Vitamin D Analogs for the Treatment of Secondary Hyperparathyroidism in Chronic Renal Failure Part II
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Other Analogs
The search for more effective vitamin D analogs for treating secondary hyperparathyroidism continues. Several have been tested in animal models.
Fan et al compared 1,25-dihydroxy-dihydrotachysterol [1,25(OH)2DHT], 1,25(OH)2D3, and OCT for their abilities to suppress PTH in the uremic rat model. Five daily doses of 1 ng of 1,25(OH)2D3 caused significant hypercalcemia and a 50% decrease in PTH.
In contrast, 1,25(OH)2DHT and OCT at doses of 25 and 50 ng were equipotent in suppressing PTH, and did not increase serum calcium.
Thus, 1,25(OH)2DHT may be another useful analog for the treatment of secondary hyperparathyroidism. Analogs with inverted stereochemistry at carbon 20 have been demonstrated to be more potent than the natural diasteromers in a number of in vitro biological assays.
Three of these were tested in the uremic rat model by Hruby et al. Following five daily injections of 250 ng/kg, CB 1093 suppressed PTH by 73% with no increase in serum calcium and with a slight decrease in serum phosphate (3.72 ± 0.65 to 2.95 ± 0.98 mM). In contrast, the same dose of 1,25(OH)2D3 reduced PTH by 82%, but part of this decrease could be attributed to the large increase in ionized calcium (1.17 ± 0.04 to 1.55 ± 0.14 mM) produced by this excessively high dose of 1,25(OH)2D3.
EB 1213 at a dose of 1250 ng/kg-d lowered PTH by 61% with no change in ionized calcium, and GS 1725 at a much lower dose of 25 ng/kg-d suppressed PTH by 80% with only a slight, but not significant, increase in ionized calcium. Clearly, there are many promising new analogs in development for the treatment of secondary hyperparathyroidism.
Mechanisms for the Selectivity of Vitamin D Analogs
The analogs developed for secondary hyperparathyroidism are slightly less active than 1,25(OH)2D3 in suppressing PTH, but are considerably less calcemic. The molecular basis for this selectivity is currently under investigation.
Conceptually, it is important to note that vitamin D compounds interact with only a few proteins in vivo.
In general, the biological profile of vitamin D compounds will be determined by their interaction with four proteins or classes of proteins:
1. the vitamin D receptor (VDR) that mediates the genomic actions;
2. the serum vitamin D binding protein (DBP) and perhaps lipoproteins that transport vitamin D compounds and control their access to the target cell;
3. the vitamin D-24-hydroxylase (24-OHase) and perhaps other minor enzymes that metabolize and/or deactivate the analogs; and
4. cell surface receptors that mediate the rapid, nongenomic actions of vitamin D compounds.
A plausible explanation for the selectivity of OCT, based on these protein interactions, has been proposed.
The mechanisms responsible for the actions of 19-norD2 and 1α(OH)D2 are not as clear.
This section discusses the current understanding of how these analogs exert their selectivity on the parathyroid glands.
22-Oxacalcitriol (OCT)
The basis for the selectivity of OCT is probably the best understood of the new vitamin D analogs.
Its affinity for the VDR is about 8 times lower than that of 1,25(OH)2D3, consistent with its lower activity in suppressing PTH.
However, its very low calcemic activity can be attributed primarily to its diminshed affinity for DBP which is approximately 500 times less than that of 1,25(OH)2D3. DBP affinity is a major effector of the pharmacokinetics of vitamin D compounds.
Studies in rats and dogs demonstrated that OCT is rapidly cleared from the circulation and achieves lower maximal levels (Cmax) in the blood when administered intraperitoneally.
Despite the lower Cmax, the amount of OCT associated with the intestinal VDR and parathyroid glands at early times post-injection were higher than those of 1,25(OH)2D3, but fell rapidly after the analog was cleared from the circulation.
This “pulse” of OCT in its target tissues elicited only transient increases in intestinal calcium transport and bone mobilization while the effects of 1,25(OH)2D3 were much more prolonged. However, in spite of the short time of residence of OCT in the parathyroid glands, the analog elicited a sustained suppression of PTH gene expression.
