Therapeutic uses of vitamin D analogues

Therapeutic Uses of Vitamin D Analogues Alex J. Brown, PhD ● The vitamin D endocrine system has been implicated in numerous biological activities throughout the body. The breadth and magnitude of vitamin D activity suggest potential therapeutic applications for the treatment of several diseases and disorders, including hyperproliferative diseases, immune dysfunction, endocrine disorders, and metabolic bone diseases. However, therapy using natural vitamin D hormone, 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) has been precluded in most cases because of the potent calcemic activity shown by this hormone. Newly developed vitamin D analogues with lower calcemic activity have been shown to retain many therapeutic properties of 1,25(OH)2D3. Molecular studies discussed in this article provide insights into the unique target cell specificity afforded by these analogues. In particular, the importance of the nuclear vitamin D receptor (VDR), serum vitamin D– binding protein, 24-hydroxylase, and membrane receptor is noted because analogue selectivity, specificity, and potency are afforded through their molecular interactions. The nuclear VDR has been isolated from a variety of target cells and tissues, suggesting that vitamin D compounds may have therapeutic potential throughout several body systems. Five vitamin D analogues have been approved for use in patients: calcipotriol (Dovonex; Leo Pharmaceuticals, Copenhagen, Denmark) for the treatment of psoriasis, 19-nor-1,25(OH)2D2 (Zemplar; Abbott Laboratories, Abbott Park, IL) for secondary hyperparathyroidism, doxercalciferol (Hectorol; Bone Care Int, Madison, WI) for reduction of elevated parathyroid hormone levels, 22-oxacalcitriol (Maxacalcitol; Chugai Pharmaceuticals, Tokyo, Japan), and alfacalcidol. Several other analogues are currently being tested in preclinical and clinical trials for the treatment of various types of cancer and osteoporosis, as well as immunosuppression. Understanding how analogues exert their selective actions may allow for the design of more effective and safer vitamin D compounds for the treatment of a wide range of clinical disorders. © 2001 by the National Kidney Foundation, Inc. INDEX WORDS: Vitamin D; analogues; hypercalcemia; hyperparathyroidism; cancer; psoriasis; osteoporosis.

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ITAMIN D HAS a central role in calcium and phosphate homeostasis by promoting the dietary absorption of calcium and facilitating bone resorption and mineralization. These classic effects of the hormone are achieved through the actions of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) on target cells of the intestine, bone, kidney, and parathyroid gland. However, vitamin D also shows multiple activities throughout several other organs and systems of the body. The vitamin D receptor (VDR) is present in numerous target cells, including cells and tissues of the skin, skeleton, digestive tract, nervous system, immune system, vasculature, heart, lungs, and various other systems throughout the body.1-16 Vitamin D performs nonclassic hormone functions within these target tissues. These vitamin D activities are achieved predominantly through interaction of the hormone with the VDR (genomic responses) and through interactions with a cell-surface–mediating receptor that causes nongenomic responses. Vitamin D has been suggested for potential therapeutic applications in the treatment of several diseases and disorders, including hyperproliferative disorders (eg, cancer and psoriasis), immune dysfunction (autoimmune disease), transplant survival, and endocrine disorders (eg, hyperparathyroidism).17 Each

of these applications is discussed in greater detail in this report. The therapeutic potential of 1,25(OH)2D3 has been limited by hypercalcemic toxicity brought on by this hormone. Conversely, vitamin D analogues have shown significantly reduced hypercalcemic toxicity at therapeutic dosages. Analogues already exist for the treatment of psoriasis (calcipotriol) and secondary hyperparathyroidism (paricalcitol injection, oral doxercalciferol, doxercalciferol injection, and 22-oxacalcitriol (OCT; Maxacalcitol; Chugai Pharmaceuticals, Tokyo, Japan]; Table 1). Other therapeutic applications are currently under investigation. This article reviews the potential for vitamin D analogue therapy for several disorders through its action on various target cells throughout the body and discusses the molecular rationale for predicting therapeutic application of vitamin D analogues, as well as mechanisms by which

From the Renal Division, Washington University School of Medicine, St Louis, MO. Address reprint requests to Alex J. Brown, PhD, Renal Division, Washington University School of Medicine, St Louis, MO 63110. E-mail: [email protected] © 2001 by the National Kidney Foundation, Inc. 0272-6386/01/3805-0504$35.00/0 doi:10.1053/ajkd.2001.28111

American Journal of Kidney Diseases, Vol 38, No 5, Suppl 5 (November), 2001: pp S3-S19

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ALEX J. BROWN Table 1.

Application

Secondary hyperparathyroidism 19-Nor-D2 OCT 1␣(OH)D2 (doxercalciferol) Psoriasis Calcipotriol OCT 1,24(OH)2D3 (tacalcitol) Cancer 19-Nor-D2 Calcipotriol EB1089 OCT 1,25(OH)2-16-ene-23-yne-F6-D3 1,25(OH)2-16-ene-23-yne-D3 1,22(S),25(OH)3-24-homo-F6-D3 25(OH)D3 PR-1906 Immunosuppression KH1060 MC1288 Osteoporosis ED-71 Osteoarthritis MC1288

Therapeutically Active Vitamin D Analogues Preclinical

Clinical

Approval

Source

X X X

X X X

X X X

Abbott Laboratories Chugai Pharmaceuticals Bone Care Int

X X

X X

X

Leo Pharmaceuticals Chugai Pharmaceuticals

X X X X X X X X X

X X X X

Leo Pharmaceuticals Leo Pharmaceuticals Chugai Pharmaceuticals Hoffman-La Roche Hoffman-La Roche

X X

Leo Pharmaceuticals Leo Pharmaceuticals

X

X

?

X

these analogues exert their selective actions on predicted target cells. Finally, this article reviews in vitro, in vivo, and clinical trials in which vitamin D analogues have been evaluated. BACKGROUND: MOLECULAR MECHANISMS OF 1,25(OH)2D3 AND ANALOGUE ACTIVITY

Vitamin D compounds exert their effects on target cells through molecular pathways that are becoming progressively better understood. Two types of pathways mediate vitamin D compound actions: the genomic pathway, involving interaction with the VDR, and the nongenomic pathway, involving interaction with a cell-surface receptor. In addition, two other types of proteins can greatly influence activities of vitamin D compounds. Metabolizing enzymes, notably vitamin D 24-hydroxylase, can inactivate 1,25(OH)2D3 and some of its analogues, but also produce stable metabolites of other analogues that retain significant biological activity. Transport proteins, including serum vitamin D–binding protein (DBP) and lipoprotein, can control the uptake of vitamin D compounds into target cells. Thus, activity of a particular vitamin D compound in vivo is

X

Chugai Pharmaceuticals

determined by the combined interactions with these four types of proteins. Structural modifications introduced into vitamin D analogues can alter these interactions to produce a unique biological profile for each compound. Understanding the structure-activity relationships for interactions may allow the design of analogues with selective therapeutic actions (Fig 1). MECHANISMS FOR THE SELECTIVITY OF VITAMIN D ANALOGUES

Analogue Selectivity Based on Conformational Changes in the VDR Most biological activities of 1,25(OH)2D3 analogues can be attributed to binding interactions with the VDR. The wide distribution of VDRs indicates that vitamin D compounds can exert their activities in a variety of target organs and cells. The VDR has been isolated from bone cells,1 skin cells,2,3 prostate-cancer cells,4 T lymphocytes from patients with tuberculosis and sarcoidosis,5 normal and neoplastic human bladder tissue,6 and human neuroblastoma cells.7 The VDR also has been isolated from mouse pulmonary adenomas8; chicken muscle tissue, myo-

