Low 25(OH) vitamin D3 levels are associated with adverse outcome in newly diagnosed, intensively treated adult acute myeloid leukemia.

BACKGROUND: Several studies have suggested that low 25(OH) vitamin D3 levels may be prognostic in some malignancies, but no studies have evaluated their impact on treatment outcome in patients with acute myeloid leukemia (AML). METHODS: Vitamin D levels were evaluated in 97 consecutive, newly diagnosed, intensively treated patients with AML. MicroRNA expression profiles and single nucleotide polymorphisms (SNPs) in the 25(OH) vitamin D3 pathway genes were evaluated and correlated with 25(OH) vitamin D3 levels and treatment outcome. RESULTS: Thirty-four patients (35%) had normal 25(OH) vitamin D3 levels (32-100 ng/mL), 34 patients (35%) had insufficient levels (20-31.9 ng/mL), and 29 patients (30%) had deficient levels (<20 ng/mL). Insufficient/deficient 25(OH) vitamin D3 levels were associated with worse relapse-free survival (RFS) compared with normal vitamin D3 levels. In multivariate analyses, deficient 25(OH) vitamin D3 , smoking, European Leukemia Network genetic group, and white blood cell count retained their statistical significance for RFS. Several microRNAs and SNPs were associated with 25(OH) vitamin D3 levels, although none remained significant after multiple test corrections; one 25(OH) vitamin D3 receptor SNP, rs10783219, was associated with a lower complete remission rate (P = .0442) and with shorter RFS (P = .0058) and overall survival (P = .0011). CONCLUSIONS: It remains to be determined what role microRNA and SNP profiles play in contributing to low 25(OH) vitamin D3 level and/or outcome and whether supplementation will improve outcomes for patients with AML.

Pharmacokinetics and metabolism of all-trans- and 13-cis-retinoic acid in pulmonary emphysema patients.

Retinoids promote lung alveolarization in animal models and were administered to patients as part of the Feasibility of Retinoid Therapy for Emphysema (FORTE) study. This FORTE substudy investigated the pharmacokinetic profiles of 2 retinoic acid isomers-all-trans-retinoic acid (ATRA) and 13-cis-retinoic acid (13-cRA)-in subjects with emphysema, evaluated strategies to overcome self-induced ATRA catabolism, and identified pharmacodynamic relationships. Comprehensive and limited pharmacokinetics were obtained at multiple visits in emphysema subjects treated with placebo (n = 30), intermittent dosing (4 days/week) with low-dose ATRA (1 mg/kg/day, n = 21), or high-dose ATRA (2 mg/kg/day, n = 25) or daily administration of 13-cRA (1 mg/kg/day, n = 40). High-dose ATRA produced the highest peak plasma ATRA Cmax. However, at follow-up, plasma ATRA C(max) was significantly decreased from baseline in subjects whose day 1 levels exceeded 100 ng/mL (P < .0001). In contrast, administration of 13-cRA produced lower plasma ATRA C(max) (<100 ng/mL), but the levels were significantly higher at follow-up than those on day 1 (P < .001). Plasma ATRA levels as determined on day 1 correlated with changes in pulmonary diffusing capacity at 6 months, consistent with concentration-dependent biologic effects (r2 = -0.25). The authors conclude that intermittent therapy with high-dose ATRA produced the greatest ATRA exposure, but alternative approaches for limiting self-induced ATRA catabolism should be sought.

In vitro and in vivo evaluation of combined calcitriol and cisplatin in dogs with spontaneously occurring tumors.

PURPOSE: Calcitriol potentiates cisplatin-mediated activity in a variety of tumor models. We examine here, the effect of calcitriol and cisplatin pre-clinically and clinically in canine spontaneous tumors through in vitro studies on tumor cells and through a phase I study of calcitriol and cisplatin to identify the maximum-tolerated dosage (MTD) of this combination in dogs with cancer and to characterize the pharmacokinetic disposition of calcitriol in dogs. METHODS: Canine tumor cells were investigated for calcitriol/cisplatin interactions on proliferation using an MTT assay in a median-dose effect analysis; data were used to derive a combination index (CI). Cisplatin was given at a fixed dosage of 60 mg/m2. Calcitriol was given i.v. and the dosage was escalated in cohorts of three dogs until the MTD was defined. Serum calcitriol concentrations were quantified by radioimmunoassay. RESULTS: In vitro, CIs < 1.0 were obtained for all combinations of calcitriol/cisplatin examined. The MTD was 3.75 microg/kg calcitriol in combination with cisplatin, and hypercalcemia was the dose-limiting toxicosis. The relationship between calcitriol dosage and either Cmax or AUC was linear. Calcitriol dosages >1.5 microg/kg achieved Cmax > or = 9.8 ng/mL and dosages >1.0 microg/kg achieved AUC > or = 45 h ng/mL. CONCLUSIONS: Calcitriol and cisplatin have synergistic antiproliferative effects on multiple canine tumor cells and high-dosages of i.v. calcitriol in combination with cisplatin can be safely administered to dogs. Cmax and AUC at the MTD 3.75 microg/kg calcitriol exceed concentrations associated with antitumor activity in a murine model, indicating this combination might have significant clinical utility in dogs.