The molecular basis for the differences in the durations of the effects in the parathyroids versus those in the intestine and bone are unclear, but the findings indicate that stimulation of intestinal calcium absorption and bone resporption are short-lived responses that require continuous exposure to vitamin D compounds.
On the other hand, even a short exposure of the parathyroid glands to vitamin D compounds leads to a prolonged suppression of PTH.
Thus, OCT appears to exert its selectivity via its rapid clearance, which exploits the differences in the biological half-lives of the desirable (PTH suppression) and undesirable (increased calcium) responses.
19-nor-1,25(OH)2D2
19-norD2 exerts its selectivity in a manner distinct from that of OCT and does not involve pharmacokinetics.
The VDR affinity of 19-norD2 is about 3 times less than that of 1,25(OH)2D3, consistent with its slightly lower potency in suppressing PTH.
Unlike OCT, 19-norD2 has relatively high DBP affinity: only three time less than that of 1,25(OH)2D3.
Not surprisingly, then, the clearance rates and tissue localization of 19-norD2 and 1,25(OH)2D3 are similar.
Thus, it would appear paradoxical that 19-norD2 has lower calcemic activity than 1,25(OH)2D3 when its VDR affinity and ability to localize to the bone and intestine are very similar to 1,25(OH)2D3.
In fact, direct measurement of the intestinal calcium transport and bone mobilization at 24 hours after a single injection of 19-norD2 or 1,25(OH)2D3 revealed equivalent, dose-dependent effects of the two compounds.
Only after chronic treatment were the differences in the effects of 19-norD2 and 1,25(OH)2D3 on intestine and bone apparent.
The analog was shown to be significantly less active in stimulating intestinal calcium absorption and bone calcium mobilization after 7 daily injections.
Again, this could not be attributed to altered pharmacokinetics with chronic treatment;there was no difference in the tissue localization of 19-norD2 and 1,25(OH)2D3 following the 7 daily injections.
These findings suggested an induced resistance to 19-norD2 with repeated treatments.
This could not be attributed to differences in intestinal VDR content, since VDR binding activity was not different following the 7-day treatment with 19-norD2 or 1,25(OH)2D3. Direct effects of 19-norD2 on osteoclast maturation and osteoclastic bone mobilization were assessed in vitro using the mouse bone marrow culture system.
It is well established that inthis model, active vitamin D compounds can promote the differentiation of osteoclast precursors present in the bone marrow to multinucleated cells expressing tartrate-resistant acid phosphatase (TRAP) and other markers of mature osteoclasts.
This action is now known to be mediated by the induction of RANKL (receptor activator of NFkB ligand) on the surface of osteoblasts.
Furthermore, when the cultures containing the mature osteoclasts and osteoblasts are plated onto bone (or dentine) slices, vitamin D compounds, via continued induction of RANKL can activate the osteoclasts to the bone surface.
The mouse bone marrow model was used to assess the relative abilities of 1,25(OH)2D3 and 19-norD2 to promote osteoclast maturation and osteoclast activity.
Incubation of freshly isolated bone marrow cells in the presence of various concentrations of 1,25(OH)2D3 and 19-norD2 for 5 days revealed no differences in the potency of these compounds to induce osteoclast formation.
However, when the these cultures were plated on dentine slices in the continued presence of various concentrations of the two vitamin D compounds, the amount of resorption in response to 19-norD2 was less than that in the 1,25(OH)2D3-treated cultures.
Both 19-norD2 and 1,25(OH)2D3 produced maximum effects at 10 nM, but the number of resorption pits and the amount of bone resorption induced by 19-norD2 was only 30% of that elicited by 1,25(OH)2D3.
Further analysis revealed that the number of osteoclasts attached to the bone slices and the area resorbed per pit were the same for 19-norD2 and 1,25(OH)2D3, indicating that the reduced bone resorption in response to 19-norD2 could be attributed to a defective initiation of resorption by the osteoclasts.
