1 Departments of Nephrology and 2 Clinical Biochemistry, St Bartholomew's and the Royal London School of Medicine and Dentistry, London, UK and 3 Leo Pharmaceuticals, Ballerup, Denmark
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Abstract |
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Methods. The effect of each sterol on parathyroid hormone (PTH) secreted by primary bovine parathyroid cells was measured. High-performance liquid chromotography and gas chromotography-mass spectrometry were used to investigate in vitro 25(OH)DHT2 metabolism. Rats were given a single intraperitoneal injection or five daily injections of each sterol, and changes in ionized calcium and PTH were measured.
Results. In vitro, all sterols suppressed PTH significantly. Calcitriol and OCT were of similar potency, but 1,25(OH)2DHT2 and 25(OH)DHT2 required higher concentrations to suppress PTH equally. We were unable to detect metabolism of 25(OH)DHT2 to 1
,25(OH)2DHT2 in vitro. In rats, a single dose of 0.5 µg/rat of calcitriol increased ionized calcium at 30 and 40 h (statistically significant at 48 h). 50 µg of OCT and 1
,25(OH)2DHT2 did not cause significant hypercalcaemia at 48 h, although 1
,25(OH)2DHT2 caused hypercalcaemia at 30 h. In contrast, 50 µg of 25(OH)DHT2 caused hypercalcaemia at 48 h but not at 30 h. Five daily doses of 0.001 µg/rat of calcitriol caused a significant rise in calcium and a 50% fall in PTH. OCT and 1
,25(OH)2DHT2 at 0.025 and 0.5 µg/rat respectively caused similar suppression of PTH but without hypercalcaemia.
Conclusion. 1,25(OH)2DHT2 and 25(OH)DHT2 are potent suppressors of PTH in vitro and in vivo. 25(OH)DHT2 may be active by virtue of its pseudo-1
-hydroxyl group. Hypercalcaemia caused by a single dose of 1
,25(OH)2DHT2 appeared to be more transient than calcitriol. Five daily doses of 1
,25(OH)2DHT2 and OCT could achieve 50% suppression of PTH without significant increments in ionized calcium. In contrast, suppression of PTH by calcitriol was associated with significant increments in ionized calcium. These data suggest that like OCT, 1
,25(OH)2DHT2 can dissociate calcaemic actions from parathyroid-suppressing actions in a manner that may be therapeutically useful.
Keywords: 1,25(OH)2DHT2; 25(OH)DHT2; calcitriol; hypercalcaemia; ionized calcium; parathyroid hormone
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Introduction |
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Dihydrotachysterol2 (DHT2) preparations have been in clinical use since the late 1930s. DHT2 is a reduced vitamin D2 analogue where the A-ring has been rotated through 180°, bringing the 3ß-hydroxyl into a pseudo-1 hydroxy position. DHT2 has high potency in some vitamin-D-resistant states including uraemia but, like calcitriol and alfacalcidol, is subject to dose-limiting calcaemic actions. We have previously shown that DHT2 is metabolized to 25-hydroxydihydrotachysterol2 (25(OH)DHT2) and 1
,25- dihydroxydihydrotachysterol2 (1
,25(OH)2DHT2) [11] and that in man DHT2 can suppress endogenously produced PTH and calcitriol with minimal effects on serum calcium [12]. Prior to the discovery of the 1
,25-dihydroxylated-DHT2 metabolite, it had been suggested that 25(OH)DHT was the biologically active metabolite of DHT2 by virtue of the psuedo-1
hydroxyl group [13] since the presence of a 1
-hydroxyl is known to be critical for high-affinity binding to the vitamin D receptor (VDR) [14]. Although 25(OH)DHT2 is likely to have calcitriol-like activity, our previous studies have shown that 1
,25(OH)2DHT2 is a more potent DHT2 metabolite that binds to the VDR 1020 times more avidly than 25(OH)DHT2, although 50100 times less than calcitriol [11]. Our previous studies have also shown that 1
-hydroxylation of 25(OH)DHT2 occurs in anephric man although the site(s) of the extrarenal 1
-hydroxylation remain unknown [15]. It is therefore possible that the main biologically active metabolite of DHT2, even when used in patients with renal failure, is attributable to the 1
,25(OH)2DHT2.
