Coleman Gross

TREATMENT OF EARLY RECURRENT PROSTATE CANCER WITH 1,25-DIHYDROXYVITAMIN D3 (CALCITRIOL)

PURPOSE Substantial experimental and epidemiological data indicate that 1,25-dihydroxyvitamin D3 (calcitriol) has potent antiproliferative effects on human prostate cancer cells. We performed an open label, nonrandomized pilot trial to determine whether calcitriol therapy is safe and efficacious for early recurrent prostate cancer. Our hypothesis was that calcitriol therapy slows the rate of rise of prostate specific antigen (PSA) compared with the pretreatment rate. MATERIALS AND METHODS After primary treatment with radiation or surgery recurrence was indicated by rising serum PSA levels documented on at least 3 occasions. Seven subjects completed 6 to 15 months of calcitriol therapy, starting with 0.5 microg. calcitriol daily and slowly increasing to a maximum dose of 2.5 microg. daily depending on individual calciuric and calcemic responses. Each subject served as his own control, comparing the rate of PSA rise before and after calcitriol treatment. RESULTS As determined by multiple regression analysis, the rate of PSA rise during versus before calcitriol therapy significantly decreased in 6 of 7 patients, while in the remaining man a deceleration in the rate of PSA rise did not reach statistical significance. Overall the decreased rate of PSA rise was statistically significant (p = 0.02 Wilcoxon signed rank test). Dose dependent hypercalciuria limited the maximal calcitriol therapy given (range 1.5 to 2.5 microg. daily). CONCLUSIONS This pilot study provides preliminary evidence that calcitriol effectively slows the rate of PSA rise in select cases, although dose dependent calciuric side effects limit its clinical usefulness. The development of calcitriol analogues with decreased calcemic side effects is promising, since such analogues may be even more effective for treating prostate cancer.

The vitamin D receptor start codon polymorphism (FokI) and bone mineral density in premenopausal American black and white women

This study examines the association between bone mineral density (BMD) and a start codon polymorphism (SCP) at the translation initiation site of the vitamin D receptor (VDR) gene. The thymine/cytosine (T/C) polymorphism in the first of two start (ATG) codons can be detected by a restriction fragment length polymorphism (RFLP) using the endonuclease FokI, which recognizes ATG as part of its restriction site. F indicates absence of the first ATG and a VDR that is shorter by three amino acids. The FokI genotype was determined in 154 premenopausal American women (72 black and 82 white) who were 20–40 years old. BMD of the total body, femoral neck, and lumbar spine were measured by dual‐energy X‐ray absorptiometry. The distribution of the SCP genotypes differed significantly by race (p < 0.001): 4% of blacks versus 18% of whites were ff homozygous and 65% of blacks versus 37% of whites were FF homozygous. There was no statistically significant interaction between race and SCP genotype in analyses of BMD at any skeletal site. In the group as a whole, the ff women had femoral neck BMD that was 7.4% lower than that of the FF women. The ff white women had total body BMD values that were 4.3% lower and femoral neck values that were 12.1% lower than FF white women. Total body and femoral neck BMD did not differ significantly by genotype in black women, and spine BMD did not differ by genotype in either race. Addition of the SCP genotype to analysis of covariance models comparing BMD of the black and white women reduced estimated differences in femoral neck BMD between the two groups by about 35%. In conclusion, the SCP polymorphism, detected with the endonuclease FokI, appears to influence peak bone density, particularly at the femoral neck. Racial differences in its distribution may explain some of the racial difference in femoral neck BMD.

The vitamin D receptor gene start codon polymorphism : A functional analysis of FokI variants

The vitamin D receptor (VDR) gene contains a start codon polymorphism (SCP) which is three codons upstream of a second start site (ATG). The SCP genotype can be determined with the restriction enzyme FokI, where “f” indicates the presence of the restriction site and the first ATG, while “F” indicates its absence. Recent evidence suggests that the ff genotype is correlated with lower bone mineral density (BMD) in some populations. The SCP results in alternate VDRs that differ structurally, with the F variant (F‐VDR) being three amino acids shorter than the f variant (f‐VDR). To determine whether there are functional differences between the f‐VDR and the F‐VDR, we studied the two VDR forms expressed in COS‐7 cells. The proteins were distinguishable from one another on Western blots by their different mobilities, confirming the larger size of f‐VDR. Ligand binding studies showed no significant differences between the affinities of the two VDR forms for [3H]‐1,25‐dihydroxyvitamin D3 ([3H]‐1,25(OH)2D3) (Kd = 131 ± 78 pM, f‐VDR; Kd = 237 ± 190 pM, F‐VDR; p = 0.24); however, a 2‐fold difference in affinity can not be discriminated by this method. There were no differences in the abilities of the two receptor forms to bind DNA as determined by electrophoretic mobility shift assays. The ability of the two VDR forms to transactivate target genes was investigated using three different vitamin D responsive luciferase reporter constructs: 24‐hydroxylase, osteocalcin, and osteopontin. In these transactivation experiments, 1,25(OH)2D3 dose‐response (0.1–10 nM) curves revealed that the ED50 values for transactivation were indistinguishable between the two VDR forms. Additionally, cultured human fibroblasts with FF,Ff, and ff genotypes had similar sensitivity to 1,25(OH)2D3 with respect to the induction of 24‐hydroxylase mRNA. In summary, we were unable to detect significant differences in ligand affinity, DNA binding, or transactivation activity between f‐VDR and F‐VDR forms. We must emphasize, however, that the sensitivity of the methods used limits our ability to detect minor differences in VDR affinity and function. In conclusion, we cannot define a mechanism whereby the SCP in the VDR might contribute to population differences in BMD.

