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Pharmacological effects of vitamin D and its analogs: recent developments Q1
Amnon C. Sintov1, Ludmilla Yarmolinsky2, Arik Dahan2 and Shimon Ben-Shabat2 1 2
Department of Biomedical Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel Department of Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel
Calcitriol, the hormonally active form of vitamin D, is well known for its diverse pharmacological activities, including modulation of cell growth, neuromuscular and immune function and reduction of inflammation. Calcitriol and its analogs exert potent effects on cellular differentiation and proliferation, regulate apoptosis and produce immunomodulatory effects. The purpose of this review is to provide information on various physiological and pharmacological activities of calcitriol and its newly discovered analogs. Special emphasis is given to skin diseases, cancer, diabetes and multiple sclerosis. A discussion is raised on the mechanisms of action of calcitriol and its analogs in various diseases, as well as on possible methods of delivery and targeting. Introduction Vitamin D includes several forms (vitamers) that are important from a physiological standpoint. Animals synthesize 7-dehydrocholesterol, the immediate precursor of cholesterol. Absorption of ultraviolet B radiation (290–315 nm) leads to a rearrangement of the 5–7-diene in the B ring of 7-dehydrocholesterol, causing ring breakage to form pre-vitamin D3 (9,10-secosterol), which is thermodynamically unstable and rearranges to cholecalciferol – the more stable vitamin D3 structure. Two more steps of oxidation lead to the vitamin D hormonally active form – calcitriol [1]. Calcitriol (1,25-dihydroxyvitamin D3) regulates various physiological processes directly, through vitamin D receptors (VDRs), or indirectly through crosstalk between proteins of signaling cascades. It is an essential factor for homeostasis of calcium and phosphorus and it has numerous biological functions, including modulation of cell proliferation, differentiation and apoptosis, neuromuscular, hormone and immune function, as well as other physiological processes [1–3]. The diverse functionalities offer a wide variety of clinical applications for vitamin D and its analogs in various diseases such as dermatological diseases, cancer, osteoporosis, inflammation and autoimmune diseases. Vitamin D deficiency in the body is of great importance for health in children as Corresponding author:. Ben-Shabat, S. ([email protected])
well as adults and elderly people [1,2]. It can lead to various malignancies of the large bowel, prostate and breasts and to diabetes mellitus type 2. The purpose of this review is to provide an overview on various physiological and pharmacological activities of calcitriol and its newly discovered synthetic analogs, and to discuss the mechanisms of their actions and possible methods for delivery and targeting.
Calcium and bone metabolism by vitamin D Calcitriol acts on various tissues and cells that are related or unrelated to homeostasis of calcium and phosphate. One of the most important roles of calcitriol is to maintain skeletal calcium balance [3–5]. Therefore, maintenance of normal serum calcium levels by vitamin D is essential to warrant the skeletal calcium balance and the optimal functioning of multiple vital processes. This constant maintenance activity occurs in several systems including the intestine (site of absorption), the kidney (site of secretion) and the skeleton, which is the largest repository of calcium in the body [4]. Calcium homeostasis is largely regulated through an integrated hormonal system and involves parathyroid hormone (PTH) [6], calcitonin and vitamin D [7], which are the primary regulators. Other hormones such as estrogen, testosterone, steroid hormones and glucagon, and minerals such as serum ionized calcium, magnesium and phosphate, as well as hormone
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1359-6446/06/$ - see front matter ß 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.drudis.2014.06.008
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receptors and calcium-sensing receptors in the presence of hormones [8], also play a part. Vitamin D primarily regulates the transcellular pathway of intestinal calcium transport, consisting of calcium entry through an apical calcium transporter, followed by intracellular buffering and an energy-dependent calcium extrusion by the plasma membrane calcium ATPase [9]. Vitamin D indirectly maintains calcium and phosphate levels for bone formation [10]. Although it affects the skeleton mainly via its actions on the intestine and the regulation of mineral homeostasis, direct effects (e.g. osteoblast differentiation and bone matrix mineralization) have also been observed [3,5,11]. In addition, regulation of the osteoclast differentiation is performed by acting on the osteoblasts through increasing the numbers of osteoblastic cells and reducing the expression of the antiosteoclastic factor osteoprotegerin [12]. Vitamin D promotes the recruitment of osteoclast precursor cells to bone resorption sites by increasing osteoclast number and enabling proper functioning of PTH to maintain serum calcium levels [13]. The molecular actions of vitamin D in bone include stimulation of osteoclastogenesis via receptor activator of nuclear factor kB ligand (RANKL) upregulation and inhibition of mineralization [14]. Increased vitamin D production restores normal serum calcium levels in three different ways: (i) by activating the vitamin-Ddependent transport system in the small intestine, increasing the absorption of dietary calcium; (ii) by increasing the mobilization of calcium from bone into the circulation; and (iii) by increasing the reabsorption of calcium by the kidneys [3]. PTH is also required to increase calcium mobilization from bone and calcium reabsorption by the kidneys. However, PTH is not required for the effect of vitamin D on the intestinal absorption of calcium.
Skin diseases and vitamin D analogs Vitamin D has several important roles in the skin (Table 1). Many in vitro and in vivo studies demonstrate dose-dependent effects of vitamin D on cell proliferation and differentiation [15–17]. Although the mechanisms that mediate the antiproliferative and pro-differentiating effects of vitamin D analogs on keratinocytes are not completely understood, it is well known that these effects are at least in part genomic and mediated by VDRs (1,25-dihydroxyvitamin D3) [15]. Vitamin D can reduce the risk of skin infection through modulating the production of various antimicrobial peptides and the cytokine response [18,19]. It is possible that vitamin D could enhance T helper type 2 responses [20]. Vitamin D deficiency is related to a high number of skin disorders, including skin cancer, autoimmune skin disorders, photodermatoses, atopic dermatitis and psoriasis [21,22].
