Vitamin D and Analogues

Chapter 83 1

Vitamin D and Analogues Glenville Jones* Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada

INTRODUCTION Vitamin D, its metabolites and analogues constitute a valuable group of compounds that can be used to modulate many aspects of osteoblast and osteoclast biology. The parent vitamin (or UV light that substitutes for any vitamin D pharmaceutical preparation as a source of the parent vitamin) has been used as a treatment for rickets and osteomalacia since its discovery in the 1920s. The first analogue of vitamin D, dihydrotachysterol, was developed for use in metabolic bone disease in the 1930s before the elucidation of the metabolism of vitamin D. In fact, it was not until the discovery of the principal metabolites: 25-hydroxyvitamin D3 (25-OH-D3) and 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3), in the early 1970s that further generations of vitamin D analogues were developed (DeLuca, 1988; Jones and Calverley, 1993; Jones et al., 1998). With the understanding of the molecular action of the hormonal form, 1α,25-(OH)2D3, has come an appreciation that it is not only a calcemic agent, regulating calcium and phosphate transport, but also a cell-differentiating agent, promoting the terminal development of a number of cell types, including the osteoclast, the enterocyte, and keratinocyte (Miyaura et al., 1981). Thus, pharmaceutical companies have striven hard over the past three decades to separate these two properties and thereby develop synthetic vitamin D analogues with specialized “calcemic” and “noncalcemic” (cell-differentiating) uses (Calverley and Jones, 1992; Bouillon et al., 1995). From this type of research has come several “low-calcemic” agents in recent years in the form of calcipotriol, OCT, 19-nor-1α,25-(OH)2D2, and 1α-OH-D2, which have found widespread use in dermatology and the treatment of secondary hyperparathyroidism. Not only do newer analogues include specialized selective vitamin D receptor (VDR) agonists but also VDR antagonists and compounds that target CYP24, a component of the calcitriol metabolism machinery that extends the life of calcitriol within the target cell. These other analogues *

Corresponding author: Glenville Jones, Department of Biochemistry, Queen’s University, Kingston, ON, Canada K7L 3N6

Principles of Bone Biology, 3rd Edition Copyright © 2008 by Academic Press. Inc. All rights of reproduction in any form reserved.

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are thus under development for use in metabolic bone diseases, osteoporosis, and cancer (Jones et al., 1998; Masuda and Jones, 2006). This chapter will review the spectrum of compounds available, possible uses of these compounds, and their potential mechanisms of action.

PHARMACOLOGICALLY IMPORTANT VITAMIN D COMPOUNDS Vitamin D compounds can be subdivided into three major groups, listed in Tables I through III and described below.

Vitamin D and Its Natural Metabolites Table I shows the structures of vitamin D3 and some of its important metabolites. Ironically, vitamin D3, the natural form of vitamin D, is not approved for use as a drug in the United States, whereas it is available as a pharmaceutical agent or as an over-the-counter supplement in virtually every other country in the world. During the late 1960s and early 1970s, most of the principal vitamin D metabolites were first isolated and identified by GC-MS and then their exact stereochemical structure determined (DeLuca, 1988). This led to chemical synthesis of the naturally occurring isomer and its testing in various biological assays in vitro and in vivo. Currently, only the compounds representing the main pathway of vitamin D activation, namely vitamin D3, 25-hydroxyvitamin D3 (25-OH-D3), and 1α,25-(OH)2D3, are synthesized and available for use as drugs.

Vitamin D Prodrugs Table II lists some of the important prodrugs of vitamin D. All of these compounds require a step (or more) of activation in vivo before they are biologically active. Included here is vitamin D2, which is derived from the fungal sterol, ergosterol, by irradiation. Because vitamin D2 is found rarely in nature and is hard to detect in humans eating 1777

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TABLE I Vitamin D and Its Natural Metabolites Vitamin D metabolites [ring structure]*

Side-chain structure

Vitamin D3 [1]

21

24

22 20

23

27

Site of synthesis

Relative VDRbinding affinity†

Relative DBPbinding affinity‡

Reference

Skin

ⱕ0.001

3,180

Mellanby, 1919 McCollum et al., 1922

Liver

0.1

66,800

Blunt et al., 1968

Kidney

100

100

Fraser & Kodicek, 1970 Holick et al., 1971

Kidney

0.02

33,900

Holick et al., 1972

Target tissues§

10

21

Holick et al., 1973

Liver ?

0.02

26,800

Suda et al., 1970

25 26

25-OH-D3 [1]

OH

1α,25-(OH)2D3 [3]

24(R),25-(OH)2D3 [1]

OH

OH OH

1α,24(R),25-(OH)3D3 [3]

25(S),26-(OH)2D3 [1]

OH OH

OH OH

*

Structure of the vitamin D nucleus (secosterol ring structure). Values reproduced from previously published data (Stern, 1981). ‡ Values reproduced from previously published data (Bishop et al., 1994) § Known target tissues included intestine, bone, kidney, skin, and the parathyroid gland. †

Vitamin D nucleus

CH2

CH2

CH3

3

3

HO

OH

[1]

nonfortified foods, we can consider it to be an artificial form of vitamin D or prodrug. Vitamin D2 is used as a substitute for the natural form, vitamin D3, in pharmaceutical preparations or over-the-counter supplements in the United States. Vitamin D2 possesses two specific modifications of the side chain (see Table II) but these differences do not preclude the same series of activation steps as vitamin D3, these giving rise to 25-OH-D2, 1α,25-(OH)2D2, and 24,25-(OH)2D2, respectively. Recently, there has been much debate in the vitamin D field, particularly in the United States where vitamin D2 is the sole agent available, about the relative utility of vitamin D2 and vitamin D3 to raise the circulating 25-OH-D level (Vieth, 2005). Evidence from research studies suggests that oral doses of vitamin D3 are significantly more effective than equivalent doses of vitamin D2 for increasing

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3

[2]

1

OH

HO

[3]

the 25-OH-D level into the sufficient range (more than 40 ng/mL) (Trang et al., 1998; Armas et al., 2003). 25-OH-D3 was developed and approved as the pharmaceutical preparation Calderol in the 1970s by Upjohn, later acquired by Organon, but was withdrawn recently and is currently unavailable. Two other prodrugs, 1α-OH-D3 and 1α-OH-D2, were synthesized in the early 1970s (Barton et al., 1973; Paaren et al., 1978) as alternative sources of 1a,25-(OH)2D3 and 1a,25-(OH)2D2, respectively, that in the process circumvent the renal 1α-hydroxylase enzyme, which was shown to be tightly regulated and prone to damage in renal disease. The final compound in the list, dihydrotachysterol (DHT) has lived a complex history as a prodrug. Originally it was believed to be “active” when converted to 25-OH-DHT by

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TABLE II Vitamin D Prodrugs Vitamin D prodrug [ring structure]*

Side-chain structure

Company

Possible target diseases

Mode of delivery

Reference

1α-OH-D3 [3]

21

Leo

Osteoporosis

Systemic

Barton et al., 1973

Genzyme

Secondary hyperparathyroidism

Systemic

Paaren et al., 1978

Dihydrotachysterol [2]

Duphar

Renal failure

Systemic

Jones et al., 1988

Vitamin D2 [1]

Various

Rickets Osteomalacia

Systemic Systemic

Fraser et al., 1973

24

22 20

23

27

25 26

1α-OH-D2 [3]

28

*

Structure of the vitamin D nucleus (secosterol ring structure).

Vitamin D nucleus

CH2

CH2

CH3

3

HO

OH

[1]

virtue of an A ring rotated 180° such that the 3β-hydroxy1 function assumes a pseudo-1α-hydroxy1 position (Jones et al., 1988). The mechanism of action of DHT has become less clear with the description of the extrarenal metabolism of 25-OH-DHT to 1α,25-(OH)2-DHT and 1β,25-(OH)2-DHT, two further metabolites that have greater biological activity than either 25-OH-DHT or DHT itself (Qaw et al., 1993).

Vitamin D Analogues Table III lists some of the most promising vitamin D analogues of 1α,25-(OH)2D3 already approved by governmental agencies or currently under development by various industrial or university research groups. Because the number of vitamin D analogues synthesized now lists in the thousands, the table is provided mainly to give a flavor of the structures experimented with thus far, the worldwide scope of the companies involved, and the broad spectrum of target diseases and uses. The first generation of calcitriol analogues included molecules with fluorine atoms placed at metabolically vulnerable positions in the side chain and resulted in highly stable and

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3

3

[2]

1

OH

HO

[3]

potent “calcemic” agents such as 26,27-F6-1α,25-(OH)2D3. A second generation of analogues focused on features that make the molecule more susceptible to clearance, such as in calcipotriol (MC903), where a C22–C23 double bond, a 24-hydroxy1 function, and a cyclopropane ring have been introduced into the side chain or in 22-oxacalcitriol (OCT) where the 22-carbon has been replaced with an oxygen atom. Both modifications have given rise to highly promising analogues marketed initially in Europe and Japan, respectively (Kragballe, 1992; Abe-Hashimoto et al., 1993). The C-24 position is a favorite site for modification and numerous analogues contain 24-hydroxy1 groups, e.g., 1α,24(S)-(OH)2D2 and 1α,24(R)-(OH)2D3 (Strugnell et al., 1995). Other analogues contain multiple changes in the side chain in combination, including unsaturation; 20-epimerization, 22-oxa replacement; and homologation in the side chain or terminal methyl groups. The resultant molecules such as EB1089 and KH1060 attracted strong attention of researchers because of their increased potency in vitro and were pursued as possible anticancer and immunomodulatory compounds, respectively.

