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NDT Advance Access originally published online on October 12, 2005
Nephrology Dialysis Transplantation 2006 21(3):644-650; doi:10.1093/ndt/gfi186
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© The Author [2005]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Original Articles: Experimental Nephrology

The vitamin D prodrugs 1{alpha}(OH)D2, 1{alpha}(OH)D3 and BCI-210 suppress PTH secretion by bovine parathyroid cells

Alex J. Brown1, Cynthia S. Ritter1, Joyce C. Knutson2 and Stephen A. Strugnell2

1 Renal Division, Washington University School of Medicine, St Louis, MO and 2 Preclinical Research, Bone Care International, Middleton, WI, USA

Correspondence and offprint requests to: Alex J. Brown, PhD, Renal Division, Washington University School of Medicine, Box 8126, 660 S. Euclid, St. Louis, MO 63110. Email: abrown{at}imgate.wustl.edu



   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Background. Active vitamin D compounds are widely used in the treatment of secondary hyperparathyroidism associated with renal failure. These compounds reduce PTH secretion through vitamin D receptor (VDR)-dependent repression of PTH gene transcription. In previous studies, 1{alpha}(OH)D3, a vitamin D prodrug, inhibited PTH secretion in cultured bovine parathyroid cells, but it was unclear whether 1{alpha}(OH)D3 itself or an active metabolite produced this inhibition.

Methods. We determined the effectiveness of the vitamin D prodrugs 1{alpha}(OH)D3, 1{alpha}(OH)D2 and 1{alpha}(OH)-24(R)-methyl-25-ene-D2 (BCI-210) at inhibiting PTH secretion in bovine parathyroid cell cultures, and examined the metabolism of [3H]1{alpha}(OH)D2 in these cells.

Results. All three prodrugs suppressed PTH secretion with approximately 10% of the activity of 1,25(OH)2D3; much higher activity than expected based on the VDR affinities of these prodrugs (0.25% of 1,25(OH)2D3). Parathyroid cells activated [3H]1{alpha}(OH)D2 to both 1,25(OH)2D2 and 1,24(OH)2D2. 1,24(OH)2D2 was detectable at 4 h, increased to a maximum at 8 h, and then decreased. In contrast, 1,25(OH)2D2 levels increased linearly with time, suggesting the presence of constitutively active vitamin D-25-hydroxylase not previously reported in parathyroid cells. The cytochrome P-450 inhibitor ketoconazole (50 µM) reduced 1{alpha}(OH)D2 metabolism to below detectable levels, but did not significantly affect suppression of PTH by 1{alpha}(OH)D2.

Conclusions. The vitamin D prodrugs 1{alpha}(OH)D3, 1{alpha}(OH)D2 and BCI-210 suppressed PTH production by cultured parathyroid cells. The ability of 1{alpha}(OH)D2 to reduce PTH despite inhibition of its metabolism suggests a direct action of this ‘prodrug’ on the parathyroid gland, but the mechanism underlying this activity is not yet known.

Keywords: BCI-210; 1{alpha}-hydroxyvitamin D2; 1{alpha}-hydroxyvitamin D3; ketoconazole; parathyroid hormone; vitamin D



   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Vitamin D therapy is widely used for the treatment of the secondary hyperparathyroidism associated with chronic renal failure [1,2]. The active hormonal form of vitamin D, 1,25-dihydroxyvitamin D, and its analogues can reduce serum PTH levels by repressing PTH gene transcription [3,4] and blocking parathyroid gland proliferation [5]. These actions are mediated by the vitamin D receptor (VDR) present in the PTH-secreting chief cells of the parathyroid glands [6–8]. High-affinity binding to the VDR generally requires hydroxyl groups at carbon 1 and at carbon 25 (or nearby carbons of the side chain). The synthetic prohormones, 1{alpha}(OH)D3 (alfacalcidol) and 1{alpha}(OH)D2 (doxercalciferol) (Figure 1), are widely used for treatment of secondary hyperparathyroidism of kidney disease, but these compounds have relatively low intrinsic affinity for the VDR compared to active compounds such as calcitriol [9], and have been thought to require systemic activation to exert their effects on the parathyroid glands.


Figure 1
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  		Fig. 1. Structures of the vitamin D prodrugs used in the current study.