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Vitamin D and its analogs have already been successfully used in the therapy of atopic dermatitis, psoriasis, vitiligo, acne and rosacea [18]. Most of the studies are devoted to treatment of psoriasis [23–27]. The highly variable nature of psoriasis and its individual presentations in patients can make choosing the most appropriate treatment difficult. Currently available synthetic vitamin D analogs that have been found to be safe and effective in topical treatment of psoriasis include calcipotriol (or calcipotriene), maxacalcitol, tacalcitol and hexafluoro-1a,25-dihydroxyvitamin D3 [23–31]. According to the literature, the rate of treatment success with vitamin D and its analogs varies from 4% to 53% [28], and many of them have significant adverse effects [23]. Calcipotriol is considered as a highly effective topical agent for the treatment of hyperproliferative skin diseases, such as psoriasis [26]. Calcipotriol exerts only negligible systemic effects on calcium homeostasis, thus eliminating the risk of the main significant side effects of hypercalciuria, hypercalcemia and bone calcium mobilization [29]. The successful introduction of clacipotriol as a safe topical antipsoriatic drug was just a prerequisite to exploring ways to improve its skin penetrability and efficacy. For example, new prodrugs that combine calcipotriol and several polyunsaturated fatty acids (PUFAs) through an ester bond were synthesized and evaluated [23]. These conjugates were capable of enhancing the penetration of vitamin D into the skin as well as inhibiting proliferation of keratinocytes in cultures [23]. Another group of prodrugs is the bioprecursor-prodrugs (e.g. alfacalcidol and doxercalciferol) which utilize the metabolic pathways to convert the prodrugs to the known active vitamin D [30]. Considering that the etiology of psoriasis is unknown, it is difficult to explain the mechanisms of action of vitamin D and its analogs [31–33]. VDR expression on keratinocytes appears to be present only in proliferating cells, and consequently the basal keratinocyte is the main VDR-containing cell in the epidermis. Variable VDR expression based on the proliferating and differentiating state of the keratinocyte, as well as local cytokine-mediated interactions, could provide an explanation for vitamin D inhibitory effects in psoriatic skin and proliferative effects in normal skin [31] (Table 1). Activation of VDR by vitamin D and its analogs might initiate diverse signal transductions that can lead to the desired pharmacological effect. Significantly higher levels of thymic stromal lymphopoietin, thymus and activation-related chemokine and C–C chemokine receptor type 4 (CCR4) expression were observed in skin samples treated with vitamin D in comparison with untreated samples. By contrast, significantly lower levels of interleukin (IL)-12/23 (p40), IL-1a, IL-1b and tumor necrosis factor (TNF)a expression were observed in the vitamin-D-treated samples versus
TABLE 1
Pharmacological effects of vitamin D and its analogs in the skin Pharmacological effect
Cell line
Refs
Modulation of growth factor and cytokine synthesis and signaling
Bone cells and keratinocytes
[31]
Induction of thymic stromal lymphopoietin and cathelicidin in psoriatic skin
In vitro: keratinocytes cells
[32]
Increasing the synthesis of platelet-derived growth factor (PDGF) promoting wound healing, and tumor necrosis factor (TNF)a promoting keratinocyte differentiation
In vitro: keratinocytes cells
[35]
Decreased synthesis of interleukin (IL)-1a and IL-6, resulting in decreased inflammation
In vitro: keratinocytes cells
[33,36]
Reduction of the size of utricles (comedolytic activity)
In vivo: rhino mouse model of acne
[38]
2
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the untreated samples. Expression of cathelicidin was elevated in vitamin-D-treated samples [32]. Cathelicidin is overexpressed in psoriatic skin lesions; however, its role in this disease has not yet been revealed. Enhanced IL-1 family members were found in human keratinocytes in psoriatic skin, and were suppressed after treatment by vitamin D and its analogs [33] (Table 1). Recent investigations have shown that atopic dermatitis results from the interplay of epidermal barrier defects, immune dysfunction and environmental triggers [34]. As mentioned previously, vitamin D is able to enhance keratinocyte differentiation, as well as have either stimulatory or suppressive effects on keratinocyte growth that is concentration dependent [15]. Vitamin D can also increase synthesis of platelet-derived growth factor (PDGF) promoting wound healing, and TNFa promoting keratinocyte differentiation [35]. Decreased synthesis of IL-1a, IL-6 and RANTES (also known as chemokine ligand 5; CCL5) secondary to vitamin D has resulted in decreased inflammation in epidermal keratinocytes [36] (Table 1). The enzyme responsible for the initial hydroxylation of vitamin D to 25-hydroxyvitamin D [cytochrome P450 (CYP)27A1] as well as the enzyme responsible for the conversion of 25-hydroxyvitamin D into the active form 1,25-dihydroxyvitamin D3 (CYP27B1) are found in keratinocytes [15]. Vitamin D has also been demonstrated to have a beneficial effect on the permeability barrier in the epidermis [37]. The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3, and its 2-methylene-19-nor analogs were evaluated for their ability to reduce the size of utricles (comedolytic activity) in a rhino mouse model of acne; the vitamin and all the analogs tested increased the skin epidermal thickness [38].
Vitamin D and its analogs in cancer treatment and prevention The role of vitamin D and its analogs in cancer treatment and prevention was investigated in different research tracks including
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clinical and epidemiological studies. According to extensive epidemiological research reports [39,40], it was found that there is a clear association between various factors responsible for vitamin D levels in the body (e.g. geography and latitude, history of sun exposure, lifestyle) and increased morbidity from cancer. It was also found that vitamin D and its analogs inhibit proliferation of cancer cells derived from multiple tissues [41–57] (Table 2). The anticancer vitamin D analogs include new calcipotriol-derived compounds [41,43], diastereomeric and geometric analogs of calcipotriol [42], seocalcitiol [44], 20-hydroxyvitamin D2 analog [50], 5-butyloxazole unit analog [55] and additional analogs containing a structurally modified side chain. The dominant side effects of vitamin D, hypercalcemia and hypercalciuria, resulted in limiting the use of vitamin D in cancer treatment [58]. To overcome this problem, more than 3000 vitamin D analogs have been synthesized in recent years [59]. Some of these analogs have been found to be more effective and less toxic than vitamin D, justifying their development and introduction to the market [60]. BGP-13 and Q2 BGP-15, calcipotriol-derived vitamin D3 analogs, are good examples of more-effective and less-toxic analogs than vitamin D [41,43]. BGP-15 is a calcipotriol-based analog where the 24-OH has been substituted by a chloride atom on the side chain of the calcipotriol molecule. BGP-13 and BGP-15 have been shown to induce apoptosis and inhibit prostate and breast cancer growth in vitro and in mice in vivo [41,43]. Antitumor effects of vitamin D and its analogs were also demonstrated in humans, when they were used either alone or in combination with other chemotherapy drugs. For example, a Phase II clinical trial that was conducted in the USA has shown that the combination of vitamin D and docetaxel could induce more than a 50% decline in the prostate cancer tumor marker, prostate-specific antigen (PSA), and improve survival of prostate cancer patients [61]. Vitamin D and its analogs exert their effects through genomic and nongenomic pathways. They operate through pharmacologically
TABLE 2
Summary of studies showing inhibition of cancer cell proliferation by vitamin D and its analogs Cancer type Breast cancer
Cancerous cells MCF-7 MCF-7, ZR-75-1 MCF-7, MDA-MB-231, T47-D MCF-7, MDA-MB-231, MCF-10A
Animal model Adult female rats
Refs [41,43] [44] [45] [46]
Lung cancer
LLC-GFP H292
Mice
[47] [48]
Skin cancer
SCC
Bladder cancer
[49]
SKMEL-188, hamster AbC1 melanoma cells
Rats
[50]
T24, UMUC3
Mice, T24 xenograft model
[51]
Mice, xenograft model
[52]
Mice, EOG xenograft model
[53]
Ovarian cancer SCOV-3, OVCAR-3, OVCAR-8
Rats, syngeneic EOG model Prostate cancer
LNCaP LNCaP, Du-145
Colonic cancer
MC38, HT29
C4-2
Q8 Leukemia
[41,43,54] [55] Mice, xenograft model
[56] [57]
HT29
Mice, xenograft model
[41,43]
HL-60
–
[42]