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TABLE III Analogues of 1α,25-(OH)2D3 Vitamin D analogue [ring structure]*

Side-chain structure

Calcitriol, 1α,25(OH)2D3 [3]

21

20

23

Possible target diseases

Mode of delivery

Reference

Roche, Duphar

Hypocalcemia Psoriasis

Systemic Topical

Baggiolini et al. (1982)

SumitomoTaisho

Osteoporosis hypoparathyroidism

Systemic Systemic

Kobayashi et al., 1982

Abbott

Secondary hyperparathyroidism

Systemic

Perlman et al., 1990

Chugai

Secondary hyperparathyroidism Psoriasis

Systemic

Murayama et al., 1986

Leo

Psoriasis Cancer

Topical Topical

Calverley, 1987

Roche

Leukemia

Systemic

Baggiolini et al., 1989

Leo

Cancer

Systemic

Binderup et al., 1991

OH

Leo

Immune diseases

Systemic

Calverley et al., 1991

OH

Deltanoids

Osteoporosis

Systemic

Shevde et al., 2002

OH

Bioxell

Prostate cancer

Systemic

Marchiani et al., 2006

OH

Chugai

Osteoporosis

Systemic

Nishii et al., 1993

27

24

22

Company

OH

25

26

26,27-F6-1α,25(OH)2D3 [3]

CF3 OH CF3

19-Nor-1α,25(OH)2D2 [5]

28

OH

22-Oxacalcitriol (OCT) [3]

O

OH

Calcipotriol (MC903) [3]

OH OH

1α,25-(OH)2-16-ene23- yne-D3 (Ro 23-7553) [6]

OH

EB1089 [3]

27a

Topical

OH 24a

20-epi-1α,25(OH)2D3 [3] 2-methylene-19-nor20-epi-1α,25-(OH)2D3 (2MD) [7] BXL-628 (formerly Ro-269228) [8] ED71 [4]

26a

1α,24(S)-(OH)2D2 [3]

OH

Genzyme

Psoriasis

Topical

Strugnell et al., 1995a

1α,24(R)-(OH)2D3 (TV-02) [3]

OH

Teijin

Psoriasis

Topical

Morisaki et al., 1975

*

Structure of the vitamin D nucleus (secosterol ring structure).

Vitamin D nucleus 16

16

CH2

CH 2

CH2

CH2

10

3

1

HO

OH

HO

2

[3]

CH83-I056875.indd 1780

OH

HO

OH

O

10

HO

OH

OH

[4]

HO

OH

HO

F

CH 2

[5]

[6]

[7]

[8]

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Chapter | 83 Vitamin D and Analogues

A few attempts have been made to modify the nucleus of calcitriol. The Roche compound 1α,25-(OH)2-16-ene23-yne-D3, touted as an antitumor compound in vivo, possesses a D-ring double bond. Declercq and Bouillon have introduced a series of biologically active analogues without the C/D rings but with a rigid backbone to maintain the spatial arrangement of the A-ring hydroxy1 groups and the side chain (Verstuyf et al., 2000). Relatively recently, the A-ring-substituted 2-hydroxypropoxy-derivative, ED71, has been tested as an antiosteoporosis drug. Other bulky modifications at the C2 position of the A ring are accommodated well by the vitamin D receptor, as indicated by retention of biological activity (Suhara et al., 2001; Shevde et al., 2002). The Abbott compound, 19-nor-1α,25-(OH)2D2, lacks a 19-methylene group and is styled upon the in vivo active metabolite, 1α,25-(OH)2 DHT2, formed from dihydrotachysterol, which retains biological activity though the C-19 methylene, is replaced by a C-19 methyl. Many other compounds have been developed with rigid or altered cistriene structures (Okamura et al., 1995) or modifications of the 1α,3β-, or 25-hydroxy1 functions, not for the purpose of developing active molecules for use as drugs, but to allow us to establish minimal requirements for biological activity in structure/activity studies (Calverley and Jones, 1992; Bouillon et al., 1995). Two recent compounds, Bioxell’s BXL-628 and Deltanoids’ 2-MD, combine modifications in the side chain with those in the nucleus. BXL-628 combines 1-fluorination, 16-ene and 23-ene unsaturations, 26,27-homologation, and 20-epimerization all found in earlier generations of analogues to make a antiproliferative agent currently in clinical trials for the treatment of prostate cancer and prostatitis (Crescioli et al., 2003; Adorini et al., 2007). Likewise, 2-MD, touted as being bone-specific, combines a novel 2-methylene substitution and the 19-nor feature with side chain 20-epimerization (Shevde et al., 2002). One series of compounds depicted in Table IV are the substituted biphenyls originally developed by Ligand, representing nonsteroidal scaffolds selected by high-throughput screening, which show weak VDR-binding but good transactivation through VDRE-driven, vitamin D-dependent genes and produce hypercalcemia in vivo (Boehm et al., 1999). This family has recently been extended by the synthesis of some highly potent, tissue-selective nonsecosteroidal VDR modulators with nanomolar affinity (e.g., LY2109866) by a research group at Eli Lilly (Ma et al., 2006). This is the first class of vitamin D mimics that lack the conventional cis-triene secosteroid structure while maintaining the spatial separation of the A-ring and side-chain hydroxy1 functions needed to bind to certain key residues of the ligand-binding pocket of the VDR. Though these nonsecosteroidal compounds exhibit a 270fold improvement of the therapeutic index over calcitriol in animal models, they are still to be tested clinically. On the contrary, Table IV also shows the structures of two

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different classes of VDR/cacitriol antagonists made by Teijin and Schering, respectively. The former compounds, most notably TEI-9647, are based on the natural metabolite 1α,25-(OH)2D3-26,23-lactone and have found clinical utility in the treatment of Paget’s disease (Ishizuka et al., 2005; Saito and Kittaka, 2006). Another group of compounds which impact the vitamin D field that are under development are the CYP24 inhibitors. By blocking CYP24A1, the main catabolic pathway within the vitamin D-target cell, these agents extend the life of the natural agonist, calcitriol, giving rise to a longer-lasting biological effect (Prosser and Jones, 2004). Sandoz/Novartis developed a group of molecules that have greater specificity toward CYP24 and CYP27B1 from the general cytochrome P450 (CYP) inhibitor, ketoconazole, which showed utility to block cell proliferation in vitro, but these compounds were discontinued after early clinical trials (Schuster, 2001). Cytochroma has synthesized a group of inhibitors based on vitamin D templates and these have currently reached phase IIB human clinical trials for the treatment of psoriasis (Posner et al., 2004; Kahraman et al., 2004). Some of these molecules show promise for use in secondary hyperparathyroidism, presumably because they counter the role of CYP24 in attenuating the effect of calcitriol on preproPTH gene suppression.

CLINICAL APPLICATIONS OF VITAMIN D COMPOUNDS The clinical usefulness of vitamin D analogues has been reviewed comprehensively by both Bikle (1992) and Bouillon et al. (1995) in overviews and also within this book. This chapter summarizes some of the highlights in this area.

Rickets and Osteomalacia When the nutritional basis of rickets and osteomalacia became apparent in the first half of the twentieth century, vitamin D (particularly vitamin D2 because it was less expensive) became the treatment of choice for these diseases. Of course, low-dose prophylactic vitamin D (400 IU) in the form of supplements to milk, margarine, and bread replaced much of the need for therapeutic vitamin D to abolish overt rickets and osteomalacia. In fact, since then full-blown vitamin D deficiency rickets (defined as plasma 25-OH-D levels below 10 ng/mL or 25 nmol/L) has become very uncommon in North America because vitamin D supplementation is required by law, whereas it was quite common before the practice of food fortification and it is still more prevalent in the world where food fortification is not permitted. On the other hand, vitamin D insufficiency (defined as plasma 25-OH-D levels in the range 10 to 40 ng/mL or 25 to 100 nmol/L) remains common in the general

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TABLE IV Miscellaneous Vitamin D Compounds Name

Structure

Name

LG190090 Ligand

Structure

LY2108491 Eli Lilly

Pharmaceuticals

O

O

O

Nonsteroidal agonist

Cl

Cl

O

Nonsteroidal agonist

Boehm et al., 1999

Ma et al., 2006

TEI-9647 Teijin

ZK159222 Schering

CH 3 CH 3 S

HO H 3C

O O

H 3C

H 3C

CH3

CH 3

OH

O

Calcitriol antagonist Dehydration product of 1α,25(R)-(OH)2D326,23(S)-lactone Saito and Kittaka, 2006 Ochiai et al., 2005 Toell et al., 2001

