Ann. N.Y. Acad. Sci. ISSN 0077-8923

A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S Issue: Steroids in Neuroendocrine Immunology and Therapy of Rheumatic Diseases

Vitamin D receptor agonists’ anti-inflammatory properties Jelena Vojinovic Faculty of Medicine, University of Nis, Nis, Serbia Address for correspondence: Professor Jelena Vojinovic, M.D., Ph.D., Bul dr Zorana Djindjica 81, Faculty of Medicine, University of Nis, 18000 Nis, Serbia. [email protected]

One century after its discovery, vitamin D has been shown to be, in fact, a pleiotropic steroid hormone, which, besides regulation of calcium homeostasis and bone turnover, has antiproliferative, prodifferentiation, antibacterial, immunomodulatory, and anti-inflammatory properties in various cells and tissues. D hormone (1␣,25(OH)2 D), regulated in an endocrine, autocrine, and paracrine manner, must be bound to the specific nuclear vitamin D receptor (VDR) to exert epigenetic and genetic effects, acting as a connection between extracellular stimuli and genomic responses of the cells. Since only high doses of hormone, provoking hypercalcemia, can achieve immunomodulatory effects, more than 3000 VDR agonists have been synthesized. Numerous experimental trials have been performed in animal models, evidencing the preventive and therapeutic potential of VDR agonists for chronic inflammatory diseases and cancer. Considering the selective anti-inflammatory effects of VDR agonists compared to glucocorticoids, sparing microbicidal functions, the fear of hypercalcemia as their only frequent side effect becomes a questionable reason for the lack of clinical studies. Keywords: vitamin D; vitamin D receptor (VDR); VDR agonist; inflammation

Introduction Almost one century after its discovery and the awarding of three Nobel Prizes,1 we have clear evidence that vitamin D is in fact a pleiotropic steroid hormone similar to other steroid hormones. Besides regulation of calcium homeostasis and bone turnover, this hormone has antiproliferative, prodifferentiation, antibacterial, immunomodulatory, and anti-inflammatory properties within the body in various cells and tissues.2–4 A question arises of whether vitamin D indeed still should be classified as a vitamin since a vitamin is defined as “a chemical substance in food which is in minute quantities essential for metabolism. Vitamins cannot usually be synthesized in the body but they occur naturally in certain foods. Insufficient supply of any particular vitamin results in a deficiency disease.” Similar as for other steroid hormones, for synthesis of D hormone it is necessary to ingest a specific diet compound, but when synthesized after the complex and in endocrine-regulated biochemical processes, it has its own endocrine, paracrine, and autocrine

control.5 Therefore, it is clear that vitamin D does not fulfill the criteria of the vitamin definition described, but rather fulfills those for a hormone (defined as a chemical substance produced in one part of the body that stimulates functional activity in another). Unfortunately, the primary classification of vitamin D into the vitamins group still deeply influences our professional perception about its function and possible therapeutic usage. Therefore, in this review we will use the term D hormone to distinguish from vitamin D3 (as a nutritional precursor compound necessary for its synthesis). Immunomodulatory properties of D hormone (1␣,25(OH)2 D) are mostly achieved only if very high doses are used, which can provoke hypercalcemia as a side effect. The discovery of the immunomodulatory and antitumor properties of D hormone prompted researchers to investigate the possibility of its use as a therapeutic agent for different autoimmune and malignant diseases.6–8 This is why during the last several decades there were many investigations with an aim to synthesize 1␣,25(OH)2 D analogs (vitamin D receptor (VDR)

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agonists) with the same biologic activity and even stronger anti-inflammatory properties but lower blood calcium–increasing capacity.9 This review will provide an overview of knowledge on vitamin D agonists during the past century, focusing on our understanding of vitamin D and the possibility to use it (or its analogs) in clinical practice. D hormone synthesis: pregenomic era of vitamin D knowledge After the discovery of vitamin D one century ago, the term vitamin D was used for cholecalcipherol according to the names given to other discovered substances at that time, as a brief name for vital amines. Unfortunately, an imprecise term given long ago is still used and influences the apprehension surrounding this biologically active compound. Actually, a substance named as a vitamin refers to one specific member among a group of steroid molecules (secosteroids) with a common A, B, C, and D ring structure derived from the cyclo-pentano-perhydro-phenanthrene ring structure (derived from cholecalcipherol in the case of vitamin D or cholesterol in the case of other steroid hormones).10 The prehormonal form of D hormone, known as vitamin D or cholecalciferol, is necessary to be ingested or mainly generated in the skin where one of the rings of the precursor molecule (7-dehydrocholesterol) is broken by ultraviolet B light (sunlight). Afterwards, to achieve the biologically active form, vitamin D must first be hydroxylated in the liver, at the carbon 25 position by 25-hydroxylase, into 25-hydroxyvitamin D3 (25(OH)D), known as calcidiol or calcifediol. Several cytochrome P450 (CYP) isoforms (including the mitochondrial CYP27A1 and the microsomal CYP2R1, CYP3A4, and CYP2J3) accomplish this hydroxylation step, but CYP2R1 is thought to be the high-affinity 25-hydroxylase.11 The 25OHD form is the most plentiful and stable metabolite of vitamin D in human serum with high affinity to bind serum vitamin D–binding protein and other albumin superfamily in the blood. As such, the 25OHD level in the serum is the best indicator of vitamin D entering the host, either by cutaneous synthesis or by ingestion in the diet. Nevertheless, this form is still not a hormone; rather, it is a prehormonal form of the natural hormone and does not exert almost any biologic activity in the body. 2

