40, 60, and 120 min after administration. Groups of three male or three female rats were given KH1060 iv at a dose of 1.2 mg/kg. Blood samples were taken from different groups 0, 5, 10, 20, 40, 60, 120, and 240 min after administration. Gøttingen minipigs (one male and one female; 9 kg) were injected into the marginal ear vein with KH1060 at a dose of 0.5 mg/kg. Blood samples were taken from each animal 0, 5, 10, 30, 45, 60, 120, and 240 min after administration. For investigation of the metabolic profile, groups of nine male or nine female mice (25 g) and groups of three male or three female rats (170 g) were given a single iv dose of 0.8 mg/kg. The dose was chosen to detect sufficient quantities of metabolites. Blood samples and livers were taken 0 and 30 min after administration. The livers were homogenized in 1 vol 0.01 M phosphate buffer (pH 7.4). All samples were analyzed using cartridge and HPLC-based procedures described previously (29, 13).

In vitro metabolism of KH1060 using liver homogenates
KH1060 was incubated in triplicate with a liver-metabolizing system, containing 100 µl postmitochondrial liver fraction (S9) from male mice, rats, and minipigs/ml in 8 mM MgCl₂, 33 mM KCl, 5 mM glucose-6-phosphate, and 4 mM NADP in 0.1 M phosphate buffer, pH 7.4. The final concentration of KH1060 was 4 x 10^-6 M. All incubations were performed at 37 C, and the samples were collected 0, 30, and 60 min after the addition of KH1060. The samples were treated and analyzed by reverse phase HPLC as described previously (13).

Competition of KH1060 and 1,25-(OH)₂D₃ for C24 oxidation enzymes
The ability of KH1060 to compete with [1ß-³H]1,25-(OH)₂D₃ for enzymes of the C24 oxidation pathway was assessed as previously described (27). Near confluence, HPK1A-ras cells were treated with 10 nM 1,25-(OH)₂D₃ to induce transcription of the enzymes of the side-chain oxidation pathway. After 18 h, cells were incubated with [1ß-³H]1,25-(OH)₂D₃ (23 nM) in the presence or absence of varying amounts of analog (0–5000 pmol) for 2 h. Nonradioactive 1,25-(OH)₂D₃ was used as a control. Radioactivity was measured in triplicate 500-µl aliquots of the aqueous fraction from the cell/medium extract by scintillation counting.

Induction of osteocalcin promoter in ROSCO cells
The relative ability of vitamin D analogs to induce vitamin D-dependent trans-activation of a chloramphenicol acetyltransferase (CAT) reporter gene using the rat osteocalcin gene promoter was performed in ROSCO cells as previously described (30). ROSCO cells were grown for 40 h in Ham’s F-12 MEM supplemented with 2% charcoal-stripped FCS. Trans-activation of the CAT gene was induced using compounds at a concentration ranging from 10^-10–10^-13 M in Ham’s F-12 MEM supplemented with 0.1% BSA. After 24 h, cells were lysed by three rounds of freeze-thawing (4 min in liquid nitrogen; 4 min in a 37 C water bath). Cell lysates were assayed for CAT activity as previously described (31), using [1-¹⁴C]acetyl-coenzyme A (2 gigabecquerels/mmol; Amersham, Slough, UK). The [¹⁴C]acetylation of chloramphenicol was measured in duplicate using a scintillation counter.