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distinct nuclear-receptor-mediated and plasma-membrane-initiated mechanisms. Vitamin D interacts with the VDRs localized in the cell nucleus to generate genomic effects [62]. The expression of VDRs varies in different situations. VDR expression is low in normal cells in several tissues, increases with malignant transformation and declines with progressive tumor growth [62]. In virgin females, for example, VDR is expressed at low levels in epithelial, stromal and immune cells of the normal mammary gland [63]. It was found that the VDR is expressed in most breast cancer cell lines, carcinogeninduced rat mammary tumors and normal breast tissue, as well as in primary breast cancer tumors. Furthermore, increased retinoid X receptor (RXR) and VDR protein levels were found in breast cancer tissue [63]. It is well known that the RXR acts as a silent partner to Q3 VDRs with its only function to increase affinity of VDR and/or RXR to its DNA recognition site. This suggests that vitamin D analogs have the potential to be developed as anticancer therapeutic agents. Following VDR activation by vitamin D, more than 60 genes are activated and lead to pro-differentiating, antiproliferative and antimetastatic effects on cells. In addition, effects on cell cycle and angiogenesis (antiangiogenic) are also occurring [62]. Vitamin D and its analogs can suppress tumorigenesis of normal cells and disruption of VDR-regulated pathways that can predispose to transformation [64]. It is known that vitamin D and its analogs can induce inhibition of cancer cell growth by regulating cell cycle progression; they can induce arrest in the G1 phase of the cell cycle in numerous cell lines that are associated with the increased expression of cyclindependent kinase inhibitors such as p21 and p27 [65]. The treatment of LNCaP cells with the vitamin D analogs BGP-13 and BGP-15 increased the number of cells in G1 phase of the cell cycle and simultaneously and significantly decreased the number of cells in S phase, thus proliferation of the treated cells was inhibited [41]. Vitamin D and its analogs are able to induce apoptosis in many cancer cells [50,66]. The ability of vitamin D to induce apoptosis is cell specific and the molecular mechanisms of this activity are not yet fully understood. The most probable mechanism of apoptosis Q4 induction is the downregulation of the antiapoptotic Bcl-2 family Q5 of proteins. Vitamin D affects the level of proapoptotic (Bax, Bac) and/or antiapoptotic (Bcl-2, Bcl-XL) proteins, turning the balance toward apoptosis rather than cell survival. In addition, vitamin D is able to enhance TNFa through caspase-dependent and caspaseindependent mechanisms [67]. For example, it was shown that procaspase-3 cleavage did not occur in MCF-7 cells after treatment with vitamin D, whereas treatment with its analogs activated caspase-3 [41]. Another function of vitamin D is its action as a pro-oxidant in cancer cells [50]. It was found that vitamin D causes an increase in the overall cellular redox potential in the cell that could translate into modulation of redox-sensitive enzymes and transcription factors that finally regulate cell cycle progression, differentiation and apoptosis [50]. Vitamin D derivatives can influence cancer cell growth and viability through modulation of growth factor signaling. There are two types of signal transducers: positive and negative. Positive regulators of the cell cycle regulate the changes necessary for cell division, whereas negative regulators control the activity of the positive regulators [68,69]. Therefore, the functional balance between these two groups of regulators, along with apoptosis, is responsible for the maintenance of normal cell number in a particular tissue [69]. It is noteworthy that, in some cell types such as acute myeloid 4
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leukemia cells, the differentiation-inducing and antiproliferative effects of calcitriol are associated with antiapoptotic and antioxidant actions [70]. Further studies are required to clarify the molecular mechanisms of the anticancer activity of vitamin D and its various analogs. From the current literature on the subject, it appears that the compound is not suitable for use in cancer as a single or primary anticancer agent but as an adjuvant in combination chemotherapy, where it can aid the actions of other cytotoxic agents, particularly those of the alkylating agents (which are not cell cycle specific). Vitamin D activity is expressed also by causing tumor regression and inhibition of tumor growth, in addition to counteracting their mutagenic effect by inducing apoptotic cell death [69]. Some evidence suggests that this vitamin could be used in high doses as a prophylactic agent for prevention of colorectal, prostate and breast cancer development [69]. Only large-scale long-term research will reveal which vitamin D analogs, either alone or in combination with other drugs, could serve as effective therapeutic agents for cancer.
Antidiabetic activity of vitamin D and its analogs Type 1 diabetes is an autoimmune disease characterized by the immune-mediated destruction of insulin-producing b cells from islets of Langerhans in the pancreas. As mentioned above, vitamin D plays a vital part in the normal functioning of the immune system and its deficiency could lead to impaired functioning of the immune system. Epidemiological studies have shown a direct correlation between the increase in the prevalence of the disease and deficiency of vitamin D. It was shown that vitamin D mediates insulin secretion and glucose uptake [71], and also regulates insulin receptor gene expression. The ability of a potent vitamin D analog: 2a-methyl-19-nor-(20S)-1a,25-dihydroxyvitamin D3 (2AMD), to prevent type 1 diabetes was determined in an Ins2 / Q6 non-obese diabetic (NOD) model. 2AMD suppresses development of type 1 diabetes, preserves islet cells and has a significant impact on b cell survival and function [72]. Lifestyle factors leading to type 2 diabetes, including obesity, aging and lack of physical activity, can also cause vitamin D deficiency. A detailed mechanism of antidiabetic activity of vitamin D in type 2 diabetes is unclear. It is only known that vitamin D regulates direct and indirect action on insulin-producing cells in the pancreas. Vitamin D can directly influence the muscle and fat cells, and can improve insulin action through reducing insulin resistance as well as reducing inflammation which is commonly present in patients with type 2 diabetes [73]. A small-scale post hoc analysis of a bone study revealed that daily calcium (500 mg) and vitamin D (700 IU) supplementation for three years prevented a further rise in fasting blood glucose in a subgroup with impaired fasting blood glucose (100–125 mg/dl) at a baseline [74].
Vitamin D and its analogs in multiple sclerosis Various genetic and environmental risk factors appear to interact and contribute to multiple sclerosis (MS), an autoimmune disease initiated by autoreactive T cells that recognize central nervous system antigens. In genetics, several human leukocyte antigen alleles (more particularly HLA-DRB1*1501) could favor the disease whereas others could be protective. Some of the genes involved in vitamin D metabolism (e.g. CYP27B1) also have a significant role
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in MS [75]. Based on epidemiological studies, three environmental risk factors were identified: (i) previous Epstein–Barr virus infection; (ii) vitamin D insufficiency; (iii) and cigarette smoking. Some clinical findings strongly suggest that vitamin D status influences the relapse rate and radiological lesions in MS patients. It was suggested that changes in vitamin D serum concentrations are correlated with MS [76]. Although the results of adequately powered randomized clinical trials using vitamin D supplementation have not yet been reported, vitamin D is a promising candidate as modulator of disease activity in MS.
Delivery and targeting of vitamin D Most orally administered drugs gain access to the blood by direct absorption through the portal vein. However, lipophilic compounds can reach the systemic circulation via the intestinal lymphatic system, an alternative absorption pathway that involves association of the lipophilic drug with the lipoprotein chylomicron inside the enterocyte. The overall bioavailability of these molecules is the sum of the portion transported via the lymphatic system and the portion absorbed through the portal blood. It has been shown that collecting the lymph through a cannula implanted in the mesenteric lymph duct reduced the systemic bioavailability of vitamin D3 from 50% to 12.5% in rats, hence revealing that 75% of the absorbed vitamin D3 was associated with lymphatic transport, and 25% was absorbed directly to the blood [77]. This unique absorption pathway possesses several significant advantages, including improved bioavailability, avoidance of first-pass hepatic metabolism, improved plasma profile of the drug and site-targeting abilities. To be transported through the lymphatic route, the drug must be highly lipophilic; a log P value above 5 and triglyceride solubility above 50 mg/ml were suggested as the borderline physicochemical characteristics. Vitamin D3 is an extremely lipophilic molecule (log P value around 9) and hence the majority of its oral bioavailability is attributable to lymphatic transport [78]. As for the analogs, their transport following oral administration is dictated by their physicochemical characteristics; more-lipophilic analogs that maintain the lipophilic nature of the vitamin could
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be transported via this unique absorption pathway. However, the more-hydrophilic analogs might not be transported through the lymphatic system. By reaching the general circulation through lymphatic transport rather than the usual portal blood, very high drug concentrations can be achieved in the lymphatics, because the volume of distribution in the lymphatic system (prior to the general circulation) is very small. It was shown that targeting of 5% of an oral valproic acid prodrug dose to the lymphatic system led to 50-fold higher lymphatic drug levels than plasma concentrations, owing to the small volume of distribution in the lymph compared with the general circulation [79]. In light of the above mentioned abilities of vitamin D and its analogs to inhibit cancer metastases spread, and because the lymphatic system plays a central part in the metastatic spread of many cancers, the delivery and targeting of vitamin D and its analogs to the lymphatic system and the consequent high lymphatic drug levels could be an added value in preventing metastases spread through the lymph.