VITAMIN D ANALOGUES

Fig 1. Structures of 1,25-(OH)2D3 and its analogues that are currently under development for various therapeutic applications.

blasts, kidney, heart, and brain cells9; rat cardiac muscle10; intestinal epithelial cells11; rat liver cells12; and central13 and peripheral14 nervous system cells. Studies have shown the presence of the VDR in rat testis and uterus, and in vivo studies indicated that germ-cell division and maturation may be associated with rat VDR of the testis.15 Animal models have shown production of 1,25(OH)2D3 in synovial fluids of arthritic joints and VDR expression at sites of cartilage erosion and in rheumatoid synovial tissues.16 The mechanism by which the VDR mediates genomic actions of vitamin D compounds is becoming clearer. On binding 1,25(OH)2D3 or an active analogue, the VDR undergoes a conformational change that allows it to interact with several other macromolecules. The first is another transcription factor, the retinoid X receptor (RXR).17 The VDR-RXR heterodimer is the ac-

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tive complex that binds to specific DNA sequences (vitamin D response elements) in target genes. Once bound to DNA, the complex recruits other factors, including steroid-receptor coactivators and other proteins of the transcriptional complex. In this way, vitamin D compounds can alter the rate of target-gene transcription. For a complete review of these mechanisms, see Peleg et al.17a Clearly, vitamin D analogues with therapeutic potential must bind to the VDR with relatively high affinity and induce a conformational change that is functionally active. Although there is a general relationship between binding affinity for the VDR and biological activity, especially with natural metabolites of vitamin D, it is now clear that some analogues have activity much greater than predicted from their VDR affinity. Peleg et al17 found that analogues with 20-epi stereochemistry had greater biological activity than predicted from their VDR-binding affinities. This interaction was the result of tighter VDR complexes with RXR, leading to enhanced binding of this heterodimer to DNA response elements in the target cell. Evidence from proteolytic enzyme digests suggested that these analogues induce a different conformational change in the VDR than 1,25(OH)2D3 (Fig 2). Considering other interactions of the ligand-bound VDR with other components of the transcriptional complex, it is possible that unique VDR conformations induced by vitamin D analogues could influence transcriptional activation in a gene-specific man-

Fig 2. Ligand-dependent changes in the VDR. Diagram of different effects of 1,25-(OH)2D3 and 20-epi1,25-(OH)2D3 on VDR conformation. Evidence suggests that 20-epi-1,25-(OH)2D3 produces a distinct conformation that increases interaction of the VDR with the RXR and subsequent DNA binding. (Data from Brown.138)

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ner, giving rise to selective actions of therapeutic utility. Analogue Specificity Based on Interactions With Serum DBP In the circulation, more than 99% of 1,25(OH)2D3 is protein bound, mostly to DBP, the major carrier of vitamin D compounds. DBP is present in significant molar excess and binds all natural vitamin D metabolites, although other proteins, such as albumin and lipoproteins, can also bind vitamin D compounds. Unlike the VDR, DBP does not require the 1-hydroxyl group. However, alterations in the vitamin D side chain greatly influence the affinity for DBP. DBP acts as a reservoir for active vitamin D compounds; thus, DBP decreases tissue accessibility and has a key role in guarding against vitamin D intoxication. DBP also enhances the circulating half-life of vitamin D compounds. Pharmacologically, DBP affinity for vitamin D analogues appears to have a major role in tissue selectivity. To date, side-chain modification in analogues has been the most typical structural change seen in 1,25(OH)2D3 analogues. In nearly all cases, this modification reduces analogue interaction with the DBP, causing the analogue to be rapidly cleared from circulation, but also enhances accessibility to the target cells. In one of the best studied examples of this pharmacokinetic mechanism of selectivity, 22-oxa1,25(OH)2D3 (OCT) affinity for DBP in rats was found to be 500 times less than that of 1,25(OH)2D3.18 As a result, OCT is rapidly cleared from the circulation and produces lower peak levels after injection.19,20 Despite lower peak levels of OCT in the blood, peak levels in the intestine and parathyroid gland were greater than those of 1,25(OH)2D3. However, OCT content in target tissues decreased quickly after its clearance from circulation, and this is responsible for the relatively short-lived effects of OCT compared with 1,25(OH)2D3 on intestinal calcium transport and bone calcium mobilization.19 In contrast to its transient effects in intestine and bone, OCT treatment produces a prolonged effect on serum parathyroid hormone (PTH) and PTH messenger RNA (mRNA). In normal rats, injection with 40 ng of OCT or 1,25(OH)2D3

ALEX J. BROWN

resulted in significantly decreased PTH mRNA levels 48 hours later.21 Similarly, in uremic dogs, a single 5-mg injection of OCT suppressed serum PTH to less than pretreatment levels for more than 69 hours after injection.22 These findings suggest that increases in intestinal calcium transport and bone mobilization are short-lived responses, whereas PTH suppression has a long half-life. Thus, it appears that pharmacokinetic properties of OCT allow this analogue to effectively suppress PTH with minimal effects on calcium transport and bone mobilization. Low DBP affinity also may be responsible for reduced absorption of vitamin D analogues through the skin. Calcipotriol, available for the treatment of psoriasis, has very low DBP affinity. Because of this, only scant amounts of the topically applied analogue are taken up into the circulation, allowing greater amounts of the analogue to be applied to psoriatic lesions without the risk for hypercalcemia.23 Although most analogues have lower affinity to DBP compared with 1,25(OH)2D3, some analogues, such as 2-(3-hydroxypropy)-1,25(OH)2D3 (ED-71), have greater affinities. These analogues tend to have longer circulating half-lives and less accessibility to target tissues. ED-71 has been shown to be an effective treatment for osteoporosis, producing greater restoration of mineral density than 1,25(OH)2D3 in ovariectomized rats. It is unclear whether the greater efficacy of ED-71 is caused by its pharmacokinetics or other properties. In some cases, altered DBP affinity cannot be shown, and other mechanisms may be implicated for analogue selectivity. Such is the case with 19-nor-1,25(OH)2D2 (19-nor-D2), for which recent in vivo studies24 showed that DBP affinity, clearance rate, and tissue localization were not significantly different from those of 1,25(OH)2D3. 24-Hydroxylase: Metabolite Activation and Degradation of Analogue Competitors Target-cell metabolism may have a role in analogue selectivity, as it does in other important steroid systems. Selectivity may be achieved by either conversion of hormones to more active metabolites or efficient degradation of hormones to inactive forms. For example, in mineralocorticoid-responsive tissue, aldosterone and glucocor-