Lack of Correlation Between Start Codon Polymorphism of the Vitamin D Receptor Gene and Bone Mineral Density in Premenopausal French Women: The OFELY Study

Previous studies have demonstrated an association between bone mineral density (BMD) and a start codon polymorphism (SCP) of the vitamin D receptor (VDR) gene in pre‐ and postmenopausal Caucasian and Japanese women. The SCP can be determined by a restriction fragment length polymorphism defined by the FokI restriction endonuclease. VDR alleles containing the FokI site are denoted by f and alleles lacking the site by F. In this study, the association between BMD and the SCP was examined in a group of 174 premenopausal French women who previously had been studied for a relationship between BMD and the VDR BsmI polymorphism. The SCP genotypes of the French women were FF 40%, Ff 44%, and ff 16% and they were independent of the BsmI genotype. BMD was measured by dual‐energy X‐ray absorptiometry at the lumbar spine, proximal femur, forearm, and total body. In contrast to previous reports, there was no association of BMD with SCP genotype in this group of Caucasian women at any site. We also measured several biochemical indices of calcium homeostasis and bone turnover. We found no statistically significant associations between SCP genotype and calcium, parathyroid hormone, or vitamin D levels. There was a 33.5% higher level of the skeletal resorption marker N‐telopeptides of type I collagen in the women with the ff genotype when compared with women with the FF genotype (p = 0.004). Other bone turnover markers failed to show an association with SCP genotype. In summary, the SCP genotype may not be associated with reduced BMD in all geographical or ethnic populations.

Hereditary vitamin D resistant rickets caused by a novel mutation in the vitamin D receptor that results in decreased affinity for hormone and cellular hyporesponsiveness.

Mutations in the vitamin D receptor (VDR) result in target organ resistance to 1alpha,25-dihydroxyvitamin D [1,25(OH)2D3], the active form of vitamin D, and cause hereditary 1,25-dihydroxyvitamin D resistant rickets (HVDRR). We analyzed the VDR of a patient who exhibited three genetic diseases: HVDRR, congenital total lipodystrophy, and persistent mullerian duct syndrome. The patient was treated with extremely high dose calcitriol (12.5 microg/d) which normalized serum calcium and improved his rickets. Analysis of [3H]1,25(OH)2D3 binding in the patients cultured fibroblasts showed normal abundance of VDR with only a slight decrease in binding affinity compared to normal fibroblasts when measured at 0 degrees C. The patients fibroblasts demonstrated 1,25(OH)2D3-induction of 24-hydroxylase mRNA, but the effective dose was approximately fivefold higher than in control cells. Sequence analysis of the patients VDR gene uncovered a single point mutation, H305Q. The recreated mutant VDR was transfected into COS-7 cells where it was 5 to 10-fold less responsive to 1,25(OH)2D3 in gene transactivation. The mutant VDR had an eightfold lower affinity for [3H]1,25(OH)2D3 than the normal VDR when measured at 24 degrees C. RFLP demonstrated that the patient was homozygous for the mutation while the parents were heterozygous. In conclusion, we describe a new ligand binding domain mutation in the VDR that causes HVDRR due to decreased affinity for 1,25(OH)2D3 which can be effectively treated with extremely high doses of hormone.

Vitamin D Receptor Polymorphisms, Bone Mineral Density, and Bone Metabolism in Postmenopausal Mexican-American Women