Northern analysis of vitamin D-induced gene expression in vitamin D target cells
The ability of vitamin D analogs and their metabolites to induce the transcription of either P450cc24 or osteopontin was examined in osteosarcoma and colon cancer cell lines. Cells grown under serum-free conditions [MEM supplemented with insulin (5 µg/ml), transferrin (5 µg/ml), and sodium selenide (5 ng/ml)] for 24 h were treated with 10^-9 M analog or vehicle (0.5% ethanol) in the presence of 1 µM ketoconazole or vehicle (0.05 M HCl). After 24 h, total RNA was isolated using the acid-guanidinium extraction procedure of Chomczynski and Sacchi (32). Total RNA (20 µg) was resolved by electrophoresis on a 1.5% formaldehyde-agarose denaturing gel and then transferred by capillary transfer to Hybond N⁺ filters (Amersham). Filters were hybridized, as previously described (33, 34), to [³²P]deoxy-CTP (111 terabecquerels/mmol; Amersham)-labeled complementary DNA (human P450cc24, rat osteopontin, or rat 18S rRNA) probes. Autoradiographs were quantified by laser densitometry, and messenger RNA (mRNA) expression was standardized relative to the expression of 18S rRNA.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Qualitative analysis of KH1060 metabolism
Generation of KH1060 metabolites in cultured human cell lines. The metabolic fate of KH1060 in a vitamin D target tissue was examined by incubating the analog with the human keratinocyte cell line HPK1A-ras. Initial HPLC separation of the total lipid extract obtained from HPK1A-ras cells incubated for 72 h with 10 µM KH1060 revealed 8 peaks that possessed the characteristic vitamin D chromophore (Fig. 2), which were resolved into a total of 21 distinct metabolites on rechromatography using Zorbax-CN. Twelve of the KH1060 metabolites were generated in sufficient quantities to be identified through chemical modification, mass spectral, and/or HPLC and GC cochromatographic analysis (Table 1). Similar metabolic profiles were observed when total lipid extracts from hepatoma cells (HepG2) incubated with KH1060 were subjected to HPLC (data not shown).

View larger version (35K): [in this window] [in a new window]   Figure 2. HPLC separation of lipid extracts from HPK1A-ras cells incubated with KH1060. HPK1A-ras cells were incubated with 10 µM KH1060 for 72 h. Total lipid extracts were separated on a Zorbax-SIL column using the solvent system HIM (91:7:2; 1 ml/min). Metabolites of KH1060 were identified based on their characteristic UV chromophore (max = 265 nm, min = 228 nm, and max/min = 1.75) and have been shaded in black. The peak shaded with cross-hatching did not have a characteristic vitamin D chromophore, but rechromatography of the peak on a Zorbax-CN column revealed that a KH1060 metabolite (Met1) was hidden underneath. The peak at 15.5 min contained three metabolites: Met10, Met11, and Met12, each of which was isolated in a quantity too small to be identified conclusively. Metabolites isolated from HPK1A-ras cells are designated Met1 to Met20, the identities of which are indicated in Table 1.

View larger version (34K): [in this window] [in a new window]   Figure 3. Mass spectra of biological and synthetic 24a-OH-KH1060 (KH1060-Met9). Trimethylsilyated derivatives of synthetic 24a-OH-KH1060 (top) or the purified metabolite generated in HPK1A-ras (KH1060-Met9; middle) or HepG2 (KH1060-Hep9; bottom) cells were analyzed by GC-MS. In each case, spectra represent the pyro-isomer derived from each metabolite, and for convenience, spectral interpretation is given with reference to the parent molecule.

View larger version (32K): [in this window] [in a new window]   Figure 4. HPLC separation of lipid extracts from hepatoma and keratinocytes incubated with KH1060 in the presence of ketoconazole. HepG2 (top) or HPK1A-ras (bottom) cells were incubated with 10 µM KH1060 in the presence of 10 µM ketoconazole or vehicle (0.05 M HCl) as described in Materials and Methods. Shaded peaks represent the difference between the amount of metabolites observed in control treated cells and that in cells treated with ketoconazole. Metabolites isolated from HPK1A-ras cells are designated Met1 to Met20. Metabolites isolated from HepG2 cells are designated Hep1 to Hep20. Number designations are equivalent, such that Met9 = Hep9.

View larger version (22K): [in this window] [in a new window]   Figure 5. Reversed-phase HPLC chromatograms of serum (A) or liver tissue (B) taken at 0 or 30 min after iv administration of 800 µg/kg KH1060 to male rats. Metabolites were extracted with acetonitrile and run directly on HPLC using a method described previously (13). Separations were achieved using a LiChrospher 100 RP-18 (5 µm, 125 x 4 mm) column and a linear water-methanol gradient from 70% to 95% methanol over 20 min at a flow rate of 2 ml/min. Detection wavelength was 264 nm. Metabolite A comigrated with 26-OH-KH1060 with a relative retention time of 0.57 and Metabolite B comigrated with 24a-OH-KH1060 with a relative retention time of 0.65. The relative retention time for KH1060 was arbitrarily set to 1.