In this study we compared the direct effects the two most active metabolites of DHT2 (25(OH)DHT2 and 1,25(OH)2DHT2) with those of calcitriol and OCT as suppressors of PTH secretion in primary cultures of parathyroid cells and on extracellular fluid calcium concentration in normal rats.
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Subjects and methods |
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Control of PTH secretion by calcium and vitamin D analogues
Cells were incubated for 24 h prior to their use in experiments. Baseline PTH secretion into media from wells were assessed prior to the start of the experiments by incubation with fresh media for 30 min or 4 h depending on the duration of the experiment. Baseline PTH secreted into the media from each of the wells were similar, indicating that differences in secretion from the experimental manipulations were not due to inherent differences between wells (data not shown).
Effect of ambient calcium on PTH secretion
In one set of experiments, to examine the response to calcium perturbation, bovine parathyroid cells were refreshed with culture media with defined calcium concentration every 30 min. Cells were initially refreshed with media containing high (2.5 mmol/l) calcium, low (0.5 mmol/l) calcium or control (1.25 mmol/l) calcium for 60 min. All cells were then washed and re-incubated in control media for a further 2 h with media changes every 30 min. Supernatants from quadruplicate cultures were stored for PTH determination at every 30-min media change. The PTH concentrations in the media from the high or low groups were compared with those of control.
Effect of vitamin D sterols on PTH secretion
The effects of various vitamin D analogues were examined as follows. Parathyroid cells were incubated for 24 h in culture media without vitamin D analogues. The culture media was changed and the supernatant was collected after a further 4-h incubation, which represented the baseline PTH secretion. Cells were then incubated for 24 h in the presence of varying concentrations of the vitamin D analogues (at concentrations 10-7 to 10-11 mol/l) or vehicle (0.0025% v/v isopropanol in media). The PTH secreted into fresh culture media containing the same sterol concentration over another 4 h incubation was measured. Bovine PTH was measured using a species-specific immunoradiometric assay (Nichols, San Capistrano, CA, USA). All samples from a single experiment were assayed in a single batch.
Metabolism of 1,25(OH)2DHT2 and 25(OH)DHT2 by bovine parathyroid cells
Parathyroid cells, isolated from bovine parathyroid glands as described earlier, were incubated with 25(OH)DHT2 or 1,25(OH)2DHT2 at concentrations of 10-7 mol/l for 4 or 24 h. Metabolites were extracted from 20 ml of media, from pooled 1-ml wells, with an equal volume of acetonitrile and the protein precipitate pelleted by centrifugation. The supernatant was diluted with 0.4 mol/l phosphate buffer (pH 10.5) applied to BondElut C18 cartridges (500 mg), washed with 8 ml H2O and 7 ml methanol : water (50/50 v/v), and metabolites eluted with 8 ml of methanol as described previously [12]. Extracts were dried down under vacuum, redissolved in high-performance liquid chromatography (HPLC) mobile phase (90/5/5, hexane/isopropanol/methanol, v/v/v), and separated on a Superspher Si60 (4 mm i.d.x119 mm length) straight phase column (Merck, Darmstadt) using a 1 ml/min flow rate. Using authentic standard to determine elution times, solvent fractions corresponding to 1
,25(OH)2DHT2 and 25(OH)DHT2 were collected for subsequent gas chromatography-mass spectrometry (GC-MS) analysis. The eluent was monitored with an UV photodiode array detector (Waters 990, Millipore, USA). Each HPLC fraction collected was evaporated to dryness and derivatized by heating with 50 µl trimethylsilylimidazole (TMSI) at 50°C for 1 h. Products were dissolved in hexane and excess TMSI was removed by passing through a small prewashed Lipidex column, as described previously [12]. To detect the presence of 1
,25(OH)2DHT2 or 25(OH)DHT2, samples were analysed by GC-MS using a 5890GC coupled to a 5970 MSD (HewlettPackard, Palo Alto, CA, USA) in selected ion monitoring mode (SIM). We have previously found this technique to be sensitive to <50 pg (data not shown).