O O

O H

Calcitriol antagonist

H

Toell et al., 2001 HO

SDZ 89-443 Sandoz/ Novartis P450 inhibitor

S CH 3 O

OH

HO

VID400 Sandoz/ Novartis

N N

N

O

OH

N H N

O

N

P450 inhibitor

Cl

Schuster et al., 2003 Cl

Schuster et al., 2003

Cl

Cl

CTA016 Cytochroma

F S O O

CYP24A1 inhibitor H

Posner et al., 2004

HO

OH

population and is being increasingly correlated with poor outcomes in several health-related areas including optimal bone mineral density (Bischoff-Ferrari et al., 2006). Vitamin D deficiency and insufficiency are also quite prevalent in the elderly and are usually treated with modest doses of 800 to 1000 IU of vitamin D (Chapuy et al., 1992). In recent years, several world, continent-wide, and national food agencies have put out new guidelines raising the recommendations for vitamin D intake for all age groups, but particularly for those in the elderly or postmenopausal category, to try to ensure adequate intakes irrespective of geographical, dietary, and sun exposure differences (National Academy of Sciences Reference Intakes, 1997; FAO/WHO Nutritional Guidelines, 2000). However, the need for the use of expensive pharmaceutical

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preparations containing calcitriol or its analogues to cure simple rickets and osteomalacia is usually not warranted. Though many of the hallmarks of rickets and osteomalacia are successfully relieved by doses of vitamin D in the range of 400 to 800 IU/day (10 to 20 µg/day), there are epidemiological data to suggest that current recommended dietary allowances (also known as DRIs) do not result in plasma 25-OH-D levels greater than 40 ng/mL which correlate with maximal bone mineral density (Holick, 2007; Bischoff-Ferrari et al., 2004) or the other health benefits of vitamin D (Bischoff-Ferrari et al., 2006). Consequently, there has been much recent debate over the optimal level of vitamin D intakes and this has led to a general view that vitamin D intakes might need to be increased above 1500 IU/day (Heaney, 2004) and possibly higher (Dawson-Hughes

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Chapter | 83 Vitamin D and Analogues

et al., 2005) in order to achieve target plasma 25-OH-D levels greater than 40 ng/mL. However, many quasigovernmental agencies have yet to translate the latest recommendations for increase into new public health guidelines.

Osteoporosis Although the etiology of this disease is complex and likely to be multifactorial (Seeman et al., 1995; Nordin, 1997), there have been consistent claims that levels of 1α,25(OH)2D3 are low in osteoporosis (Riggs and Melton, 1992; Eastell and Riggs, 2005). In addition, the recent debate over VDR genotypes correlating with bone mineral density (Morrison et al., 1994; Whitfield et al., 2000; Uitterlinden et al., 2005) suggests some genetically inherited basis involving vitamin D exists leading to increased susceptibility to osteoporosis. As a consequence it is not surprising that clinical trials of 1α-OH-D3 (Orimo et al., 1987), 1α-OH-D2 (Gallagher et al., 1994), and 1α,25(OH)2D3 (Gallagher et al., 1989; Ott and Chestnut, 1989; Tilyard et al., 1992) have been undertaken. Modest gains in bone mineral density and reductions in fracture rates are reported in many of these studies, and this subject has been reviewed by Seeman et al. (1995). With the demonstration that ovariectomy results in enhanced production of osteoclastogenic cytokines such as interleukin-6, tumor necrosis factor (TNF), and interleukin1 as well as cytokine-mediated osteoclast recruitment and increased bone resorption has come a clearer understanding of the molecular processes underlying postmenopausal osteoporosis (Manolagas and Jilka, 1995; Teitelbaum and Ross, 2003). Theories focusing on osteoblast/osteoclast communication led to the discovery of receptor activator of nuclear factor κB (RANK), its ligand RANKL, and the decoy receptor, osteoprotegerin, and how agents such as 1α,25-(OH)2D3 can influence osteoclastogenesis and bone resorption (Aubin and Bonnelye, 2000). Although 1α,25-(OH)2D3 treatment might be expected to exacerbate the excessive bone-resorptive component of osteoporosis, the vitamin D hormone also raises plasma Ca2⫹ levels and stimulates synthesis of bone matrix formation in osteoblasts. In fact, Raisz and coworkers (Hock et al., 1986) have shown that pharmacological doses of 1α,25-(OH)2D3 administered to rats, in great excess over the doses used in osteoporosis, result in hypercalcemia and nephrocalcinosis that is accompanied by a hyperosteoid or undermineralized condition in the long bones. Although small doses of vitamin D (800 to 1000 IU) have proven effective in treating vitamin D deficiency accompanying osteoporosis and even reduce fracture rates (Chapuy et al., 1992), the use and effectiveness of active vitamin D metabolites in the treatment of osteoporosis are controversial. Nevertheless, the experience seems to have been that beneficial effects can be observed and bone loss reduced, but at the expense of occasional hypercalcemia.

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In North America, where dietary Ca intakes and absorption rates are higher, this has led to intolerable side effects and the discontinuation of the use of 1α,25-(OH)2D3 and 1α-OH-D3 for the treatment of osteoporosis. In the United Kingdom, Australia, Italy, Japan, New Zealand, and 16 other countries in rest of the world, these drugs are approved or side effects tolerated. Nevertheless, some pharmaceutical companies have sought to develop “milder” but “longer-lived” calcitriol analogues for use in osteoporosis. ED-71 represents such an analogue, which by virtue of an A-ring substituent at C-2 and tighter-binding affinity to DBP has a longer t1/2 in the plasma (Nishii et al., 1993). ED-71 has performed well at restoring bone mass without causing hypercalcemia in long-term studies involving ovariectomized rats (Okano et al., 1991) and in phase I and II clinical trials (Matsumoto and Kubodera, 2007). Another bone-specific analogue with potential for treatment of osteoporosis, 2-MD (Shevde et al., 2002) is at a relatively early stage of development.

Renal Osteodystrophy Chronic renal disease (CKD) is accompanied by the gradual loss of renal 25-OH-D3-1α-hydroxylase (CYP27B1) activity over the five-stage natural history of the disease which culminates in dialysis (stage 5). As early as stage 2 of CKD, the 1α-hydroxylase declines leading to reduced plasma levels of 1α,25-(OH)2D3, which results in hypocalcemia and secondary hyperparathyroidism. Unchecked, these biochemical events, together with the other sequelae of renal failure such as phosphate retention, can result in renal osteodystrophy. Active vitamin D analogues, such as 1α-OH-D3 and 1α,25-(OH)2D3, raise plasma Ca2⫹ concentrations and, in addition, lower PTH levels by direct suppression of PTH gene transcription at the level of the PTH gene promoter. Slatopolsky and colleagues (Delmez et al., 1989) showed that intravenous infusion of “active” vitamin D preparations results in a more effective suppression of plasma PTH levels without such a profound increase in plasma [Ca2⫹] in end-stage renal disease. Subsequent work has employed “low-calcemic” vitamin D analogues such as OCT or 19-nor-1α,25-(OH)2D2 as substitutes for the more calcemic natural hormone (Brown et al., 1989; Slatopolsky et al., 1995). More recently, the Food and Drug Administration (FDA) approved both oral and intravenous, 1α-OH-D2 (trade name, Hectorol) for the treatment of secondary hyperparathyroidism at earlier stages 3 and 4 of the disease. In clinical trials, 1α-OH-D2 effectively suppressed PTH in renal failure patients with very few incidences of hypercalcemia and hyperphosphatemia (Frazao et al., 2000). Recently, oral formulations of 19-nor-1α,25-(OH)2D2 were also approved. In 2003, a body of leading nephrologists released guidelines (KDOQI, 2003) recommending more aggressive use of vitamin D preparations and “active” vitamin D analogues in the treatment of secondary hyperparathyroidism in CKD. KDOQI guidelines suggested that treatment as early as

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stage 3 [glomerular filtration rate (GFR) less than 60] might benefit the patient by limiting the extreme rises in plasma PTH levels and preventing the parathyroid gland resistance to vitamin D treatment often observed in end-stage renal disease (ESRD). KDOQI guidelines also recognized the high frequency of vitamin D deficiency (25-OH-D less than 10 ng/mL) and vitamin D insufficiency (25-OH-D 10 to 30 ng/mL) in the CKD and ESRD population (Gonzalez et al., 2004) and recommended an initial attempt at vitamin D repletion with escalating doses of vitamin D2 prior to administration of “active” vitamin D analogue replacement therapy. Currently, both oral and intravenous formulations of various active vitamin D analogues are available for use in stage 3, 4, and 5 patients to take over, if and when vitamin D repletion fails to regulate PTH levels. The emergence of the potential importance of the extrarenal 1α-hydroxylase in normal human physiology has led to a reevaluation of the vitamin D repletion and “active” hormone replacement arms of the CKD therapy (Jones, 2007). The value of the vitamin D repletion is now seen as providing the substrate 25-OH-D for both the renal 1α-hydroxylase, which is the main determinant of circulating 1α,25-(OH)2D3, and the extrarenal 1α-hydroxylase, which is postulated to augment 1α,25-(OH)2D3 synthesis for local or paracrine actions around the body. Although the decline of the renal enzyme during CKD is well established, the fate of the extrarenal 1α-hydroxylase in the face of uremia is largely a matter of conjecture. Evidence from anephric patients treated with large doses of 25-OH-D3 (Dusso, et al., 1988) suggests that the extrarenal enzyme survives in CKD patients, arguing that provision of a source of 25-OH-D to vitamin D-deficient and -insufficient patients throughout all stages of CKD is warranted. It also argues for the more judicious use of “active” vitamin D analogues as hormone replacement therapy layered on top of conventional vitamin D repletion therapy. Early attempts at this type of combined vitamin D/“active” vitamin D analogue approach in a pediatric population have resulted in a more efficient PTH control without many of the usual problems of soft-tissue calcification observed in patients treated only with active vitamin D analogues (Briese et al., 2006; Fournier et al., 2007).