The calcidiol form of vitamin D (25OHD) is then transported through the bloodstream to the proximal tubule of the kidney, where it is hydrolyzed at the 1␣ position to form the final biologically active form of D hormone calcitriol (1␣,25(OH)2 D), by the enzyme 25-hydroxyvitamin D-1␣-hydroxylase (CYP27B1). The activity of this enzyme is increased by parathyroid hormone (PTH) secreted by the parathyroid gland, which is the pivotal activator of CYP27B1 in proximal tubule cells.12 Thereafter, the synthesized calcitriol becomes the real D hormone with full biological activity similar to other steroid hormones. For many years it was believed that regulation of calcium homeostasis within the body and a positive influence on bone turnover are the only or crucial roles of this hormone and the reasons why it was considered to be the vitamin necessary for bone health. This scheme remains correct, but it is now understood that many tissues, particularly macrophages in all tissues and various epithelia, are able to express 1␣-hydroxylase and to synthesize active D hormone locally.13 D hormone synthesized locally in the tissues or present in the blood acts on numerous cells and tissues throughout the body, in an endocrine, autocrine, and paracrine manner, and serves as a connection between extracellular stimuli and genomic responses of the cells.14 This is why measurement of 1␣,25(OH)2 D in serum, unfortunately, provides little insight into D hormone status within the tissues because of its tight physiologic control by serum calcium levels and PTH, and very short half-life. On the other hand, it is of great importance for physicians to understand that measurement of 25(OH)D levels (as the only standardized tool to estimate vitamin D status) is actually only the reflection of the balance between food and/or supplement vitamin D diet intake and its utilization in the local tissues as the active D hormone (especially immune cells in the state of chronic inflammation). This is probably the best explanation for numerous clinical and epidemiological studies showing a connection between low vitamin D status and numerous immune and malignant diseases.15,16 VDR: genetic and epigenetic era of knowledge Steroid hormones are among the most fundamental signaling molecules in nature and are responsible for the regulation of development, reproduction,

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metabolism, and responses to environmental triggers.17 It took nearly 20 years to clone the gene for the VDR and to characterize it as a nuclear receptor for 1␣,25(OH)2 D, after receptors for all other nuclear hormones, such as estradiol, testosterone, cortisol, and retinoic acid, had been cloned and found to belong to the same transcription factor family. The signaling pathway of steroid hormones is via cellular and nuclear hormone receptors that respond to hormones based on steroid scaffolds (estrogen receptor (ER), progesterone receptor, androgen receptor (AR), glucocorticoid receptor, thyroid receptor, and VDR). It is of essential importance to be aware that all of these hormones influence bone formation and immune regulation. Steroid nuclear receptors, when bound to their agonist hormone, bind to chromatin at hormone response elements to affect the expression of downstream genes and mediate chromatin remodeling, epigenetic modifications, receptor recycling, and ultimately gene expression.18 It is recognized that only 1␣,25(OH)2 D and VDR agonists have high affinity to bind VDRs19 and, when bound, heterodimerize with the retinoic acid X receptor. Afterwards, this complex binds to the vitamin D–responsive element and acts as a transcriptional factor to enhance or repress gene transcription.20 It has been estimated that more than 2000 genes are directly or indirectly controlled by this transcriptional complex.21 The VDR gene shows the highest expression in metabolic tissues, such as kidneys, bone, and intestine, but low to moderate expression in nearly all other of the approximately 250 different human tissues. The actions of 1␣,25(OH)2 D, such as its role in calcium homeostasis and bone mineralization, but also its cell and immune-regulatory effects, are mediated by the gene regulatory actions of VDRs.22 This is the major pathway that orchestrates D hormone endocrine effects within the body. In contrast to other nuclear receptors for steroid hormones, there is only one gene coding VDRs. The VDR gene is located on chromosome 12 (12q13.11), and it was shown that some of its polymorphisms may be connected with the presence and/or severity of many rheumatic diseases.23,24 It seems that the VDR gene itself can influence some chronic diseases and VDR-bound agonists can influence the expression and activity of numerous genes. Although VDRs are transcription factors acting in the nucleus, VDR protein in the cytosol is associated with sarcoplas-

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mic reticulum Ca2+ -ATPase in plasma membranes, which can explain some of the rapid, nongenomic actions of 1␣,25(OH)2 D, such as the uptake of calcium.25 The last decade could be named the epigenetic era of knowledge since numerous recent studies suggested that epigenetic processes are responsible for tissue-specific gene expression during differentiation and may play a key role in adaptive responses to nutritional and environmental factors. Gene regulation, influenced by the VDR agonist complex, appears to be modulated by dual modifications of histone acetylation and DNA methylation, implicating D hormone and synthetic VDR agonists as potent genetic and epigenetic regulators.26 The cell nucleus is a complex of genomic DNA and nucleosomes, referred to as chromatin, which prevents access of DNA-binding proteins, such as transcription factors, to their genomic targets.27 This chromatin potential is essential for long-lasting regulatory decisions, such as terminal differentiation of cells. Epigenetic alterations consist of heritable modifications of the DNA on the basis of reversible posttranslational modification of histone proteins, such as acetylation and methylation, that are directed by a class of coregulatory proteins with either histone acetyltransferase, histone deacetylase (HDAC), histone methyltransferase, or demethylase activity.28,29 VDR bound to its ligand (agonist) initiates creation of a complex between nuclear receptors and gene coactivators, leading to local opening of chromatin, and induces mRNA synthesis of target genes through mediator proteins and general transcription factors. On the contrary, if only VDRs are in contact with corepressors and HDACs, they will locally repress chromatin and inactivate target genes.30 Gene regulation is modulated by dual modifications of histone acetylation and DNA methylation caused by the VDR–agonist complex and it appears to be the major mechanism by which D hormone regulates gene expression and numerous inflammatory functions within the body. The strong epigenetic potential of VDR agonists makes them desirable treatment options in numerous inflammatory and malignant diseases. VDR agonists To achieve genetic and epigenetic effects, it is necessary for D hormone as a natural compound to be present in high concentrations in cells and tissues.