Calcaemic effect of vitamin D analogues in rats
Single-dose experiments
Ten groups of four female Wistar rats fed on normal rat chow (containing 0.7% calcium, 0.5% phosphorus, and 600 iu/kg vitamin D) were given a single intraperitoneal dose of either calcitriol (at 0.05, 0.5, or 5.0 µg), OCT or 1,25(OH)2DHT2 (at 0.5, 5.0, or 50 µg) in 0.6% (v/v ethanol) in 0.2 ml of ethylene glycol. Control animals were injected with 0.2 ml ethylene glycol alone. Each group of rats was matched for weight. Blood ionized calcium concentrations were determined from duplicate tail capillary samples, measured 30 and 48 h after injection. The experiment was conducted over 2 days and the mean ionized calcium for each group of rats was compared with the controls of that day. Ionized calcium was measured using a 634 ISE Ca2+/pH Analyser (Ciba Corning, MA, USA).
Five daily dosing regimen
Female Wistar rats maintained on rat chow as above, were given five daily i.p. injections of vitamin D analogues dissolved in ethylene glycol. Twenty-four hours after the last dose (i.e. on day 6), tail blood samples for ionized calcium were taken under sedation with fentanyl and midazolam, and animals were exsanguinated. Serum was stored at -70°C until analysis for rat intact PTH concentration using a two-site radioimmunometric assays (Nichols, San Capistrano, CA, USA). The inter- and intra-assay coefficient of variation (CV) was less than 3%. Serum calcitriol was measured using a combined immunoextraction and [123I] radio-immunoassay system (IDS, Tyne and Wear, UK). The intra-assay CV was less than 6%.
Statistics
Data were analysed by ANOVA. Group means were compared using Student's t-test. P<0.05 was considered to represent significance.
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Results |
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Effect of vitamin D analogues on PTH secretion
In these experiments the media PTH concentrations were expressed relative to control values. Figure 2a shows the results from two pooled experiments where the effect on PTH secretion by calcitriol or OCT was compared directly (n=7 for each concentration of sterol). Calcitriol and OCT at concentrations greater than or equal to 10-10 mol/l significantly suppressed PTH secretion in a dose-dependent manner and to a similar extent. PTH concentration in the media fell to 77±3% and 82±4% of control values in the presence of 10-10 mol/l calcitriol or 10-10 mol/l OCT respectively. There was no significant difference between the suppression of PTH secretion by calcitriol or OCT over the concentration range 10-11 to 10-7 M (n.s. by ANOVA).
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The suppression of PTH by 1,25(OH)2DHT2 and 25(OH)DHT2 was also compared over the same concentration range in two separate experiments. The pooled results (n=8) are shown in Figure 2c
. Both metabolites had a dose-dependent effect on PTH secretion, significantly suppressing PTH at concentrations of 10-8 mol/l or greater (PTH concentration in the presence of 10-8 mol/l concentrations of 1
,25(OH)2DHT2 or 25(OH)DHT2 were 72±4 and 77±2% of control respectively, P<0.05). Over the concentration range 10-7 to 10-11 mol/l, there was no difference between the effects of 1
,25(OH)2DHT2 and 25(OH)DHT2 (n.s. by ANOVA).
Metabolism of 1,25(OH)2DHT2 and 25(OH)DHT2 by parathyroid cells
HPLC separation of extracted media from the cells incubated with 1,25(OH)2DHT2 gave rise to a peak at 11.96 min with the characteristic UV spectrum and retention time consistent with that of authentic 1
,25(OH)2DHT2. Similarly, HPLC separation of the extract isolated from cells incubated with 25(OH)DHT2 also eluted as a single peak during HPLC purification. The retention time of this peak (3.66 min) and the UV absorption spectrum (absorption maxima at 241, 251 and 261 nm) were identical to that of authentic 25(OH)DHT2, but there was no evidence of a peak at 11.96 min during HPLC separation, which would correspond to 1
,25(OH)2DHT2. Furthermore, GC-MS analysis of the HPLC solvent fraction at this time revealed no evidence for the formation of the 1
,25(OH)2DHT2 metabolite.