Psoriasis and Cancer The demonstration that 1α,25-(OH)2D3 is an antiproliferative, prodifferentiating agent for certain cell types in vivo and many cell lines in vitro suggested that vitamin D analogues might offer some relief in hyperproliferative disorders such as psoriasis and cancer. Early psoriasis trials with 1α,25-(OH)2D3 were moderately successful but plagued with hypercalcemic side effects. Modifications to the protocol included: (1) administration of calcitriol overnight when intestinal concentrations of [Ca2⫹] were low, (2) substitution of “low-calcemic” analogues for the calcitriol.

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According to Holick (1995), oral calcitriol is an effective treatment for psoriasis when administered using an overnight protocol. However, by far the most popular treatment for psoriasis is the topical administration of the “lowcalcemic” analogue calcipotriol, formulated as an ointment (Kragballe, 1992). When given orally, calcipotriol is ineffective because it is rapidly broken down (Binderup and Bramm, 1988). When given topically as an ointment, calcipotriol survives long enough to cause improvement in more than 75% of patients (Kragballe et al., 1991). Both 1α,25(OH)2D3 and calcipotriol are effective in psoriasis because they block hyperproliferation of keratinocytes, increase differentiation of keratinocytes, and help suppress local inflammatory factors through their immunomodulatory properties. Calcipotriol has now been marketed worldwide for use in psoriasis for more than 15 years. The success of calcipotriol has spawned the development of second-generation analogues. Several hundreds of vitamin D analogues have been tested in vitro and in vivo with some degree of success in controlling the growth of tumor cells offering potential for use as anticancer drug therapies (reviewed extensively in Masuda and Jones, 2006). Many vitamin D compounds are extremely effective antiproliferative or pro-differentiation agents in vitro using a variety of mechanisms involving gene expression of cell division and pro-apototic genes to produce their effects. Preclinical studies in laboratory animals have also resulted in promising data (Masuda and Jones, 2006). For example, in mice inoculated with fulminant leukemia, moderate leukemia, or slowly progressive leukemia, the Roche compound 1α,25-(OH)2-16-ene23-yne-D3 administered at 1.6 μg q.o.d. was significantly more effective than 0.1 μg q.o.d. 1α,25-(OH)2D3 at increasing survival time even though the 1α,25-(OH)2D3-treated group developed mild hypercalcemia and the analoguetreated animals remained normocalcemic (Zhou et al., 1990). With the analogue EB1089, the promising antiproliferative effects observed in vitro and in the NMU-induced mammary tumor and in LNCaP prostate cancer xenograft models (Colston et al., 2003; Blutt et al., 2000) were also extended into the clinic. Early trials in limited numbers of breast cancer patients have been followed up with more extensive ongoing phase II and phase III clinical trials in a number of different cancers (Gulliford et al., 1998; Evans et al., 2002; Dalhoff et al., 2003). Several other analogues have entered clinical trials for the treatment of a variety of hyperproliferative diseases, usually involving VDR-positive tumors (see Masuda and Jones, 2006). Many trials are still ongoing including the testing of BXL-628 (see Table III) in prostate-related diseases (Crescioli et al., 2003) Despite the enormous promise of vitamin D analogues as anticancer agents, this has yet to result in an approved vitamin D analogue for use in any type of cancer (Masuda and Jones, 2006). The principal problem in anticancer studies involving orally administered vitamin D compounds is

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hypercalcemia. Though the newer analogues appear to be less calcemic than calcitriol itself, they still retain some ability to raise serum calcium; they are not “noncalcemic” as is sometimes claimed. Another problem emerging from experience with clinical trials is that effective doses needed to retard cell growth (~1 nM or higher) cannot be attained in vivo because of low bioavailability (Beer et al., 2004; Trump et al., 2004). One of the principal determinants of tumor cell vitamin D analogue levels is the catabolic enzyme CYP24A1, which is upregulated in vitamin D-target cells, and limits the effective drug concentration reached. Another approach to effective vitamin D therapy in cancer patients is the potential use of CYP24-inhibitors (see Table IV). Nevertheless, it remains uncertain whether we will ever develop a vitamin D compound sufficiently devoid of calcemic activity while retaining sufficient antiproliferative activity to be valuable in cancer and also deliver it to target cells in appropriate concentrations

Immunosuppression The immunosuppressive properties of 1α,25-(OH)2D3 and its analogues have been the subject of several excellent reviews (Bouillon et al., 1995; Van Etten, 2000; Mathieu and Adorini, 2002). 1α,25-(OH)2D3 is believed to work by regulation of the expression of various cytokines, particularly those involved in suppressing inflammation and which raise the Th2/Th1 ratio. The hormone also stimulates the transcription of a natural bacterial peptide, cathelicidin, which kills Mycobacterium tuberculosis resulting in increased resistance to tuberculosis (Wang et al., 2004; Holick, 2007). The spectrum of effects exhibited by 1α,25(OH)2D3 and its analogues on the immune system results in beneficial effects on a wide variety of autoimmune diseases. Researchers have demonstrated the ability of calictriol to suppress the onset of experimental encephalitis (Lemire and Clay, 1991) and type I diabetes in NOD mice (Mathieu et al., 1995), and to work synergistically with cyclosporine to provide immunosuppression in transplantation medicine (Mathieu et al., 1994a). This latter development has led to some optimism that coadministration of a vitamin D analogue with cyclosporin can reduce the dosage of the latter drug and minimize the serious side effects associated with its use. Several studies (Mathieu et al., 1995, 1994b; Veyron et al., 1993) have focused on the immunosuppressive effects of Leo drugs KH1060 and 20-epi-1α,25-(OH)2D3, both of which contain the 20-S side-chain configuration. Recent generations of compounds such as BXL-628, that contain multiple modifications found in the Leo Pharma drugs, are being tested in prostatitis, an inflammation of the prostate (Adorini, 2007). Again, it remains unclear whether analogues that show promise in immunological studies will prove to be effective immunomodulators in the clinic.

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CRITERIA THAT INFLUENCE PHARMACOLOGICAL EFFECTS OF VITAMIN D COMPOUNDS Activating Enzymes It has been shown by using in vitro models that some vitamin D compounds lacking 1α-hydroxylation (e.g., 24(R),25-(OH)2D3) are capable of interacting with the vitamin D receptors (VDRs) and transactivating reporter genes but this occurs only at high concentrations of ligand (Uchida et al., 1994). It seems unlikely that these concentrations will be reached in vivo except in hypervitaminosis D. Consequently, most of the compounds described in Tables I and II lack lack vitamin D biological activity unless they are activated in vivo. This is particularly the case for the parent vitamin D3 itself, for its main circulating form 25-OH-D3 or for any of the prodrugs listed in Table II. Vitamins D2 and D3 depend on both the liver 25-hydroxylase and kidney 1α-hydroxylase enzyme systems in order to be activated, whereas most prodrugs require only a single step of activation. Indeed, the 1α-OH-D drugs were designed to overcome the tightly regulated 1α-hydroxylase step that is easily damaged in chronic renal failure. In essence, prodrugs depend on the weakly regulated 25-hydroxylase step in the liver for activation. In recent years, the cytochrome P450 originally thought to be responsible for 25-hydroxylation of vitamin D3, CYP27A1, has been cloned and shown to be a bifunctional polypeptide that executes both activation of vitamin D3 and the 27-hydroxylation of cholesterol during bile acid biosynthesis (Okuda et al., 1995). However, the CYP27A1 enzyme has a relatively low affinity for vitamin D, does not 25-hydroxylate vitamin D2, and when mutated results in cerebrotendinous xanthomatosis not rickets. Consequently, another “physiologically relevant” 25-hydroxylase may exist and there are now several candidate P450s (Prosser and Jones, 2004), the main one being CYP2R1 (Cheng and Russell, 2003), a high-affinity microsomal enzyme with known human mutations that cause rickets, that has been recently shown to 25-hydroxylate the prodrug 1α-OH-D2 (Jones et al., 2006). However, it is clear that the mitochondrial CYP27A1 efficiently 25-hydroxylates 1α-OH-D3 to give 1a,25-(OH)2D3 (Guo et al., 1993); and is present in a variety of tissues as well as the liver (e.g., kidney and bone). In fact, studies using cultured bone cells and even keratinocytes in vitro are able to demonstrate synthesis of 1α,25-(OH)2D3 from 1α-OH-D3 (Ichikawa et al., 1995; Jones et al., 1999a) or 1α,24-(OH)2D2 from 1α-OH-D2 (Masuda et al., 2006). If these findings can be extrapolated to the in vivo situation, the implications of this work are that in CYP27A1, vitamin D target cells may have some ability to synthesize the active form from a prodrug without the need for the hormone to enter the bloodstream. The ability of extrarenal tissues to 1α-hydroxylate various 25-hydroxylated metabolites and analogues has

