1α,25-Dihydroxyvitamin D₃ [1α,25(OH)₂VD₃¹], the active and hormonal form of vitamin D₃, is a potent negative regulator of parathyroid gland function, with effects on both PTH mRNA production and PTH secretion as well as parathyroid cell proliferation (Chertow et al., 1975; Mayer and Hurst, 1978; Cantly et al., 1985; Silver et al., 1985, 1986; Russell et al., 1986).

26,26,26,27,27,27-Hexafluoro-1α,25(OH)₂ vitamin D₃ [F₆-1α,25(OH)₂VD₃], a fluorinated derivative of 1α,25(OH)₂VD₃ has been reported to be several times as potent as the parent compound at increasing intestinal calcium transport and bone calcium (Tanaka et al., 1984; Kiriyama et al., 1991; Inaba et al., 1993). The inhibitory effect of F₆-1α,25(OH)₂VD₃ on PTH secretion from parathyroid cells also exceeds that of the parent compound (Katsumata et al., 1996; Tsushima et al., 1996; Imanishi et al., 1999). F₆-1α,25(OH)₂VD₃ has been used clinically for the treatment of secondary hyperparathyroidism in cases of chronic renal failure and for the control of hypoparathyroidism (Nakatsuka et al., 1992; Akiba et al., 1998; Inoue and Fujimi, 1998; Morii et al., 1998). The mechanism of action of 1α,25(OH)₂VD₃ involves a specific intracellular receptor (vitamin D receptor, VDR), which binds 1α,25(OH)₂VD₃ with a high affinity and modulates the transcription of vitamin D responsive genes in the parathyroid glands (Naveh-Many et al., 1990; Demay et al., 1992; Brown et al., 1992b, 1995; Hellman et al., 1999). F₆-1α,25(OH)₂VD₃ is considered to act by the same pathway.

We earlier demonstrated that radioactivity was mainly found in the vitamin D target tissues (i.e., in the kidneys and small intestine) and is locally distributed in the methaphysis of bone in rats dosed with [1β-³H]F₆-1α,25(OH)₂VD₃ (Komuro et al., 1996b, 1998). ST-232 (26,26,26,27,27,27-hexafluoro-1α,23S,25-trihydroxyvitamin D₃), a 23S-hydroxylated metabolite, accounted for the majority of the compound administered (Komuro et al., 1996a,c, 1998), and we further reported that F₆-1α,25(OH)₂VD₃ can be transformed to ST-232 in the kidneys and small intestine on the basis of in vitro metabolic studies (Komuro et al., 1999). Moreover, ST-232, the main metabolite of F₆-1α,25(OH)₂VD₃ in target tissues, is reported to retain biological activity (Katsumata et al., 1996), whereas the 23S-hydroxylated metabolite of 1α,25(OH)₂VD₃ was a less active compound (Horst et al., 1984).

It has been demonstrated that F₆-1α,25(OH)₂VD₃ is metabolized to ST-232 by rat or human mitochondrial 24-hydroxylase expressed in Escherichia coli (Hayashi et al., 1998; Sakaki et al., 2003), and we have reported that the enzyme in the small intestine is induced by F₆-1α,25(OH)₂VD₃ itself (Komuro et al., 1999). These findings suggest that the enhanced biological activity of F₆-1α,25(OH)₂VD₃ is due to its metabolism to the bioactive metabolite, ST-232, and its persistence as this form.

With regard to the parathyroids, Imanishi et al. (1999) reported in vitro metabolism of F₆-1α,25(OH)₂VD₃ in primary cultured bovine parathyroid cells. The present study was performed to compare [1β-³H]F₆-1α,25(OH)₂VD₃ with [1β-³H]1α,25(OH)₂VD₃ with regard to the distribution and metabolism in the parathyroid glands after intravenous dosing.

Materials and Methods

Materials. Radioactive [1β-³H]F₆-1α,25(OH)₂VD₃ (code TRQ 6721 or 8742; 640 or 555 GBq/mmol; Fig. 1) and [1β-³H]1α,25(OH)₂VD₃ (code TRQ 7362, 618 GBq/mmol; Fig. 1) were purchased from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK).