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The use of cholecalcipherol or calcitriol to accomplish this induces hypercalcemia as a side effect. This is why pharmaceutical companies focused on the development of more than 3000 D hormone analogs (agonists), with the majority of them carrying a modification in their aliphatic side chain.31 Development of agonists is based on knowledge about the specificity of biochemical mechanisms of VDR binding with natural D hormone or its analogs (VDR agonists). Binding is a result of specific interactions of D hormone or its agonists with the ligandbinding pocket within the ligand-binding domain (LBD) of the VDR. The LBD is a member of a highly conserved DNA-binding domain (DBD) protein family and forms a complex structure when bound to VDRs.32 Biochemical and biophysical methods showed that there is a significant stabilization of the LBD in the presence of specific ligand, resulting in a more compact and stabile structure.33 Natural D hormone, as a specific ligand, is bound via interaction of its three hydroxyl groups to a pair of amino acids inside the tertiary structure of the formed complex with the LBD. In the VDR–LBD complex, the A-ring of D hormone or its agonist adopts a ␤-chair conformation with the 1-OH and 3-OH groups in equatorial and axial orientations, respectively.34 This is likely why it was shown that VDR agonists with an OH group in 1␣ position have the highest specificity of binding to VDR.19 There are several biochemically different types of VDR agonists, which are going to be described briefly, but for a detailed description the reader is referred to an informative review by Carlsberg and Molnar.22 Most D hormone analogs (VDR agonists) carry only minor modifications compared to the natural hormone having the same secosteroidal structure and forming the same agonistic VDR conformation. Nevertheless, there are several different groups of analogs that can be conditionally divided as steroidal or nonsteroidal (Table 1).35–55 The first steroidal 16-ene VDR agonist, synthesized more than 20 years ago, demonstrated inhibition of leukemic cell growth without modification of intestinal calcium absorption. Note that 16-ene analogs, developed later as a group, presented an ability to accumulate in the circulation by escaping the C23-hydroxylation step (inducing compound degradation), but still retaining a nonhypercalcemic effect.56 Elocalcitol (BXL-628, 1␣-fluoro-25-hydroxy-16,23E-diene4

26,27-bishomo-20-epi-vitamin D3 ), another 16-ene modified analog, exerted positive effects and were proposed as a treatment option for benign prostatic hyperplasia.57 Semi-steroidal analogs are the second group synthesized that either have significant changes in their A-, C-, or D-rings, such as CD ring deletion, or carry substantial additions, such as the second side chain. The most important representatives from this group are the 20-epi analogs MC1288 (20epi-1␣,25(OH)2 D3 ) and KH1060 (1␣,25(OH)2 (20S)-22-oxa-24,26,27-trihomovitamin D3 ). These analogs were the first synthetic ligands that were proven to cocrystallize with VDRs,58 while their binding with VDRs did not result in significant variations of the LBD conformation, usually induced by the natural ligand. The same result was obtained when VDR–LBD was crystallized in complex with the other synthesized secosteroid analogs, such as MC903 (calcipotriol), EB1089 (seocalcitol), and TX522 (14-epi conformation).59 Calcipotriol is the most important representative from this group since it is the only one registered for the topical treatment of psoriasis (chronic autoimmune inflammatory disease). Numerous studies using these VDR analogs in experimental animal models have shown their enhanced antiproliferative and anti-inflammatory properties in vitro and in vivo, such as in type I diabetes (T1D) and inflammatory bowel disease (IBD).40,41 A similar group of analogs, based on similar stereochemistry, was synthesized later on using the 20-cyclopropyl modification. These subtypes of compounds have shown higher potency than the natural D hormone in inhibiting the proliferation of cancer cell lines and production of proinflammatory cytokines, such as interferon (IFN)-␥ or tumor necrosis factor (TNF)-␣, and a stronger potency in primary VDR target gene induction with less hypercalcemic ability.60 Nonsteroidal VDR ligands have been synthesized, and even efficiently used clinically, for several members of the steroid hormone nuclear receptors, such as flutamine for the AR and raloxifene for the ER. Following the same analogy and using new medicinal chemistry approaches, which improved stability and efficacy, the new type of VDR analogs named phenyl-furan were synthesized as nonsteroidal VDR agonists.61 In this group are D hormone analogs (agonists) with two side chains, represented by Gemini, which has identical side chains that, despite its

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Table 1. VDR agonist subtype representatives and their effects on diseases

VDR agonist

Disease

Steroidal alfacalcidol

osteoporosis, chronic renal insufficiency, rheumatoid arthritis6, 35 breast cancer, prostate cancer, colon cancer, thyroid carcinoma36