A phase I pharmacokinetic and pharmacodynamic study of intravenous calcitriol in combination with oral gefitinib in patients with advanced solid tumors.

PURPOSE: In preclinical models, calcitriol and the tyrosine kinase inhibitor gefitinib are synergistic and modulate extracellular signal-regulated kinase (Erk) and Akt pathways. Therefore, we conducted a phase I study of calcitriol and gefitinib to determine the maximum tolerated dose (MTD) of this combination. EXPERIMENTAL DESIGN: Calcitriol was given i.v. over 1 h on weeks 1, 3, and weekly thereafter. Gefitinib was given at a fixed oral daily dose of 250 mg starting at week 2 (day 8). Escalation occurred in cohorts of three patients until the MTD was defined. Pharmacokinetic studies were done for calcitriol and gefitinib. Serial skin biopsies were done to investigate epidermal growth factor receptor (EGFR) pathway pharmacodynamic interactions. RESULTS: Thirty-two patients were treated. Dose-limiting hypercalcemia was noted in two of four patients receiving 96 mug/wk of calcitriol. One of seven patients developed dose-limiting hypercalcemia at the MTD 74 mug/wk calcitriol dose level. The relationship between calcitriol dose and peak serum calcitriol (C(max)) and systemic exposure (AUC) was linear. Mean (+/-SD) serum calcitriol C(max) at the MTD was 6.68 +/- 1.42 ng/mL. Gefitinib treatment inhibited EGFR, Akt, and Erk phosphorylation in the skin. Calcitriol did not have consistent effects on skin EGFR or its downstream elements. The combination of gefitinib and calcitriol did not modulate tumor EGFR pathway in patients with serial tumor biopsies. CONCLUSIONS: High doses of weekly i.v. calcitriol can be administered safely in combination with gefitinib. Calcitriol concentrations achieved at the MTD 74 mug calcitriol exceed in vivo concentrations associated with antitumor activity in preclinical models.

CYP24 splicing variants are associated with different patterns of constitutive and calcitriol-inducible CYP24 activity in human prostate cancer cell lines.

24-Hydroxylase (CYP24) activity modulates in vitro and in vivo calcitriol metabolism and biologic effects. We have investigated, in human PC3, DU145 and LNCaP prostate cancer cell lines, the relationship of CYP24 single nucleotide polymorphisms (SNPs) and splicing and the variable patterns of baseline and calcitriol-inducible CYP24 activity. DU145 cells exhibit baseline CYP24 activity that is further induced by calcitriol. Baseline and inducible CYP24 activity were barely detectable in LNCaP cells. In PC3, baseline CYP24 activity was undetectable but induced by calcitriol. A different pattern of SNPs was identified at positions 24, 46, 146 and 198 in the intron between exons 9 and 10 of CYP24 gene in each cancer cell line. DU145 displayed baseline CYP24 splicing between exon 9 and exon 11; splicing was only observed in calcitriol treated LNCaP cells. Untreated PC3 had a mixed picture (splicing and no splicing); only the spliced form was seen after calcitriol treatment. These results demonstrate that calcitriol treatment modulates CYP24 splicing, and suggests that differences in CYP24 splicing are associated with different patterns of CYP24 activity.

Monocyte fructose 1,6-bisphosphatase and cytidine deaminase enzyme activities: potential pharmacodynamic measures of calcitriol effects in cancer patients.