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always been a controversial story. However, it was widely accepted that extrarenal 1α-hydroxylase activity is associated with certain granulomatous conditions (e.g., sarcoidosis) (Adams and Gacad, 1985). Currently, there is little information for why the enzyme is overexpressed in sarcoidosis. In these patients, 25-OH-D can be converted to 1α,25-(OH)2D, a step that, unlike the renal case, is not subject to tight regulation and thus potentially more likely to result in hypercalcemia. Exposure of such patients to sunlight or administration of 25-OH-D can result in excessive plasma levels of 1α,25-(OH)2D. The cloning of the cytochrome P450 representing the 1α-hydroxylase (officially named CYP27B1) (St. Arnaud et al., 1997; Takeyama et al., 1997) has been followed by confirmation that the cytochrome can be expressed extrarenally in skin and lung cancer cells (Fu et al., 1997; Jones et al., 1999b). This has extended over the past decade with further studies of CYP27B1 mRNA levels using real-time PCR and specific anti-CYP27B1 antibodies (Hewison and Adams, 2005) to show the widespread distribution of this enzyme in many normal tissues as well as pathological situations. As alluded to earlier, the concept of the extrarenal 1α-hydroxylase suggests that this enzyme plays an important physiological as well as pathological role (Jones, 2007) and this has in turn raised the level of importance given to ensuring maintenance of adequate 25-OH-D levels by vitamin D or direct 25-OH-D3 supplementation rather than just by calcitriol hormone replacement. Most of the calcitriol analogues listed in Table III are thought to be active as such, not requiring any step of activation prior to their action on the transcriptional machinery or in nongenomic pathways. It remains a theoretical possibility, though, that the biological activity of one of these parent analogues could be altered by enzyme systems in vivo, either by the generation of a more potent metabolite or by giving rise to a less active but more long-lived catabolite.

Vitamin D-Binding Protein The vitamin D-binding protein (DBP) serves several functions including providing transport for a lipid-soluble vitamin D analogue. Most of the analogues of calcitriol, designed to date, contain modifications to the side chain and this is usually detrimental to binding to DBP. Several analogues, for example, calcipotriol or OCT, have very weak affinities for DBP, reduced by two to three orders of magnitude relative to 1α,25-(OH)2D3. This property has important implications for metabolic clearance rates, delivery to target cells, and tissue distribution (Bouillon et al., 1991; Kissmeyer et al., 1995). Detailed studies with one analogue, OCT, have shown it to bind primarily to β-lipoprotein and exhibit an abnormal tissue distribution in vivo, with abnormally high concentrations (ng/g tissue) in

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the parathyroid gland (Tsugawa et al., 1991). It was thus proposed that this unusual distribution may make OCT a useful systemically administered drug with a selective advantage in the treatment of hyperparathyroidism. Another vitamin D analogue with a modified side chain is 20-epi-1α,25-(OH)2D3, where the 20-S configuration of the side chain is opposite to the normal 20-R configuration. The DBP binding affinity of this analogue is virtually unmeasurable because it does not displace [3H]25-OH-D3 from the plasma-binding protein (Dilworth et al., 1994). Confirmation that this is indeed the case comes from GH-reporter gene transactivation assays where 20-epi1α,25-(OH)2D3 transactivates equally well in COS cells incubated in the presence and absence of fetal calf serum (as a source of DBP). On the other hand, 1α,25-(OH)2D3induced GH reporter gene expression is sensitive to DBP in the external growth medium, requiring 2-fold less hormone in the absence of DBP as in its presence (Dilworth et al., 1994). It therefore appears that analogues that bind DBP less well than 1α,25-(OH)2D3 derive a target cell advantage over the natural hormone, if they are able to find alternative plasma carrier proteins to transport them to their target cells. However, these same alternative plasma carriers presumably result in changes in the tissue distribution and hepatic clearance of analogues over the natural metabolites of vitamin D. The recent development of a DBP-knockout mouse (Safadi et al., 1999) suggests that 25-OH-D3 clearance is more rapid in the absence of DBP. The availability of the model permits the study of alternate vitamin D analogue transport mechanisms in an in vivo setting.

Vitamin D Receptor/RXR/VDRE Interactions Previous chapters in this book have established that 1α,25(OH)2D3 is able to work through a VDR-mediated genomic mechanism to stimulate transcriptional activity at vitamin D-dependent genes. Considerable progress has been made recently toward delineating the precise conformational changes that take place when the natural ligand binds to the VDR (Wurtz et al., 1997; Rochel et al., 2001); and the nature of the postligand binding transcriptional events that occur, particularly the nature of the coactivator proteins involved (Rachez and Freedman et al., 2000; Kato et al., 2000). These developments have improved our thinking about how and where analogues might act differently from 1α,25-(OH)2D3 in the transcriptional cascade. Whether 1α,25-(OH)2D3 works through other non-VDR-mediated mechanisms to produce physiologically relevant effects is a question that currently remains unproven, but this question is also important to our understanding of the pharmacological effects of vitamin D analogues. Much evidence exists to support the viewpoint that vitamin D analogues mimic 1α,25-(OH)2D3 and use a genomic mechanism. The first clue that vitamin D analogues can

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work through a VDR-mediated transcriptional mechanism came 25 years ago from the bone resorption studies reported by Stern in her classic review “Monolog on Analogs”(1981). Stern showed that there exists a strong correlation between chick intestinal VDR binding of an analogue and its potency in a [45Ca] rat bone resorption assay. This suggests that a vitamin D analogue is only as good as its affinity for the VDR. Over the past 25 years since Stern’s article, there have been many claims that VDR-binding affinity is not the only factor in determining biological activity of a given analogue but also that VDR-binding affinity is not even the major factor, transactivation activity stemming from a series of parameters such as conformation of the ligand/VDR complex, binding of the RXR partner, stability of the VDR/RXR/ ligand complex, or even the nature of the coactivator proteins recruited to the complex. Examples of these apparent discrepancies between VDR affinity and biological activity will be provided later but it should be pointed out that some of these discrepancies are almost certainly explained by other considerations such as DBP binding or pharmacokinetics. Preliminary results with the analogues KH1060, EB1089, and 20-epi-1α,25-(OH)2D3 (Binderup et al., 1991) suggested that they might be active in immunoregulatory roles at concentrations orders of magnitude below their affinities for the VDR (e.g., at as low as 10⫺15 M for KH1060, whereas it binds VDR at 10⫺11M). More recent results (Yang and Freedman, 1999; Dilworth et al., 1994, 1997) suggest that 20-epi compounds including KH1060 are consistently only one to two orders of magnitude more potent than 1α,25(OH)2D3 in gene transactivation assays and in differentiation assays, a difference that could be explained by fine-tuning the transcriptional model of analogue action (e.g., by including pharmacokinetic considerations) rather than discarding the genomic hypothesis altogether. The majority of researchers are keeping an open mind on this subject and are searching for differences in the newly delineated transcriptional machinery that might explain qualitative and quantitative differences between 1α,25-(OH)2D3 and its analogues. Over the past decade it has been clearly established that the liganded VDR functions transcriptionally as a vitamin D-VDR-RXR heterodimer (Macdonald et al., 1993; reviewed in Haussler et al., 1998) and not as a VDR-VDR homodimer (Carlberg, 1995). The role of the RXR ligand is still controversial, many studies suggesting that pan RAR and RXR ligands such as 9-cis-retinoic acid inhibit VDRRXR heterodimer formation, whereas other studies demonstrate the synergistic effects of pure RXR ligands (so-called rexinoids) and 1α,25-(OH)2D3 on VDR-RXR-driven transcription at a CYP24-VDRE (Zou et al., 1997). Whether vitamin D analogues might differ from 1α,25-(OH)2D3 and act transcriptionally through VDR-VDR homodimers or other VDR-nuclear transcription factor heterodimers are ideas that have been considered as theoretical possibilities, and in some cases even shown to occur weakly in vitro, but largely dismissed as occurring in vivo.

