Low Calcemic Activity Research Articles

Advances in the Administration of Vitamin D Analogues to Support Bone Health and Treat Chronic Diseases.

Vitamin D (VD) exerts a wide variety of biological actions in addition to its well-known roles in calcium homeostasis. Nutritional VD deficiency induces rachitic abnormalities in growing children and osteomalacia in adults, and it has been proposed to underlie the onset and development of multiple non-communicable chronic diseases. Therefore, the administration of VD or synthetic VD analogues represents a promising therapeutic strategy; indeed, VD and a VD agonist have shown clinical promise in mitigating osteoporosis and symptoms of insufficient calcium intake. However, even though high doses of VD analogues have shown pre-clinical efficacy against several diseases, including cancers, they have not yet had wide-spread clinical success. This difference may be due to limitation of clinical doses in light of the inherent calcemic action of VD. An approach to overcome this problem involves the development of VD analogues with lower calcemic activity, which could be administered in high doses to attenuate the onset and progress of disease. In a similar strategy, selective estrogen receptor modulators have had success as anti-osteoporosis drugs, and they have shown benefit for other estrogen target organs by serving as partial antagonists or agonists of estrogen receptor α. It is thus conceivable to generate synthetic partial antagonists or agonists for the VD receptor (VDR) that would exert beneficial effects on bone and other VD target organs. In this review, we discuss the molecular basis of the development of such synthetic VDR ligands from the viewpoint of roles of VDR in gene regulation.

Lithocholic acid-based design of noncalcemic vitamin D receptor agonists

The hypercalcemic effects of the hormone 1α,25-dihydroxyvitamin D3 (calcitriol) and most of known vitamin D metabolites and analogs call for the development of non secosteroidal vitamin D receptor (VDR) ligands as new selective and noncalcemic agonists for treatment of hyperproliferative diseases. We report on the in silico design and stereoselective synthesis of six lithocholic acid derivatives as well as on the calcemic activity of a potent LCA derivative and its crystallographic structure in complex with zVDR LBD. The low calcemic activity of this compound in comparison with the native hormone makes it of potential therapeutic value. Structure-function relationships provide the basis for the development of even more potent and selective lithocholic acid-based VDR ligands.

Synthesis and Biological Evaluation of Cyclopropylamine Vitamin D‐Like CYP24A1 Inhibitors

AbstractCYP24A1 hydroxylase plays a key role in tuning the levels and function of 1,25(OH)2D3, and inhibitors of CYP24 may be used for the treatment of a wide variety of clinical conditions. In the present work we describe the synthesis and biological properties of a small series of novel cyclopropylamine vitamin D‐like CYP24A1 inhibitors, designed as structural analogues of our previously described cyclopropylamine vitamin D‐like highly selective CYP24A1 inhibitor, CPA1. When tested in a cell‐free assay, two of these compounds, namely VN‐23 and SAP‐3, were found to be potent inhibitors of CYP24A1, with compound VN‐23 exhibiting selectivity with respect to CYP27B1 almost comparable to that of the lead compound CPA1. Interestingly, although compound SAP‐3, is only moderately selective with respect to CYP27B1, it is almost as potent as 1,25(OH)2D3 in binding for the vitamin D receptor (VDR) and transcriptional activity, while showing low calcemic activity in vivo.

Vitamin D analogues: Potential use in cancer treatment

The vitamin D receptor (VDR) is a member of the thyroid-steroid family of nuclear transcription factors. Following binding of the active form of vitamin D, i.e., 1,25(OH)2D3 (also known as calcitriol) and interaction with co-activators and co-repressors, VDR regulates the expression of several different genes. Although relatively little work has been carried out on VDR in human cancers, several epidemiological studies suggest that low circulating levels of vitamin D are associated with both an increased risk of developing specific cancer types and poor outcome in patients with specific diagnosed cancers. These associations apply especially in colorectal and breast cancer. Consistent with these findings, calcitriol as well as several of its synthetic analogues have been shown to inhibit tumor cell growth in vitro and in diverse animal model systems. Indeed, some of these vitamin D analogues with low calcemic inducing activity (e.g., EB1089, inecalcitol, paricalcitol) have progressed to clinical trials in patients with cancer. Preliminary results from these trials suggest that these vitamin D analogues have minimal toxicity, but clear evidence of efficacy remains to be shown. Although evidence of efficacy for mono-treatment with vitamin D analogues is currently lacking, several studies have reported that supplementation with calcitriol or the presence of high endogenous circulating levels of vitamin D enhances response to standard therapies.