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Fig. 1.

Chemical structures of [1β-³H]F₆-1α,25(OH)₂VD₃, its metabolites and [1β-³H]1α,25(OH)₂VD₃(*, tritium-labeled position).

Nonradioactive F₆-1α,25(OH)₂VD₃ (lot no. 80201) was obtained from Sumitomo Pharmaceuticals (Osaka, Japan). Nonradioactive ST-232 [23S-hydroxy-F₆-1α,25(OH)₂VD₃, lot no. 508CR001] and ST-233 [23-oxo-F₆-1α,(OH)VD₃, lot no.410211], the metabolites of F₆-1α,25(OH)₂VD₃ detected in the kidneys (Komuro et al., 1996a), intestine (Komuro et al., 1996a), and bones (Komuro et al., 1998) of rats dosed with [1β-³H]F₆-1α,25(OH)₂VD₃, were also obtained from Sumitomo Pharmaceuticals (Fig. 1). All other reagents and solvents were of the best grade commercially available in standard catalogs.

Animals. Fourteen-week-old male Wistar rats were purchased from Japan SLC (Shizuoka, Japan) and were used at the age of 15 weeks. During experiment 1, the rats had free access to tap water and MF laboratory food (Oriental Yeast Co., Ltd., Tokyo, Japan). During experiment 2, the rats received tap water and CRF-1 laboratory food (Oriental Yeast Co., Ltd.) ad libitum. The rats were given [1β-³H]F₆-1α,25(OH)₂VD₃ or [1β-³H]1α,25(OH)₂VD₃ at a dose of 10 μg/kg. The administration was performed intravenously in a volume of 0.5 ml/kg b.w. using ethanol/saline (1:1, v/v) as the vehicle. The dose levels were higher than those in clinical use but were chosen as a compromise to give detectable parathyroid gland levels of the test compound.

Experiment 1, Distribution Studies. At 6, 24, and 48 h postdosing, 8 to 10 rats per time point for each dose were anesthetized with diethyl ether, and blood was collected from the abdominal vein. After killing, the thyroid glands including parathyroid glands were resected, and the parathyroid glands were removed under a microscope (experiment 1: SZH, Olympus, Tokyo, Japan; experiment 2: SZ4045, Olympus). The liver, kidneys, intestine, bones, and skin were also resected, and serum was separated from blood by centrifugation. The thyroid glands excluding the parathyroid glands, kidneys, and small intestine were homogenized with similar amounts of saline. Tissues, tissue homogenates, and serum of each animal were removed for oxidation in a sample oxidizer (Tri-Carb model 387; PerkinElmer Life Sciences, Boston, MA).

Experiment 2, Metabolism Studies. The parathyroid glands of six rats were pooled and homogenized with saline using both a digital-homogenizer (Iuchi, Osaka, Japan) and a sonic-homogenizer (Sonifier 250; Branson UnltrasonicsCorporation, Danbury, CT) until no particles were observed. Homogenized samples were transferred to microtubes and extracted three times with 1 ml of ethanol. The extracts were combined and dried under a stream of nitrogen. For radioactivity profiling, the reconstituted samples were applied to high-performance liquid chromatography (HPLC) equipment. To estimate the recovery, parathyroid glands of three additional rats were pooled and treated as described above, and the extracts and the residues were dissolved with ethanol and a tissue solubilizer, NCS-2 (PerkinElmer Life Sciences), for radioactivity counting. The serum of six rats was pooled, 100-μl aliquots were filtered [Sun-prep 4(T)-HV, 0.45 mm, 4-mm i.d.; Millipore Corporation, Bedford, MA] and combined with 100 μl of saline used for washing the filter, and then the filtration samples were applied to HPLC.