VITAMIN D ANALOGUES

ticoids are bound with equal affinity to the aldosterone-mediating receptor. However, efficient degradation of glucocorticoids renders them unavailable to the receptor, resulting in aldosterone selectivity. Other examples, such as the metabolic pathway involving thyroxine conversion, show how selectivity is achieved through metabolic activation of hormones. Analogues may exert selective activity by the same mechanisms. Vitamin D and its analogues induce vitamin D 24-hydroxylase, which catalyzes the major pathway of cellular metabolism for these compounds. This metabolism involves a series of oxidation reactions occurring at carbons 23 and 24, culminating in oxidative cleavage of the side chain. Although the side-chain cleavage product is inactive, intermediary metabolites retain some biological activity, although they have lower VDR affinity and less activity than the parent compound. Thus, side-chain metabolism by vitamin D 24-hydroxylase is generally believed to have a role primarily in attenuating the activity of vitamin D compounds. Most of the analogues under development are altered in the side-chain portion of the molecule, which can influence the rate of metabolism and therefore activity within the target cell. Structural modifications that enhance side-chain metabolism decrease activity, whereas modifications that slow metabolism enhance activity. A study by Zhao et al25 showed in vitro how these metabolic differences could exert differential biological activities. 1,25(OH)2D3 and several analogues were examined for their antiproliferative effects on cultured MCF-7 breast-cancer cells. The addition of a cytochrome P-450 inhibitor that blocks 24-hydroxylase activity (ketoconazole) caused a reduction in the median effective dose for each analogue. The degree of reduction in median effective dose varied among analogues, likely the result of differences in catabolism rates by 24-hydroxylase.25 Two other analogues, EB1089 and 20-epi-1,25(OH)2D3, have VDR affinities similar to that of 1,25(OH)2D3, but much greater activity. At least part of this disparity may be attributed to lower metabolism rates and accumulation of active intermediates of the analogues.26-28 Cell-specific differences in metabolism of an analogue relative to 1,25(OH)2D3 may produce

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selective actions at the tissue or cell level. For example, similar biological activities of OCT and 1,25(OH)2D3 are seen in parathyroid cells,29 and the two compounds are degraded at similar rates. However, in keratinocytes, OCT is less active than 1,25(OH)2D3 and is degraded more rapidly.30 In monocytes, OCT is degraded more slowly than 1,25(OH)2D3.31 Thus, differences in catabolism could produce cell-specific effects of vitamin D analogues. The enzyme 24-hydroxylase may be responsible for converting some analogues to active metabolites that accumulate in target cells. This has been shown with both in vitro32 and in vivo33 studies. For example, 24-hydroxylase converts the analogue 1,25(OH)2-16-ene-D3 to an active 24-oxo intermediate.34 However, further oxidation occurs very slowly, with the active analogue intermediate retaining significant biological activity. The analogue 20-epi1,25(OH)2D3 experiences a similar interruption in side-chain catabolism, with the active 24-oxo metabolite accumulating in target cells, most likely retaining considerable biological activity.31 In another example, the analogue KH1060 and its 24- and 26-hydroxylated metabolites are individually more potent in inhibiting cell proliferation compared with 1,25(OH)2D3. These findings indicate that these potent metabolites may contribute to the overall activity of the parent compound in slowing cell proliferation.35 Additional pathways of metabolism, such as epimerization of carbon 3, may contribute favorably to overall biological activity of certain analogues, as seen in the investigation by Reddy.36 Investigation of tissue specificity in which the 3␤-hydroxyl is changed to the 3␣ configuration found that this epimerization occurs in keratinocytes, parathyroid cells, osteoblastic cells, and colon-cancer (Caco-2) cells. However, this epimerization is absent in kidney and myeloidleukemia (HL-60) cells. In these in vitro studies of parathyroid cells, 3-epi-1,25(OH)2D3 had nearly the same PTH-suppressing potency as 1,25(OH)2D3. However, 3-epi-1,25(OH)2D3 is catabolized more slowly by 24-hydroxylase and may accumulate in parathyroid cells, possibly causing the prolonged effects of 1,25(OH)2D3 on PTH seen in vivo.36

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Nongenomic Activity Mediated by a Cell-Surface Receptor In vitro studies showed that 1,25(OH)2D3 controls gene transcription through interaction with the VDR and also interacts at the cell membrane, causing rapid, apparently nongenomic responses in target cells. These rapid effects of 1,25(OH)2D3 on calcium transport have been shown in several in vitro systems, including nongenomic activities seen in perfused duodenum,37 phosphate fluxes in isolated enterocytes,38 alkaline phosphatase in duodenum,39 cyclic guanosine monophosphate in kidney cortex,40 and duodenum.41 Phosphoinositide metabolism was shown in isolated enterocytes,42 myocytes,43 and parathyroid cells.44 In addition, nongenomic responses to 1,25(OH)2D3 led to increases in cytosolic calcium levels within minutes after addition, too rapid to involve changes in gene transcription. Increased intracellular calcium levels with 1,25(OH)2D3 treatment have been observed in a number of cell types, including osteoblasts,45 parathyroid cells,46 human myeloid-leukemia cells,47 enterocytes,48 and myocytes.48 The role of the nongenomic pathway in the overall response to 1,25(OH)2D3 remains controversial. Numerous studies suggested that these nongenomic actions may not be critical to the biological activity of 1,25(OH)2D3,49-54 including inhibition of cell proliferation.51,55 Conversely, it was proposed that nongenomic events may modulate the genomic actions of 1,25 (OH)2D3,56,57 possibly by rapidly activating protein kinases that phosphorylate the VDR and alter its activity. Phosphorylations of the VDR by protein kinases A and C have been shown to decrease VDR activity somewhat, whereas phosphorylation by casein kinase may potentially enhance VDR activity. Thus, protein kinase C and possibly A may serve to attenuate the genomic actions of 1,25(OH)2D3 because these enzyme activities are rapidly activated by vitamin D in many target cells. It is possible that differential activation of inhibitory kinases could alter the VDR and therefore influence the genomic activity of an analogue. The ligand specificity for membrane receptors that mediates nongenomic actions is clearly different from that of the VDR.58-60 Vitamin D analogues may vary widely in their abilities to

ALEX J. BROWN

activate nongenomic pathways within target cells, which could ultimately affect their genomic activities. Thus, membrane receptors represent new pharmacological targets for vitamin D analogues that could produce cell- or gene-specific effects. TARGETS OF VITAMIN D ANALOGUE THERAPIES

The previous discussion of structural features and molecular interactions shows potential mechanisms by which vitamin D analogues may exert target-cell selectivity. In general, a successful analogue must retain high activity in the therapeutic target, but have low calcemic and phosphatemic activities. For most applications, use of 1,25(OH)2D3 has been precluded by the potent calcemic activity shown by this hormone. The remainder of this article explores various targets of analogue therapy. Secondary Hyperparathyroidism Patients with chronic renal failure often develop hyperparathyroidism secondary to phosphate retention and reduced serum 1,25(OH)2D3 levels. Elevated PTH levels can lead to highturnover bone disease and osteitis fibrosa. Prevention and treatment of this disorder require control of serum phosphate and replacement therapy with 1,25(OH)2D3. However, the use of calciumbased phosphate binders to retard dietary phosphate absorption combined with 1,25(OH)2D3 treatment poses a significant risk for hypercalcemia. In addition, 1,25(OH)2D3 is a phosphatemic hormone that can contribute to hyperphosphatemia in renal patients. Another form of renal osteodystrophy, low-turnover (adynamic) bone disease, is becoming increasingly common in these patients and has been attributed to inappropriately low levels of PTH arising from overtreatment with 1,25(OH)2D3 and calcium. Thus, it is clear that analogues are needed that effectively suppress PTH, but with less risk for producing hypercalcemia and hyperphosphatemia. The first analogue shown to meet these criteria, in experimental animal models of renal failure, was OCT.61 This analogue from Chugai Pharmaceuticals contains an oxygen in place of carbon 22 of the side chain (Fig 1). Early in vitro studies using parathyroid cell cultures showed that OCT reduced PTH secretion as effectively as 1,25(OH)2D3.21 Subsequent in vivo studies