Antitumor effects of vitamin D analogs on hamster and mouse melanoma cell lines in relation to melanin pigmentation.

Deregulated melanogenesis is involved in melanomagenesis and melanoma progression and resistance to therapy. Vitamin D analogs have anti-melanoma activity. While the hypercalcaemic effect of the active form of Vitamin D (1,25(OH)2D3) limits its therapeutic use, novel Vitamin D analogs with a modified side chain demonstrate low calcaemic activity. We therefore examined the effect of secosteroidal analogs, both classic (1,25(OH)2D3 and 25(OH)D3), and novel relatively non-calcemic ones (20(OH)D3, calcipotriol, 21(OH)pD, pD and 20(OH)pL), on proliferation, colony formation in monolayer and soft-agar, and mRNA and protein expression by melanoma cells. Murine B16-F10 and hamster Bomirski Ab cell lines were shown to be effective models to study how melanogenesis affects anti-melanoma treatment. Novel Vitamin D analogs with a short side-chain and lumisterol-like 20(OH)pL efficiently inhibited rodent melanoma growth. Moderate pigmentation sensitized rodent melanoma cells towards Vitamin D analogs, and altered expression of key genes involved in Vitamin D signaling, which was opposite to the effect on heavily pigmented cells. Interestingly, melanogenesis inhibited ligand-induced Vitamin D receptor translocation and ligand-induced expression of VDR and CYP24A1 genes. These findings indicate that melanogenesis can affect the anti-melanoma activity of Vitamin D analogs in a complex manner.

Paricalcitol, a vitamin d receptor activator, inhibits tumor formation in a murine model of uterine fibroids.

We examined the antitumor and therapeutic potentials of paricalcitol, an analog of 1,25-dihydroxyvitamin D3 with lower calcemic activity, against uterine fibroids using in vitro and in vivo evaluations in appropriate uterine fibroid cells and animal models. We found that paricalcitol has potential to reduce the proliferation of the immortalized human uterine fibroid cells. For the in vivo study, we generated subcutaneous tumors by injecting the Eker rat-derived uterine leiomyoma cell line (ELT-3) rat uterine fibroid-derived cell line in athymic nude mice supplemented with estrogen pellets. These mice were administered with vehicle versus paricalcitol (300 ng/kg/d) or 1,25-dihydroxyvitamin D3 (500 ng/kg/d) for 4 consecutive weeks, and the data were analyzed. We found that while both paricalcitol and 1,25-dihydroxyvitamin D3 significantly reduced fibroid tumor size, the shrinkage was slightly higher in the paricalcitol-treated group. Together, our results suggest that paricalcitol may be a potential candidate for effective, safe, and noninvasive medical treatment option for uterine fibroids.

New vitamin D analogs as potential therapeutics in melanoma

Extensive evidence shows that the active form of vitamin D3 – 1α,25-dihydroxyvitamin D3 – plays an important role in cancer prevention, has tumorostatic activity and may potentially be used in therapy for melanoma. Vitamin D3 and its analogs (secosteroids) exert multiple effects on cancer cells, including inhibition of cell growth and induction of differentiation. Activity of secosteroids depends on multiple cellular factors, including expression of the vitamin D receptor. Despite its endogenous origin, the key drawback for the use of pharmacologically effective doses of 1α,25-dihydroxyvitamin D3 is its hypercalcemic effect leading to profound toxicity. The solution may lie in properties of vitamin D3 analogs with modified side chains, which demonstrate low calcemic activity but conserve the anti-tumor properties. Noncalcemic vitamin D compounds were found to be potent in multiple studies that mandate further clinical testing. Finally, recent studies revealed alternative metabolic pathways for secosteroids and new targets in the cells, which opens up new therapeutic possibilities.