The HPLC apparatus was equipped with a solvent delivery pump (l-6300; Hitachi, Tokyo, Japan), an ultraviolet detector (SPD-10A, 254 nm; Shimadzu, Kyoto, Japan), and a data-acquisition system (805 data station; Waters, Tokyo, Japan), with a reversed-phase ODS column (Sumipax ODS A-212, 5 μm, 6.0 mm i.d. × 150 mm; Sumika Chemical Analytical Service, Osaka, Japan) at a flow rate of 1.5 ml/min. The mobile phase used was acetonitrile/water/tetrahydrofuran (55:40:5, v/v/v). The effluents were collected in 20-s fractions, and the radioactivity of each fraction was measured and profiled. Identification of metabolites was performed by cochromatography with F₆-1α,25(OH)₂VD_3, ST-232, and ST-233 standards. The relative proportions of metabolites were calculated from the total radioactivity of the sample in each radiochromatogram.

Radioactivity Measurements. The radioactivity of each sample was determined with a liquid scintillation spectrometer (system 387; PerkinElmer Life Sciences).

Results and Discussion

Parathyroid, Serum, and Other Tissue Levels of Radioactivity. Data for parathyroid, serum, and other tissue levels of radioactivity 6, 24, and 48 h after single intravenous administrations of [1β-³H]F₆-1α,25(OH)₂VD₃ and [1β-³H]1α,25(OH)₂VD₃ to rats are summarized in Table 1. With [1β-³H]F₆-1α,25(OH)₂VD₃, the values at 6, 24, and 48 h postdosing were relatively constant at 73.6, 64.9, and 71.3 ng Eq/g, respectively, whereas they decreased with time in the [1β-³H]1α,25(OH)₂VD₃ case. The differences at 6, 24, and 48 h postdosing were 2.5-, 8.4-, and 14.6-fold, respectively.

Radioactivity in the serum of rats dosed intravenously with [1β-³H]F₆-1α,25(OH)₂VD₃ was decreased with time. In the intestine and kidneys, the highest values were observed at 24 h postdosing, whereas in the other tissues, they were apparent at 6 h postdosing. The radioactivities in kidneys and intestine at 48 h postdosing were 85.7- and 20.1-fold, respectively, higher than that in serum. In the group given [1β-³H]1α,25(OH)₂VD₃ administration, the radioactivity levels in serum and tissues declined relatively rapidly, and maximum levels of radioactivity were observed at 6 h postdosing.

Metabolites in Parathyroid and Serum with [1β-³H]F₆-1α,25(OH)₂VD₃ Administration. Radio-HPLC profiles of parathyroid and serum 6, 24, and 48 h after a single intravenous administration of [1β-³H]F₆-1α,25(OH)₂VD₃ are shown in Fig. 2, and data for metabolites are summarized in Table 2. The recovery by pretreatment procedure was over 95% of total radioactivity. Therefore, the composition percentage on HPLC is very similar to that on total radioactivity. The metabolite patterns allowed clear assignment by comparison with authentic standards. At 6 h postdosing, the unchanged compound was mainly detected at about 74.2% on HPLC. At the 24- and 48-h time points, over half of the radioactivity was observed as ST-232 (50.5 and 75.0% on HPLC, respectively); then ST-233, the 23-oxo form, was additionally apparent at 48 h.

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Fig. 2.

HPLC profiles of parathyroid extracts of rats 6, 24, and 48 h after intravenous administration of [1β-³H]F₆-1α,25(OH)₂VD₃at a dose of 10 μg/kg.

The metabolite patterns of serum allowed clear assignment by comparison with authentic standards. At 6 h postdosing, the unchanged compound was mainly detected at about 90.6% on HPLC and then decreased with time. However, no metabolites were evident (Fig. 3). The large amount of radioactivity eluting at solvent front of the serum profiles seems to be tritiated water, because our previous data shows that the ratios of serum concentration of radioactivity assayed by wet method and dry method at 24 h after doing of rats was 0.51 (Komuro et al., 1996b).

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Fig. 3.

HPLC profiles of serum of rats 6, 24, and 48 h after intravenous administration of [1β-³H]F₆-1α,25(OH)₂VD₃at a dose of 10 μg/kg.