22-oxacalcitol (OCT) 16-ene 1,25(OH)2– 16-ene-23-ene-D3 Semi-steroidal 20-epi 1,25(OH)2– 20-epi-D3 KH 1060 (1,25(OH)2– 20-epi-22-oxa-24,26,27trishomo-D3 ) seocalcitol EB 1089 (1,25(OH)2 -diene-24,26,27-trihomo-D3 ) elocalcitol (1␣-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epivitamin D3 ) MC1288 (20-epi-1␣,25(OH)2 D3 ) calcipotriol MC 903 20-cyclopropyl BXL-62 (1alpha,25(OH)(2)-16-ene-20-cyclopropyl-vitamin D3 )

Non-steroidal agonists diphenylmethane derivatives bis-aromatic molecules phenyfuran analogues54 analogues with two side chains (Gemini)

significantly increased volume (25%), have high affinity to bind VDRs.62 There have been numerous experimental trials in animal models investigating the therapeutic possibilities and safety of numerous different VDR agonists, with promising results. A preventive and therapeutic capacity of VDR agonists has been shown in animal models of systemic lupus erythematosus in MRL–lpr/lpr mice, experimental allergic encephalomyelitis, collagen-induced arthritis, Lyme arthritis, IBD, and autoimmune diabetes (T1D).47,63–67 Most clinical trials with natural D hormone or its natural precursors, and VDR agonists (such as alphacalcidol), have focused on bone or musculoskeletal functions, but they have also shown preventive and therapeutic potential for ex-

HL-60 myeloid leukemia37

diabetes40 inflammatory bowel disease (IBD)41 breast cancer42, 43 pancreatic cancer44 idiopathic detrusor overactivity45 inflammatory myopathies46 collagen-induced arthritis47 clinical use in psoriasis48 anti-inflammatory properties49 IBD50 benign prostatic hyperplasia51 osteoporosis52 immunomodulation53 breast cancer55

traskeletal chronic diseases, such as cardiovascular and autoimmune diseases, cancer, diabetes, and infections. For example, alphacalcidol is the first ever synthesized D hormone analog that differs from natural hormone only by lacking a 25(OH) group, and it is not naturally produced. It has shown beneficial effects on disease activity and immunoregulation in patients with active rheumatoid arthritis.6,68,69 Recently, it has been shown that, despite previous beliefs, it acts directly on inflammatory cytokine production without needing to be additionally hydroxylated at the 25 position.70 As a drug, it has been, for decades, registered and efficaciously used to treat primary and glucocorticoidinduced osteoporosis, yet it is not used as an adjunct treatment in patients with rheumatoid arthritis.

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Unfortunately, most of the investigations using VDR analogs that successfully dissociated beneficial immune regulatory effects from undesired side effects on calcium metabolism have not progressed beyond the preclinical stage and only a few VDR agonists are presently on the market as drugs to treat immune or malignant diseases. In most cases, VDR agonists chosen for development were not those that have shown to be most efficacious in in vitro and in vivo assays, but were those that showed in vivo the lowest calcemic potential. This, believed to be the safest selection approach, did not provide the most effective drug for the patients suffering different chronic diseases. VDR agonists and inflammation VDRs are expressed in most types of immune cells, especially in all antigen presenting cell (APC) types, such as macrophages and dendritic cells (DCs), as well as in both CD4+ and CD8+ T cells and B cells,71 indicating the importance of natural or synthetic VDR agonists as immunoregulators. Moreover, macrophages and DCs have an ability to overexpress hydroxyvitamin D-1␣-hydroxylase (CYP27B1). After stimulation induced by proinflammatory signals, such as IFN-␥ or NF-␬B, these innate immunity cells actively synthesize the active D hormone from 25(OH)D substrate.72 This is a unique D hormone property compared to other steroid hormones, indicating that it is naturally aimed to be an important immunoregulatory molecule (expressing a cytokine-type mode of action). The D hormone markedly modulates the phenotype and functions of APCs, particularly DCs, which is why it was postulated that D hormone is a physiological controller of immune responses and possibly involved in maintaining tolerance to selfantigens.73 In macrophages and monocytes, VDR agonists positively influence their own effects by increasing the expression of VDRs and the cytochrome P450 protein CYP27B1. Certain Toll-like receptor– mediated signals can also increase the expression of VDRs. Note that 1,25(OH)2 D also induces monocyte proliferation and the expression of interleukin (IL)-1 and cathelicidin (an antimicrobial peptide) by macrophages, thereby contributing to innate immune responses to some bacteria. VDR agonists inhibit in myeloid DCs, but not in plasmacytoid DCs, the expression of surface costimulatory