Common polymorphisms in the vitamin D receptor (VDR) gene have been shown to correlate with bone mineral density (BMD). However, attempts to replicate the original findings in other populations have yielded variable results. These disparities may reflect ethnic or environmental differences in the expression of the VDR effect upon BMD. We examined a relatively ethnically homogeneous group of 103 healthy postmenopausal Caucasian women of Mexican descent living in Northern California. We determined the VDR genotype and measured the BMD at the lumbar spine and femoral neck by dual‐energy X‐ray absorptiometry, as well as several biochemical indices of mineral metabolism. The prevalence of the BB genotype, associated in previous studies with the lowest BMD, was 8% and highly linked to the tt genotype. Absolute and age‐adjusted BMD at both hip and spine showed a trend toward lower BMD in the BB, AA, and tt genotypes, but this trend did not achieve statistical significance. There were no consistent intergroup differences in change in BMD over 2 years of follow‐up, nor in mean serum concentrations of 25‐hydroxyvitamin D, 1,25‐dihydroxyvitamin D, osteocalcin, or total urinary pyridinolines. Intact parathyroid hormone concentrations were significantly higher in subjects with the AA genotype, with a trend toward higher values in those with the BB and tt genotypes as well. Our data suggest that there may be a decrease in BMD associated with the B, A, and t alleles, but the intergroup difference in BMD is 0.2–0.5 standard deviations (SD) at the lumbar spine and 0.3 SD at the femoral neck, decreases that are smaller than previously reported. Given the relatively low prevalence of the BB/tt genotype in Mexican‐American Caucasians, a larger sample would be required to detect a significant association between VDR alleles and differences in BMD of the magnitude suggested by our data. We conclude that a genotype effect of this magnitude, if present, would be clinically relevant, but the impact on BMD is too small to detect with statistical significance in a study of this size.

Global clinical response in Cushing's syndrome patients treated with mifepristone

Mifepristone, a glucocorticoid receptor antagonist, improves clinical status in patients with Cushings syndrome (CS). We examined the pattern, reliability and correlates of global clinical response (GCR) assessments during a 6‐month clinical trial of mifepristone in CS.

Suramin, hydrocortisone, and retinoic acid modify inhibitory effects of 1,25-dihydroxyvitamin D3 on prostatic epithelial cells

The proliferation of prostatic epithelial cells is regulated by the complex interplay of numerous growth-stimulatory and growth-inhibitory factors. 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] has recently been identified as a potent inhibitor of the growth of prostatic epithelial cells. Epidemiologic studies indicate that vitamin D deficiency may be a risk factor for the development of clinical prostate cancer, possibly due to increased growth and reduced differentiation of prostatic cells in an environment with decreased 1,25(OH)(2)D(3). The application of vitamin D or analogs in chemotherapy against prostate and other cancers is being explored by several investigators. In order to use vitamin D most efficaciously in a clinical setting, it may be beneficial to learn more about the interaction of 1,25(OH)(2)D(3) with other factors that regulate prostatic epithelial cellular growth. In this study, we examined the effect of the proliferative status of cultured cells on their ability to respond to 1,25(OH)(2)D(3), and found that minimally proliferative cells were equally as responsive to 1,25(OH)2D3 as actively dividing cells. We noted no apparent interaction of 1,25(OH)(2)D(3) with epidermal growth factor, insulin-like growth factor, cholera toxin, or transforming growth factor-β, but we did find synergistic inhibitory effects of 1,25(OH)(2)D(3) with suramin and retinoic acid. Perhaps most noteworthy was the dramatic increase in potency of 1,25(OH)(2)D(3) that occurred upon deletion of hydrocortisone from the culture medium. Our in vitro studies indicate that combination therapy of vitamin D analogs with suramin, vitamin A analogs, or anti-glucocorticoids might be considered for prostate cancer.

Chapter 9 – Vitamin D: Biology, Action, and Clinical Implications

Publisher Summary Vitamin D is one of the major regulators of calcium homeostasis in the body and is critically important for normal mineralization of bone. The active hormone, 1α,25-dihydroxyvitamin D [1,25(OH)2D], is produced by sequential hydroxylations of vitamin D in the liver (25- hydroxylation) and the kidney (1-hydroxylation). 1,25(OH)2D, acting through the vitamin D receptor (VDR), acts by a genomic mechanism identical to the classical steroid hormones to regulate target gene transcription. In the past decade, it has become increasing clear that vitamin D has many additional functions that implicate the hormone in a wide array of actions relating to bone formation as well as to other areas unrelated to bone or mineral metabolism, including anti-proliferative, pro-differentiating, and immune–suppressive activities. This chapter describes the basic biology of vitamin D, including its metabolism, physiology, mechanism of action, and its diverse functions in the body, including those actions that relate to mineral metabolism as well as the newer actions. Several potential mechanisms have been put forward to implicate vitamin D in the development of osteoporosis. First, the possibility that polymorphisms within the gene encoding the VDR contribute substantially to genetic differences in osteoporosis risk has been raised. The basis for this genetic effect on osteoporosis risk is presumably as a hereditary factor affecting “peak bone mass,” but the mechanism is unknown. The second mechanism proposed is an age-related decline in renal 1,25(OH)2D production, due in part to a diminished renal response to PTH and reduced intestinal calcium absorption. There appears to be a defect in the renal response to PTH so that older women with osteoporosis require greater amounts of PTH to stimulate 1,25(OH)2D production. Third, a relative decrease in circulating 1,25(OH)2D has been considered a contributing factor in the development of senile osteoporosis. A low vitamin D state from an inadequate diet and decreased exposure to sunlight as people age, especially in the house-bound elderly, contribute to malabsorption of calcium and vitamin D “insufficiency” in the elderly.