Concluding remarks and future perspectives The current collected data could suggest that vitamin D and its analogs have a promising therapeutic potential, especially for prevention and treatment of various skin diseases, cancer development, diabetes and MS. Additional information on vitamin D research is needed from an interdisciplinary perspective. There is a need for long-term clinical studies with relevant outcomes, including bone health, immune function, autoimmune disorders and chronic disease prevention. In addition, there is a need for more clarification of the side effects such as hypercalcemia and hypercal- Q7 ciuria. Further research to help understand the outcomes of vitamin D and its analogs is required in terms of metabolic partitioning and mobilization of key vitamin D metabolites to ensure safety for longterm vitamin D intake. There is still much that remains to be elucidated before the various mechanisms of therapeutic activity of vitamin D and its analogs can be thoroughly understood. More clinical studies are warranted to establish effectiveness in treatment of the aforementioned diseases.
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22 Reichrath, J. (2007) Vitamin D and the skin: an ancient friend, revisited. Exp. Dermatol. 16, 618–625 23 Ben-Shabat, S. et al. (2005) Vitamin D3-based conjugates for topical treatment of peoriasis: synthesis, antiproliferative activity, and cutaneous penetration studies. Pharm. Res. 22, 50–58 24 O’Neill, J.L. and Feldman, S.R. (2010) Vitamin D analogue-based therapies for psoriasis. Drugs Today 46, 351–360 25 Tre´mezaygues, L. and Reichrath, J. (2011) Vitamin D analogs in the treatment of psoriasis: where are we standing and where will we be going? Dermatoendocrinology 3, 180–186 26 McCormack, P.L. (2011) Calcipotriol/betamethasone dipropionate: a review of its use in the treatment of psoriasis vulgaris of the trunk, limbs and scalp. Drugs 16, 709–730 27 Oquendo, M. et al. (2012) Topical vitamin D analogs available to treat psoriasis. Skinmed 10, 301–304 28 Devaux, S. et al. (2012) Adherence to topical treatment in psoriasis: a systematic literature review. J. Eur. Acad. Dermatol. Venereol. 3, 61–67 29 Binderup, L. and Bramm, E. (1988) Effects of a novel vitamin D analogue MC903 on cell proliferation and differentiation in vitro and on calcium metabolism in vivo. Biochem. Pharmacol. 37, 889–895 30 Kubodera, N. (2009) A new look at the most successful prodrugs for active vitamin D (D hormone): alfacalcidol and doxercalciferol. Molecules 14, 3869–3880 31 Gurlek, A. et al. (2002) Modulation of growth factor/cytokine synthesis and signaling by 1alpha, 25-dihydroxyvitamin D(3): implications in cell growth and differentiation. Endocr. Rev. 23, 763–786 32 Sato-Deguchi, E. et al. (2012) Topical vitamin D3 analogues induce thymic stromal lymphopoietin and cathelicidin in psoriatic skin lesions. Br. J. Dermatol. 167, 77–84 33 Balato, A. et al. (2013) Interleukin-1 family members are enhanced in psoriasis and suppressed by vitamin D and retinoic acid. Arch. Derm. Res. 305, 255–262 34 Paller, A.S. (2012) Latest approaches to treating atopic dermatitis. Chem. Immunol. Allerg. 96, 132–140 35 Geilen, C.C. et al. (1997) 1a,25-Dihydroxyvitamin D3 induces sphingomyelin hydrolysis in HaCaT cells via tumor necrosis factor alpha. J. Biol. Chem. 272, 8997– 9001 36 Fujita, H. et al. (2007) The direct action of 1alpha, 25(OH)2-vitamin D3 on purified mouse Langerhans cells. Cell Immunol. 245, 70–79 37 Bickle, D.D. et al. (2010) Differential regulation of epidermal function by VDR coactivators. J. Steroid Biochem. Mol. Biol. 121, 308–313 38 Nieves, N.J. et al. (2010) Identification of a unique subset of 2-methylene-19-nor analogs of vitamin D with comedolytic activity in the rhino mouse. J. Invest. Derm. 130, 2359–2367 39 Freedman, D.M. et al. (2007) Prospective study of serum vitamin D and cancer mortality in the United States. J. Natl. Cancer Inst. 99, 1594–1602 40 Shao, T. et al. (2012) Vitamin D and breast cancer. Oncologist 17, 36–45 41 Berkovich, L. et al. (2010) Induction of apoptosis and inhibition of prostate and breast cancer growth by BGP-15, a new calcipotriene-derived vitamin D3 analog. Anticancer Drugs 21, 609–618 42 Milczarek, M. et al. (2013) Synthesis and biological activity of diastereomeric and geometric analogs of calcipotriol, PRI-2202 and PRI-2205, against human HL-60 leukemia and MCF-7 breast cancer cells. Cancers (Basel) 5, 1355–1378 43 Berkovich, L. et al. (2013) Inhibition of cancer growth and induction of apoptosis by BGP-13 and BGP-15, new calcipotriene-derived vitamin D3 analogs, in vitro and in vivo studies. Invest. New Drugs 31, 247–255 44 Vink-van Wijngaarden, T. et al. (1996) Inhibition of insulin and insulin-like growth factor-I-stimulated growth of human breast cancer cells by 1,25-dihydroxyvitamin D3 and the vitamin D3 analogue EB1089. Eur. J. Cancer 5, 842–848 45 Wang, Q. et al. (2000) 1,25-Dihydroxyvitamin D3 and all-trans-retinoic acid sensitize breast cancer cells to chemotherapy-induced cell death. Cancer Res. 60, 2040–2048 46 Richard, C.L. et al. (2010) Involvement of 1,25D3-MARRS (membrane associated, rapid response steroid-binding), a novel vitamin D receptor, in growth inhibition of breast cancer cells. Exp. Cell Res. 316, 695–703 47 Nakagawa, K. et al. (2005) 1a,25-Dihydroxyvitamin D(3) is a preventive factor in the metastasis of lung cancer. Carcinogenesis 26, 429–440 48 Zhang, Q. et al. (2012) CYP24 inhibition preserves 1a,25-dihydroxyvitamin D(3) anti-proliferative signaling in lung cancer cells. Mol. Cell Endocrinol. 355, 153–161 49 Ma, Y. et al. (2008) 1a,25-Dihydroxyvitamin D3 potentiates cisplatin antitumor activity by p73 induction in a squamous cell carcinoma model. Mol. Cancer Ther. 7, 3047–3055 50 Slominski, A.Y. et al. (2011) 20-Hydroxyvitamin D2 is a noncalcemic analog of vitamin D with potent antiproliferative and prodifferentiation activities in normal and malignant cells. Am. J. Physiol. Cell Physiol. 300, 526–541