In vitro bone resorption in response to 19-norD2 and 1,25(OH)2D3 was also examined by Balint et al.
They found no significant difference in the potencies of the two compounds in stimulating calcium efflux from isolated neonatal calvaria.
The apparent disparity may be attibuted to the relatively short exposure time of 48 hours, since, as noted above, differences in the in vivo bone mobilizing effects of 19-norD2 and 1,25(OH)2D3 require more than 24 hours to become apparent.
Since the actions of vitamin D compounds on bone resorption are thought to be mediated through actions on the osteoblast, Finch et al examined the effects of 19-norD2 on other vitamin D actions in the osteoblastic cell line MG63.
No differences were observed in the induction of osteocalcin, alkaline phosphatase activity or VDR content, or in the suppression of cell proliferation by 19-norD2 and 1,25(OH)2D3.
Furthermore, the rate of catabolism of 19-norD2 in the bone marrow cultures was not different from that of 1,25(OH)2D3, and VDR content was not differentially affected by 19-norD2 treatment.
Based on our current understanding of vitamin D-mediate bone resorption, these finding would suggest that the reduced bone resorbing activity of 19-norD2 may be due to a reduced ability to induce RANKL in the osteoblast, a hypothesis currently under investigation.
1α(OH)D2
The basis for the low calcemic activity of 1α(OH)D2 is much less understood.
Early studies with 1α(OH)D2 showed that it was much less toxic than 1α(OH)D3 when the compounds were administered chronically.
Paradoxically, the stimulation of calcium transport and bone mobilization by 1α(OH)D2 and 1α(OH)D3 were not different.
The reason for the lower toxicity of 1α(OH)D2 remains unclear.
Unlike the other vitamin D analogs, 1α(OH)D2 is actually a pro-hormone that is converted, under physiologic conditions,mainly to 1,25(OH)2D2 which has been reported to have the same potency as 1,25(OH)2D3 in stimulating calcium transport in vivo and bone mobilization in vivo and in vitro. However, Mawer et al demonstrated that vitamin D2, but not vitamin D3, could be converted to 1,24(OH)2D2, a metabolite with potent cell differentiation activity, but much lower calcemic activity than 1,25(OH)2D2 or 1,25(OH)2D3.
It has been proposed that 1α(OH)D2, but not 1α(OH)D3, may be 24-hydroxylated within target cells to produce an active metabolite (1,24(OH)2D2) that is less toxic.
This hypothesis remains to be tested.
The pharmacokinetics of falecalcitriol differ from those of 1,25(OH)2D3.
The fluorine atoms at the end of the side chain have been shown to impede catabolism of this molecule. Unlike 1,25(OH)2D3, which is hydroxylated at carbons 23 and 24 and then oxidatively cleaved to calcitroic acid, falecalcitriol is metabolized only partially metabolized, primarily to its 23-hydroxylated metabolite [1,23,25(OH)3-26,27-F6-D3] in vivo and in various cell culture systems.
This intermediate retains considerable biological activity.
The fluorine atoms also affect binding to DBP; falecalcitriol has a slightly lower affinity for DBP and is cleared a bit more rapidly than 1,25(OH)2D3.
Despite the shorter circulating half-life and 3-fold lower affinity for the VDR, falecalcitriol has prolonged and potent biological activity in vivo, presumably due to its slower target tissue metabolism.
Whether this analog exerts a selective action on the parathyroid glands is not known.
Future Perspectives
Less calcemic vitamin D analogs that can effectively suppress PTH in chronic renal failure patients offer safer alternatives to 1,25(OH)2D3 in the treatment of secondary hyperparathyroidism and the resulting renal osteodystrophy.
The currently available analogs represent only the initial attempts at modifying the vitamin D molecule to produce more selective drugs.
As we learn more about the how these modifications influence the interactions of vitamin D analogs with the VDR, DBP, the 24-hydroxylase and the membrane receptor and, in turn, the biological profile observed in vivo, it should be possible to design new analogs with greater specificity and a larger margin of safety for treatment of renal osteodystrophy and other diseases as well.
