Current Pharmaceutical Design - Volume 6, Issue 7, 2000

The wide range of activities now attributed to 1,25(OH)2 D 3 has suggested numerous potential therapeutic applications for this vitamin D hormone, including inhibiting growth of various type of cancer. Unfortunately, the potent calcemic activity of the natural hormone has precluded its use in most cases. Vitamin D analogs with higher therapeutic indices offer renewed hope for treatment of malignancies. The promising analogs currently under study were selected from hundreds of candidates by in vitro screening followed by in vivo testing. The mecha-nism( s) responsible for the greater effectiveness of most of these compounds is not known. Our current understanding of vitamin D physiology and biochemistry suggests that the biological profile of an analog would be determined primarily by its interaction with four classes of proteins: 1) the nuclear vitamin D receptor (VDR) that mediates transcriptional regulation; 2) the metabolic enzymes, primarily the vitamin D-24-hydroxylase but possibly others 3) serum transporters, mainly vitamin D binding protein (DBP), and perhaps lipoproteins; and 4) a new class of receptors that reside in the plasma membrane and mediate rapid, nongenomic responses. This article discusses how the manner in which analogs associate with these proteins can potentially produce selective actions at the tissue, cell and gene level. A thorough understanding of the influence of these analog-protein interactions on the biological profile of vitamin D analogs will be invaluable for the design of future analogs with enhanced target specificity.

The role of 1,25-dihydroxyvitamin D 3 (1,25-(OH)2 D 3 ) as a regulator of cell growth and differentiation is well recognized. Currently, 1,25-(OH)2 D 3 and vitamin D analogs are being evaluated for their therapeutic potential in the treatment of hyperproliferative disorders like cancer. In the present review, we will discuss several processes that might be involved in 1,25-(OH)2 D 3 - and vitamin D analog-mediated suppression of cancer cell growth. The effects on tumor cell proliferation, differentiation, apoptosis, angiogenesis, metastases, and parathyroid hormone-related peptide secretion will be highlighted. In addition, combination therapy with other tumor effective drugs will be addressed. Furtermore, we will focus on the potential drawbacks and the possible side effects of vitamin D compounds in the treatment of cancer.

In the first section, the general three-dimensional structure of the ligand-binding domain (LBD) of nuclear receptors (NR) was briefly described on the basis of their x-ray crystal structures. Emphasis was placed on the three major conformations of NR-LBD and their role in the transactivation function. In the second part, the structure-function relationship of vitamin D was analyzed based on the ligand struc-ture, in particular by using systematic conformational analysis as a tool. On the basis of the conformational analysis of the vitamin D side chain and studies using conformationally restricted synthetic vitamin D analogs, we suggested the active space region concept of vitamin D The vitamin D side-chain region was grouped into five regions (A, G, EA, EG and F). Activity orders, in terms of the spatial region, found by these studies are as follows: Affinity for vitamin D receptor (VDR), Affinity for vitamin D binding protein (DBP), Target gene transactivation, Cell differentiation, Bone calcium mobilization, Intestinal calcium absorption, In the third section, homology modeling of VDR-LBD and docking of the natural ligand, 1,25-(OH)2 D 3 , into the ligand binding cavity of the model are described. Amino acid residues forming hydrogen bonds with the biologically important 1alpha - and 25-OH groups were identified: 1alpha-OH forms a pincer-type hydrogen bond with R274 and S237 and 25-OH with H397. This VDR-LBD/1,25-(OH)2 D 3 docking model was firmly substantiated by mutation analysis. Using this VDR model, the structure-function relationship of highly potent vitamin D analogs was discussed.

Vitamin D analogs in which the triene moiety is replaced by an aromatic ring have been synthesized and their ability to bind to the vitamin D receptor investigated.