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Adding to the complexity of the target cell action of 1α,25-(OH)2D3, and thus that of vitamin D analogues, is the type and context of the VDRE involved (Haussler et al., 1998). One possibility is that vitamin D analogues could show selectivity for certain genes based on the type of VDRE within their promoter. Morrison and Eisman (1991) showed that a noncalcemic analogue such as calcipotriol is easily capable of transactivating a calcemic VDRE such as the human osteocalcin promoter-VDRE placed in front of the CAT reporter gene and stably transfected into ROS 17/2 cells provided that it can get into the target cell. One interpretation of this experiment is that a noncalcemic analogue with good VDR affinity is just as calcemic as 1α,25(OH)2D3 if it can be delivered to the target cell. Another idea put forward by Morrison and Eisman (1991) is that noncalcemic analogues may be capable of stimulating both cell-differentiating and calcemic genes but that the former genes require only a short pulse of analogue to effect a switch in the cell cycle, whereas the latter genes require a sustained concentration of the vitamin D ligand. The concentration of 1α,25-(OH)2D3 may be sustained in vivo by renal synthesis and some protection by DBP, whereas systemically administered noncalcemic analogues reach a high initial concentration but do not bind DBP and are rapidly metabolized and cleared. This hypothesis remains to be adequately tested. Carlberg et al. (1994) has also tested the idea that other vitamin D analogues (EB1089 and KH1060) might favor one specific VDRE using the mouse osteopontin gene VDRE (DR3-type) and the same human osteocalcin VDRE that Morrison and Eisman used. Carlberg et al. (1994) found that 1α,25-(OH)2D3 and the two analogues are unable to differentiate between the two different types of VDRE. Though many VDREs have been postulated in the literature, the direct repeat-3 spacer type (DR3) of VDRE seems to be the sequence that is gaining widespread acceptance as the most physiologically relevant (Haussler et al., 1998; Jones et al., 1998). Whether other more exotic DR4, DR6, or inverted palindrome (IP9) nucleotide sequences are recognized by the analogue-VDR/RXR complex in vivo still remains unclear (Carlberg, 1995). Even with the DR-3 type of VDRE, the gene and cell context seems to be important in determining the transactivation produced by the vitamin D analogue. The work of Williams’ laboratory (Brown et al., 1994; Williams et al., 1995; Kane et al., 1996) suggests that 1α,25-(OH)2D3, KH1060, and EB1089 show different patterns of gene activation in bone marrow, osteoblastic cells (ROS17/2, ROS25/1, and UMR106), and intestine (HT29 and CaCo-2) that appear to be gene and cell-specific. Part of the explanation for gene- and tissue-specific effects probably lies in the influence of neighboring response elements to the VDRE and the binding of tissue-specific transcription factors at these sites. More recent work (Lin et al., 2002) using expression profiling (gene array) to investigate the differences in gene expression exhibited by squamous

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1788 carcinoma cells (SCC25) in response to EB1089 and 1α,25(OH)2D3 concluded that the two agents did not have qualitatively different effects. In fact, the differences in gene expression between EB1089 and 1α,25-(OH)2D3 were the result of potency differences and nullified by coadministration of a general P450 inhibitor such as ketoconazole, implying that they were the result of excessive metabolism of 1α,25-(OH)2D3. On the other hand, the laboratories of Lian and Stein have elegantly demonstrated (Guo et al., 1997) that the response to 1α,25-(OH)2D3-VDR-RXR complex to the gene promoter of the osteocalcin gene in osteoblasts depends on occupancy of an adjoining YY1binding site that allows for temporal changes in responsiveness to 1α,25-(OH)2D3. Though this work explains why 1α,25-(OH)2D3 might have gene- and tissue-specific effects, it does not explain the analogue-specific differences in Williams’ work, whereas the work of Lin et al. suggests that there are differences of potency not selectivity. As alluded to earlier, the emergence of important new information about (1) the structure of the ligand-binding domain of the VDR and (2) coactivator characterization and involvement in the 1α,25-(OH)2D3-VDR-RXR transcriptional machinery have opened up additional possibilities about where vitamin D analogues might differ in their action from the natural ligand. Evidence suggests that the “Trap Door Hypothesis” for retinoid binding to RAR/RXR (Renaud et al., 1995) also applies to 1α,25-(OH)2D3 binding to VDR. In this model, 1α,25-(OH)2D3 binding to a central binding pocket triggers a dramatic conformational change of helix 12, a domain close to the C terminus of the VDR, such that it moves from a position on the exterior of the VDR to one within the interior of the receptor, thereby closing the access channel to the ligand-binding pocket. In the process, amino acid residues of the AF-2 domain that are hidden in the unliganded VDR become exposed in the liganded VDR and are now available to interact with coactivator proteins. The recruitment of coactivators to the 1α,25(OH)2D3-VDR-RXR subsequently leads to the recruitment of other transcription factors which result in chromatin remodeling and gene transcription (Whitfield et al., 2005). In the execution of this work, members of Moras’ laboratory have modeled the ligand-binding pocket of VDR and shown it to be able to accommodate with ease several other analogues depicted in Table III (Rochel et al., 2001). Though many of the active vitamin D analogues, especially the 20-epi analogues, have bulky side-chain substituents or radically different side-chain orientations (Yamamoto et al., 1999), the pocket appears to have a great reserve capacity for binding (Rochel et al., 2001). As a result, from modeling alone it is difficult to forecast radical changes in VDR conformations as a result of binding to these different analogues. Nevertheless, there is some indirect evidence, most notably from experiments measuring susceptibility to protease digestion, that subtle differences do occur in VDR-RXR-containing transcription complexes when different ligands are used

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(Peleg et al., 1995; Carlberg et al., 1995; Van den Bemd et al., 1996). Binding of 20-epi-analogues (e.g., MC1288 and KH1060) to the VDR results in increased resistance to protease digestion compared with 1α,25-(OH)2D3, which has been interpreted as evidence for differences in accessibility of protease to cleavage sites (Peleg et al., 1995). Interestingly, there appears to be a direct correlation between transactivation activity of an analogue and the resistance of the VDR-transactivation complex to protease, a relationship that applies to different analogues and even to metabolites from a single analogue (Peleg et al., 1995; Liu et al., 1997; Van den Bemd et al., 2000). Because the rearrangement in helix 12 of the VDR brings about exposure of the AF-2 domain and this is critical to coactivator binding, it might be expected that subtle conformational differences in VDR observed for different vitamin D analogues might also be reflected in differences in coactivator recruitment. Consequently, several groups have looked for qualitative differences in the pattern of coactivators recruited or quantitative differences in the strength of RXR heterodimerization or coactivator binding following ligand binding. Liu et al., (2001) used a series of AF-2 domain mutants to reach the conclusion that the conformational changes occurring in the VDR upon hormone or 20-epi-1α,25-(OH)2D3 binding have a bigger impact on RXR-heterodimerization than on coactivator recruitment. This is in complete contrast to the work of Freedman and coworkers, who have shown repeatedly (Cheskis et al., 1995; Yang and Freedman, 1999) that analogue binding [20-epi-1α,25-(OH)2D3 or 1α,25-(OH)216-ene, 23-yneD3] to VDR results in no difference in RXR-heterodimerization compared with binding of 1α,25(OH)2D3. Instead, Freedman’s group reports that the ability of a various analogues to transactivate vitamin D-dependent genes or to stimulate differentiation of cells is best correlated with their ability to recruit the coactivator, DRIP-205, one of the many components of the DRIP complex isolated by Freedman’s group (Rachez et al., 1999; Freedman and Reszka, 2005). Among the other coactivators/transcription factors implicated in vitamin D analogue action is GRIP-1 (TIF-2), which has been purported to have a particular propensity to interact with the analogue OCT (Takeyama et al., 1999). In another study by Issa et al. (2002), a broad panel of vitamin D analogues showed that GRIP-1 was more consistently recruited at levels closer to that of 1α,25(OH)2D3 than was another coactivator AIB-1. Work by Peleg et al. (2003) offers an insight into the purported bone tissue selectivity of the Roche analogue Ro 26-9228 (see Table III, renamed BXL-628) which recruits GRIP-1 in osteoblasts but not CaCo-2 colon cancer cells; though these authors may now need to explain why BXL-628 is now being pursued clinically in prostatic diseases rather than osteoporosis as was originally attempted. Thus, it appears that there is a fairly strong basis for the hypothesis that differences in the biopotency advantage of certain vitamin D analogues over 1α,25-(OH)2D3 are caused in part by

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changes in the recruitment of dimerization partner and/or coactivators, but there is no clear consensus on which of these coactivator proteins is the important one or if these different coactivators can explain tissue/cell selectivity.