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51 Ma, Y. et al. (2010) 1,25D3 enhances antitumor activity of gemcitabine and cisplatin in human bladder cancer models. Cancer 116, 3294–3303 52 Zhang, X. et al. (2006) Vitamin D receptor is a novel drug target for ovarian cancer treatment. Curr. Cancer Drug Targets 3, 229–244 53 Moore, R.G. et al. (2012) Efficacy of a non-hypercalcemic vitamin-D2 derived anticancer agent (MT19c) and inhibition of fatty acid synthesis in an ovarian cancer xenograft model. PloS One 7, 3143–3150 54 Wang, W.L. et al. (2012) 1,25-Dihydroxyvitamin D(3) modulates lipid metabolism in prostate cancer cells through miRNA mediated regulation of PPARA. J. Steroid Biochem. Mol. Biol. 136, 247–251 55 Stio, M. et al. (2011) The novel vitamin D analog ZK191784 inhibits prostate cancer cell invasion. Anticancer Res. 31, 4091–4098 56 Bhatia, V. et al. (2009) EB1089 inhibits the parathyroid hormone-related proteinenhanced bone metastasis and xenograft growth of human prostate cancer cells. Mol. Cancer Ther. 8, 1787–1798 57 Milczarek, M. et al. (2013) Combined colonic cancer treatment with vitamin D analogs and irinotecan or oxaliplatin. Anticancer Res. 33, 433–444 58 Jones, G. (2008) Pharmacokinetics of vitamin D toxicity. Am. J. Clin. Nutr. 88, 582– 586S 59 Eduardo-Canosa, S. et al. (2010) Design and synthesis of active vitamin D analogs. J. Steroid Biochem. Mol. Biol. 12, 7–12 60 Cheung, F.S. et al. (2012) Current progress in using vitamin D and its analogs for cancer prevention and treatment. Expert Rev. Anticancer Ther. 6, 811–837 61 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. 6, 669–674 62 Kim, S.H. et al. (2012) Characterization of vitamin D receptor (VDR) in lung adenocarcinoma. Lung Cancer 77, 265–271 63 Zinser, G.M. and Welsh, J. (2004) Vitamin D receptor status alters mammary gland morphology and tumorigenesis in MMTV-neu mice. Carcinogenesis 12, 2361–2372 64 Kemmis, C.M. and Welsh, J. (2008) Mammary epithelial cell transformation is associated with deregulation of the vitamin D pathway. J. Cell Biochem. 105, 980– 988 65 Nemazannikova, N. et al. (2013) Role of vitamin D metabolism in cutaneous tumour formation and progression. J. Pharm. Pharmacol. 65, 2–10 66 Matthews, D. et al. (2010) Genomic vitamin D signaling in breast cancer: insights from animal models and human cells. J. Steroid Biochem. Mol. Biol. 121, 362–367 67 Narawaez, C.J. and Welsh, J. (2001) Role of mitochondria and caspases in vitamin Dmediated apoptosis of MCF-7 breast cancer cells. J. Biol. Chem. 276, 9101–9107 68 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 69 Chakraboiti, C.K. (2011) Vitamin D as a promising anticancer agent. Indian J. Pharmacol. 43, 113–120 70 Danilenko, M. et al. (2003) Carnosic acid potentiates the antioxidant and prodifferentiation effects of 1alpha, 25-dihydroxyvitamin D3 in leukemia cells but does not promote elevation of basal levels of intracellular calcium. Cancer Res. 63, 1325–1332 71 Manna, P. and Jain, S.K. (2012) Vitamin D up-regulates glucose transporter 4 (GLUT4) translocation and glucose utilization mediated by cystathionine-g-lyase (CSE) activation and H2S formation in 3T3L1 adipocytes. J. Biol. Chem. 287, 42324– 42332 72 Kiekhaefer, C.M. et al. (2011) 2a-Methyl-19-nor-(20S)-1,25-dihydroxyvitamin D(3) protects the insulin 2 knockout non-obese diabetic mouse from developing type 1 diabetes without hypercalcaemia. Clin. Exp. Immunol. 166, 325–332 73 Holick, M.F. (2004) Vitamin D: importance in the prevention of cancers, type 1 diabetes, heart disease, and osteoporosis. Am. J. Clin. Nutr. 79, 362–371 74 Pittas, A.G. et al. (2007) The effects of calcium and vitamin D supplementation on blood glucose and markers of inflammation in nondiabetic adults. Diabetes Care 30, 980–986 75 Pozuelo-Moyano, B. and Benito-Leon, J. (2013) Vitamin D and multiple sclerosis. Rev. Neurol. 56, 243–251 76 Slavov, G.S. et al. (2013) Vitamin D immunomodulatory potential in multiple sclerosis. Folia Med. (Plovdiv) 55, 5–9 77 Dahan, A. and Hoffman, A. (2005) Evaluation of a chylomicron flow blocking approach to investigate the intestinal lymphatic transport of lipophilic drugs. Eur. J. Pharm. Sci. 24, 381–388 78 Dahan, A. and Hoffman, A. (2006) Use of a dynamic in vitro lipolysis model to rationalize oral formulation development for poor water soluble drugs: correlation with in vivo data and the relationship to intra-enterocyte processes in rats. Pharm. Res. 23, 2165–2174 79 Dahan, A. et al. (2008) The oral absorption of phospholipid prodrugs: in vivo and in vitro mechanistic investigation of trafficking of a lecithin-valproic acid conjugate following oral administration. J. Control. Release 126, 1–9
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Pharmacological effects of vitamin D and its analogs: recent developments Q1
Amnon C. Sintov1, Ludmilla Yarmolinsky2, Arik Dahan2 and Shimon Ben-Shabat2 1 2
Department of Biomedical Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel Department of Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel
Calcitriol, the hormonally active form of vitamin D, is well known for its diverse pharmacological activities, including modulation of cell growth, neuromuscular and immune function and reduction of inflammation. Calcitriol and its analogs exert potent effects on cellular differentiation and proliferation, regulate apoptosis and produce immunomodulatory effects. The purpose of this review is to provide information on various physiological and pharmacological activities of calcitriol and its newly discovered analogs. Special emphasis is given to skin diseases, cancer, diabetes and multiple sclerosis. A discussion is raised on the mechanisms of action of calcitriol and its analogs in various diseases, as well as on possible methods of delivery and targeting. Introduction Vitamin D includes several forms (vitamers) that are important from a physiological standpoint. Animals synthesize 7-dehydrocholesterol, the immediate precursor of cholesterol. Absorption of ultraviolet B radiation (290–315 nm) leads to a rearrangement of the 5–7-diene in the B ring of 7-dehydrocholesterol, causing ring breakage to form pre-vitamin D3 (9,10-secosterol), which is thermodynamically unstable and rearranges to cholecalciferol – the more stable vitamin D3 structure. Two more steps of oxidation lead to the vitamin D hormonally active form – calcitriol [1]. Calcitriol (1,25-dihydroxyvitamin D3) regulates various physiological processes directly, through vitamin D receptors (VDRs), or indirectly through crosstalk between proteins of signaling cascades. It is an essential factor for homeostasis of calcium and phosphorus and it has numerous biological functions, including modulation of cell proliferation, differentiation and apoptosis, neuromuscular, hormone and immune function, as well as other physiological processes [1–3]. The diverse functionalities offer a wide variety of clinical applications for vitamin D and its analogs in various diseases such as dermatological diseases, cancer, osteoporosis, inflammation and autoimmune diseases. Vitamin D deficiency in the body is of great importance for health in children as Corresponding author:. Ben-Shabat, S. ([email protected])
well as adults and elderly people [1,2]. It can lead to various malignancies of the large bowel, prostate and breasts and to diabetes mellitus type 2. The purpose of this review is to provide an overview on various physiological and pharmacological activities of calcitriol and its newly discovered synthetic analogs, and to discuss the mechanisms of their actions and possible methods for delivery and targeting.