Quantitative analysis of KH1060 metabolism
Pharmacokinetic analysis of KH1060 in vivo and in liver postmitochondrial (S9) fractions in vitro. Mice, rats, and minipigs were given high doses of KH1060 to determine the rate of disappearance of the analog in vivo. Much lower doses would have been more physiologically and pharmacologically relevant, but were not feasible due to our dependence upon a UV-based HPLC assay with a detection limit of 10 ng/ml for analysis of serum samples. Thus, the serum half-life [t_1/2(ß)] of KH1060 was approximately 30 min in all species, but was almost twice as long in the female rats and the male minipig (Table 2). In contrast, 1,25(OH)₂D₃ had a t_1/2 of 82 min in a female minipig and 138 min in male rats, suggesting that KH1060 is metabolized at least 3 times faster than 1,25-(OH)₂D₃ in vivo.

View this table: [in this window] [in a new window]   Table 2. In vitro and in vivo pharmacokinetic analysis of KH1060 and 1,25-(OH)2D3 in mice, rats, and minipigs

Pharmacokinetic analysis of KH1060 in HPK1A-ras cells in vitro. The stability of KH1060 in vitamin D target cells was examined by measuring the disappearance of the analog compared with 1,25-(OH)₂D₃ over a 72-h period (Fig. 6). The t_1/2 of both KH1060 and 1,25-(OH)₂D₃ in HPK1A-ras cells was about 12 h, whereas only 10% of either of the substrates remained unchanged in the target cells after 48 h. The formation of several metabolites was also measured over a period of 72 h (Fig. 6, inset). Octanor-20-OH-KH1060 (Met8) and 26-OH-KH1060 (Met16) first appeared as early as 6 h, then plateaued for the remaining portion of the 72-h period. 24-OH-KH1060 (Met13) was generated at a much slower rate than Met16 and Met8, but continued to accumulate during the rest of the incubation period, implying that this metabolite is not subject to further metabolism in HPK1A-ras cells. 24a-OH-KH1060 (Met9) was also detected as early as 6 h, but did not accumulate to the same extent as other metabolites.

View larger version (26K): [in this window] [in a new window]   Figure 6. Time course of the metabolism of KH1060 and 1,25-(OH)2D3 using HPK1A-ras cells. HPK1A-ras cells were incubated with 10 µM analog for various times between 0 and 72 h. The graph depicts the disappearance of substrate vs. time; the inset shows the formation of several KH1060 metabolites vs. time. Metabolites were quantitated based on integration of UV peaks at an absorbance of 265 nm. Each point is the mean ± SE of three incubations.

View larger version (25K): [in this window] [in a new window]   Figure 7. Competitive inhibition of [1ß-3H]calcitroic acid production using nonradioactive KH1060 or 1,25-(OH)2D3 in HPK1A-rascells. [1ß-3H]1,25-(OH)2D3 was incubated with HPK1A-ras cells in the presence of vehicle alone or varying concentrations 1,25-(OH)2D3 (box) or KH1060 (triangle) as outlined in the Materials and Methods. Each point in the figure is the mean ± SE of three flasks counted in triplicate.

View larger version (22K): [in this window] [in a new window]   Figure 8. Induction of a vitamin D-inducible CAT reporter gene in ROSCO cells. ROSCO cells were grown for 40 h in the presence of 2% charcoal-stripped FCS. Cells were then treated with varying concentration (10-13–10-10 M) of 1,25-(OH)2D3, KH1060, KH1060-Met9, -Met13, - Met16, -Met20, or vehicle (0.1% ethanol) for 24 h. Cells were assayed for CAT activity as described in the Materials and Methods. Each point is the mean of duplicate incubations.