Synthesis and biological activities of vitamin D-like inhibitors of CYP24 hydroxylase

Selective inhibitors of CYP24A1 represent an important synthetic target in a search for novel vitamin D compounds of therapeutic value. In the present work, we show the synthesis and biological properties of two novel side chain modified 2-methylene-19-nor-1,25(OH) 2D 3 analogs, the 22-imidazole-1-yl derivative 2 (VIMI) and the 25- N-cyclopropylamine compound 3 (CPA1), which were efficiently prepared in convergent syntheses utilizing the Lythgoe type Horner–Wittig olefination reaction. When tested in a cell-free assay, both compounds were found to be potent competitive inhibitors of CYP24A1, with the cyclopropylamine analog 3 exhibiting an 80–1 selective inhibition of CYP24A1 over CYP27B1. Addition of 3 to a mouse osteoblast culture sustained the level of 1,25(OH) 2D 3, further demonstrating its effectiveness in CYP24A1 inhibition. Importantly, the in vitro effects on human promyeloid leukemia (HL-60) cell differentiation by 3 were nearly identical to those of 1,25(OH) 2D 3 and in vivo the compound showed low calcemic activity. Finally, the results of preliminary theoretical studies provide useful insights to rationalize the ability of analog 3 to selectively inhibit the cytochrome P450 isoform CYP24A1.

MAPK Expression Profile in AML cells ex Vivo changes with Differentiation Induced by 1,25-Dihydroxyvitamin D3 and Its Analogs PRI-1906 and PRI-2191

Abstract 5009 Background:The physiological form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), has well-established anti-cancer activity, but its potential clinical use is limited by hypercalcemia. Efforts to overcome this difficulty include structural modifications of 1,25D, as well as enhancement of its activity by other compounds such as antioxidants. The PRI analogs (termed “deltanoids”) have been shown to have low calcemic activity in animals, and while the PRI-1906 (24-ene-1,25-dihydroxyvitamin D2) and PRI-2191 ([24R]-1,24-dihydroxyvitamin D3) have greater activity on established Acute Myeloid Leukemia (AML) cell lines than their congeners, their relative potency on differentiation of AML blasts is not entirely clear. We therefore studied the effects of these analogs on leukemic blasts obtained from AML patients with different FAB subtypes, and combined them in some cases with Carnosic Acid (CA), a natural benzenediol abietane diterpene found in rosemary (Rosemarinus officinalis) and common sage (Salvia offinalis). Methods:Leukemia blasts from patients with newly diagnosed, untreated AML were obtained from the peripheral blood after formal consent on an IRB approved protocol. Cells were isolated by Histopaque centrifugation, washed, and placed in tissue culture. 1,25D, PRI-1906, or PRI-2191 (each 10 nM), and CA (10 uM) were added, and cells cultured for the times shown, and then harvested for analysis. Results:Cells from 8 patients were studied (1 patient with FAB-M1, 1 patient with FAB-M2, 1 patient with FAB-M2 with mixed myeloid and lymphoid lineage; 1 patient with FAB-M3; 1 patient with FAB-M4; 3 patients with FAB-M5). Using expression of CD11b/CD14 by immunophenotyping as markers of differentiation, the 1,25D and the deltanoids each induced modest differentiation in AML FAB-M1, with greater differentiation in response to the combination of a deltanoid plus CA. Similar differentiation effects were seen in AML FAB-M2, and more dramatic effects in AML FAB-M3. Differentiation was also seen in AML FAB-M4. In AML FAB-M5, 1,25D and deltanoids induced differentiation, but CA appeared to add little to this process in 2 of the patient's samples. One patient with AML FAB-M5 did show significant differentiation in response to CA as well as 1,25D; of note, that patient had a t(11;19) involving the MLL-1 gene, with normal FLT-3 genotype). In 6 out of the 8 cases, the G1 arrest increased when AML cells were treated with CA alone, but the effect was not enhanced by CA combined with 1,25D or its analogs. MAP Kinase mRNA array analysis showed upregulation of MAP Kinase pathway molecules in response to 1,25D and the deltanoids. Conclusions: 1The analogs of 1,25D, PRI-1906 and PRI-2191 induce cell cycle arrest and differentiation of AML cells ex vivo, and carnosic acid can enhance this effect. 2. As previously shown for 1,25D, MAPK signaling network is an important regulator of this process. 3.Since the PRI analogs are less calcemic than 1,25D, and carnosic acid has no effect on calcium homeostasis in AML cells, the potential therapeutic ratio is higher for the analogs combined with carnosic acid enhancer. Thus, these compounds may be considered as candidates for chemoprevention/ differentiation therapy of AML. (Supported by NIH Grant 1RO1-CA044722-21 to GPS). Disclosures:No relevant conflicts of interest to declare.