PURPOSE: To determine, in peripheral blood monocytes (PBM), whether the enzymatic activities of fructose 1,6-bisphosphatase (FBPase), cytidine deaminase (CDDase) and 24-hydroxylase (CYP24), enzymes regulated by calcitriol are useful pharmacodynamic (PD) measures of calcitriol effects in cancer patients. METHODS: Cancer patients enrolled in a phase I clinical trial of calcitriol and carboplatin were studied. Baseline and calcitriol-induced changes in FBPase, CDDase and CYP24 activities were measured in PBM collected before, 6, 24, and 48 h after administration of calcitriol, prior to carboplatin, in doses ranging from 4 to 11 mug daily for 3 consecutive days (QDx3). Normal FBPase, CYP24 and CDDase activities were measured in PBM from untreated healthy volunteers. RESULTS: Baseline activities in PBM from cancer patients and healthy volunteers were (median and range): 1.0 (0.0-43.5) and 4.4 (3.1- 8.2) nmol/min/mg protein for FBPase (P = 0.002); 2.5 (0.9-9.3) and 0.8 (0.4-2.0) fmol/h/10(6) cells for CYP24 (P = 0.016), and 5.6 (2.5-22.3) and 6.6 (1.1-47.4) nmol/min/mg protein for CDDase (P > 0.05), respectively. All calcitriol doses achieved peak serum calcitriol levels > x3 the physiological levels, increased cancer patient PBM FBPase activity to normal levels and decreased CDDase activity to undetectable levels within 48 h, with no significant change in CYP24 activity. These enzyme activity changes were not associated with hypercalcemia. CONCLUSIONS: Calcitriol treatment-induced increase in FBPase and decrease in CDDase activities in cancer patient PBM are potential early and sensitive non-hypercalcemia PD measures of calcitriol effects.

Feasibility of retinoids for the treatment of emphysema study.

BACKGROUND: Retinoids promote alveolar septation in the developing lung and stimulate alveolar repair in some animal models of emphysema. METHODS: One hundred forty-eight subjects with moderate-to-severe COPD and a primary component of emphysema, defined by diffusing capacity of the lung for carbon monoxide (Dlco) [37.1 +/- 12.0% of predicted] and CT density mask (38.5 +/- 12.8% of voxels <- 910 Hounsfield units) [mean +/- SD] were enrolled into a randomized, double-blind, feasibility study at five university hospitals. Participants received all-trans retinoic acid (ATRA) at either a low dose (LD) [1 mg/kg/d] or high dose (HD) [2 mg/kg/d], 13-cis retinoic acid (13-cRA) [1 mg/kg/d], or placebo for 6 months followed by a 3-month crossover period. RESULTS: No treatment was associated with an overall improvement in pulmonary function, CT density mask score, or health-related quality of life (QOL) at the end of 6 months. However, time-dependent changes in Dlco (initial decrease with delayed recovery) and St. George Respiratory Questionnaire (delayed improvement) were observed in the HD-ATRA cohort and correlated with plasma drug levels. In addition, 5 of 25 participants in the HD-ATRA group had delayed improvements in their CT scores that also related to ATRA levels. Retinoid-related side effects were common but generally mild. CONCLUSIONS: No definitive clinical benefits related to the administration of retinoids were observed in this feasibility study. However, time- and dose-dependent changes in Dlco, CT density mask score, and health-related QOL were observed in subjects treated with ATRA, suggesting the possibility of exposure-related biological activity that warrants further investigation.

The antitumor efficacy of calcitriol: preclinical studies.

Studies in our laboratory demonstrate that vitamin D (1,25 dihydroxycholecalciferol or calcitriol) has significant antitumor activity in vitro and in vivo in murine and human squamous cell, prostate, lung, pancreatic and myeloma model systems. Calcitriol induces G0/G1 arrest, modulates p27 and p21, the cyclin-dependent kinase (cdk) inhibitors implicated in G1 arrest, and induces cleavage of caspase 3, PARP and the mitogen-activated protein kinase (MEK) in a caspase-dependent manner. Calcitriol also decreases phospho-Erk (P-Erk) and phospho-Akt (P-Akt), kinases that regulate cell survival pathways and up-regulate the pro-apoptotic signaling molecule, MEKK-1. Glucocorticoids enhance calcitriol-mediated activities pre-clinically in vitro and in vivo. Dexamethasone (dex) significantly potentiated the antitumor effect of calcitriol and decreased calcitriol-induced hypercalcemia. Both in vitro and in vivo, dex increased vitamin D receptor (VDR) ligand binding in the tumor while decreasing binding in intestinal mucosa, the site of calcium absorption. These studies demonstrated that calcitriol has significant antiproliferative activity in a number of pre-clinical model systems and form the groundwork for on-going clinical studies investigating calcitriol as an anticancer agent.

Pharmacokinetics of liquid calcitriol formulation in advanced solid tumor patients: comparison with caplet formulation.