VITAMIN D ANALOGUES

confirmed that normal rats injected with a single 40-ng dose of OCT showed a 60% to 80% reduction in pre-pro PTH mRNA levels in parathyroid glands, levels similar to those obtained with the same dose of 1,25(OH)2D3. Studies of uremic rats showed that PTH suppression by OCT is accompanied by less calcemic activity than with 1,25(OH)2D3. The impact of OCT therapy on bone also has been examined. A study of nephrectomized dogs showed that OCT significantly reduced serum PTH levels. OCT reversed abnormal bone formation and improved mineralization lag time, but did not significantly alter the level of bone turnover.62 Importantly, there was no development of low-turnover (adynamic) bone disease. Similar positive findings were reported in the uremic rat model. Most recently, bone histomorphometric analysis in a small group of renal patients showed that OCT reduced bone turnover and fibrosis.63 Thus, by effectively reducing PTH levels, OCT was able to ameliorate high-turnover bone disease and did not produce adynamic bone. This selective action of parathyroid glands suggested that OCT would be an effective treatment for secondary hyperparathyroidism. Clinical trials of this analogue have been completed, but little information has been published on its effectiveness in patients, especially compared with 1,25(OH)2D3.63,64 OCT has been approved for the treatment of secondary hyperparathyroidism in Japan.

Fig 3. Suppression of PTH by 19-norD2 in uremic rats. Partially nephrectomized rats were maintained for 1 month on a high (0.9% P) phosphate diet to promote development of secondary hyperparathyroidism. The rats were then injected IP every other day for 8 days with the specified dose of 1,25(OH)2D3 or 19-norD2. Serum was analyzed for ionized calcium and PTH 24 hours after the last injection. PTH values are expressed as the percentage of pretreatment levels; serum calcium levels are posttreatment. Data are expressed as the mean ⴞ SEM (n ⴝ 12 to 15).

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Unlike 1,25(OH)2D3, the analogue 19-nor1,25(OH)2D2 (19-nor-D2) lacks the exocyclic carbon 19 and has a vitamin D2 instead of vitamin D3 side chain (Fig 1). Developed by Abbott Laboratories (Abbott Park, IL), 19-nor-D2 is now available as Zemplar (paricalcitol injection) for the treatment of secondary hyperparathyroidism. Efficacy of 19-nor-D2 was found in initial studies to be similar to that of 1,25(OH)2D3 in suppressing PTH secretion. In vivo studies using uremic rats found 19-nor-D2 to be effective in decreasing serum PTH levels with little or no hypercalcemia65 (Fig 3). 19-Nor-D2 treatment also inhibited parathyroid gland hyperplasia in uremic rats66,67 through the induction of the cell-cycle inhibitor p21 and suppressing expression of transforming growth factor-␣ in parathyroid glands. Contrary to the effect of 1,25(OH)2D3, which increases the intestinal VDR, 19-nor-D2 suppresses the intestinal VDR, a possible explanation for the absence of calcemic activity in these studies.68 A recent report examining effects of 19-nor-D2 on bones of uremic rats showed that the analogue could both prevent and reverse osteitis fibrosa in these animals.69 Clinical trials with 19-nor-D2, discussed in more detail in other articles in this supplement, showed that this analogue can safely reduce PTH levels in patients with renal failure.70 Doxercalciferol (1␣[OH]D2), another analogue for the treatment of elevated PTH levels associated with secondary hyperparathyroidism,

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is marketed as Hectorol by Bone Care Int (Madison, WI). In clinical trials, 1␣(OH)D2 reduced PTH levels in patients with renal failure without markedly increasing levels of hypercalcemia.71 Studies of hemodialysis patients with secondary hyperparathyroidism have shown that intermittent oral therapy with 1␣(OH)D2 effectively suppressed intact PTH (iPTH), with acceptable levels of mild hypercalcemia and hyperphosphatemia.72 In two 24-week trials of oral doxercalciferol, 91 of 99 hemodialysis patients with initial iPTH levels of 400 pg/mL showed greater than 50% suppression of iPTH.73 Intravenous doxercalciferol showed similar efficacy to the oral product in a 12-week open-label trial with an 8-week washout period, but less hypercalcemia and hyperphosphatemia.74 The low calcemic activities of these newer analogues may also allow their use for treatment of primary hyperparathyroidism. This application has proven impractical with 1,25(OH)2D3 because of its tendency to worsen hypercalcemia in these patients. Although the analogues described have not been shown to be more effective than 1,25(OH)2D3 in suppressing PTH in vivo or in vitro, they are of great importance because their reduced tendency to cause hypercalcemia. Whether analogues can be modified to exert greater suppressive activity than 1,25(OH)2D3 remains under investigation. Therapeutic Targets: The Digestive System Colon cancer. Epidemiological evidence has suggested that as UV light exposure or vitamin D levels decrease, the risk for developing certain noncutaneous neoplasms, including colon cancer, increases.75-77 Support for this inverse correlation comes from in vitro studies that confirmed the presence of the VDR in a number of cancercell lines and showed growth inhibition in these cell lines as a result of 1,25(OH)2D3 treatment.1,5-9 In vivo studies showed that 22(S)-24-homo26,27-hexafluoro-1,22,25(OH)3D3 inhibited both growth and invasiveness of human colon-cancer cells placed in immunocompromised mice; cell growth resumed after withdrawal of analogue treatment.78 In a colon-cancer model using rats, a different vitamin D analogue, 1,25(OH)2-16-ene23-yne-26,27-hexafluoro-D3, induced a threefold

ALEX J. BROWN

reduction in tumor incidence with no change in serum calcium or phosphorus levels both before and during azoxymethane induction of tumors.79 In carcinogen-induced colon and small-intestine cancer in rats, OCT was shown to inhibit cell growth.80 Other in vivo studies of nude mice showed that EB1089 (seocalcitol) caused growth reduction of 41% to 49% in the LoVo coloncancer cell line without hypercalcemia.81 In a recent phase I human study of EB1089, tolerable dose estimations were determined to be 7 mg/ m2/d. In this study of 36 patients with advanced breast and colon cancer, 6 patients on treatment for more than 90 days showed stabilization of the disease, with significant reductions in hypercalcemia compared with 1,25(OH)2D3.82 Pancreatic cancer. Initial studies of animals showed the potential of vitamin D analogues in the treatment of pancreatic cancers, particularly when these compounds were used in combination with existing chemotherapies.83-85 These therapeutic effects were observed in xenograft studies examining the effects of 1,25(OH)2D3 or OCT on several pancreatic cell lines.86 Results of these in vivo studies suggested that both compounds could effectively block the proliferation of three of nine cell lines. OCT, because of its lower calcemic activity, may be preferable for treatment of pancreatic cancer. Liver cancer. Early clinical results of an antiproliferative study of liver cancer showed that EB1089 retained the low calcemic activity previously observed in animal models, and this analogue was especially active in the induction of tumor regression in hepatocellular carcinoma, in which complete remission was obtained.87 Therapeutic Targets: Bone and the Skeletal System Osteoporosis. The importance of vitamin D to bone metabolism has led to investigation of the potential benefits of 1,25(OH)2D3 for treatment of osteoporosis. 1,25(OH)2D3 and its precursor, 1␣(OH)D3, were shown to increase bone mineral density in many studies.88-94 Enhanced calcium absorption may be the mechanism for these observed increases; the frequency of hypercalcemia observed in these studies certainly supports this hypothesis. Although bone mineral density increased, this finding did not always