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molecules, such as major histocompatibility complex class II, CD40, CD80, and CD86. In addition, they decrease IL-12 production by DCs while inducing the production of IL-10, and block B cell proliferation, plasma cell differentiation, and immunoglobulin production.74,75 VDR expression in T cells is only upregulated following activation, and VDR agonists were shown to inhibit antigen-induced T cell proliferation and cytokine production. In T cells, 1,25(OH)2 D decreases the production of IL-2, IL-17, and IFN-␥ , and attenuates the cytotoxic activity and proliferation of CD4+ and CD8+ T cells.76 Treatment with VDR agonists also inhibits IL-17 production, a proinflammatory cytokine shown recently to be produced by pathogenic T cells in various models of chronic inflammation and immune-mediated tissue injury, including organ-specific autoimmunity in the brain, heart, synovium, and intestines, and allergic disorders of the lung and skin, as well as microbial infections of the intestines and nervous system.77 Interestingly, IL-17 production is induced by IL-23 while p40 chain is strongly inhibited by VDR agonists.78,79 Note that 1,25(OH)2 D might also promote the development of forkhead box protein 3 (FoxP3)+ regulatory T (Treg ) cells and IL-10–producing T regulatory type 1 (TR1) cells.80 VDR agonists have also been shown to enhance the development of TH 2 cells via a direct effect on naive CD4+ cells. This could account for the beneficial effect of VDR agonists in the treatment of some inflammatory conditions. D hormone capability to skew T cells toward the TH 2 pathway had been suggested but could not be confirmed by other studies. Nevertheless, TH 2 cells can be targets of VDR agonists but this depends on their activation and differentiation status.81 Thus, VDR agonists can apparently upregulate, downregulate, or have no effect on IL-4 production and, consequently, on TH 2 cell development, illustrating the complexity of immunoregulatory pathways. Chronic inflammation could be a risk factor for the development of many cancers.82 VDR agonists’ influence on inflammation and their antiproliferative and prodifferentiating effects raise the possibility of their use as anticancer agents.83,84 In cancer tissue, there is an evident presence of inflammation characterized by the presence of inflammatory cells and the overexpression of inflammatory mediators,

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such as cytokines, chemokines, prostaglandins (PGs), and reactive oxygen and nitrogen.85 Proinflammatory mediators activate angiogenic switches usually under the control of vascular endothelial growth factor and thereby promote tumor angiogenesis, metastasis, and invasion. Recent research suggests that VDR agonists exert anti-inflammatory actions in addition to their other known antiproliferative actions, such as: (1) suppression of the expression of COX-2, the enzyme that synthesizes PGs; (2) downregulation of the expression of PG receptors that are essential for their signaling; and (3) upregulation of the expression of 15-PGDH (the enzyme that inactivates PGs).86 Recently, it was discovered that VDR agonists decrease expression of aromatase, the enzyme essential for catalysis of peripheral estrogen synthesis from androgens present in cancer tissues (breast and prostate cancer). Inflammatory cytokines (TNF-␣, IL-6) are also strong enhancers of aromatase activity.87 Therefore, VDR agonists exert an inhibitory effect on breast cancer growth by a direct repression of aromatase transcription and as an indirect effect due to a reduction of the levels and biological activity of PGs (especially PGE21).88 According to this, Krishnan et al.86,89 hypothesized that the combination of calcitriol and nonsteroidal anti-inflammatory drugs would exhibit additive/synergistic activity to inhibit prostate cancer cell growth and that VDR agonists in combination with aromatase inhibitors could improve disease outcome in breast cancer. These hypotheses are supported with several findings in clinical trials.90,91 Immunomodulatory actions of natural D hormone have been confirmed for different synthetic VDR agonists in numerous in vitro and in vivo experimental studies. In most of them, anti-inflammatory properties of the VDR agonists used were the best explanation for beneficial preventive or therapeutic effects obtained in animal models of chronic inflammatory diseases or cancers. Conclusion The vitamin D story lasts more than century, with accumulating knowledge and exciting discoveries, but, unfortunately, professional understanding and clinical applicability have not improved. Glucocorticoids, as synthetic analogs of natural steroid hormone, have been used in medicine for decades and are considered to be powerful immunosuppressive

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agents. Because of their nonselective immunosuppression and metabolic actions, patients using them as their only treatment option for chronic inflammatory disease suffer many severe side effects. Considering the selective immunomodulatory effects of VDR agonists, sparing microbicidal functions, the fear of hypercalcemia as their only frequent side effect becomes a questionable reason for the lack of studies in humans. This review has summarized significant scientific background about VDR agonists’ anti-inflammatory potential, which is more extensive than for many drugs in clinical use. A shortage of randomized clinical trials among 3000 VDR agonists synthesized is confusing and could be a consequence of, for example, a lack of understanding or interest of pharmaceutical companies and/or the professional community. We can only hope that this will last less than decades until we learn how to use them, as we learned for glucocorticoids. Conflicts of interest The author declares no conflicts of interest. References 1. Nobelprize.org. 2013. The Nobel Prize in Physiology or Medicine 1903. Nobel Media AB 2013. Accessed April 5, 2014. http://nobelprize.org/nobel_prizes/ medicine/laureates/1903/finsen-bio.html. 2. Haussler, M.R. et al. 2010. The nuclear vitamin D receptor controls the expression of genes encoding factors which feed the “Fountain of Youth” to mediate healthful aging. J. Steroid. Biochem. Mol. Biol. 121: 88–97. 3. Arnson, Y. et al. 2007. Vitamin D and autoimmunity new etiological and therapeutical considerations. Ann. Rheum. Dis. 66: 1137–1142. 4. Pludowski, P. et al. 2013. Vitamin D effects on musculoskeletal health, immunity, autoimmunity, cardiovascular disease, cancer, fertility, pregnancy, dementia and mortality— a review of recent evidence. Autoimmun Rev. 12: 976– 989. 5. Wacker, M. & M.F. Holick. 2013. Vitamin D—effects on skeletal and extraskeletal health and the need for supplementation. Nutrients 5: 111–148. 6. De Luca, H. 2004. Overview of general physiologic features and functions of vitamin D. Am. J. Clin. Nutr. 80: 1689S– 1696S. 7. Andjelkovic, Z. et al. 1999. Disease modifynig and immunoregulatory effects of high oral dose 1␣(OH)D3 in rheumatoid arthritis patients. Clin. Exp. Rheumatol. 17: 59–62. 8. Antico, A. et al. 2012. Can supplementation with vitamin D reduce the risk or modify the course of autoimmune diseases? A systematic review of the literature. Autoimmun Rev. 12: 127–136.