Calcium and bone metabolism by vitamin D Calcitriol acts on various tissues and cells that are related or unrelated to homeostasis of calcium and phosphate. One of the most important roles of calcitriol is to maintain skeletal calcium balance [3–5]. Therefore, maintenance of normal serum calcium levels by vitamin D is essential to warrant the skeletal calcium balance and the optimal functioning of multiple vital processes. This constant maintenance activity occurs in several systems including the intestine (site of absorption), the kidney (site of secretion) and the skeleton, which is the largest repository of calcium in the body [4]. Calcium homeostasis is largely regulated through an integrated hormonal system and involves parathyroid hormone (PTH) [6], calcitonin and vitamin D [7], which are the primary regulators. Other hormones such as estrogen, testosterone, steroid hormones and glucagon, and minerals such as serum ionized calcium, magnesium and phosphate, as well as hormone
www.drugdiscoverytoday.com 1 Please cite this article in press as: A.C. Sintov, et al., Pharmacological effects of vitamin D and its analogs: recent developments, Drug Discov Today (2014), http://dx.doi.org/10.1016/ j.drudis.2014.06.008
1359-6446/06/$ - see front matter ß 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.drudis.2014.06.008
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receptors and calcium-sensing receptors in the presence of hormones [8], also play a part. Vitamin D primarily regulates the transcellular pathway of intestinal calcium transport, consisting of calcium entry through an apical calcium transporter, followed by intracellular buffering and an energy-dependent calcium extrusion by the plasma membrane calcium ATPase [9]. Vitamin D indirectly maintains calcium and phosphate levels for bone formation [10]. Although it affects the skeleton mainly via its actions on the intestine and the regulation of mineral homeostasis, direct effects (e.g. osteoblast differentiation and bone matrix mineralization) have also been observed [3,5,11]. In addition, regulation of the osteoclast differentiation is performed by acting on the osteoblasts through increasing the numbers of osteoblastic cells and reducing the expression of the antiosteoclastic factor osteoprotegerin [12]. Vitamin D promotes the recruitment of osteoclast precursor cells to bone resorption sites by increasing osteoclast number and enabling proper functioning of PTH to maintain serum calcium levels [13]. The molecular actions of vitamin D in bone include stimulation of osteoclastogenesis via receptor activator of nuclear factor kB ligand (RANKL) upregulation and inhibition of mineralization [14]. Increased vitamin D production restores normal serum calcium levels in three different ways: (i) by activating the vitamin-Ddependent transport system in the small intestine, increasing the absorption of dietary calcium; (ii) by increasing the mobilization of calcium from bone into the circulation; and (iii) by increasing the reabsorption of calcium by the kidneys [3]. PTH is also required to increase calcium mobilization from bone and calcium reabsorption by the kidneys. However, PTH is not required for the effect of vitamin D on the intestinal absorption of calcium.
Skin diseases and vitamin D analogs Vitamin D has several important roles in the skin (Table 1). Many in vitro and in vivo studies demonstrate dose-dependent effects of vitamin D on cell proliferation and differentiation [15–17]. Although the mechanisms that mediate the antiproliferative and pro-differentiating effects of vitamin D analogs on keratinocytes are not completely understood, it is well known that these effects are at least in part genomic and mediated by VDRs (1,25-dihydroxyvitamin D3) [15]. Vitamin D can reduce the risk of skin infection through modulating the production of various antimicrobial peptides and the cytokine response [18,19]. It is possible that vitamin D could enhance T helper type 2 responses [20]. Vitamin D deficiency is related to a high number of skin disorders, including skin cancer, autoimmune skin disorders, photodermatoses, atopic dermatitis and psoriasis [21,22].
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Vitamin D and its analogs have already been successfully used in the therapy of atopic dermatitis, psoriasis, vitiligo, acne and rosacea [18]. Most of the studies are devoted to treatment of psoriasis [23–27]. The highly variable nature of psoriasis and its individual presentations in patients can make choosing the most appropriate treatment difficult. Currently available synthetic vitamin D analogs that have been found to be safe and effective in topical treatment of psoriasis include calcipotriol (or calcipotriene), maxacalcitol, tacalcitol and hexafluoro-1a,25-dihydroxyvitamin D3 [23–31]. According to the literature, the rate of treatment success with vitamin D and its analogs varies from 4% to 53% [28], and many of them have significant adverse effects [23]. Calcipotriol is considered as a highly effective topical agent for the treatment of hyperproliferative skin diseases, such as psoriasis [26]. Calcipotriol exerts only negligible systemic effects on calcium homeostasis, thus eliminating the risk of the main significant side effects of hypercalciuria, hypercalcemia and bone calcium mobilization [29]. The successful introduction of clacipotriol as a safe topical antipsoriatic drug was just a prerequisite to exploring ways to improve its skin penetrability and efficacy. For example, new prodrugs that combine calcipotriol and several polyunsaturated fatty acids (PUFAs) through an ester bond were synthesized and evaluated [23]. These conjugates were capable of enhancing the penetration of vitamin D into the skin as well as inhibiting proliferation of keratinocytes in cultures [23]. Another group of prodrugs is the bioprecursor-prodrugs (e.g. alfacalcidol and doxercalciferol) which utilize the metabolic pathways to convert the prodrugs to the known active vitamin D [30]. Considering that the etiology of psoriasis is unknown, it is difficult to explain the mechanisms of action of vitamin D and its analogs [31–33]. VDR expression on keratinocytes appears to be present only in proliferating cells, and consequently the basal keratinocyte is the main VDR-containing cell in the epidermis. Variable VDR expression based on the proliferating and differentiating state of the keratinocyte, as well as local cytokine-mediated interactions, could provide an explanation for vitamin D inhibitory effects in psoriatic skin and proliferative effects in normal skin [31] (Table 1). Activation of VDR by vitamin D and its analogs might initiate diverse signal transductions that can lead to the desired pharmacological effect. Significantly higher levels of thymic stromal lymphopoietin, thymus and activation-related chemokine and C–C chemokine receptor type 4 (CCR4) expression were observed in skin samples treated with vitamin D in comparison with untreated samples. By contrast, significantly lower levels of interleukin (IL)-12/23 (p40), IL-1a, IL-1b and tumor necrosis factor (TNF)a expression were observed in the vitamin-D-treated samples versus
TABLE 1
Pharmacological effects of vitamin D and its analogs in the skin Pharmacological effect
Cell line
Refs
Modulation of growth factor and cytokine synthesis and signaling
Bone cells and keratinocytes
[31]
Induction of thymic stromal lymphopoietin and cathelicidin in psoriatic skin
In vitro: keratinocytes cells
[32]
Increasing the synthesis of platelet-derived growth factor (PDGF) promoting wound healing, and tumor necrosis factor (TNF)a promoting keratinocyte differentiation
In vitro: keratinocytes cells
[35]
Decreased synthesis of interleukin (IL)-1a and IL-6, resulting in decreased inflammation
In vitro: keratinocytes cells
[33,36]
Reduction of the size of utricles (comedolytic activity)
In vivo: rhino mouse model of acne
[38]
2
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the untreated samples. Expression of cathelicidin was elevated in vitamin-D-treated samples [32]. Cathelicidin is overexpressed in psoriatic skin lesions; however, its role in this disease has not yet been revealed. Enhanced IL-1 family members were found in human keratinocytes in psoriatic skin, and were suppressed after treatment by vitamin D and its analogs [33] (Table 1). Recent investigations have shown that atopic dermatitis results from the interplay of epidermal barrier defects, immune dysfunction and environmental triggers [34]. As mentioned previously, vitamin D is able to enhance keratinocyte differentiation, as well as have either stimulatory or suppressive effects on keratinocyte growth that is concentration dependent [15]. Vitamin D can also increase synthesis of platelet-derived growth factor (PDGF) promoting wound healing, and TNFa promoting keratinocyte differentiation [35]. Decreased synthesis of IL-1a, IL-6 and RANTES (also known as chemokine ligand 5; CCL5) secondary to vitamin D has resulted in decreased inflammation in epidermal keratinocytes [36] (Table 1). The enzyme responsible for the initial hydroxylation of vitamin D to 25-hydroxyvitamin D [cytochrome P450 (CYP)27A1] as well as the enzyme responsible for the conversion of 25-hydroxyvitamin D into the active form 1,25-dihydroxyvitamin D3 (CYP27B1) are found in keratinocytes [15]. Vitamin D has also been demonstrated to have a beneficial effect on the permeability barrier in the epidermis [37]. The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3, and its 2-methylene-19-nor analogs were evaluated for their ability to reduce the size of utricles (comedolytic activity) in a rhino mouse model of acne; the vitamin and all the analogs tested increased the skin epidermal thickness [38].