 
An unpublished study by Olgaard and coworkers demonstrated a direct suppression of PTH secretion by 1{alpha}(OH)D3 in parathyroid tissue culture [10]. This initial study did not investigate whether suppression of PTH by 1{alpha}(OH)D3 required conversion to a more active metabolite. We have shown that parathyroid cells possess vitamin D hydroxylase (CYP24) activity [11,12], and may therefore be capable of activating vitamin D prodrugs such as 1{alpha}(OH)D3 in situ. A recent report by Correa et al. [13] confirmed the existence of CYP24 expression in human parathyroid tissue as well as the presence of mRNAs for both 25-hydroxyvitamin D 1{alpha}-hydroxylase (CYP27B1) and vitamin D 25-hydroxylase (CYP27A1). Although hydroxylase enzyme activity was not demonstrated, these findings suggest the possibility that the PTH suppressive activity of 1{alpha}(OH)D3 and 1{alpha}(OH)D2 may be due, at least in part, to conversion to active metabolites in parathyroid cells.

In the present study, we have examined the in vitro suppression of PTH secretion by several vitamin D prodrugs lacking side chain hydroxyl groups. [3H]-1{alpha} (OH)D2 was used to follow the metabolism and potential activation of 1{alpha}-hydroxyvitamin D compounds by parathyroid cells. The effect of blocking metabolism with the cytochrome P450 inhibitor ketoconazole on the ability of the 1{alpha}(OH)D2 to suppress PTH was assessed. Demonstration of direct effects on the parathyroid glands of vitamin D prodrugs could have important implications for the design of vitamin D analogues for the treatment of secondary hyperparathyroidism.



   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Preparation of dispersed bovine parathyroid cells
Dispersed bovine parathyroid cells were prepared as previously described [14]. Briefly, bovine parathyroid glands (obtained from MBH Enterprises, Tampa, FL, USA) were trimmed of extraneous fatty tissue, sliced to 0.5 mm thickness with a tissue slicer (Stadie Riggs; Thomas Scientific, Swedesboro, NJ) and placed in a mixture of DME:Ham's F-12 medium (50:50) containing 0.5 mM calcium and collagenase (3000 U/ml of collagenase XI-S; Sigma, St Louis, MO, USA). The suspension (10 ml media per gram of tissue) was agitated in a shaking water bath at 37°C for 90 min. Periodic passage of the mixture through the tip of a 10 ml pippette assisted in the disaggregation. The digested tissue was washed three times with serum-free culture medium containing DME:Ham's F-12 (50:50), 1 mM CaCl2, 15 mM Hepes, 100 IU/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml insulin, 5 µg/ml holo-transferrin, 2 mM glutamine, 1% non-essential amino acids and 0.1% bovine serum albumin (fraction V).

Dispersed cells were plated in 12-well culture plates at 0.2 x 106/well in the above medium containing 4% newborn calf serum. After 24 h, the medium was replaced with serum-free medium. The medium was changed every 2–3 days of culture.

Assessment of PTH suppression activity
Primary cultures of bovine parathyroid cells were grown to confluency in serum-free medium as previously described [15], and then treated for 72 h with 0.1, 1.0, 10 and 100 nM 1,25(OH)2D3, 1{alpha}(OH)D3, 1{alpha}(OH)D2 or BCI-210 with daily changes of medium. The cells were then changed into fresh medium and the amount of PTH secreted during a 4 h incubation was determined using an ELISA kit (Immutopics, San Capistrano, CA).

To determine the effects of ketoconazole, an inhibitor of metabolism, on PTH, confluent monolayers were treated with 1{alpha}(OH)D2 (100 nM) for 48 h with media changes every 12 h. RNA was harvested from the cells using RNAzol B (Cinna/Biotecx), resolved on 1.2% agarose/formaldehyde gels and transferred to nylon membrane (Zeta-Probe, Bio-Rad). PTH mRNA and 18S rRNA were measured by hybridizing the membranes with riboprobes described previously [16]. The data are expressed as the ratio of PTH mRNA to 18S rRNA.

Metabolism
To determine if 1{alpha}(OH)D2 is metabolized to a more active compound in parathyroid cells, [3H]1{alpha}(OH)D2 (100 nM, 1 µCi) was incubated with confluent monolayers of bovine parathyroid cells. After 0, 4, 8 or 24 h, the reactions were terminated by the addition of one volume of methanol containing 0.5 nmol of 1,25(OH)2D2 and 1,24(OH)2D2. The cells plus medium were extracted by a modification of the method of Bligh and Dyer [17]. The metabolites of 1{alpha}(OH)D2 in the organic phase were resolved by normal phase HPLC using hexane:isopropanol:methanol (91:7:2). Radioactive peaks eluting with standard 1,25(OH)2D2 or 1,24(OH)2D2 were further resolved by reverse phase HPLC (ODS-IP column, Beckman-Coulter) using methanol:water (4:1) as mobile phase.