The non linear relationship between calcitriol (1,25-D(3)) dose and AUC in cancer patients suggests that the commercially available caplet 1,25-D(3) formulation (Rocaltrol) cannot achieve the high systemic exposure associated with antitumor activity in animal models. The primary objective of this analysis was to determine whether a liquid 1,25-D(3) formulation had a more favorable pharmacokinetic profile. This analysis was based on the results obtained in 2 phase I clinical studies seeking to determine the maximum tolerated dose of 1,25-D(3) administered in combination with either dexamethasone or paclitaxel daily for three consecutive days weekly. Data were available for 12 patients treated with the caplet formulation at doses ranging from 12 microg to 21 microg, and for 16 patients treated with the liquid formulation at doses ranging from 13 microg to 36 microg; data for 19 patients were available at doses for which both formulations were used. There were no differences in C(max) and AUC(0-24 h) between the two formulations (P > 0.17) As was noted with the caplet formulation, dose-related proportional increases in C(max) and AUC(0-24 h) were not observed with liquid 1,25-D(3) at doses > or = 13 microg (P > 0.83). We conclude that the commercially available liquid 1,25-D(3) formulation offers no PK advantage over caplet formulation.

Pharmacokinetics of high-dose oral calcitriol: results from a phase 1 trial of calcitriol and paclitaxel.

OBJECTIVES: The data reported are from a trial designed to determine, in patients with advanced cancer, the maximum tolerated dose and pharmacokinetics of calcitriol when administered with paclitaxel, an agent whose antitumor activity in in vitro and in vivo studies has been shown to be enhanced by calcitriol. An additional goal was to evaluate the relationship between calcitriol dose and hypercalcemia. METHODS: Calcitriol was given orally for 3 consecutive days each week, and paclitaxel (80 mg/m(2)) was given intravenously weekly. Thirty-six patients were treated in cohorts composed of 3 to 9 patients, at escalating dose levels of calcitriol. The starting dose of calcitriol was 4 microg for 3 consecutive days each week, and the maximum dose administered was 38 microg for 3 consecutive days each week. The preparation of calcitriol used in this trial was a commercially available caplet (0.5 microg per caplet). Serum calcitriol concentrations were measured by radioimmunoassay. Detailed assessments of calcitriol pharmacokinetics were performed in 26 patients. RESULTS: There was substantial interpatient variation in peak serum calcitriol concentrations (C(max)), time to reach C(max), and area under the concentration versus time curve (AUC). Serum calcitriol AUC was not proportional to calcitriol dose (P =.0014). AUC for the 24-hour period after calcitriol administration [AUC (0-24)] at 38 microg was only 4 times that at 4 microg, instead of the 9.5-fold increase expected for a proportional relationship. Calcitriol plasma concentrations of 600 to 1440 pg/mL were achieved. No dose-limiting toxicity occurred in this trial. CONCLUSIONS: Despite variability in absorption, very high doses of calcitriol can be safely administered with paclitaxel. The high calcitriol serum concentrations achieved in this study approach those that, both in vitro and in vivo, potentiate the cytotoxicity of taxanes and platinum analogs.

A limited sampling method for the estimation of serum calcitriol area under the curve in cancer patients.

Pharmacokinetic (PK) data from 34 cancer patients receiving 2 to 10 micrograms of calcitriol subcutaneously (s.c.) were used to develop a limited sampling method for predicting serum calcitriol area under curve (AUC) based on three samples instead of the full complement of 12 to 16 samples. Serum calcitriol levels were measured by 1, 25-dihydroxyvitamin D3-[I125] radioimmunoassay. Individual patient-corrected serum calcitriol AUC0-12 h was calculated by the trapezoidal rule after subtracting the pretreatment serum calcitriol level. PK data were split into "training" and "evaluation" sets based on calcitriol dose and chronological order of enrollment. Linear regression models of log-corrected AUC0-12 h versus individual log calcitriol serum levels in the hour 1, 2, 3, 4, 6, and 8 samples were established using the training data set of 17 patients. The fit was tested on the evaluation data set of 17 patients using mean squared error (MSE) as the fit criterion. The best single time point predictor of log AUC0-12 h was the log serum (calcitriol) at hour 6 (MSE = 0.0061). The best prediction of log AUC0-12 h using two time points was found to involve hour 6 and hour 2 (MSE = 0.0018). The prediction equation for the latter model was as follows: Log AUC = 1.125 + 0.3756.log (calcitriol) at hour 2 + 0.5859.log (calcitriol) at hour 6. This limited sampling method was further evaluated in 83 cancer patients treated with 4 to 38 micrograms of oral (p.o.) calcitriol; observed and predicted calcitriol AUC0-12 h were highly correlated (r > or = 0.90, p = 0.0001). These results show that serum calcitriol AUC0-12 h after s.c. and p.o. calcitriol administration is accurately estimated using pretreatment and 2- and 6-hour blood samples.