Vitamin D and its analogs in cancer treatment and prevention The role of vitamin D and its analogs in cancer treatment and prevention was investigated in different research tracks including
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clinical and epidemiological studies. According to extensive epidemiological research reports [39,40], it was found that there is a clear association between various factors responsible for vitamin D levels in the body (e.g. geography and latitude, history of sun exposure, lifestyle) and increased morbidity from cancer. It was also found that vitamin D and its analogs inhibit proliferation of cancer cells derived from multiple tissues [41–57] (Table 2). The anticancer vitamin D analogs include new calcipotriol-derived compounds [41,43], diastereomeric and geometric analogs of calcipotriol [42], seocalcitiol [44], 20-hydroxyvitamin D2 analog [50], 5-butyloxazole unit analog [55] and additional analogs containing a structurally modified side chain. The dominant side effects of vitamin D, hypercalcemia and hypercalciuria, resulted in limiting the use of vitamin D in cancer treatment [58]. To overcome this problem, more than 3000 vitamin D analogs have been synthesized in recent years [59]. Some of these analogs have been found to be more effective and less toxic than vitamin D, justifying their development and introduction to the market [60]. BGP-13 and Q2 BGP-15, calcipotriol-derived vitamin D3 analogs, are good examples of more-effective and less-toxic analogs than vitamin D [41,43]. BGP-15 is a calcipotriol-based analog where the 24-OH has been substituted by a chloride atom on the side chain of the calcipotriol molecule. BGP-13 and BGP-15 have been shown to induce apoptosis and inhibit prostate and breast cancer growth in vitro and in mice in vivo [41,43]. Antitumor effects of vitamin D and its analogs were also demonstrated in humans, when they were used either alone or in combination with other chemotherapy drugs. For example, a Phase II clinical trial that was conducted in the USA has shown that the combination of vitamin D and docetaxel could induce more than a 50% decline in the prostate cancer tumor marker, prostate-specific antigen (PSA), and improve survival of prostate cancer patients [61]. Vitamin D and its analogs exert their effects through genomic and nongenomic pathways. They operate through pharmacologically
TABLE 2
Summary of studies showing inhibition of cancer cell proliferation by vitamin D and its analogs Cancer type Breast cancer
Cancerous cells MCF-7 MCF-7, ZR-75-1 MCF-7, MDA-MB-231, T47-D MCF-7, MDA-MB-231, MCF-10A
Animal model Adult female rats
Refs [41,43] [44] [45] [46]
Lung cancer
LLC-GFP H292
Mice
[47] [48]
Skin cancer
SCC
Bladder cancer
[49]
SKMEL-188, hamster AbC1 melanoma cells
Rats
[50]
T24, UMUC3
Mice, T24 xenograft model
[51]
Mice, xenograft model
[52]
Mice, EOG xenograft model
[53]
Ovarian cancer SCOV-3, OVCAR-3, OVCAR-8
Rats, syngeneic EOG model Prostate cancer
LNCaP LNCaP, Du-145
Colonic cancer
MC38, HT29
C4-2
Q8 Leukemia
[41,43,54] [55] Mice, xenograft model
[56] [57]
HT29
Mice, xenograft model
[41,43]
HL-60
–
[42]
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distinct nuclear-receptor-mediated and plasma-membrane-initiated mechanisms. Vitamin D interacts with the VDRs localized in the cell nucleus to generate genomic effects [62]. The expression of VDRs varies in different situations. VDR expression is low in normal cells in several tissues, increases with malignant transformation and declines with progressive tumor growth [62]. In virgin females, for example, VDR is expressed at low levels in epithelial, stromal and immune cells of the normal mammary gland [63]. It was found that the VDR is expressed in most breast cancer cell lines, carcinogeninduced rat mammary tumors and normal breast tissue, as well as in primary breast cancer tumors. Furthermore, increased retinoid X receptor (RXR) and VDR protein levels were found in breast cancer tissue [63]. It is well known that the RXR acts as a silent partner to Q3 VDRs with its only function to increase affinity of VDR and/or RXR to its DNA recognition site. This suggests that vitamin D analogs have the potential to be developed as anticancer therapeutic agents. Following VDR activation by vitamin D, more than 60 genes are activated and lead to pro-differentiating, antiproliferative and antimetastatic effects on cells. In addition, effects on cell cycle and angiogenesis (antiangiogenic) are also occurring [62]. Vitamin D and its analogs can suppress tumorigenesis of normal cells and disruption of VDR-regulated pathways that can predispose to transformation [64]. It is known that vitamin D and its analogs can induce inhibition of cancer cell growth by regulating cell cycle progression; they can induce arrest in the G1 phase of the cell cycle in numerous cell lines that are associated with the increased expression of cyclindependent kinase inhibitors such as p21 and p27 [65]. The treatment of LNCaP cells with the vitamin D analogs BGP-13 and BGP-15 increased the number of cells in G1 phase of the cell cycle and simultaneously and significantly decreased the number of cells in S phase, thus proliferation of the treated cells was inhibited [41]. Vitamin D and its analogs are able to induce apoptosis in many cancer cells [50,66]. The ability of vitamin D to induce apoptosis is cell specific and the molecular mechanisms of this activity are not yet fully understood. The most probable mechanism of apoptosis Q4 induction is the downregulation of the antiapoptotic Bcl-2 family Q5 of proteins. Vitamin D affects the level of proapoptotic (Bax, Bac) and/or antiapoptotic (Bcl-2, Bcl-XL) proteins, turning the balance toward apoptosis rather than cell survival. In addition, vitamin D is able to enhance TNFa through caspase-dependent and caspaseindependent mechanisms [67]. For example, it was shown that procaspase-3 cleavage did not occur in MCF-7 cells after treatment with vitamin D, whereas treatment with its analogs activated caspase-3 [41]. Another function of vitamin D is its action as a pro-oxidant in cancer cells [50]. It was found that vitamin D causes an increase in the overall cellular redox potential in the cell that could translate into modulation of redox-sensitive enzymes and transcription factors that finally regulate cell cycle progression, differentiation and apoptosis [50]. Vitamin D derivatives can influence cancer cell growth and viability through modulation of growth factor signaling. There are two types of signal transducers: positive and negative. Positive regulators of the cell cycle regulate the changes necessary for cell division, whereas negative regulators control the activity of the positive regulators [68,69]. Therefore, the functional balance between these two groups of regulators, along with apoptosis, is responsible for the maintenance of normal cell number in a particular tissue [69]. It is noteworthy that, in some cell types such as acute myeloid 4
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leukemia cells, the differentiation-inducing and antiproliferative effects of calcitriol are associated with antiapoptotic and antioxidant actions [70]. Further studies are required to clarify the molecular mechanisms of the anticancer activity of vitamin D and its various analogs. From the current literature on the subject, it appears that the compound is not suitable for use in cancer as a single or primary anticancer agent but as an adjuvant in combination chemotherapy, where it can aid the actions of other cytotoxic agents, particularly those of the alkylating agents (which are not cell cycle specific). Vitamin D activity is expressed also by causing tumor regression and inhibition of tumor growth, in addition to counteracting their mutagenic effect by inducing apoptotic cell death [69]. Some evidence suggests that this vitamin could be used in high doses as a prophylactic agent for prevention of colorectal, prostate and breast cancer development [69]. Only large-scale long-term research will reveal which vitamin D analogs, either alone or in combination with other drugs, could serve as effective therapeutic agents for cancer.