The results of our studies on the biological activity of side-chain modified analogues of vitamin D are reviewed. These analogues appeared to be effective in induction of cell differentiation, inhibition of tumour cell proliferation in vitro and in increasing of antitumour effect of cytostatics. On the other hand, inhibition of cytostatic-induced apoptosis by these compounds was observed. The mechanism of the antiproliferative effect of calcitriol analogues in vitro is discussed. The induction of antigens CD14 and CD11b expression and phagocytic activity of HL-60 cells after exposure to these compounds is related to their effect on cell differentiation. The differentiation of the HL-60 leukaemia cells induced by side-chain modified analogues of calcitriol increases their sensitivity to the antiproliferative effect of cisplatin, doxorubicin and genistein, despite of that this pretreatment causes resistance of these cells to cytostatics-induced apoptosis. We observed a synergistic antiproliferative effect of the combined therapy using analogues of calcitriol with subsequent treatment with the above-mentioned cytostatics. This effect was measured as a significant decrease of the ID 50 values for each cytostatic applied after pretreatment of the tumour cells with the calcitriol analogues.The results presented suggest that the improved therapeutic effect may be achieved also in vivo by the combined application of the analogues (without calcemic activity) of calcitriol with antitumour agents.

The introduction of oxygen atoms into different positions of the vitamin D side chain is described. By combining the arising 23-oxa and 25-oxa elements with other structural modifications (19-nor, iso-19-nor, 20-methyl, 20-ene, 20,21-cyclo) calcitriol analogs with remarkable levels of dissociation between beneficial acitivities on cell growth regulation and undesired hypercalcemia were identified. Structure-activity relations are elaborated in a very systematic outline of the Schering drug finding program in this particular class of vitamin D compounds.

Growing interests have been focused on the development of hybrid-analogs with modifications of the A-ring and the side chain of 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). An exocyclic methylene group at C-10, a hydroxy group at C-1 and a hydroxy group at C-3 play a crucial role in the expression of biological activities of 1alpha,25(OH)2D3. However, relationship between the functional groups and activities has not been fully understood. We have synthesized and evaluated biological activities of several singly dehydroxylated A-ring analogs of 19-nor-1alpha,25(OH)2D3 and 19-nor-22-oxa-1alpha,25(OH)2D3. All of them have an extremely low binding affinity for vitamin D receptor (VDR). Some of them lack the 1alpha-hydroxy group that is considered to be essential for VDR-mediated gene expression, have greater or equivalent potencies to 1alpha,25(OH)2D3 for inducing differentiation and cell cycle G0-G1 arrest of human promyelocytic leukemia cells as well as for the transactivation of target genes including a rat 25-hydroxyvitamin D3-24-hydroxylase gene promoter and a human osteocalcin gene promoter in transfected mammalian cells. The assessment of a ligand/VDR/Retinoid X receptor complex formation using a two-hybrid luciferase assay revealed that the liganded VDR has high potency to form a heterodimer, but this could not explain the high biological potency of the 19-nor analogs. Other reason(s) including an interaction with transcriptional cofactors should be considered to explain the mechanism of action of 19-nor analogs.

In this article are reviewed available experimental and clinical studies on vitamin D analogs, and molecular and cellular mechanisms of their antineoplastic activity. In more detail are discussed the antiproliferative and pro-differentiative effects, inhibition of tumor-induced angiogenesis and induction of apoptosis. The use of vitamin D analogs is however hampered by their toxicity. In various experimental systems it was shown that the activities of vitamin D analogs can be enhanced by combined application with retinoids or other biological active compounds, such as cytokines and growth factors. Retinoids and vitamin D analogs were found to have synergistic inhibitory effects on tumor cell proliferation and angiogenic capability, and both agents applied simultaneously are efficacious in small doses. Thus combined therapy could find application in clinical practice. There are up to now only very limited data on the treatment of cutaneous malignancies with vitamin D analogs, and it appears that a combined therapy, preferably with retinoids, could be more beneficial. The new synthetic, more potent and less calcemic analogs might find wide application in chemotherapy of premalignant and early malignant cutaneous tumors, and could be especially useful for chemoprevention in the high-risk groups, eg. xeroderma pigmentosum, organ transplant recipients, arsenical keratoses and others.