To determine the efficiency of ketoconazole to inhibit the metabolism of 1{alpha}(OH)D2, confluent monolayers were treated with [3H]1{alpha}(OH)D2 (100 nM, 1 µCi) for 48 h with media changes every 12 h. Medium from each plate was placed in a separate container after each media change; at the end of the incubation, pooled media from each individual plate was extracted and normal phase HPLC performed as above to detect the presence of 1,25(OH)2D2 and 1,24(OH)2D2. Radioactivity in the aqueous phase of the extraction was measured to assess the amount of conversion to side chain-cleaved metabolites.



   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Suppression of PTH in vitro
The ability of the 1{alpha}-hydroxy analogues to suppress PTH was assessed in vitro in cultures of bovine parathyroid cells. The structures of the analogues tested are shown in Figure 1 and their effects on PTH secretion are shown in Figure 2. The 1{alpha}-hydroxy compounds, 1{alpha}(OH)D2, 1{alpha}(OH)D3 and BCI-210 were all effective in reducing PTH secretion, with potencies about 10% of 1,25(OH)2D3. The high activity of BCI-210, which was designed to be resistant to both 24- and 25-hydroxylation, suggests that the 1{alpha}-hydroxy compounds either do not require activation or that this compound is activated by an alternative pathway such as 26-hydroxylation.


Figure 2
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  		Fig. 2. Effect of vitamin D analogues of steady-state secretion of PTH by bovine parathyroid cells. Confluent cultures of bovine parathyroid cells were incubated with the specified concentration of the vitamin D analogue for 72 h with media changes every 24 h. Fresh medium was added and the PTH secreted into the medium during a 4-h period was determined by radioimmunoassay and normalized to cell protein. EtOH refers to vehicle (ethanol) control. Data are given as mean ± SD (n = 5–6 except control where n = 11). *P<0.05 vs control, ANOVA with pairwise multiple comparisons (Dunn's Method).

 
Metabolism of 1{alpha}(OH)D2 in bovine parathyroid cells
To determine if the 1{alpha}-hydroxy compounds may be converted to more active metabolites, parathyroid cells were incubated with [3H]1{alpha}(OH)D2 for 0, 4, 8 and 24 h, and the cells plus medium were analysed for more polar metabolites. The HPLC profiles of a representative sample at each time point are shown in Figure 3A. There was a progressive loss of radioactive 1{alpha}(OH)D2 with the appearance of tritiated peaks co-eluting with 1,24(OH)2D2 and 1,25(OH)2D2 on normal phase HPLC. The amount of 1,24(OH)2D2 increased up to 8 h after substrate addition and then declined, while the amount of 1,25(OH)2D2 increased linearly for the entire 24 h incubation.


Figure 3
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  		Fig. 3. Metabolism of [3H]1{alpha}(OH)D2 by bovine parathyroid cells. Confluent cultures of bovine parathyroid cells were incubated with [3H]1{alpha}(OH)D2 (100 nM, 1 µCi) for 0, 4, 8 or 24 h. Cell plus medium were extracted and resolved by normal phase HPLC. Radioactivity in each fraction was determined by scintillation counting. The elution postition of internal standards for 1{alpha}(OH)D2, 1,25(OH)2D2 and 1,24(OH)2D2 are shown. Panels A and B represent the same data presented with different scales on the y-axis.

 
To confirm the identities of these metabolites, the putative 1,24(OH)2D2 and 1,25(OH)2D2 peaks were isolated and rechromatographed on reverse phase HPLC (data not shown). The radioactive peaks co-eluted with the internal standards, consistent with their identities as 1,24(OH)2D2 and 1,25(OH)2D2. A third metabolite peak in the normal phase HPLC profile was observed at fraction 38, in the position expected for 1,26(OH)2D2, but the lack of a standard prevented its further analysis. There was also a progressive increase in radioactivity in the methanol strip, indicating the presence of even more polar metabolites.

The amount of the metabolites present represented a very small proportion of the intitial [3H]1{alpha}(OH)D2 as illustrated in Figure 3B, in which the scale of the y-axis of Figure 3A is expanded. The HPLC analysis indicates that about 1% of the input 10–7 M [3H]1{alpha}(OH)D2, equivalent to approximately 10–9 M 1,25(OH)2D2, was present at 24 h after substrate addition when 1,25(OH)2D2 levels were highest. In addition, we found these dihydroxylated metabolites predominantly in the medium rather than the cells (data not shown).