Calcaemic effect of vitamin D analogues in vivo
Twenty-five groups of four rats were injected intraperitoneally with a single dose of each vitamin D analogue of interest, or vehicle (control). At the end of the experiment, there were no significant changes in intra- and inter-group rat weights (data not shown). Blood ionized calcium concentrations were compared with the mean value from the control group. Vehicle-treated rats from this and other experiments (n=90) had a mean (±SD) blood ionized calcium concentration of 1.40 (0.06) mmol/l. Figure 3 shows the effect after a single intraperitoneal injection in normal rats at 30 and 48 h. Calcitriol at a dose of 0.5 µg increased ionized calcium at 30 h and this reached statistical significance at 48 h. At a dose of 5 µg a significant incremental rise of ionized calcium was found at 30 h and this was sustained at 48 h (Figure 3b
). In this experiment, 30 h after injecting vehicle (control), ionized calcium concentration was 1.49±0.03 mmol/l, whereas rats injected with 0.5 or 5 µg calcitriol had mean ionized calcium concentrations of 1.55±0.05 (P=n.s.) and 1.61±0.05 mmol/l (P<0.05) respectively. After 48 h, control rats had ionized calcium concentrations of 1.42±0.03 mmol/l, but rats injected with 0.5 or 5 µg calcitriol were significantly hypercalcaemic (1.51±0.02 and 1.58±0.03 mmol/l, both P<0.01). In contrast, OCT at doses of 0.5, 5 and 50 µg caused no significant changes in ionized calcium at either 30 h (Figure 3a
) or 48 h (Figure 3b
). 1
,25(OH)2DHT2, caused a significant increase of ionized calcium only at the highest dose (50 µg) after 30 h (1.60±0.04 mmol/l vs vehicle=1.49± 0.03 mmol/l, P<0.05) although this was not sustained at 48 h (1.47±0.03 mmol/l vs vehicle=1.42± 0.03 mmol/l, P=n.s.). In contrast, rats treated with 25(OH)DHT2 experienced no significant increment of ionized calcium at 30 h even at the highest dose of 50 µg, but a significant incremental rise was found at 48 h (
iCa was 0.22±0.02 mmol/l, P<0.01).
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PTH suppression in normal rats by vitamin D analogues
We administered doses of calcitriol, OCT, and 1,25(OH)2DHT2 required to suppress PTH by 50%. These were 0.001, 0.025, and 0.05 µg/rat/day respectively. At these doses, OCT and 1
,25(OH)2DHT2 significantly suppressed PTH, but there were no changes in ionized calcium. In contrast, 50% suppression of PTH by calcitriol caused significant hypercalcaemia (Figure 4
).
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Discussion |
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It has been postulated that in the case of OCT, dissociation of PTH suppression and hypercalcaemia may be related to the low affinity of OCT for plasma vitamin D binding protein (DBP) [17], favouring high free concentrations and a short plasma half- life [18]. It is therefore noteworthy that 25-hydroxydihydrotachysterol3 and 1,25-dihydroxydihydrotachysterol3 have also been shown to have low affinity for DBP [11], although the affinity of the DHT2 metabolites has not been studied.
Recent data emphasizing the lesser calcaemic effect of vitamin D2 analogues compared with their vitamin D3 counterparts [19] might be an alternative explanation for the same phenomenon occurring with DHT2 metabolites. The ability of 1-hydroxyvitamin D2 to suppress secondary hyperparathyroidism with apparent minimal calcaemic effect in haemodialysis patients [19] has been attributed to the formation of 1
,24S-dihydroxyvitamin D2 as well as 1
,25- dihydroxyvitamin D2 [20,21]. In contrast, 1
-hydroxyvitamin D3 is not 24-hydroxylated in this manner and is metabolized only to calcitriol. Subsequent 24-hydroxylation of calcitriol inactivates the vitamin D3 sterol. However, the metabolites of DHT2 used in these experiments are 25-hydroxylated, and there is no potential for generating a 1,24 dihydroxy-DHT2 metabolite. This route of metabolism is unlikely to be a reason for any dissociation of calcaemic from PTH-suppressing activity by 1
,25(OH)2DHT2.