View larger version (37K): [in this window] [in a new window]   Figure 9. Analysis of vitamin D inducible mRNA expression in cultured cell lines. (A)(B) Northern analyses from UMR-106 cells and Caco-2 cells. Total RNA was isolated from UMR-106, Caco-2, ROS 17/2.8 and ROS 25/1 cells treated with 10-9 M inducer (1, 25-(OH)2D3, KH1060, or metabolites) in the presence or absence of ketoconazole as described in the Materials and Methods. Northern blots from UMR-106 (A) and Caco-2 (B) cells were probed for P450cc24 and osteopontin mRNA expression respectively. After exposure to autoradiographic film, blots were stripped and hybridized with an 18s rRNA specific probe to determine the relative amount of total RNA transferred to the nylon membrane.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The extremely potent vitamin D analog KH1060 was rapidly metabolized in vivo and in vitro through similar pathways. Although we observed a maximum of four metabolites at detectable levels in vivo, the two that were tentatively identified were also those metabolites found in high abundance in vitro, namely 24a(S)-OH-KH1060 and 26-OH-KH1060. Using the cultured human keratinocyte cell line, HPK1A-ras, which permits an increased sensitivity, KH1060 was found to be metabolized to over 20 metabolites that lie on a variety of pathways (Fig. 10). Using extensive HPLC and GC-MS, we have identified the majority of these metabolites as side-chain hydroxylated derivatives with extra hydroxyl functions at carbons 24, 24a, 26, and 26a, which resemble intermediates in the catabolism of 1,25-(OH)₂D₃ (36) and vitamin D analogs such as 1,24(S)-(OH)₂D₂ (37), EB1089 (38, 39), and OCT (35). In addition, we found several truncated metabolites, observed for other 22-oxa-derivatives such as OCT (35). These included 20-OH- and 20-oxo-truncated forms that probably arise from either C23 or C20 hydroxylation giving a unstable hemiacetal that eliminates carbons C23 to C27a (35).

View larger version (17K): [in this window] [in a new window]   Figure 10. Proposed pathways for the catabolism of KH1060 in vitamin D target cells.

Despite its slow rate of metabolism via the C24 oxidation pathway, KH1060 was metabolized at a rate equal to or much faster than that of 1,25-(OH)₂D₃ in a variety of species in vivo and cell types in vitro. One obvious explanation for this rapid rate of metabolism of KH1060 lies in the susceptibility of KH1060 to enzymatic modification at a number of different sites by both vitamin D-specific and nonspecific pathways. The enzymes responsible are sensitive to ketoconazole, confirming the involvement of cytochrome P450 isoforms. Of the alternate pathways, 26-OH-KH1060 is the most abundant metabolite formed in the target cell line HPK1A-ras. Hydroxylation of an analog at C26 has been reported for the vitamin D analogs EB1089 (38, 39) and OCT (35) and in biosynthesis of the 26,23-lactones of 25-hydroxyvitamin D₃ and 1,25-(OH)₂D₃ (42). The 26-hydroxylase enzyme involved remains unknown.

The remarkable biological activity reported for KH1060 (6) in the face of the extensive metabolism of the analog in vivo and in vitro is all the more puzzling. It has been suggested that the increased trans-activation ability of some vitamin D analogs might be due to tighter ligand-VDR-RXR-DNA binding (15), although this explanation largely ignores the metabolic instability of KH1060 described here. Another possibility is the fact that KH1060 is responsible for only part of the transcriptional activity ascribed to it, with the rest being due to its immediate metabolites. As illustrated in Fig. 9, we found excellent retention of biological activity of KH1060 in the metabolites 26-OH-KH1060 (KH1060-Met16) and 24a(S)-OH-KH1060 (KH1060-Met9), which were only slightly inferior to KH1060 and still superior to 1,25-(OH)₂D₃ at in-ducing gene expression of the stably transfected VDRE-dependent CAT reporter. Furthermore, the biological activities of KH1060, 26-OH-KH1060, and 24a(S)-OH-KH1060 were not significantly affected by the presence of DBP (data not shown). This is consistent with data suggesting that inversion of the vitamin D₃ side-chain greatly reduces DBP-binding ability (27, 13), making the cellular uptake of analogs less affected by the medium composition (14, 27). As with the natural hormone, 24-hydroxylation of KH1060 gives a compound, 24-OH-KH1060, with low biological activity.