Effect of ketoconazole on 1{alpha}(OH)D2 metabolism and PTH suppression
The necessity of side chain hydroxylation in the suppression of PTH by 1{alpha}(OH)D2 was examined using the competitive cytochrome P450 inhibitor ketoconazole. Pilot studies indicated that at least 50 µM ketoconazole was required to completely block 1{alpha}(OH)D2 metabolism, and that the inhibitory effect could not be sustained for 24 h necessitating addition of fresh medium containing ketoconazole every 12 h for 48 h. The efficiency of inhibition of 1{alpha}(OH)D2 metabolism by ketoconazole was assessed by adding 100 nM [3H]1{alpha}(OH)D2 (or vehicle) with or without 50 µM ketoconazole every 12 h over a 48 h treatment period. The medium was collected after each 12 h time period and analysed for water-soluble radioactivity (primarily calcitroic acid) as a measure of efficiency of inhibition. As shown in Figure 4, the rate of 1{alpha}(OH)D2 metabolism in the absence of ketoconazole increased with each time period, presumably owing to the progressive induction of the 24-hydroxylase. Metabolism was completely blocked by the presence of ketoconazole; water-soluble radioactivity was the same as the no cell control at the end of each time period. Furthermore, HPLC analysis of cell and medium extracts demonstrated that no detectable dihydroxylated metabolites were produced in the presence of ketoconazole. Figure 5 shows representative HPLC profiles of combined cell plus medium extracts from cells at the end of the final incubation period. These findings clearly show that 1,24(OH)2D2 and 1,25(OH)2D2 were not produced when ketoconazole was replenished every 12 h.


Figure 4
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  		Fig. 4. Effect of ketoconazole on 1{alpha}(OH)D2 metabolism. Confluent cultures of bovine parathyroid cells were incubated with [3H]1{alpha}(OH)D2 (100 nM, 1 µCi) for 48 h in the absence (solid bars) or presence (hatched bars) of 50 µM ketoconazole with media changes every 12 h. Medium removed at the end of each 12 h period and the final cell monolayer extracted [17]. Radioactivity remaining in the aqueous phase after three extractions was quantified by scintillation counting. Data are expressed as mean±SD (n = 3). *P<0.05 vs no cell control. **P<0.05 vs ketoconazole-treated cells.

 

Figure 5
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  		Fig. 5. Effect of ketoconazole on the conversion of 1{alpha}(OH)D2 to 1,24(OH)2D2 and 1,25(OH)2D2. Confluent cultures of bovine parathyroid cells were incubated as described in Figure 4 in the absence (solid line) or presence (dashed line) of ketoconazole. HPLC profiles of extracts from cells plus medium prepared at the end of the last 12 h period are shown. The profiles are representative of triplicate samples.

 
We next examined the PTH mRNA levels in cells treated with 1{alpha}(OH)D2 (or vehicle) with or without ketoconazole under conditions identical to those above. As shown in Figure 6, 1{alpha}(OH)D2 alone decreased PTH mRNA levels by 50% comparable to the reduction in PTH secretion shown in Figure 2. Interestingly, ketoconazole alone also reduced PTH mRNA by an as yet unknown mechanism. In the presence of ketoconazole, 1{alpha}(OH)D2 further suppressed PTH mRNA by about 55%, as effectively as in the absence of ketoconazole. Similar results were obtained in two additional experiments in different cell preparations treated in the same manner.


Figure 6
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  		Fig. 6. Effects of ketoconazole on suppression of PTH by 1{alpha}(OH)D2. Confluent cultures of bovine parathyroid cells were incubated for 48 h with 100 nM 1{alpha}(OH)D2 in the absence or presence of the specified concentration of 50 µM ketoconazole with media changes every 12 h. PTH mRNA and 18S rRNA were determined by northern blot analysis. Data are given as mean± SD (n = 5–6). *P<0.05 vs corresponding non-1{alpha}(OH)D2-treated control.