Abstract 3529: Evaluation of PRI vitamin D analogues, alone or with enhancers, as antileukemia agents

Abstract The physiological form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D) has well established anti-cancer activity, but its potential use in the clinic is limited by the danger of life-threatening hypercalcemia. Efforts to overcome this difficulty include structural modifications of 1,25D, as well as enhancement of its activity by other compounds. In this study we have combined these two approaches by comparison of the efficacy of the induction of differentiation in AML using analogs synthesized in the Warsaw Pharmaceutical Research Institute(PRI), alone or with its enhancers carnosic acid (CA), a plant antioxidant, and SB202190 (SB), an inhibitor of p38MAPK α, β. The PRI analogs have previously been shown to have low calcemic activity in animals, and while the analogs PRI-1906 (24-ene-1,25-dihydroxy vitamin D2) and PRI-2191([24R]-1,24-dihydroxyvitamin D3) had greater activity on established AML cell lines than their congeners, their relative potency on AML blasts was not entirely clear. We therefore studied an additional cohort of 20 patients with AML, and when sufficient cell numbers were available the analogs were supplemented with CA and SB to form a “differentiation cocktail” for study ex vivo. In this series we found that there was variability in the differentiation responses between individual patients, but the majority (∼90 %) of the blast samples responded to 1,25D and to both analogs. The intensity of the response was greatly increased when the “differentiation cocktail” was used rather than the analog alone, and while there was a similarity between the potencies of PRI-1906 and PRI-2191, the most robust responses in this series were seen when PRI-1906, alone or with CA and SB, was used. The strength of responses correlated with the enhancement of VDR expression in treated cells studied by Western blots. We conclude that PRI analogs 1906 and 2191 supplemented with CA and SB merit further scrutiny as candidates for chemoprevention/ differentiation therapy of AML. (Supported by USAPH NIH grant RO1 CA 44722-20 from NCI to GPS, the ISF grant 778/07 to MD, by Wroclaw Research Center EIT+ Operational Programme Innovative Economy, 1.1.2; POIG 01.01.02-02-003/08-00 to EM, and by the Foundation for Polish Science to EG). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3529. doi:10.1158/1538-7445.AM2011-3529

The Importance of Stereochemistry on the Actions of Vitamin D

The seco-steroid hormone 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)] is the most potent natural metabolite of vitamin D(3) and regulates primarily calcium and phosphate homeostasis, but also as a regulator of specific differentiation and of the immune system. Most, if not all, of the biological actions of 1α,25(OH)(2)D(3) are mediated through its specific receptor, the vitamin D receptor (VDR), which is a member of the nuclear receptor superfamily acting as a ligand-dependent transcription factor with coactivators. 1α,25(OH)(2)D(3) has significant therapeutic potential in the treatment of osteoporosis, rickets, secondary hyperparathyroidism, psoriasis, and renal osteodystrophy. However, the use of 1α,25(OH)(2)D(3) itself is limited because it induces significant hypercalcemia. Vitamin D is a highly flexible molecule and a very large number of analogs have been synthesized by industry and academia in an attempt to provide beneficial therapeutic agents with low calcemic activity. Chemical modifications of every portion of the vitamin D(3) molecule (the A, C, and D rings, the 17β-aliphatic side chain, and the 5,6,7,8-diene moiety) have been reported, with the most of the interesting analogs resulting from a combination of several modifications. The three-dimensional structure of both rat and human VDR-LBD have provided significant information for our understanding of the structure-function relationship (SFR) of vitamin D and some synthetic analogs. In this review, we focus on the current understanding of the relationship between selected stereochemical modifications of key structural components (i.e. A-ring, CD-ring and Side-chain) of the 1α,25(OH)(2)D(3) molecule and their effect on biological potency and selectivity. Based on current information, suggestions for the structure-based design of therapeutically valuable vitamin D analogs will conclude the review.