The parathyroid gland is the overall regulatory organ for general calcium homeostasis. F₆-1α,25(OH)₂VD₃ is reported to have one-third the affinity of 1α,25(OH)₂VD₃ for binding to vitamin D-binding protein and one-fourth for VDR, increasing blood-ionized calcium in parathyroidectomized rats to normal levels (Katsumata et al., 1996). In addition, F₆-1α,25(OH)₂VD₃ administered orally was found to reduce increasing plasma PTH concentrations and PTH mRNA levels in parathyroid-thyroid tissues with therapeutic effects on aberrant bone metabolism in 5/6 nephrectomized rats at lower doses than with 1α,25(OH)₂VD₃ (Tsushima et al., 1996). Based on these results, the compound was evaluated for the biological potency and the applicability for clinical control of hypoparathyroidism, secondary hyperparathyroidism, and osteodystrophy in patients with chronic renal failure at lower doses than that of 1α,25(OH)₂VD₃ (Nakatsuka et al., 1992; Akiba et al., 1998; Inoue and Fujimi, 1998; Morii et al., 1998).

In the present study, to clarify the distribution of radioactivity in the parathyroid glands of rats dosed with [1β-³H]F₆-1α,25(OH)₂VD₃ or [1β-³H]1α,25(OH)₂VD₃, we separated the parathyroid glands from the thyroid glands, allowing demonstration of specific distribution and accumulation of radioactivity in the parathyroid glands. Levels of radioactivity 6, 24, and 48 h after single intravenous administration of [1β-³H]F₆-1α,25(OH)₂VD₃ were consistently higher in the parathyroid glands than in the serum, with a distribution pattern that is similar to that in kidneys and small intestine, the target organs of this compound. In contrast, a marked decrease with time was observed for the [1β-³H]1α,25(OH)₂VD₃ case. The main form of [1β-³H]F₆-1α,25(OH)₂VD₃ in parathyroid glands at 6 h postdosing was determined to be the unchanged compound, whereas at 24 and 48 h postdosing, ST-232, a biologically active metabolite, predominated. In serum, the unchanged compound was mainly detected at 6 h postdosing and then decreased with time, but no metabolites were detected. This again demonstrates similarity for the parathyroid glands with the kidneys, small intestine, and bone, rather than for the serum.

Parathyroid glands are reported to contain VDR (Naveh-Many et al., 1990; Brown et al., 1992b, 1995; Demay et al., 1992; Hellman et al., 1999), and therefore the observed distribution of radioactivity may reflect binding of [1β-³H]F₆-1α,25(OH)₂VD₃ and [1β-³H]1α,25(OH)₂VD₃. Brown et al. (1992a, 1999) earlier reported that in parathyroid cells, as in other target tissues, 1α,25(OH)₂VD₃ was degraded by side chain oxidation, suggesting that the inducible 24-hydroxylase is responsible. Recently, reverse-transcription polymerase chain reaction and immunohistochemical analyses showed expression of 1α-hydroxylase in both normal and pathological parathyroid tissue, and the findings imply that in addition to feedback control by circulating 1α,25(OH)₂VD₃ levels, parathyroid cells may also be influenced by local 1α-hydroxylase activity with possible growth-regulatory effects (Segersten et al., 2002). Under basal conditions, a non-small cell lung carcinoma expressed only CYP1α and showed 1α-hydroxylase enzyme activity, but when treated with 1α,25(OH)₂VD₃, it began to express CYP24 and to exhibit 24-hydroxylase enzyme activity (Jones et al., 1999). Although the existence of 24-hydroxylase in the parathyroid glands remains to be confirmed, a similar situation might also prevail in this site.

In conclusion, the present study demonstrated local retention of [1β-³H]F₆-1α,25(OH)₂VD₃ and the bioactive metabolite ST-232 in parathyroid glands after intravenous administration. The findings may indicate that one of the reasons for the higher potency of F₆-1α,25(OH)₂VD₃ than 1α,25(OH)₂VD₃ in parathyroid glands is linked to differences in distribution and metabolism.