 


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The parathyroid glands were definitively identified as a vitamin D target tissue soon after characterization of the active form of vitamin D, 1,25(OH)2D3 [6–8]. The glands contain a high content of VDR, and 1,25(OH)2D3 has been shown to repress PTH gene transcription [3,4] and inhibit parathyroid cell proliferation [5] through a VDR-dependent mechanism. The secondary hyperparathyroidism that develops in patients with chronic renal failure is believed to be attributable in part to the reduction of renal synthesis of 1,25(OH)2D3 and loss of suppression of parathyroid gland function by 1,25(OH)2D3. Vitamin D therapy is widely used to treat hyperparathyroidism in these patients. Although 1,25(OH)2D3 and its synthetic precursor, 1{alpha}(OH)D3 (alfacalcidol), have been successfully employed for this purpose for many years, the natural vitamin D hormone is a potent stimulator of intestinal calcium absorption and often produces hypercalcaemia, especially in patients receiving calcium-based phosphate binders to control hyperphosphataemia. This has led to the development of vitamin D analogues that retain the suppressive actions of 1,25(OH)2D3 on the parathyroid glands but have less calcaemic activity. Several analogues are now available for treatment of secondary hyperparathyroidism in renal failure patients, including 19-nor-1,25(OH)2D2 (paricalcitol), 22-oxa-1,25(OH)2D3 (maxacalcitol), 1{alpha}(OH)D3 (alfacalcidol), 1{alpha}(OH)D2 (doxercalciferol) and 1,25(OH)2-26,27-F6-D3 (falecalcitriol) [18,19]. Vitamin D prodrugs such as doxercalciferol owe their reduced calcaemic activity at least in part to the pharmacokinetics of their activation, which leads to sustained serum levels of active compound at more physiological concentrations than can be produced by administration of active compounds. Recent studies indicate that 1{alpha}(OH)D3 produces a disproportionately large degree of PTH suppression relative to calcitriol, based on observed serum levels of active metabolites, suggesting either a direct suppressive effect of such prodrugs on the parathyroid, or activation in situ [20].

To further clarify the structural requirements for effective vitamin D analogues in controlling parathyroid gland function, we investigated the ability of synthetic vitamin D prodrugs to directly suppress PTH synthesis and secretion. As reported by Nielsen et al., 1{alpha}(OH)D3 was able to suppress PTH release from cultured bovine parathyroid cells. A similar suppression was observed in the present study with 1{alpha}(OH)D2 and BCI-210. BCI-210 is a novel prodrug with a 24 (R) methyl group and a 25–27 double bond that is designed to resist both 24- and 25-hydroxylation (Figure 1). All of these prodrugs produced less PTH suppression than an equivalent dose of 1,25(OH)2D3. Nonetheless, these findings clearly indicate that parathyroid cells can respond to vitamin D compounds lacking side chain hydroxyl groups, suggesting that systemic activation (i.e. 24- or 25-hydroxylation) of these compounds in vivo may not be necessary for suppression of PTH.

Vitamin D compounds such as 1{alpha}(OH)D3 that lack a side chain hydroxyl group have relatively low affinity for the VDR (approximately 400-fold lower than calcitriol) [9]. One possibility, then, is that the activity of 1{alpha}(OH)D3, 1{alpha}(OH)D2 and BCI-210 in parathyroid cells is attributable to hydroxylated metabolites produced intracellularly i.e. target cell activation. Incubation of parathyroid cells with [3H]1{alpha}(OH)D2 led to the formation of both [3H]1,25(OH)2D2 and [3H]1,24(OH)2D2. We have previously demonstrated the presence of vitamin D-24-hydroxylase (CYP24) activity in parathyroid cells [11,15]. The amount of 1,24(OH)2D2 produced was maximal at 8 h and then decreased. This could be attributed to the drop in 1{alpha}(OH)D2 substrate concentration to lower levels at which 1,24(OH)2D2 is not formed efficiently [14] and/or to further metabolism of 1,24(OH)2D2 by CYP24 [11,12]. The 24-hydroxylase can oxidize carbons 24 and 23 of the vitamin D side chain, leading to more polar metabolites with limited VDR affinity and PTH suppressing activity [11], and eventually to side chain cleavage and inactivation [21].

Production of 1,25(OH)2D2 from 1{alpha}(OH)D2 in parathyroid cells occurred in a linear fashion for the entire incubation period. The observation that parathyroid cells are capable of 25-hydroxylation of vitamin D compounds has not been previously reported. However, Correa et al. [13] recently reported the presence of transcripts for CYP27A1, a cytochrome P450 capable of 25-hydroxylating vitamin D, in human parathyroid glands. We confirmed the presence of these transcripts in human, rat and bovine parathyroid glands (data not shown). CYP27A1 may be responsible for the observed production of 1,25(OH)2D2 in parathyroid cells. However, CYP27A1 does not 25-hydroxylate 1{alpha}(OH)D2 efficiently [22], suggesting that at least one other vitamin D 25-hydroxylase is also present in these cells.