Target Cell Catabolic Enzymes In recent years, much evidence has accumulated to support the hypothesis that 1α,25-(OH)2D3 is subject to target cell catabolism and side-chain cleavage to calcitroic acid via a 24-oxidation pathway (Makin et al., 1989). The cloning of CYP24, the cytochrome P450 involved, has confirmed that it is vitamin D-inducible because its gene promoter contains a VDRE, carries out multiple steps in the side-chain modification process, and is present in many (if not all) vitamin D-target cells (Akiyoshi-Shibata et al., 1994; Prosser and Jones, 2004). We have postulated that the purpose of this catabolic pathway is to desensitize the target cell to continuing hormonal stimulation by 1α,25-(OH)2D3 (Lohnes and Jones, 1992). Recently, support for this hypothesis has emerged when St. Arnaud’s group engineered a CYP24-knockout mouse that exhibits 50% lethality at weaning, death resulting from hypercalcemia and nephrocalcinosis (St. Arnaud, 1999). Surviving mice show an inability to rapidly clear a bolus dose of 1α,25-(OH)2D3 from the bloodstream and tissues (Masuda et al., 2005) and a metabolic bone disease reminiscent of the excessive osteoid bone pathology observed in rodents given excessive amounts of 1α,25-(OH)2D3 (Hock et al., 1986). Recent work with this model has shown that the bone defect is probably caused by excessive 1α,25-(OH)2D3 levels because crossing the CYP24-knockout mouse with the VDR-knockout mouse results in a phenotype without the bone defect (St. Arnaud et al., 2000). Given the demonstrated importance of CYP24 to 1α,25-(OH)2D3 clearance, one must ask the question of whether vitamin D analogues might be subject to the same catabolic processes that determine their pharmacokinetics? If not, what other drugcatabolizing systems are present within vitamin D-target cells to inactivate the vitamin D analogue? Certainly there are vitamin D analogues such as calcipotriol, OCT, EB1089, and KH1060 that are metabolized by vitamin D-target cells to clearly defined and unique metabolites (Masuda et al., 1994, 1996; Shankar et al., 1997; Dilworth et al., 1997), which resemble products of the 24-oxidation pathway for 1α,25-(OH)2D3 or which are unique to the particular analogue. Furthermore, some of these metabolites are products only of vitamin D target cells and are D-inducible, implying that CYP24 is involved in their formation, and this has been confirmed with some analogues such as calcipotriol (Jones et al., 2006). Moreover, in the case of several analogues blocked at C-24 and subject to metabolism elsewhere on the side chain, the direct involvement of CYP24 is strongly implicated or proven. Examples where CYP 24 involvement is strongly suspected include 23-hydroxylation

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of 26,27-hexafluro-1α,25-(OH)2D3 (Sasaki et al., 1995); 26-hydroxylation of 24-difluro-1α, 25-(OH)2D3 (Miyamoto et al., 1997); 26-hydroxylation of 1α, 25-(OH)2-16ene-23yne-D3 (Satchell and Norman, 1995); and 26- and 28-hydroxylation of 1α, 25-(OH)2D2 (Rao et al., 1999; Shankar et al., 2001). Because many of these same products are observed in vitro and in vivo and because pharmacokinetic parameters often parallel target cell metabolic parameters (Kissmeyer et al., 1995; Jones, 1997), one concludes that target cell metabolism of vitamin D analogues must contribute to the pharmacokinetics and biological activity observed in vitro and in vivo. In fact, there is little doubt that the poor performance of some promising vitamin D analogues during in vivo testing is because of their poor metabolic stability. Accordingly, greater attention to the metabolic potential of in vitro testing systems and/or greater use of defined target cell (and hepatic) metabolic systems is warranted. One factor regarding target cell metabolism considered in recent years is the possibility that vitamin D analogues might be activated rather than catabolized by the same enzymes (Siu-Caldera et al., 1999; Swami et al., 2003). Although this is potentially more important for prodrugs (see Table II), the generation of large numbers of metabolites from such analogues as KH1060 (Dilworth et al., 1997) or the formation of long-lived metabolites such as 26,27-hexafluro-1α,23,25(OH)3D3 from 26,27-hexafluro-1α,25-(OH)2D3 (Sasaki et al., 1995) complicates the picture. In most cases, however, this issue can be resolved on pharmacokinetic grounds.

Other Factors Hepatic Clearance of Vitamin D Analogues The poor DBP-binding properties of many side-chain modified calcitriol analogues open up the possibility of alternative plasma carriers and accelerated degradation. The liver plays a major role in such metabolic clearance and a small number of detailed studies performed to date have included in vitro incubation with liver preparations. Calcipotriol (Sorensen et al., 1990), OCT (Masuda et al., 1996), EB1089 (Kissmeyer et al., 1997), and KH1060 (Rastrup-Andersen et al., 1992) are all subject to metabolism by liver enzymes. One such liver enzyme capable of 23- and 24-hydroxylation of 1α,25-(OH)2D3, and possibly some of its analogues, is the abundant general cytochrome P450, CYP3A4 (Xu et al., 2006). Indeed, this enzyme is upregulated by 1α,25(OH)2D3 in duodenum suggesting that a physiologically relevant loop exists (Thummel et al., 2001). Because, over the years, there have been frequent reports of drug-induced osteomalacia associated with coincidental use of anticonvulsants (e.g., diphenylhydantoin) or barbiturates and vitamin D preparations (e.g., Onodera et al., 2002), the direct association between CYP3A4 and 1α,25-(OH)2D3 is potentially important to explain the putative accelerated clearance of vitamin D metabolites (Gascon-Barre et al., 1984).

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These cytochrome P450 enzymes give rise to intermediate polarity molecules or truncated metabolites, which can be further glucuronidated and excreted in bile (e.g., OCT; Kobayashi et al., 1991). A recent study has defined UGT1A3 as the isoform of UDP-gluronosyltransferase involved in glucuronidation of the 23-hydroxylated metabolite of the analogue 26,27-F6-1α,25-(OH)2D3 (Kasai et al., 2005), whereas UG1A4 appears to be the isoform involved in conjugation of 1α,25-(OH)2D3 (Hashizume et al., 2008). Few, if any, studies have separately considered the rate of catabolism or glucuronidation relative to 1α,25-(OH)2D3. However, data are available comparing the in vivo rate of metabolic clearance of vitamin D analogues with 1α,25-(OH)2D3, though inevitably this probably measures a few in vitro parameters, such as the rate of both hepatic and target cell metabolism, in addition to the affinity of DBP binding within a single in vivo parameter. Thus, in lieu of detailed in vitro metabolic analyses, the t1/2 of the vitamin D analogue is a useful term for indicating the general survival of the vitamin D drug in vivo. Data for this parameter have been published for some of the most interesting analogues (Kissmeyer et al., 1995).

Nongenomic Actions of Vitamin D Analogues The nongenomic actions of 1α,25-(OH)2D3 have been reviewed elsewhere (Norman et al., 1992; Bouillon et al., 1995) and were described in detail in Chapter 35 and will not be repeated here. One analogue purported to discriminate between genomic and nongenomic actions is 1β,25-(OH)2D3, the epimeric form of 1α,25-(OH)2D3, which is an antagonist of nongenomic but not genomic actions (Bouillon et al., 1995). The membrane VDR initially described by Nemere et al. (1994) and identified as annexin II (Baran et al., 2000) may be involved in mediating putative nongenomic effects. It will be interesting to see whether an annexin-II knockout mouse will possess a distinct phenotype that will aid in delineating the nongenomic actions of vitamin D in the same way that the VDR-knockout mouse has aided our understanding of the genomic actions. In recent years, further attempts to purify and identify the putative membrane receptor have resulted in a newly-named membrane-associated response system (MARRS) in chick intestinal cells (Rohe et al., 2005) that may explain rapid nongenomic actions (Norman, 2005). But at this point in time, little work has been performed on the specificity of the vitamin D-binding site of membrane VDR/annexin II or MARRS complex and thus the possibility that the nongenomic actions/membrane VDR might explain vitamin D analogue actions seems premature.

Part | III Pharmacological Mechanisms of Therapeutics

criteria that are important to vitamin D analogue action. This in turn allows us to put forward a model for how vitamin D analogues may work in vivo. This is depicted in Figure 1. As a general model, it allows for consideration of both prodrugs (those requiring 25-hydroxylation by CYP27A1 or CYP2R1; those requiring 1α-hydroxylation by the kidney or extrarenal 1α-hydroxylase) and 1α,25-(OH)2D3 analogues. This model therefore makes a distinction between those target cells that express an extrarenal 1α-hydroxylase (CYP27B1) and therefore have the ability to make and respond to their own “local” 1α,25-(OH)2D3 and those that simply respond to circulating hormonal 1α,25-(OH)2D3 through their VDR with altered transcription. This model features a conventional VDR-RXR heterodimer working through a DR-3 type VDRE in most genes. Crucial characteristics for each new analogue (all measurable in vitro) are in our opinion: 1. affinity for DBP, 2. affinity for VDR, 3. ability to recruit RXR and coactivators followed by transactivation of genes, 4. rate of target cell metabolism (reflected partly in pharmacokinetic measurements), and 5. rate of hepatic clearance (reflected partly in pharmacokinetic measurements). All parameters contribute significantly to the overall biological activity. Target cell distribution differences might be expected for those analogues that do not bind DBP and this model does not explain the differences observed at different genes within the same or different cells. However, it might be useful to outline broad expectations for a new compound. Based on this model one would predict that those analogues that have good VDR binding affinity but slow rates of target cell metabolism owing to side-chain blocks, such as strategically placed fluorine atoms or double bonds, might be more active than 1α,25-(OH)2D3 in vitro and perhaps also in vivo. Hexa-fluoro-1α,25-(OH)2D3 and EB1089 are such compounds; looking alike in vitro and differing in vivo owing to differences in DBP binding and metabolism. On the other hand, a compound such as calcipotriol binds VDR moderately well but is rapidly metabolized in both the liver and the vitamin D target cell; is very active in vitro or when applied to the skin topically; but is inactive when administered orally and is forced to enter the bloodstream and pass the liver to get to its target site. It remains to be seen if this general model can be applied to all vitamin D analogues (e.g., the 20-epi- superanalogues such as KH1060, which has a very complex metabolic picture but very high biological activity in vitro) or must be adapted.