VITAMIN D ANALOGUES

translate to the important parameter of reduced fractures.91-94 New vitamin D analogues in development hold the promise of increased bone formation and reduced bone resorption without associated hypercalcemia. Preclinical trials with one compound, ED-71, showed increased bone mass after ovariectomy in a rat model.95 Other in vivo research with ED-71 found that through increased bone formation, reduced resorption, and enhanced intestinal calcium absorption, the analogue was able to counter glucocorticoid-induced osteopenia.96 Human clinical trials with ED-71 are underway. Therapeutic Targets: Muscle and Skin Psoriasis. Vitamin D is converted to its active form by the skin, and active forms of 1,25(OH)2D3 have a vital role in skin-tissue function through their effects on proliferation and differentiation in keratinocytes. Development of new 1,25(OH)2D3 analogues initially focused on the treatment of psoriasis, a skin disorder resulting from problems in epidermal differentiation. Calcipotriol, the first analogue to be introduced for this purpose, was developed by Leo Pharmaceutical Products (Copenhagen, Denmark).97 Structural modifications of calcipotriol include joining carbons 26 and 27 of the vitamin D side chain to form a cyclopropyl ring, with the hydroxyl group at carbon 24 and a double bond at carbon 22 (Fig 1). Calcipotriol binds the VDR with affinity similar to that of 1,25(OH)2D3, and the ability of calcipotriol to facilitate terminal differentiation of proliferating keratinocytes, modulate release of immune mediators by keratinocytes, and suppress the immune system also were similar.98-100 However, calcipotriol is differentiated from 1,25(OH)2D3 in its effects on urinary calcium levels and bone mass. Both orally and intraperitoneally, calcipotriol was substantially less likely (200 times less potent) to increase urinary calcium levels or decrease bone mass in normal rats. This finding suggested a possible topical application of calcipotriol for the treatment of psoriasis, and its efficacy has been shown in clinical trials.101 Very little of the compound reaches the circulation because of its low affinity for DBP, and in vivo studies suggested the analogue is very quickly metabolized. Calci-

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potriol is marketed as Psorcutun, Daivonex, and Dovonex and is currently approved for the treatment of psoriasis in Europe and the United States. Other vitamin D analogues, including 1,24(OH)2D3 (tacalcitol), have been investigated as topical psoriasis treatments.102 1,24(OH)2D3 inhibits keratinocyte proliferation as effectively as 1,25(OH)2D3, but because it is only slightly less calcemic, it may prove to be less selective than calcipotriol.103 OCT, which is more active than 1,25(OH)2D3 in terminally differentiating keratinocytes in vitro,104 also is currently being tested for its efficacy in the treatment of psoriasis. Recent in vitro studies showed that OCT was able to inhibit the proliferation of murine T lymphocytes stimulated by con-A and suppress interleukin-8 and interleukin-6 production by keratinocytes. OCT also inhibited activator protein-1 (AP-1) and nuclear factor-␬B activation in the suppression of the inflammatory process.105 In addition, a recent study of OCT and 1,25(OH)2D3 showed that these agents caused cell degeneration on normal and psoriatic epidermis in organ culture by the process of necrosis, rather than apoptosis. This is the first report of cell degeneration as a direct effect of 1,25(OH)2D3.106 Therapeutic Targets: Blood and the Vascular System Leukemia. Among its other actions, it has long been recognized that 1,25(OH)2D3 modulates hematopoiesis, with effects on the differentiation and proliferation of myeloid leukemia cells. In a study evaluating treatment with 1,25(OH)2D3, patients with myelodysplastic syndrome, who may go on to develop acute myelogenous leukemia, failed to realize clinical benefit, and most experienced hypercalcemia.107,108 Results obtained with the analogue 1␣(OH)D3109,110 confirmed earlier findings, pointing to the need for clinically effective but less calcemic analogues. In a preclinical study evaluating the analogue 1,25(OH)2-16-ene-23-yne-D3, mice inoculated with the myeloid-leukemia cell line WEHI3BD⫹ survived significantly longer at doses that failed to produce hypercalcemia.111 These encouraging findings suggest that further investigation of vitamin D compounds may prove beneficial for leukemia therapy, particularly the study of those

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analogues showing potential for hematopoietic activity. Therapeutic Targets: Immune and Lymphatic Systems Immunologic disorders. Immunosuppressive properties of 1,25(OH)2D3 have spawned interest in its potential applications to increase transplant graft survival and treat autoimmune diseases. However, the high doses of 1,25(OH)2D3 necessary for immune suppression in vivo would cause hypercalcemia; therefore, less calcemic analogues are needed. Animal models for autoimmune disease (experimental autoimmune encephalomyelitis, thyroiditis, and nephritis) have been used to measure the ability of vitamin D compounds to alter the progression of autoimmune disease.112 In these models, use of 1,25(OH)2D3 to treat autoimmune disorders in vivo was, as predicted, severely limited by hypercalcemia, with peak effectiveness attained when compounds were administered before the induction of experimental autoimmunity. The knowledge that 1,25(OH)2D3 activity is potentiated by cyclosporine may be exploited so that analogues with greater immune suppression and less hypercalcemic activity may be developed for combined use with cyclosporine or other immunosuppressants. Transplant rejection. Prevention of graft rejection may provide yet another application of vitamin D–mediated immunosuppression. The administration of various vitamin D compounds significantly prolonged survival of skin,113-115 heart,116-118 and kidney allografts,112,119 indicating tremendous potential in this area. In addition, combining vitamin D analogues (KH1060 and MC1288) with cyclosporine A potentiated allograft survival.114,120 These findings suggest that analogue immunosuppressive properties may permit reduced use of cyclosporine A and provide alternative therapies to glucocorticoid combination regimens. Therapeutic Targets: Genitourinary and Reproductive Organs Prostate cancer. Cancer of the prostate is the most commonly diagnosed non-skin malignancy in American men. As a consequence, the finding that 1,25(OH)2D3 blocks the growth of certain prostatic cell lines121 is of substantial interest. In addition, the potential benefit of vitamin D

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therapy for prostate cancer is suggested by the ability of 1,25(OH)2D3 to cause irreversible growth inhibition in primary cultures derived from prostate tumors.122 Chen et al4 investigated effects of two analogues, 25(OH)D3 and 19-norD2, comparing their antiproliferative activity to that of 1,25(OH)2D3 in primary cultures and cell lines of human prostate-cancer cells, as well as their relative activities in the transactivation of the VDR in androgen-insensitive PC-3 cell lines. In vitro studies showed that 1,25(OH)2D3 and 19-nor-D2 caused similar dose-dependent inhibition in the cell and primary cultures. 25(OH)D3 had inhibitory effects similar to those of 1,25(OH)2D3, indicating that prostate cells express the 1␣-hydroxylase and therefore are capable of activating 25(OH)D3 to 1,25(OH)2D3. Additional support for the potential benefit of vitamin D therapy in prostate cancer was found in several in vivo studies. Tumor weight reductions were noted in mice inoculated with prostatecell lines LNCaP or PC-3 and treated with 1,25(OH)2D3.121 Tumor incidence was decreased by 42% and 45% in two studies using 1,25(OH)2D3 and 1,25(OH)2-16-ene-23-ynehexafluoro-D3 before tumor induction.123 Treatment with the vitamin D analogue EB1089 (seocalcitol), which causes less hypercalcemia than 1,25(OH)2D3, also produced significant growth reduction.124 Based on these encouraging preliminary animal studies, human clinical trials were initiated. A phase II trial was run in 11 patients with hormone-refractory metastatic prostate cancer; however, 1,25(OH)2D3 was unable to reduce tumor mass at hypercalcemic doses.125 Konety et al126 reported results of in vitro studies of prostatecancer cells in which greater growth inhibition was observed with a combination of EB1089 and 9-cis retinoic acid, suggesting that retinoids may have a modulating effect on 1,25(OH)2D3. Therefore, vitamin D therapy combined with other agents may be effective in treating this type of cancer. Recent studies also have drawn a weak but general connection between polymorphisms in the VDR and susceptibility of prostate cancer. The study of three separate VDR polymorphisms in a population of 132 sporadic cases of prostate cancer suggested that VDR polymorphisms may