Antidiabetic activity of vitamin D and its analogs Type 1 diabetes is an autoimmune disease characterized by the immune-mediated destruction of insulin-producing b cells from islets of Langerhans in the pancreas. As mentioned above, vitamin D plays a vital part in the normal functioning of the immune system and its deficiency could lead to impaired functioning of the immune system. Epidemiological studies have shown a direct correlation between the increase in the prevalence of the disease and deficiency of vitamin D. It was shown that vitamin D mediates insulin secretion and glucose uptake [71], and also regulates insulin receptor gene expression. The ability of a potent vitamin D analog: 2a-methyl-19-nor-(20S)-1a,25-dihydroxyvitamin D3 (2AMD), to prevent type 1 diabetes was determined in an Ins2 / Q6 non-obese diabetic (NOD) model. 2AMD suppresses development of type 1 diabetes, preserves islet cells and has a significant impact on b cell survival and function [72]. Lifestyle factors leading to type 2 diabetes, including obesity, aging and lack of physical activity, can also cause vitamin D deficiency. A detailed mechanism of antidiabetic activity of vitamin D in type 2 diabetes is unclear. It is only known that vitamin D regulates direct and indirect action on insulin-producing cells in the pancreas. Vitamin D can directly influence the muscle and fat cells, and can improve insulin action through reducing insulin resistance as well as reducing inflammation which is commonly present in patients with type 2 diabetes [73]. A small-scale post hoc analysis of a bone study revealed that daily calcium (500 mg) and vitamin D (700 IU) supplementation for three years prevented a further rise in fasting blood glucose in a subgroup with impaired fasting blood glucose (100–125 mg/dl) at a baseline [74].
Vitamin D and its analogs in multiple sclerosis Various genetic and environmental risk factors appear to interact and contribute to multiple sclerosis (MS), an autoimmune disease initiated by autoreactive T cells that recognize central nervous system antigens. In genetics, several human leukocyte antigen alleles (more particularly HLA-DRB1*1501) could favor the disease whereas others could be protective. Some of the genes involved in vitamin D metabolism (e.g. CYP27B1) also have a significant role
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in MS [75]. Based on epidemiological studies, three environmental risk factors were identified: (i) previous Epstein–Barr virus infection; (ii) vitamin D insufficiency; (iii) and cigarette smoking. Some clinical findings strongly suggest that vitamin D status influences the relapse rate and radiological lesions in MS patients. It was suggested that changes in vitamin D serum concentrations are correlated with MS [76]. Although the results of adequately powered randomized clinical trials using vitamin D supplementation have not yet been reported, vitamin D is a promising candidate as modulator of disease activity in MS.
Delivery and targeting of vitamin D Most orally administered drugs gain access to the blood by direct absorption through the portal vein. However, lipophilic compounds can reach the systemic circulation via the intestinal lymphatic system, an alternative absorption pathway that involves association of the lipophilic drug with the lipoprotein chylomicron inside the enterocyte. The overall bioavailability of these molecules is the sum of the portion transported via the lymphatic system and the portion absorbed through the portal blood. It has been shown that collecting the lymph through a cannula implanted in the mesenteric lymph duct reduced the systemic bioavailability of vitamin D3 from 50% to 12.5% in rats, hence revealing that 75% of the absorbed vitamin D3 was associated with lymphatic transport, and 25% was absorbed directly to the blood [77]. This unique absorption pathway possesses several significant advantages, including improved bioavailability, avoidance of first-pass hepatic metabolism, improved plasma profile of the drug and site-targeting abilities. To be transported through the lymphatic route, the drug must be highly lipophilic; a log P value above 5 and triglyceride solubility above 50 mg/ml were suggested as the borderline physicochemical characteristics. Vitamin D3 is an extremely lipophilic molecule (log P value around 9) and hence the majority of its oral bioavailability is attributable to lymphatic transport [78]. As for the analogs, their transport following oral administration is dictated by their physicochemical characteristics; more-lipophilic analogs that maintain the lipophilic nature of the vitamin could
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be transported via this unique absorption pathway. However, the more-hydrophilic analogs might not be transported through the lymphatic system. By reaching the general circulation through lymphatic transport rather than the usual portal blood, very high drug concentrations can be achieved in the lymphatics, because the volume of distribution in the lymphatic system (prior to the general circulation) is very small. It was shown that targeting of 5% of an oral valproic acid prodrug dose to the lymphatic system led to 50-fold higher lymphatic drug levels than plasma concentrations, owing to the small volume of distribution in the lymph compared with the general circulation [79]. In light of the above mentioned abilities of vitamin D and its analogs to inhibit cancer metastases spread, and because the lymphatic system plays a central part in the metastatic spread of many cancers, the delivery and targeting of vitamin D and its analogs to the lymphatic system and the consequent high lymphatic drug levels could be an added value in preventing metastases spread through the lymph.
Concluding remarks and future perspectives The current collected data could suggest that vitamin D and its analogs have a promising therapeutic potential, especially for prevention and treatment of various skin diseases, cancer development, diabetes and MS. Additional information on vitamin D research is needed from an interdisciplinary perspective. There is a need for long-term clinical studies with relevant outcomes, including bone health, immune function, autoimmune disorders and chronic disease prevention. In addition, there is a need for more clarification of the side effects such as hypercalcemia and hypercal- Q7 ciuria. Further research to help understand the outcomes of vitamin D and its analogs is required in terms of metabolic partitioning and mobilization of key vitamin D metabolites to ensure safety for longterm vitamin D intake. There is still much that remains to be elucidated before the various mechanisms of therapeutic activity of vitamin D and its analogs can be thoroughly understood. More clinical studies are warranted to establish effectiveness in treatment of the aforementioned diseases.
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