The amount of 1,25(OH)2D2 and 1,24(OH)2D2 in the parathyroid cells cultures is relatively small at all times examined (Figure 3B). Furthermore, when ketoconazole was added to the cells every 12 h over a 48 h period, no 1,24(OH)2D2 or 1,25(OH)2D2 were detected at the end of the final 12 h treatment period. In addition, the aqueous counts, a measure of side chain cleavage via the C24 oxidation pathway, in the ketoconazole-treated samples were the same as those found with substrate incubated under identical conditions without cells for a 12 h period, indicating virtually no metabolism of the 1{alpha}(OH)D2. This complete block of metabolism did not prevent the reduction of PTH mRNA by 1{alpha}(OH)D2.

The ability of 1{alpha}(OH)D2, and probably the other vitamin D ‘prodrugs’, to suppress PTH without activation raises the question as to how this suppression is produced, given the relatively low VDR affinity of these compounds. One possibility is that these compounds accumulate in the parathyroid and reach intracellular concentrations sufficient to directly activate VDR and subsequently suppress PTH production. High intracellular levels of 1{alpha}(OH)D2 were, in fact, observed in the metabolism studies, supporting this possibility. Intraparathyroid accumulation may also explain the clinical observation that, although patients who received equal doses of intravenous or oral 1{alpha}(OH)D2 gave rise to equal serum levels of the active metabolite 1,25(OH)2D2, the dose of intravenous 1{alpha}(OH)D2 required for equivalent PTH suppression was 40% of the oral dose [23]. This clinical data is consistent with the possibility that the initial high serum levels of 1{alpha}(OH)D2 produced by intravenous administration deliver high levels of 1{alpha}(OH)D2 to the parathyroid glands, enhancing the effectiveness of PTH suppression. Further studies will be necessary to confirm this possibility.

An alternative explanation for the observed PTH-lowering ability of 1{alpha}(OH)D2 is interaction with a novel receptor. Recent studies by Panda et al. using VDR and 1{alpha}-hydroxylase null mice have suggested the possibility that the high concentrations of 1,25(OH)2D3 may act independently of the VDR to block parathyroid hyperplasia [24]. VDR null mice did not develop parathyroid hyperplasia when fed a rescue diet containing 2.0% calcium, 1.25% phosphorus, 20% lactose and 2.2 U/g vitamin D3, whereas mice lacking both VDR and 1{alpha}-hydroxylase had enlarged parathyroid glands. The presence of a non-VDR receptor that is activated by high 1,25(OH)2D3 was proposed. This as yet hypothetical receptor may mediate the effects of vitamin D compound lacking side chain hydroxyl groups including those investigated in the present study. One or both of these two alternate mechanisms may explain the high potency of (20S)-1{alpha}-hydroxy-2-methylene-19-nor-bishomopregnacalciferol (2M-bisP), an analogue lacking a side chain, to effectively lower PTH levels in both normal [25] and uraemic rats [26].

The present study also revealed that ketoconazole itself can inhibit PTH, an observation that has not been previously reported. The addition of 1{alpha}(OH)D2 produced a similar percentage reduction in PTH regardless of presence of ketoconazole, indicating both that the two compounds act via distinct mechanisms and that the mechanism utilized by 1{alpha}(OH)D2 was not significantly impaired by ketoconazole. The mechanism for the suppression by ketoconazole is under investigation.

In summary, we have shown that PTH synthesis and secretion in cultured parathyroid cells is inhibited by exogenous vitamin D prodrugs lacking side chain hydroxyl groups. This suppression was not diminished when metabolism was blocked with the cytochrome P450 inhibitor ketoconazole indicating that side chain oxidation may not be required. This direct effect may be attributed to accumulation of 1{alpha}(OH)D2 in the parathyroid to high intracellular levels that activate the vitamin D receptor or, possibly, to interaction with a novel receptor. Further studies are necessary to define the mechanism(s) involved. These findings may have important implications for the design of vitamin D analogues for treatment of secondary hyperparathyroidism.



   Acknowledgments
 
Supported by a grant from Bone Care International.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Received for publication: 17. 6.05
Accepted in revised form: 7. 9.05


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