Three‐step hydroxylation of vitamin D3 by a genetically engineered CYP105A1

Our previous studies revealed that the double variant of cytochrome P450 (CYP)105A1, R73V/R84A, has a high ability to convert vitamin D(3) to its biologically active form, 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)], suggesting the possibility for R73V/R84A to produce 1α,25(OH)(2)D(3). Because Actinomycetes, including Streptomyces, exhibit properties that have potential advantages in the synthesis of secondary metabolites of industrial and medical importance, we examined the expression of R73V/R84A in Streptomyces lividans TK23 cells under the control of the tipA promoter. As expected, the metabolites 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1α,25(OH)(2)D(3) were detected in the cell culture of the recombinant S. lividans. A large amount of 1α,25(OH)(2)D(3), the second-step metabolite of vitamin D(3), was observed, although a considerable amount of vitamin D(3) still remained in the culture. In addition, novel polar metabolites 1α,25(R),26(OH)(3)D(3) and 1α,25(S),26(OH)(3)D(3), both of which are known to have high antiproliferative activity and low calcemic activity, were observed at a ratio of 5:1. The crystal structure of the double variant with 1α,25(OH)(2)D(3) and a docking model of 1α,25(OH)(2)D(3) in its active site strongly suggest a hydrogen-bond network including the 1α-hydroxyl group, and several water molecules play an important role in the substrate-binding for 26-hydroxylation. In conclusion, we have demonstrated that R73V/R84A can catalyze hydroxylations at C25, C1 and C26 (C27) positions of vitamin D(3) to produce biologically useful compounds.

Efficacy of a potent and safe vitamin D receptor agonist for the treatment of inflammatory bowel disease

Deficiency in 1α,25-dihydroxyvitamin D 3 (1,25D 3) has been suggested as an important environmental factor for immuno-mediated disorders including inflammatory bowel diseases (IBD), comprising Crohn's disease and ulcerative colitis, both characterized by chronic intestinal inflammation. Administration of vitamin D receptor (VDR) agonists can ameliorate spontaneous and induced animal models of colitis, but hypercalcemia is a dose-limiting adverse event. Previous work in our laboratory has identified 1α,25(OH) 2-16-ene-20-cyclopropyl-vitamin D 3 (BXL-62) as a potent anti-inflammatory VDR agonist with a low calcemic activity. In the present study, we confirm the marked anti-inflammatory properties of BXL-62 and show its capacity to induce VDR primary response genes, like CYP24A1 and CAMP, at lower concentrations than 1,25D 3, in PBMCs from IBD patients. Its higher anti-inflammatory potency compared to 1,25D 3 was demonstrated by the significantly more potent inhibition in PBMCs and in lymphocyte-enriched lamina propria mononuclear cells of the pro-inflammatory cytokines TNF-α, IL-12/23p40, IL-6 and IFN-γ, both at mRNA and protein level. The therapeutic efficacy of intra-rectal administration of BXL-62 in experimental IBD is shown by its beneficial effects, significantly higher than 1,25D 3, to induce recovery of clinical symptoms of colitis at normocalcemic doses in mice undergoing dextran sodium sulfate-induced colitis. These results confirm the therapeutic efficacy of VDR agonists in experimental colitis, and suggest BXL-62 as a promising compound for IBD treatment.

Lithocholic acid derivatives act as selective vitamin D receptor modulators without inducing hypercalcemia

1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], a vitamin D receptor (VDR) ligand, regulates calcium homeostasis and also exhibits noncalcemic actions on immunity and cell differentiation. In addition to disorders of bone and calcium metabolism, VDR ligands are potential therapeutic agents in the treatment of immune disorders, microbial infections, and malignancies. Hypercalcemia, the major adverse effect of vitamin D(3) derivatives, limits their clinical application. The secondary bile acid lithocholic acid (LCA) is an additional physiological ligand for VDR, and its synthetic derivative, LCA acetate, is a potent VDR agonist. In this study, we found that an additional derivative, LCA propionate, is a more selective VDR activator than LCA acetate. LCA acetate and LCA propionate induced the expression of the calcium channel transient receptor potential vanilloid type 6 (TRPV6) as effectively as that of 1alpha,25-dihydroxyvitamin D(3) 24-hydroxylase (CYP24A1), whereas 1,25(OH)(2)D(3) was more effective on TRPV6 than on CYP24A1 in intestinal cells. In vivo experiments showed that LCA acetate and LCA propionate effectively induced tissue VDR activation without causing hypercalcemia. These bile acid derivatives have the ability to function as selective VDR modulators.