PROPOSED MOLECULAR MECHANISMS OF ACTION OF VITAMIN D COMPOUNDS Building on the data acquired from a variety of in vitro tests performed over the past 20 years and described briefly in the previous section of this chapter, one is able to identify those

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FUTURE PROSPECTS A number of researchers remain optimistic that the unraveling of the genomic (or nongenomic) mechanism of action

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Liver Vitamin D3 25-OH-D3

Nucleus

1␣,25-(OH)2D3

Kidney

Calcitroic acid

Nucleus CYP27A1

DBP

2R1

CYP

Megalin/cubulin

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1

CYP27B

1

CYP27B

ⴙ

CYP24

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Vitamin D-dependent genes

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mRNA

Extra-renal 1␣-hydroxylating target cells

CYP24

A1

Cytokines Calbindins

p21

RXR/VDR Vitamin D-dependent genes

Osteopontin VDRE

mRNA

Calbindins

Normal target cells FIGURE 1 Current concepts of the activation, mechanism of action, and catabolism of vitamin D. The model incorporates a plasma-binding protein (DBP) that acts as a carrier of vitamin D metabolites and analogues; activating enzymes (CYPs) involved in activation of vitamin D or prodrug; target cell transcriptional machinery (VDR, RXR, coactivators) involved in biological actions of 1α,25-(OH)2D3 or its analogues; and target cell catabolic enzyme system (CYP24A1) involved in degradation of 1α,25-(OH)2D3 or its analogue. The figure shows the metabolism of vitamin D in the context of the cells involved. Clockwise: (Top Left) Hepatocyte showing some of the candidate cytochrome P450s shown to 25-hydroxylate vitamin D and its prodrugs; note that VDR is believed to be absent from liver cells. (Top Middle) Proximal Tubular Cell showing the key elements in the uptake of 25-OH-D3 and its conversion to 1α,25-(OH)2D3. Megalin/cubulin are cell surface receptors that execute endocytosis of the DBP/25-OH-D3 complex, whereas CYP27B1 is the main component of the 1α-hydroxylase, responsible for synthesis of circulating 1α,25-(OH)2D3. (Lower Right) Conventional Target Cell that lacks megalin/cubulin and takes up only the free ligand, 1α,25-(OH)2D3, but not the DBP originally involved in transporting the ligand to the target cell. The key elements of the transcriptional machinery are shown, including VDR/RXR as well as representative gene products such as cell division protein p21, the bone matrix protein osteopontin, the calcium transport protein calbindin, and the autoregulatory protein CYP24A1. The role of the highly inducible CYP24A1 is to convert the hormone (or analogue) into inactive degradation products, such as calcitroic acid, which enter plasma and are excreted in bile. (Lower Left) Target Cell that expresses extrarenal 1α-hydroxylase (CYP27B1) and the megalin/cubulin machinery to take up 25-OH-D3, and thus is capable of making 1α,25-(OH)2D3 locally. The cell can also respond in a likewise manner to the conventional target cell because it also possesses the VDR and other transcriptional machinery. The expectation is that cells involved in cell differentiation or controlling cell division require higher concentrations of 1α,25-(OH)2D3 in order to modulate a different set of genes, and the CYP27B1 boosts local production to augment that circulating 1α,25-(OH)2D3 arriving from the kidney in the bloodstream. Under normal physiological processes, locally produced 1α,25-(OH)2D3 would not enter the general circulation, though in pathological conditions (e.g., sarcoidosis) this could occur. At this time, it is not clear how many cell types can be considered simple target cells and how many possess the CYP27B1 and megalin/cubulin to allow for local production of hormone.

of 1α,25 (OH)2D3 will reveal new approaches by which the vitamin D signaling cascade can be exploited. Certainly, the significant progress made in characterizing the coactivator proteins and the rest of the transcriptional apparatus will continue. One is able to predict fairly confidently from success in related steroid hormone fields that a fully functional vitamin D-dependent in vitro reconstituted VDRRXR transcriptional system, devoid of the complications of metabolic enzymes, will be the perfect model to test the transactivation activity of future vitamin D analogues. It seems likely that this approach will allow us to dissect out the exact features that give certain analogues a transcriptional advantage to provide increased potency and/or selectivity over 1α,25-(OH)2D3.

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Studies of the vitamin D-binding pockets of VDR, DBP, and the three (or more) vitamin D-related cytochrome P450s will continue to be a major goal now that all these specific proteins have been cloned, overexpressed, and crystallized. Although the ligand-binding domains of the nuclear receptors have been studied, the full-length proteins are beyond the current limits of NMR or x-ray crystallography. It is also likely that technical problems with these procedures will be overcome shortly and the full-length proteins can be tackled. The initial work of the Moras group (Rochel et al., 2001) on the ligand-binding domain of the VDR will be extended to new analogues and there will also be a growing focus on the other major proteins in the vitamin D signal transduction pathway.

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The wide availability of recombinant proteins for hundreds of cytochromes P450 from species across the phylogenetic tree, including 58 CYPs in the human genome, has allowed for the elucidation of some crystal structures and also modeling studies of the enzymes involved in vitamin D metabolism (Prosser et al., 2006; Hamamoto et al., 2006; Masuda et al., 2007). Current models are starting to reveal key substrate side-chain contact residues (e.g., Ala326 within CYP24A1) associated with hydroxylation (Prosser et al., 2007). The membrane-associated region of cytochromes P450 has posed problems for expression and crystallization but enormous strides have been made based on models built with x-ray data from soluble prokaryotic isoforms and with truncated mammalian isoforms such as CYP2C5 (Williams et al., 2000) and the first crystal structures of vitamin D-related CYPs e.g., CYP2R1 are now emerging (Strushkevich et al., 2008). Access to full-length CYP241 and CYP27B1 has also permitted a more efficient search for potential inhibitors. Such specific inhibitors of CYP241 and CYP27B1 (Schuster et al., 2001, 2003; Muralidharan et al., 1997; Posner et al., 2004) may be of value in blocking 1α,25-(OH)2D3 catabolism or synthesis in certain clinical conditions where excessive breakdown is suspected. In general, modeling of VDR and cytochromes P450 is expected to lead to more rational vitamin D analogue design to take advantage of structural idiosyncrasies of all of these key proteins. Meanwhile, the not-so-rational synthesis of new analogues is likely to continue. The list of applications for vitamin D compounds continues to increase (reviewed in Jones et al., 1998; Holick, 2007; Jones, 2007). These applications have been further rationalized with the availability of VDR knockout mice to demonstrate vitamin D-dependent processes (Yoshizawa et al., 1997; Li et al., 1997). Elucidation of the mechanism by which 1α,25-(OH)2D3 and its analogues regulate the cell cycle and proliferation remains an important priority of the field. Current applications of vitamin D analogues still fall mainly into calcium-related and cell-proliferative/differentiating arenas but the “rediscovery” of the wide effects of vitamin D deficiency and insufficiency has reinvigorated the whole field. The goal of developing analogues that can completely separate the “calcemic” and “nonclassical” properties of 1α,25-(OH)2D3 has not yet been fully realized. However, some promising compounds have been synthesized and interesting idiosyncrasies of their biological actions have surfaced (e.g., tissue, cell, gene, and VDRE differences). It remains to be seen whether these differences can be exploited. On the other hand, it must be stated that if vitamin D analogues work only through a VDR-mediated genomic mechanism, it is difficult to appreciate how the “calcemic” properties of 1α,25-(OH)2D3 can ever be fully resolved from the “cell-differentiating” properties given that pharmacokinetic differences have provided only a partial separation. On a more optimistic front, it can be stated that since the first/second editions of this book were published,

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several new vitamin D analogues (OCT, 1α,24-(OH)2D3, 1α-OH-D2, and 19-nor-1α,25-(OH)2D2) have received governmental approval to be used in the treatment of various clinical conditions worldwide. We must remain upbeat that more vitamin D analogues will be developed and important new applications of vitamin D, particularly in the area of bone biology, remain to be uncovered.

ACKNOWLEDGMENTS The author is supported by the Canadian Institutes of Health Research. He also thanks David Prosser for his help in assembly of the tables and figures contained in this chapter.

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Chapter | 83 Vitamin D and Analogues

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