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9. Adorini, L. 2002. 1,25-Dihydroxyvitamin D3 analogs as potential therapies in transplantation. Curr. Opin. Investig. Drugs. 3: 1458–1463. 10. De Luca, H.F. 1974. Vitamin D: the vitamin and the hormone. Fed. Proc. 33: 2211–2219. 11. Cheng, J.B. et al. 2004. Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase. Proc. Natl. Acad. Sci. U. S. A. 101: 7711–7715. 12. Jurutka, P.W. et al. 2007. Vitamin D receptor: key roles in bone mineral pathophysiology, molecular mechanism of action, and novel nutritional ligands. J. Bone Miner. Res. 22: V2–V10. 13. Adams, J.S. & M. Hewison. 2010. Update in vitamin D. J. Clin. Endocrinol. Metab. 95: 471–478. 14. Mora, R. et al. 2008. Vitamin effects on the immune system: vitamins A and D take centre stage. Nat. Rev. Immunol. 8: 685–698. 15. Cutolo, M. et al. 2011. Vitamin D endocrine system involvement in autoimmune rheumatic diseases. Autoimmun. Rev. 11: 84–87. 16. Bjelakovic, G. et al. 2011. Vitamin D supplementation for prevention of mortality in adults. Cochrane Database Syst. Rev. 7: CD007470. 17. Margolis, R.N. & S. Christakos. 2010. The nuclear receptor superfamily of steroid hormones and vitamin D gene regulation. Ann. N. Y. Acad. Sci. 1192: 208–214. 18. Lonard, D.M. et al. 2007. Nuclear receptor coregulators and human disease. Endocr. Rev. 28: 575–587. 19. Eduardo-Canosa, S. et al. 2010. Design and synthesis of active vitamin D analogs. J. Steroid. Biochem. Mol. Biol. 121: 7–12. 20. Norman, A.W. 2006. Vitamin D receptor: new assignments for an already busy receptor. Endocrinology 147: 5542–5548. 21. Nagpal, S. et al. 2005. Noncalcemic actions of vitamin D receptor ligands. Endocr. Rev. 26: 662–687. 22. Carlberg, C. & F. Moln´ar. 2012. Current status of vitamin D signaling and its therapeutic applications. Curr. Topics Med. Chem. 12: 1–20. 23. Ranganathan, P. 2009. Genetics of bone loss in rheumatoid arthritis—role of vitamin D receptor polymorphisms. Rheumatology 48: 342–346. 24. Maalej, A. et al. 2005. Association study of VDR gene with rheumatoid arthritis in the French population. Genes Immun. 6: 707–711. 25. Huhtakangas, J.A. et al. 2004. The vitamin D receptor is present in caveolae enriched plasma membranes and binds 1␣,25(OH)2-vitamin D3 in vivo and in vitro. Mol. Endocrinol. 18: 2660–2671. 26. Hossein-Nezhad, A. & M.F. Holick. 2012. Optimize dietary intake of vitamin D: an epigenetic perspective. Curr. Opin. Clin. Nutr. Metab. Care 15: 567–579. 27. Razin, A. 1998. CpG methylation, chromatin structure and gene silencing-a three-way connection. EMBO J. 17: 4905– 4908. 28. Narlikar, G.J. et al. 2002. Cooperation between complexes that regulate chromatin structure and transcription. Cell 108: 475–487. 29. Vojinovic, J. & N. Damjanov. 2011. HDAC inhibition in rheumatoid arthritis and juvenile idiopathic arthritis. Mol. Med. 7: 397–403.

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30. Takeyama, K.I. & S. Kato. The vitamin D3 1alphahydroxylase gene and its regulation by active vitamin D3. Biosci. Biotechnol. Biochem. 75: 208–213. 31. Carlberg, C. 2003. Molecular basis of the selective activity of vitamin D analogues. J. Cell. Biochem. 88: 274–281. 32. Chawla, A. 2001. Nuclear receptors and lipid physiology: opening the X-files. Science 294: 1866–1870. 33. Nagy, L. & J.W. Schwabe. 2004. Mechanism of the nuclear receptor molecular switch. Trends Biochem. Sci. 29: 317– 324. 34. Bouillon, R. et al. 1995. Structure-function relationships in the vitamin D endocrine system. Endocrine. Rev. 16: 200– 257. 35. Schacht, E. & J.D. Ringe. 2012. Alfacalcidol improves muscle power, muscle function and balance in elderly patients with reduced bone mass. Rheumatol Int. 32: 207–215. 36. Pernalete, N. et al. 1991. The activity of 22-oxacalcitol in osteoblast-like (ROS17/2.8) cells. Endocrinology 129: 778– 784. 37. Dore, B.T. et al. 1993. Interaction of retinoic acid and vitamin D3 analogs on HL-60 myeloid leukemic cells. Leuk. Res. 17: 749–757. 38. Kumgai, T. et al. 2003. Vitamin D2 analog 19-nor-1,25dihydroxyvitamin D2: antitumor activity against leukemia, myeloma and colon cancer. J. Natl. Cancer. Inst. 95: 896–905. 39. Koeffler, H.P. et al. 2005. Vitamin D(2) analog (Paricalcitol; Zemplar) for treatment of myelodysplastic syndrome. Leuk. Res. 29: 1259–1262. 40. Mathieu, C. et al. 1995. Prevention of type I diabetes in NOD mice by nonhypercalcemic doses of a new structural analog of 1,25-dihydroxyvitamin D3, KH1060. Endocrinology 136: 866–872. 41. Stio, M. et al. 2001. Suppressive effect of 1,25dihydroxyvitamin D3 and its analogues EB 1089 and KH 1060 on T lymphocyte proliferation in active ulcerative colitis. Biochem. Pharmacol. 61: 365–371. 42. Colston, K.W. et al. 1992. EB1089: a new vitamin D analogue that inhibits the growth of breast cancer cells in vivo and in vitro. Biochem. Pharmacol. 44: 2273–2280. 43. Mathiasen, I.S. et al. 1999. Apoptosis induced by vitamin D compounds in breast cancer cells is inhibited by Bcl-2 but does not involve known caspases or p53. Cancer. Res. 59: 4848–4856. 44. Evans, T.R. et al. 2002. A phase II trial of the vitamin D analogue Seocalcitol (EB1089) in patients with inoperable pancreatic cancer. Br. J. Cancer. 86: 680–685. 45. Digesu, G.A et al. 2012. Phase IIb, multicenter, double-blind, randomized, placebo-controlled, parallel-group study to determine effects of elocalcitol in women with overactive bladder and idiopathic detrusor overactivity. Urology 80: 48–54. 46. Di Luigi, L. et al. 2013. The vitamin D receptor agonist BXL01–0029 as a potential new pharmacological tool for the treatment of inflammatory myopathies. PLoS One 8: e77745. 47. Larsson, P. et al. 1998. A vitamin D analogue (MC 1288) has immunomodulatory properties and suppresses collageninduced arthritis (CIA) without causing hypercalcaemia. Clin. Exp. Immunol. 114: 277–283. 48. Segaert, S. & M. Ropke. 2013. The biological rationale for use of vitamin d analogs in combination with corticosteroids for