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have a role in the disease and serve as markers for prostate cancer.127 Breast cancer. Vitamin D analogue therapy has the potential to arrest breast cancer. In vitro, preclinical, and clinical studies are currently in progress to evaluate potential analogue therapies for breast cancer. The demonstrated in vitro ability of 1,25(OH)2D3 to inhibit cell proliferation and promote differentiation of breast-cancer cells led to investigation of the therapeutic potential of vitamin D analogues for breast cancer.17 Both healthy and cancerous breast tissues contain the VDR128; VDR content and VDR-binding activity are normal in malignant tissue, and VDR content has not been related to breast cancer survival. However, disease-free survival time after initial treatment may be reduced in the absence of tumor VDR. Animal studies suggest that vitamin D therapy may have some efficacy in breast cancer. In rats, reductions in the growth rate of nitrosomethylurea-induced mammary tumors were noted after treatment with 0.5-mg/kg doses of 1␣(OH)D3, which is converted to 1,25(OH)2D3 in vivo. However, greater doses of 1␣(OH)D3 produced hypercalcemia despite a low-calcium diet.129 1,25(OH)2D3, as well as the 20-epi analogues KH1060, KH1049, and MC1301, were not effective, although the analogue EB1089 (seocalcitol) developed by Leo Pharmaceuticals significantly slowed tumor progression at a dose of 0.5 mg/ kg/d without hypercalcemia. Although greater suppression was achieved at 2.5 mg/kg, hypercalcemia occurred.129 In MCF-7 breast-cancer cells in nude mice, EB1089 was shown to reduce tumor growth by fourfold compared with untreated mice. Induction of apoptosis markers correlated with reduced tumor growth.130 In another study, EB1089 markedly reduced the total number of bone metastases, mean surface area of osteolytic lesions, and tumor burden within bone when tested in nude mice injected with the human breast cancer cell line MDA-MB-23. Longitudinal analysis showed that mice treated with EB1089 had a marked increase in survival and developed fewer bone lesions over time.131 Another analogue, OCT, worked synergistically with tamoxifen, effectively blocking the growth of human breast-cancer cells implanted in athymic mice.83 The growth of rat mammary tumors induced by 7,12-dimeth[a]anthracene was

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also inhibited by OCT.132 Serum calcium levels were unaffected by therapeutic doses of the analogue in either study. 1␣,25-Dihydroxy-16-ene23-yne-26,27-hexafluoro-vitamin D3, in combination with tamoxifen, also reduced tumor burden in nitrosomethylurea-treated rats. By itself, the analogue was still able to increase tumor latency and reduce tumor incidence.84 Effective doses of 1␣,25-dihydroxy-16-ene-23-yne-26,27-hexafluorovitamin D3 were nonhypercalcemic. A crucial development for antiestrogen-treated patients with breast cancer is the finding that two different human breast-cancer cell lines resistant to antiestrogen therapy were more sensitive to treatment with EB1089 than the parent MCF-7 cell line. This is an important finding, given that most patients with breast cancer treated with antiestrogens eventually develop resistance to treatment.133 Clinical trials for vitamin D therapy for patients with breast cancer remain rare. However, daily topical application of 100 mg of calcipotriol in a group of 14 patients found a 50% reduction in lesion diameter in three patients and a lesser response in another patient.134 Vitamin D analogues are currently being studied in clinical trials; no published data are available to date. Therapeutic Targets: Joints Osteoarthritis. In experiments using rats with collagen-induced arthritis, MC1288 was able to suppress previously established collagen-induced arthritis through the analogue’s immunomodulatory properties without inducing hypercalcemia. MC1288 was able to decrease the incidence and severity of arthritis, and treatment with this analogue diminished serum levels of rat collagen antibodies (CII).135 Similarly, mitogeninduced proliferation of lymph-node cells from rat CII-immunized animals was reduced.135 Genetic analysis also linked the VDR to osteoarthritis. A study of 851 men showed that adjacent genes, the COL2A1 and VDR genes, are involved in radiographic osteoarthritis. The COL2A1 genotype was associated with narrowing of joint space, whereas the VDR genotype was associated with osteophytes.136 Therapeutic Targets: The Pulmonary System Lung cancer. Although VDRs have been detected in alveolar T lymphocytes of patients with

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ALEX J. BROWN

pulmonary granulomatous disease (peripheral T lymphocytes were VDR negative),5 the application of analogues for lung cancer has shown little promise to date. Analogues with side-chain modifications, PRI-1906, PRI-1907, PRI-1909, PRI2191, PRI-2192, PRI-2193, and PRI-2194, were tested against a spectrum of various human cancer-cell lines using the MTT-tetrazolium technique.137 All analogues tested, except PRI-1909, showed antiproliferative activity against cell lines of human breast cancer, but none of these analogues showed antiproliferative activity against other human cancer-cell lines tested, including those originating from lung, colon, prostate, urinary bladder, ovary, pancreas, stomach, and kidney. Antiproliferative activity in these cell lines did not exceed the experimental cutoff of 20%. CONCLUSION

This article presents evidence for the potential of vitamin D therapy for treatment of a number of disorders. These targets of classic and nonclassic vitamin D endocrine activity include cells of the skin and nervous, skeletal, reproductive, and digestive systems, as well as many other tissues. In most cases, the high calcemic activity of 1,25(OH)2D3 has precluded its use. New vitamin D analogues with lower calcemic activity have been shown to retain many of the therapeutic effects of 1,25(OH)2D3. Currently, analogues are in use for treatment of psoriasis (calcipotriol) and secondary hyperparathyroidism (paricalcitol, doxercalciferol, OCT, and alfacalcidol). Other analogues are in preclinical and clinical trials for various types of cancer, autoimmune disease and transplant rejection, and osteoporosis. Finally, a clearer understanding of molecular mechanisms that allow these compounds to exert therapeutic activity with less hypercalcemia may lead to the development of new vitamin D analogues with greater specificity. REFERENCES 1. van Leeuwen JP, van den Bemd GJ, van Driel M, Buurman CJ, Pois HA: 24,25-Dihydroxyvitamin D3 and bone metabolism. Steroids 66:375-380, 2001 2. Babina M, Krautheim M, Grutzkau A, Henz BM: Human leukemic (HMC-1) mast cells are responsive to 1alpha, 25-dihydroxyvitamin D3: Selective promotion of ICAM-3 expression and constitutive presence of vitamin D3 receptor. Biochem Biophys Res Commun 273:1104-1110, 2000