Catalytic asymmetric synthesis and anticancer effects of the novel non-calcemic analog of vitamin D, 2α-fluoro-19-nor-22-oxa-1α,25-dihydroxyvitamin D 3 in metastatic lung carcinoma

1α,25-Dihydroxyvitamin D 3 (1α,25-D 3) has potent antiproliferative and anti-invasive properties in vitro in cancer cells. However, the major limitation to its clinical use is that it causes hypercalcemia. Therefore, vitamin D analogs with potent cell regulatory effects but with weaker calcemic effects than 1α,25-D 3 are required. Among them, 22-oxa-1α,25-D 3 and 19-nor-1α,25-D 3 have anti-cancer effects with relatively low calcemic effects. Modifications at the C-2α position of the A-ring also produced analogs with a unique biological profile. Not only the side-chain but also the A-ring modification thus generates a unique analog with potent cell regulatory effects and low calcemic activity as well. We report here that the hybrid 1α,25-D 3 analog, synthesized via the highly regio- and stereo-selective ring opening 2α-fluorination and catalytic asymmetric carbonyl-ene cyclization, with 2α-fluoro, 19-nor, and 22-oxa modification exhibits unique cell regulatory activities against the development of metastatic lung carcinoma.

Potent, Selective and Low-Calcemic Inhibitors of CYP24 Hydroxylase: 24-Sulfoximine Analogues of the Hormone 1α,25-Dihydroxyvitamin D3

A dozen 24-sulfoximine analogues of the hormone 1alpha,25-dihydroxyvitamin D(3) were prepared, differing not only at the stereogenic sulfoximine stereocenter but also at the A-ring. Although these sulfoximines were not active transcriptionally and were only very weakly antiproliferative, some of them are powerful hydroxylase enzyme inhibitors. Specifically, 24-(S)-NH phenyl sulfoximine 3a is an extremely potent CYP24 inhibitor (IC(50) = 7.4 nM) having low calcemic activity. In addition, this compound shows high selectivity toward the CYP24 enzyme in comparison to CYP27A1 (IC(50) > 1000 nM) and CYP27B (IC(50) = 554 nM).

Synthesis of 21-nor-22-oxa-1α,25-dihydroxyvitamin D 3 derivatives in quest of a drug with low calcemic activity

Two 21-nor-22-oxa-1α,25-dihydroxyvitamin D 3 derivatives have been synthesized in quest of a drug with lower calcemic activity than Maxacalcitol, 22-oxa-1α,25-dihydroxyvitamin D 3, being used as antihyperparathyroidism and antipsoriatic drug. Of two 21-nor products obtained, the product carrying one carbon elongated side chain with diethylcarbinol moiety has been found to exhibit comparable differentiation-inducing activity to Maxacalcitol with much lower exhibition of calcemic activity.

Toxicity and antineoplastic effect of (24 R)-1,24-dihydroxyvitamin D 3 (PRI-2191)

Many efforts have been made to obtain active and less toxic Vitamin D analogs for new clinical applications. The results of previous studies demonstrated the efficacy and safety of topical treatment of psoriasis with one of these analogs, 1,24-dihydroxyvitamin D 3, tacalcitol (1,24-(OH) 2D 3). In the present study, we evaluated the toxicity and antitumor effect of this analog. Lethal toxicity of 1,24-(OH) 2D 3 after s.c. injection was significantly lower than that of calcitriol. No significant differences were observed in the toxicity of the analogs when administered p.o. Calcium levels in the serum of mice treated with calcitriol were significantly higher (111%) than those in mice treated with 1,24-(OH) 2D 3 (89%) at 5 day after the first s.c. (10 μg/kg/day) administration in comparison to the control (healthy, untreated animals). Oral administration increased the calcium level by 78% for calcitriol and only to 47% over the control for 1,24-(OH) 2D 3. Parallel administration of clodronate prevented the calcitriol- and 1,24-(OH) 2D 3-induced lethal toxicity and also prevented increase in calcium levels. Single therapy with calcitriol did not affect tumor growth in the 16/C mouse mammary cancer model. In contrary, 1,24-(OH) 2D 3 alone reduced tumor volume to 41% of control. Cisplatin alone did not affect growth of 16/C tumor in these conditions. The growth of tumors in the presence of cisplatin was inhibited by 1,24-(OH) 2D 3 but not by calcitriol. Interestingly, the inhibition of tumor growth in cisplatin-treated mice by 1,24-(OH) 2D 3 was greater, than that observed in mice treated with this analog alone. In conclusion, 1,24-(OH) 2D 3 revealed higher antitumor and lower calcemic activity and toxicity than calcitriol. Application of biphosphonates along with Vitamin D analogs is sufficient to overcome the calcemic and toxic side effects of the proposed treatment.