Previously we have demonstrated that 25(OH)DHT3 and 1,25(OH)2DHT3 are extensively metabolized in vitro (in UMR-106 rat osteoblast cell lines) and in the rat [22,23]. We were unable to demonstrate in vitro metabolism of 25(OH)DHT2 to 1
,25(OH)2DHT2 in these studies using similar methods, although we did observe more polar metabolites of both 25(OH)DHT2 and 1
,25(OH)2DHT2 following incubation with parathyroid cells. These polar metabolites are likely to represent 24-oxidation products (data not shown). Unfortunately there was insufficient material for their characterization. The observed PTH suppression by 25(OH)DHT2 indicates that this metabolite is biologically active and, by implication, capable of binding to the VDR without first undergoing 1
-hydroxylation. This is compatible with our previous studies showing that 25(OH)DHT2 binds to the VDR; 25(OH)DHT2 was found to bind to VDR extracted from bovine thymus and chick intestine, although with an affinity 1000 times less than calcitriol and 724 times less than 1
,25(OH)2DHT2 [11]. Those studies also examined the ability of the DHT3 metabolites to activate transcription within COS-1 cells which had been transfected with a rat osteocalcin vitamin D-responsive element coupled to a growth hormone reporter gene. In that model, both 1
,25(OH)2DHT3 and 25(OH)DHT3 increased transcription of growth hormone; 1
,25(OH)2DHT3 was only 10 times less active than calcitriol, whereas 25(OH)DHT3 was 25 times less active than calcitriol, findings compatible with the present observation that 25(OH)DHT2 is an active metabolite capable of suppressing PTH secretion by parathyroid cells. It is therefore likely that the 3ß-hydroxyl group of the 25(OH)DHT2 metabolite is acting as a pseudo-1
-hydroxyl group in this setting.
Although these results show that 1-hydroxylation of 25(OH)DHT2 is not required to confer activity, we have previously established that 1
-hydroxylation occurs even in anephric man; we identified 1,25(OH)2DHT2 in patients with renal failure who were treated with DHT2 [15]. The site of 1
-hydroxylation of 25(OH)DHT2 has not been established. Hep 3B hepatoma cells were not able to metabolize 25(OH)DHT2 to 1
,25(OH)2DHT2 [11], and the present studies demonstrate that parathyroid cells do not detectably metabolize 25(OH)DHT2 to 1
,25(OH)2DHT2. Thus the site(s) of the extrarenal 1
-hydroxylation of 25(OH)DHT2 remains unknown.
In vitro, we found that 10-fold higher concentration of 1,25(OH)2DHT2 relative to calcitriol, resulted in similar PTH suppression (the linear regression lines for the effect of the two sterols on PTH secretion was shifted by approximately one log order). By contrast, in single-dosing studies, hypercalcaemic equivalence was in the range of 10-to 100-fold, and in the multiple dosing studies, equivalent PTH suppression was achieved by 1
,25(OH)2DHT2 at a dose 50-fold greater than calcitriol. It is possible that these differences in potency may be related to difference in catabolism of the sterols in these experiments. The known low binding affinity of DHT metabolites to DBP may enhance their rate of clearance in vivo. As with OCT, rapid clearance from the circulation may explain the phenomenon of dissociation of PTH suppressing activity from hypercalcaemic activity.
Our studies have clearly demonstrated for the first time that both 1,25(OH)2DHT2 and 25(OH)DHT2 have powerful inhibitory effects on PTH secretion by bovine parathyroid cells. We conclude that the direct effect of 25(OH)DHT2 supports the hypothesis that the 3ß-hydroxyl group of DHT acts as a pseudo-1
hydroxyl group, and 25(OH)DHT2 does not require further activation to exert biological activity. The magnitude of the suppression of PTH secretion in vitro is similar to that achieved by calcitriol, though higher concentrations of the sterols were required. The effects of the DHT2 metabolites on blood ionized calcium were different. Single-dose studies of 1
,25(OH)2DHT2 caused a smaller and more transient effect on serum calcium than calcitriol. Multiple dosing studies suggested that 1
,25(OH)2DHT2 can dissociate the PTH suppressing activity from the hypercalcaemic activity. We hypothesize that, like OCT, this phenomenon is possibly to be due to pharmacokinetic properties of 1
,25(OH)2DHT2. In particular, low binding of 1
,25(OH)2DHT2 to DBP may cause its rapid clearance from the circulation and account for its transient hypercalcaemic activity. DHT2 remains a licensed pharmaceutical and the presented results suggest that further studies of the ability of DHT2 and its metabolites to dissociate PTH suppression from calcaemia in clinical uraemia are warranted.
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Acknowledgments |
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Notes |
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References |
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