C 2014 New York Academy of Sciences. Ann. N.Y. Acad. Sci. xxxx (2014) 1–10 

Vojinovic

49.

50.

51.

52.

53.

54. 55.

56.

57.

58.

59.

60. 61.

62.

63.

64.

the topical treatment of plaque psoriasis. J. Drugs Dermatol. 12: 129–137. Laverny, G. et al. 2009. Synthesis and anti-inflammatory properties of 1alpha,25-dihydroxy-16-ene-20-cyclopropyl24-oxo-vitamin D3, a hypocalcemic, stable metabolite of 1alpha,25-dihydroxy-16-ene-20-cyclopropyl-vitamin D3. J. Med. Chem. 52: 2204–2213. Laverny, G. et al. 2010. Efficacy of a potent and safe vitamin D receptor agonist for the treatment of inflammatory bowel disease. Immunol. Lett. 131: 49–58. Penna, G. et al. 2009. Human benign prostatic hyperplasia stromal cells as inducers and targets of chronic immunomediated inflammation. J. Immunol. 182: 4056–4064. Kashiwagi, H. et al. 2011. Novel nonsecosteroidal vitamin D3 carboxylic acid analogs for osteoporosis, and SAR analysis. Bioorg. Med. Chem. 19: 4721–4729. Per¨akyl¨a, M. et al. 2005. Gene regulatory potential of nonsteroidal vitamin D receptor ligands. Mol. Endocrinol. 19: 2060–2073. Gajewski, R. et al. 2006. Phenyfuran compounds as vitamin D receptor modulators. World Patent 051936. So, J.Y. et al. 2013. Targeting CD44-STAT3 signaling by Gemini vitamin D analog leads to inhibition of invasion in basallike breast cancer. PLoS One 8: 54020. Norman, A. et al. 1990. Structure-function studies on analogues of 1 alpha,25-dihydroxyvitamin D3: differential effects on leukemic cell growth, differentiation, and intestinal calcium absorption. Cancer Res. 50: 6857–6864. Maggi, M. et al. 2006. Pre-clinical evidence and clinical translation of benign prostatic hyperplasia treatment by the vitamin D receptor agonist BXL-628 (Elocalcitol). J. Endocrinol. Invest. 29: 665–674. Tocchini-Valentini, G. et al. 2001. Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands. Proc. Natl. Acad. Sci. U. S. A. 98: 5491–5496. Tocchini-Valentini, G. et al. 2004. Crystal structures of the vitamin D nuclear receptor liganded with the vitamin D side chain analogues calcipotriol and seocalcitol, receptor agonists of clinical importance. Insights into a structural basis for the switching of calcipotriol to a receptor antagonist by further side chain modification. J. Med. Chem. 47: 1956– 1961. Uskokovic, M. et al. 2006. C-20 cyclopropyl vitamin D3 analogs. Curr. Top. Med. Chem. 6: 1289–1296. Asano, L. et al. 2013. Structural basis for vitamin D receptor agonism by novel non-secosteroidal ligands. FEBS Lett. 587: 957–963. Herdick, M. et al. 2000. Response element- and coactivatormediated conformational change of the vitamin D3 receptor permits sensitive interaction with agonists. Mol. Pharmacol. 57: 1206–1217. Cantorna, M.T. et al. 2000. 1,25-Dihydroxycholecalciferol prevents and ameliorates symptoms of experimental murine inflammatory bowel disease. J. Nutr. 130: 2648– 2652. Lemire, J.M. & D.C. Archer. 1991. 1,25-dihydroxyvitamin D3 prevents the in vivo induction of murine experimental autoimmune encephalomyelitis. J. Clin. Invest. 87: 1103– 1107.