3. Li XY, Boudjelal M, Xiao JH, Peng ZH, Asuru A, Kang S, Fisher GJ, Voorhees JJ: 1,25-Dihydroxyvitamin D3 increases nuclear vitamin D3 receptors by blocking ubiquitin/ proteasome-mediated degradation in human skin. Mol Endocrinol 13:1686-1694, 1999 4. Chen TC, Schwartz GG, Burnstein KL, Lokeshwar BL, Holick MF: The in vitro evaluation of 25-hydroxyvitamin D3 and 19-nor-1,25-dihydroxyvitamin D2 as therapeutic agents for prostate cancer. Clin Cancer Res 6:901-908, 2000 5. Biyoudi-Vouenze R, Cadranel J, Valeyre D, Milleron B, Hance AJ, Soler P: Expression of 1,25(OH)2D3 receptors on alveolar lymphocytes from patients with pulmonary granulomatous diseases. Am Rev Respir Dis 143:1367-1380, 1991 6. Konety BR, Lavelle JP, Pirtskalaishvili G, Dhir R, Meyers SA, Nguyen TS, Hershberger P, Shurin MR, Johnson CS, Trump DL, Zeidel ML, Getzenberg RH: Effects of vitamin D (calcitriol) on transitional cell carcinoma of the bladder in vitro and in vivo. J Urol 165:253-258, 2001 7. Celli A, Treves C, Stio M: Vitamin D receptor in SH-SY5Y human neuroblastoma cells and effect of 1,25dihydroxyvitamin D3 on cellular proliferation. Neurochem Int 34:117-124, 1999 8. Zhong K, Chua BH, Palmer KC: Vitamin D3 receptor expression in N-ethylnitrosourea-induced mouse pulmonary adenomas. Am J Respir Cell Mol Biol 11:480-486, 1994 9. Zanello SB, Collins ED, Marinissen MJ, Norman AW, Boland RL: Vitamin D receptor expression in chicken muscle tissue and cultured myoblasts. Horm Metab Res 29:231-236, 1997 10. Walters MR, Wicker DC, Riggle PC: 1,25-Dihydroxyvitamin D3 receptors identified in the rat heart. J Mol Cell Cardiol 18:67-72, 1986 11. Armbrecht HJ, Boltz MA, Hodam TL, Kumar VB: Differential responsiveness of intestinal epithelial cells to 1,25-dihydroxyvitamin D3: Role of protein kinase C. J Endocrinol 169:145-151, 2001 12. Duncan WE, Whitehead D, Wray HL: A 1,25dihydroxyvitamin D receptor-like protein in mammalian and avian liver nuclei. Endocrinology 122:2584-2589, 1988 13. Veenstra TD, Prufer K, Koenigsberger C, Brimijoin SW, Grande JP, Kumar R: 1,25-Dihydroxyvitamin D3 receptors in the central nervous system of the rat embryo. Brain Res 804:193-205, 1998 14. Cornet A, Baudet C, Neveu I, Baron-Van Evercooren A, Brachet P, Naveilhan P: 1,25-Dihydroxyvitamin D3 regulates the expression of VDR and NGF gene in Schwann cells in vitro. J Neurosci Res 53:742-746, 1998 15. Osmundsen BC, Huang HF, Anderson MB, Christakos S, Walters MR: Multiple sites of action of the vitamin D endocrine system: FSH stimulation of testis 1,25-dihydroxyvitamin D3 receptors. J Steroid Biochem 34:339-343, 1989 16. Tetlow LC, Woolley DE: The effects of 1alpha,25dihydroxyvitamin D3 on matrix metalloproteinase and prostaglandin E2 production by cells of the rheumatoid lesion. Arthritis Res 1:63-70, 1999 17. Peleg S, Sastry M, Collina WS, Viahop JE, Norman AW: Distinct conformational changes induced by 20-epi analogues of 1␣,25-dihydroxyvitamin D3 are associated with

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enhanced activation of the vitamin D receptor. J Biol Chem 270:10551-10558, 1995 17a. Peleg S: Molecular basis for differential action of vitamin D analogs, in Feldman D, Glovieux F, Pike JW (eds): Vitamin D. New York, NY, Academic, 1997, pp 1011-1025 18. Okano T, Tsugawa N, Masuda S, Takeuchi A, Kobayashi T, Nishii Y: Protein-binding properties of 22-oxa1alpha,25-dihydroxyvitamin D3, a synthetic analogue of 1alpha,25-dihydroxyvitamin D3. J Nutr Sci Vitaminol 35:529533, 1989 19. Kobayashi T, Okano T, Tsugawa N, Masuda S, Takeuchi A, Nishii Y: Metabolism and transporting system of 22-oxacalcitriol. Contrib Nephrol 91:129-133, 1991 20. Brown AJ, Finch J, Grieff M, Ritter C, Kubodera N, Nishii Y, Slatopolsky E: The mechanism for the disparate actions of calcitriol and 22-oxacalcitriol in the intestine. Endocrinology 133:1158-1164, 1993 21. Brown AJ, Ritter CR, Finch JL, Morrissey J, Martin KJ, Murayama E, Nishii Y, Slatopolsky E: The noncalcemic analogue of vitamin D, 22-oxacalcitriol, suppresses parathyroid hormone synthesis and secretion. J Clin Invest 84:728732, 1989 22. Brown AJ, Finch JL, Lopez-Hilker S, Dusso A, Ritter C, Pernalete N, Slatopolsky E: New active analogues of vitamin D with low calcemic activity. Kidney Int 38:S22S27, 1990 (suppl 29) 23. Binderup L: Comparison of calcipotriol with selected metabolites and analogues of vitamin D3: Effects on cell growth regulation in vitro and calcium metabolism in vivo. Pharmacol Toxicol 72:240-244, 1993 24. Brown AJ, Finch J, Takahashi F, Ritter CS, Slatopolsky E: Distinct mechanisms for the selective actions of two vitamin D analogues, 19-nor-1,25(OH)2D2 and 22-oxa1,25(OH)3D3, on the parathyroid glands. J Am Soc Nephrol 8:571A, 1997 (abstr) 25. Zhao J, Tan BK, Marcelis S, Verstuyf A, Bouillon R: Enhancement of antiproliferative activity of 1 alpha,25dihydroxyvitamin D3 (analogues) by cytochrome P450 enzyme inhibitors is compound and cell-type specific. J Steroid Biochem Mol Biol 57:197-202, 1996 26. Shankar VN, Dilworth FJ, Makin HL, Schroeder NJ, Trafford DJ, Kissmeyer AM, Calverley MJ, Binderup E, Jones G: Metabolism of the vitamin D analogue EB1089 by cultured human cells: Redirection of hydroxylation site to distal carbons of the side-chain [published erratum in Biochem Pharmacol 53:1946, 1997]. Biochem Pharmacol 53: 783-793, 1997 27. Hansen CM, Maenpaa PH: EB1089, a novel vitamin D analogue with strong antiproliferative and differentiationinducing effects on target cells. Biochem Pharmacol 54:11731179, 1997 28. Dilworth FJ, Calverley MJ, Makin HL, Jones G: Increased biological activity of 20-epi-1,25-dihydroxyvitamin D3 is due to reduced catabolism and altered protein binding. Biochem Pharmacol 47:987-993, 1994 29. Brown AJ, Berkoben M, Ritter C, Kubodera N, Nishii Y, Slatopolsky E: Metabolism of 22-oxacalcitriol by a vitamin D-inducible pathway in cultured parathyroid cells. Biochem Biophys Res Commun 189:759-764, 1992 30. Bikle DD, Abe-Hashimoto J, Su MJ, Felt S, Gibson