Tissue distribution and activity studies of 1,24-dihydroxyvitamin D 2, a metabolite of vitamin D 2 with low calcemic activity in vivo

The active vitamin D compound 1α,24( S)-dihydroxyvitamin D 2 (1,24(OH) 2D 2) is under development as a therapy for disorders including cancer and secondary hyperparathyroidism. 1,24(OH) 2D 2 is a potent inhibitor of cell proliferation in vitro and, relative to calcitriol (1,25(OH) 2D 3), has reduced calcemic activity in vivo. To examine the mechanisms underlying this reduced calcemic activity, we studied the tissue distribution in rats of radiolabeled 1,24(OH) 2D 2 or 1,25(OH) 2D 3 over 24 h. Serum levels of 1,24(OH) 2D 2 were lower than those of 1,25(OH) 2D 3 at all time points; however, tissue levels of radiolabeled compounds followed different patterns. In duodenum and kidney, 1,24(OH) 2D 2 and 1,25(OH) 2D 3 rose to similar levels at early time points; 1,24(OH) 2D 2 levels then declined more rapidly. In bone marrow, 1,24(OH) 2D 2 and 1,25(OH) 2D 3 were present at similar levels at all time points. In liver, 1,24(OH) 2D 2 levels were two-fold higher than 1,25(OH) 2D 3 at 1 h post-injection, declining to similar levels by 8 h. In vitamin D-deficient rats, doses of 1,24(OH) 2D 2 30-fold higher than 1,25(OH) 2D 3 were required to produce equal stimulation of intestinal calcium absorption. In the same deficient animals, 1,24(OH) 2D 2 and 1,25(OH) 2D 3 were nearly equipotent at stimulating bone calcium mobilization. In cultured bone cells, 1,24(OH) 2D 2 and 1,25(OH) 2D 3 were equipotent at stimulating osteoclast formation and bone resorption. In summary, the reduced calcemic activity of 1,24(OH) 2D 2 may result from altered pharmacokinetics relative to 1,25(OH) 2D 3, resulting in relatively rapid decreases in 1,24(OH) 2D 2 levels and activity in target organs such as intestine. Further studies will be necessary to confirm these findings and to confirm the clinical utility of 1,24(OH) 2D 2.

Vitamin D analogues for secondary hyperparathyroidism.

Secondary hyperparathyroidism (2HPT), a common disorder in patients with chronic renal failure, develops in response to phosphate retention and low serum 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3), calcitriol). Replacement therapy with calcitriol or its precursor 1alpha-hydroxyvitamin D(3) (1alphaOHD(3), alfacalcidol) often produces hypercalcaemia, especially when combined with calcium-based phosphate binders. In addition, these vitamin D compounds can aggravate the hyperphosphataemia in these patients. Several vitamin D analogues have been developed that retain the direct suppressive action of 1,25(OH)(2)D(3) on the parathyroid glands but have less calcaemic activity, thereby offering a safer and more effective means of controlling 2HPT. 1,25-Dihydroxy-19-norvitamin D(2) (19-norD(2)) and 1alpha-hydroxyvitamin D(2) (1alphaOHD(2)) are available in the US and 1,25-dihydroxy-22-oxavitamin D(3) (22-oxacalcitriol, OCT) and 1,25-dihydroxy-26,26,26,27,27,27-hexafluorovitamin D(3) (1,25(OH)(2)26,27F6 D(3), falecalcitriol) have been approved for use in Japan. Animal studies have demonstrated that OCT and 19-norD(2) have a wider therapeutic window for suppression of parathyroid hormone (PTH) because of their lower calcaemic and phosphataemic activities. The low calcaemic activity of OCT has been attributed to its rapid clearance, which prevents sustained effects on intestinal calcium absorption and bone resorption, but still allows a prolonged suppression of PTH gene expression and parathyroid cell growth. The calcaemic activity of 19-norD(2) diminishes with the duration of treatment by as yet unknown mechanisms. The lower toxicity of 1alphaOHD(2), compared with 1alphaOHD(3), has also been noted with chronic, but not acute administration, perhaps due to differential metabolism. The unique actions of falecalcitriol may also result from an altered metabolism. A clear understanding of the molecular basis for the selectivity of vitamin D analogues on parathyroid function may allow the design of even more effective analogues.