KH1060 metabolites also exhibited strong transactivation ability for vitamin D-dependent genes within their native context. Vitamin D-inducible mRNA expression in cancer cell lines demonstrated that KH1060, 26-OH-KH1060, and 24a(S)-OH-KH1060 all possess significant biological activity. In all cell lines tested, these three metabolites had activities more than or equal to that of 1,25-(OH)₂D₃, except in ROS 17/2.8 cells in the absence of ketoconazole. The high efficacy of 1,25-(OH)₂D₃ in ROS 17/2.8 cells in the absence of ketoconazole probably lies in the fact that these cells lack C24 oxidation enzyme activity (43) and P450cc24 mRNA expression (44), making the natural hormone more stable than KH1060 or its metabolites in this cell line. Ketoconazole had little effect on the efficacy of vitamin D-dependent mRNA induction by KH1060 or its metabolites. In contrast, vitamin D-dependent mRNA induction by 1,25-(OH)₂D₃ was significantly increased by ketoconazole treatment in UMR-106 and Caco-2 cells, two cell lines that have been shown to contain C24 oxidation enzyme activity (43, 45, 34). These data reinforce the view that the rate at which 1,25-(OH)₂D₃ and its analogs are metabolized is an important parameter leading to attenuation of their biological activity. As KH1060 can be converted into stable metabolites, which are only slightly less biologically active, this is presumably the reason why significant increases in mRNA induction were not observed in our assay systems when we compared ketoconazole-treated and -untreated cells. Whether the observed differences in biological activity of metabolites in different tissues (e.g. 26a-OH-KH1060) are due to multiple conformations of the VDR or to differences in the complement of catabolic enzymes within those tissues is worthy of further inves-tigation.

The results presented in this paper suggest that future analog studies should consider two additional factors when measuring the efficacy of vitamin D compounds in target cell assays: 1) Is the reference compound, 1,25-(OH)₂D₃, or the analog of interest selectively metabolized in the bioassay system being studied?; 2) Is the biological activity measured in the system attributable to the analog itself or to one (or more) of its metabolites? Only after these questions have been answered satisfactorily or circumvented by the judicious use of inhibitors or the exclusion of catabolic enzymes in a cell-free system can the biological activity of two analogs such as KH1060 and 1,25-(OH)₂D₃ be accurately compared. From the stability and biological activity data presented here for KH1060, it cannot be assumed that the parent molecule is always responsible for the biological activity ascribed to it in various in vitro systems, especially given its short t_1/2in vivo and extensive catabolism in vitro. Although a full understanding of the mechanism of action of KH1060 remains elusive at this time (46), this report establishes the complexity of the metabolism of KH1060, the wide range of cytochromes P450 involved in this process, and a possible role for KH1060 metabolites in the overall biological effects of the analog.

View larger version (27K): [in this window] [in a new window]   Figure 11. Effect of inhibin immunoneutralization on gonadotropin subunit mRNA concentrations and serum gonadotropins in adult female rats. Treatment groups include intact animals () and rats given either inhibin antiserum (|og) or NSS (). Animals were killed 2 or 12 h after inhibin antisera or NSS. *, P < 0.05 compared with intact group. Values are the mean ± SEM (n = 6–8/group).

	Acknowledgments

	Footnotes

 
¹ Presented in part in abstract form at the Eighteenth Annual Meeting of the American Society for Bone and Mineral Research, Seattle, Washington, September 1996 (47 ). This work was supported by Grant MA-9475 (to G.J.) from the Medical Research Council of Canada.

² Recipient of an Ontario Graduate Studentship.

³ Recipient of a Medical Research Council (UK) Clinician Scientist Fellowship.

Received April 24, 1997.

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