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DF, Pillai S: 22-Oxa calcitriol is a less potent regulator of keratinocyte proliferation and differentiation due to decreased cellular uptake and enhanced catabolism. J Invest Dermatol 105:693-698, 1995 31. Kamimura S, Gallieni M, Kubodera N, Nishii Y, Brown AJ, Slatopolsky E, Dusso A: Differential catabolism of 22-oxacalcitriol and 1,25-dihydroxyvitamin D3 by normal human peripheral monocytes. Endocrinology 133:27192723, 1993 32. Siu-Cldera ML, Clark JW, Santos-Moore A, Peleg S, Liu YY, Uskokovic MR, Sharma S, Reddy GS: 1Alpha,25dihydroxy-24-oxo-16-ene vitamin D3, a metabolite of a synthetic vitamin D3, is equipotent to its parent in modulating growth and differentiation of human leukemic cells. J Steroid Biochem Mol Biol 59:405-412, 1996 33. Lemire JM, Archer DC, Reddy GS: 1,25-Dihydroxy24-oxo-16ene-vitamin D3, a renal metabolite of the vitamin D analogue 1,25-dihydroxy-16ene-vitamin D3, exerts immunosuppressive activity equal to its parent without causing hypercalcemia in vivo. Endocrinology 135:2818-2821, 1994 34. Reddy GS, Clark JW, Tserng KY, Uskokovic MR, McLane JA: Metabolism of 1,25(OH)2-16-ene-D3 in kidney: Influence of structural modification of D-ring on side-chain metabolism. Bioorg Med Chem Lett 3:1879-1884, 1993 35. Dilworth FJ, Williams GR, Kissmeyer AM, Nielsen JL, Binderup E, Calverley MJ, Makin HL, Jones G: The vitamin D analog, KH1060, is rapidly degraded both in vivo and in vitro via several pathways: Principal metabolites generated retain significant biological activity. Endocrinology 138:5485-5496, 1997 36. Reddy GS: Target tissue specific metabolism of 1alpha,25(OH)2D3 through A-ring modification, in Norman AW (ed): Tenth Workshop on Vitamin D, Strasbourg, France. Berlin, Germany, de Gruytes, 1997, p 65 (abstr) 37. Nemere I, Yoshimoto Y, Norman AW: Calcium transport in perfused duodena from normal chicks: Enhancement within fourteen minutes of exposure to 1,25-dihydroxyvitamin D3. Endocrinology 115:1476-1483, 1984 38. Karsenty G, Lacour B, Ulmann A, Pierandrei E, Drueke T: Early effects of vitamin D metabolites on phosphate fluxes in isolated rat enterocytes. Am J Physiol 248: G40-G45, 1985 39. Bachelet M, Ulmann A, Lacour B: Early stimulation of alkaline phosphatase activity in response to 1alpha,25dihydroxycholecalciferol. Biochem Biophys Res Commun 89:694-700, 1979 40. Vesely DL, Juan D: Cation-dependent vitamin D activation of human renal cortical guanylate cyclase. Am J Physiol 246:E115-E120, 1984 41. Guillemant J, Guillemant S: Early rise in cyclic GMP after 1,25-dihydroxycholecalciferol administration in the chick intestinal mucosa. Biochem Biophys Res Commun 93:906-911, 1980 42. Lieberherr M, Grosse B, Duchambon P, Drueke T: A functional cell surface type receptor is required for the early action of 1,25-dihydroxyvitamin D3 on the phosphoinositide metabolism in rat enterocytes. J Biol Chem 264:2040320406, 1989 43. Morelli S, de Boland AR, Boland RL: Generation of inositol phosphates, diacylglycerol and calcium fluxes in

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myoblasts treated with 1,25-dihydroxyvitamin D3. Biochem J 289:675-679, 1993 44. Bourdeau A, Atrnani F, Grosse B, Lieberherr M: Rapid effects of 1,25-dihydroxyvitamin D3 and extracellular Ca2⫹ on phospholipid metabolism in dispersed porcine parathyroid cells. Endocrinology 127:2738-2743, 1990 45. Lieberherr M: Effects of vitamin D3 metabolites on cytosolic free calcium in confluent mouse osteoblasts. J Biol Chem 262:13168-13173, 1987 46. Sugimoto T, Ritter C, Ried I, Morrissey J, Slatopolsky E: Effect of 1,25-dihydroxyvitamin D3 on cytosolic calcium in dispersed parathyroid cells. Kidney Int 33:850854, 1988 47. Hruska KA, Bar-Shavit Z, Malone JD, Teitelbaum S: Ca2⫹ priming during vitamin D-induced monocytic differentiation of a human leukemia cell line. J Biol Chem 263:1603916044, 1988 48. Lucas PA, Roullet C, Duchambon P, Lacour B, Drueke T: Rapid stimulation of calcium uptake by isolated rat enterocytes by 1,25(OH)2D3. Pflugers Arch 413:407-413, 1989 49. Khoury R, Ridall AL, Norman AW, Farach-Carson MC: Target gene activation by 1,25-dihydroxyvitamin D3 in osteosarcoma cells is independent of calcium influx. Endocrinology 135:2446-2453, 1994 50. Khoury RS, Weber J, Farach-Carson MC: Vitamin D metabolites modulate osteoblast activity by Ca⫹2 influxindependent genomic and Ca⫹2 influx-dependent nongenomic pathways. J Nutr 125:S1699-S1703, 1995 (suppl 6) 51. Norman AW, Bouillon R, Farach-Carson MC, Bishop JE, Zhou LX, Nemere I, Zhao J, Muralidharan KR, Okamura WH: Demonstration that 1beta,25-dihydroxyvitamin D3 is an antagonist of the nongenomic but not genomic biological responses and biological profile of the three A-ring diastereomers of 1alpha,25-dihydroxyvitamin D3. J Biol Chem 268:20022-20030, 1993 52. Jurutka PW, Terpening CM, Haussler MR: The 1,25dihydroxy-vitamin D3 receptor is phosphorylated in response to 1,25-dihydroxy-vitamin D3 and 22-oxacalcitriol in rat osteoblasts, and by casein kinase II, in vitro. Biochemistry 32:8184-8192, 1993 53. Zhou LX, Norman AW: 1Alpha,25(OH)2-vitamin D3 analogue structure-function assessment of intestinal nuclear receptor occupancy with induction of calbindin-D28K. Endocrinology 136:1145-1152, 1995 54. Farach-Carson MC, Abe J, Nishii Y, Khoury R, Wright GC, Norman AW: 22-Oxacalcitriol: Dissection of 1,25(OH)2D3 receptor-mediated and Ca2⫹ entry-stimulating pathways. Am J Physiol 263:F705-F711, 1993 55. Hedlund TE, Moffatt KA, Miller GJ: Stable expression of the nuclear vitamin D receptor in the human prostatic carcinoma cell line JCA-1: Evidence that the antiproliferative effects of 1alpha, 25-dihydroxyvitamin D3 are mediated exclusively through the genomic signaling pathway. Endocrinology 137:1554-1561, 1996 56. Baran DT: Nongenomic actions of the steroid hormone 1,25-dihydroxyvitamin D3. J Cell Biochem 56:303306, 1994 57. Baran DT, Sorensen AM, Shalhoub V, Owen T, Stein G, Lian J: The rapid nongenomic actions of 1,25-dihydroxyvitamin D3 modulate the hormone-induced increments in osteo-

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