VDR agonists’ anti-inflammatory properties

65. Cantorna, M.T. et al. 1996. 1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis. Proc. Natl. Acad. Sci. U. S. A. 93: 7861–7864. 66. Nataf, S. et al. 1996. 1,25 Dihydroxyvitamin D3 exerts regional effects in the central nervous system during experimental allergic encephalomyelitis. J. Neuropathol. Exp. Neurol. 55: 904–914. 67. Casteels, K. et al. 1998. Prevention of autoimmune destruction of syngeneic islet grafts in spontaneously diabetic nonobese diabetic mice by a combination of a vitamin D3 analog and cyclosporine. Transplantation 65: 1225–1232. 68. Hein, G. & P. Oelzner. 2000. Vitamin D metabolites in rheumatoid arthritis: findings–hypotheses—consequences. Z. Rheumatol. 59: 28–32. 69. Yamauchi, Y. et al. 1989. A double blind trial of alfacalcidol on patients with rheumatoid arthritis (RA). Ryumachi 29: 11–24. 70. Vojinovic, J. et al. 2012. Alphacalcidol (D-hormone analog) modulate inflammatory cytokine production without conversion to calcitriol. Ann. Rheum. Dis. 1(Suppl 3.): 672. 71. Veldman, C.M. et al. 2000. Expression of 1,25dihydroxyvitamin D(3) receptor in the immune system. Arch. Biochem. Biophys. 374: 334–338. 72. van Etten, E. & C. Mathieu. 2005. Immunoregulation by 1,25-dihydroxyvitamin D3: basic concepts. J. Steroid. Biochem. Mol. Biol. 97: 93–101. 73. Adorini, L. et al. 2004. Pharmacological induction of tolerogenic dendritic cells and regulatory T cells. Semin. Immunol. 16: 1271–1234. 74. Adams, J.S. & M. Hewison. 2008. Unexpected actions of vitamin D: new perspectives on the regulation of innate and adaptive immunity. Nat. Clin. Pract. Endocrinol. Metab. 4: 80–90. 75. Liu, P.T. et al. 2006. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science 311: 1770–1773. 76. Adorini, L et al. 2008. Control of autoimmune diseases by the vitamin D endocrine system. Nat. Clin. Pract. Rheumatol. 4: 404–412. 77. Colin, E.M. et al. 2010. 1,25-dihydroxyvitamin D3 modulates Th17 polarization and interleukin-22 expression by memory T cells from patients with early rheumatoid arthritis. Arthritis Rheum. 62: 132–142. 78. Sternberg, Z. 2012. Autonomic disfunction: a unifying multiple sclerosis theory linking chronic cerebrospinal venous insufficiency, vitamin D3, and Epstein-Barr virus. Autoimmun. Rev. 12: 250–259. 79. Steinman, L. 2007. A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage. Nat. Med. 13: 139–145. 80. Tang, J. et al. 2009. Calcitriol suppresses antiretinal autoimmunity through inhibitory effects on the Th17 effector response. J. Immunol. 182: 4624–4632. 81. Daniel, C. et al. 2008. Immune modulatory treatment of trinitrobenzene sulfonic acid colitis with calcitriol is associated with a change of a T helper (Th) 1/Th17 to a Th2 and regulatory T cell profile. J. Pharmacol. Exp. Ther. 324: 23–33.

C 2014 New York Academy of Sciences. Ann. N.Y. Acad. Sci. xxxx (2014) 1–10 

9

VDR agonists’ anti-inflammatory properties

Vojinovic

82. Di Rosa, M. et al. 2013. Vitamin D3 insufficiency and colorectal cancer. Crit. Rev. Oncol. Hematol. 88: 594–612. 83. Allavena, P. et al. 2008. Pathways connecting inflammation and cancer. Curr. Opin. Gen. Develop. 18: 3–10. 84. Guo, J. et al. 2013. 25(OH)2 D3 inhibits hepatocellular carcinoma development through reducing secretion of inflammatory cytokines from immunocytes. Curr. Med. Chem. 20: 4131–4141. 85. Mantovani, A. et al. 2008. Cancer-related inflammation. Nature 454: 436–444. 86. Krishnan, A.V. & D. Feldman. 2010. Molecular pathways mediating the anti-inflammatory effects of calcitriol: implications for prostate cancer chemoprevention and treatment. Endocr. Relat. Cancer 17: R19–R38. 87. Villagio, B. et al. 2012. 1,25-dihydroxyvitamin D3 downregulates aromatase expression and inflammatory cytokines

10

88.

89.

90.

91.

in human macrophages. Clin. Exp. Rheumatol. 30: 934– 938. Cutolo, M. 2012. The challenges of using vitamin D in cancer prevention and prognosis. Isr. Med Assoc. J. 14: 637– 639. Krishnan, A.V. et al. 2012. The potential therapeutic benefits of vitamin D in the treatment of estrogen receptor positive breast cancer. Steroids 77: 1107–1112. Beer, T.M. et al. 2007. Double-blinded randomized study of high-dose calcitriol plus docetaxel compared with placebo plus docetaxel in androgen-independent prostate cancer: a report from the ASCENT Investigators. J. Clin. Oncol. 25: 669–674. Crew, K.D. 2013. Vitamin D: are we ready to supplement for breast cancer prevention and treatment? ISRN Oncol. doi: 10.1155/2013/483687.

C 2014 New York Academy of Sciences. Ann. N.Y. Acad. Sci. xxxx (2014) 1–10