Serum vitamin D metabolites in colorectal cancer patients receiving cholecalciferol supplementation: correlation with polymorphisms in the vitamin D genes.

Cholecalciferol (D(3)) supplementation results in variable increases in serum 25(OH)D(3) levels, however, the influence of genetic polymorphisms on these variable responses is unclear. We measured serum 25(OH)D(3), 24,25(OH)(2)D(3), 1,25(OH)2D(3) and VDBP levels in 50 colorectal cancer (CRC) patients before and during 2,000 IU daily oral D(3) supplementation for six months and in 263 archived CRC serum samples. Serum PTH levels and PBMC 24-OHase activity were also measured during D(3) supplementation. TagSNPs in CYP2R1, CYP27A1, CYP27B1, CYP24A1, VDR, and GC genes were genotyped in all patients, and the association between these SNPs and serum vitamin D(3) metabolites levels before and after D(3) supplementation was analyzed. The mean baseline serum 25(OH)D(3) level was less than 32 ng/mL in 65 % of the 313 CRC patients. In the 50 patients receiving D(3) supplementation, serum levels of 25(OH)D(3) increased (p = 0.008), PTH decreased (p = 0.036) and 24,25(OH)(2)D(3), 1,25(OH)(2)D(3), VDBP levels and PBMC 24-OHase activity were unchanged. GC SNP rs222016 was associated with high 25(OH)D(3) and 1,25(OH)(2)D(3) levels at baseline while rs4588 and rs2282679 were associated with lower 25(OH)D(3) and 1,25(OH)(2)D(3) levels both before and after D(3) supplementation. CYP2R1 rs12794714 and rs10500804 SNPs were significantly associated with low 25(OH)D(3) levels after supplementation but not with baseline 25(OH)D(3). Our results show that D(3) supplementation increased 25(OH)D(3) levels in all patients. GC rs4588 and rs2283679 SNPs were associated with increased risk of vitamin D(3) insufficiency and suboptimal increase in 25(OH)D(3) levels after D(3) supplementation. Individuals with these genotypes may require higher D(3) supplementation doses to achieve vitamin D(3) sufficiency.

Oral bioavailability of DN101, a concentrated formulation of calcitriol, in tumor-bearing dogs.

PURPOSE: High-dose calcitriol (1,25-dihydroxyvitamin D(3)) has antineoplastic activity against a range of tumors and potentiates chemotherapeutic agents. In an earlier canine study, the MTD of intravenous (i.v.) calcitriol was 3.75 µg/kg, but polysorbate-associated hypersensitivity reactions were common. Use of commercially available oral calcitriol is limited by the absence of a formulation of suitable strength to allow administration of a reasonable number of caplets. This study evaluated the bioavailability of DN101, a concentrated oral calcitriol formulation specifically developed for anticancer applications. METHODS: An open-label, single-dose, 2-way crossover study was conducted. Dogs randomly received a single 3.75 µg/kg dose of calcitriol either i.v. or oral (as DN101), followed by cisplatin (60 mg/m(2)). Three weeks later, the alternate form of calcitriol was given prior to another dose of cisplatin. Dogs received antihistamines and corticosteroids prior to both treatments. Food was withheld for 12 h before and after therapy. Serum calcitriol concentrations were measured by radioimmunoassay. RESULTS: Ten tumor-bearing dogs received both i.v. and oral calcitriol. Six dogs experienced hypersensitivity reactions during i.v. calcitriol. Sequence of calcitriol administration (day-1 vs. day-21) by either i.v. or oral routes had no effect on the major calcitriol pharmacokinetic parameters. Oral calcitriol resulted in significantly lower values for AUC (P = 0.05) and prolonged T (1/2) (P = 0.003) when compared to i.v. Calcitriol oral bioavailability was highly variable among dogs (mean ± SEM, 71 ± 12.6%). CONCLUSIONS: This study demonstrates that a high-dose formulation of calcitriol has a moderate bioavailability in dogs, but inter-individual variability in PK parameters is similar to that observed in people. With this bioavailability, serum concentrations of calcitriol that exhibit antitumor activity in a preclinical murine model were achieved in some dogs. Exploration of methods to minimize variation in calcitriol systemic exposure is warranted.

Calcitriol enhances gemcitabine anti-tumor activity in vitro and in vivo by promoting apoptosis in a human pancreatic carcinoma model system.

Gemcitabine is the standard care chemotherapeutic agent to treat pancreatic cancer. Previously we demonstrated that calcitriol (1, 25-dihydroxycholecalciferol) has significant anti-proliferative effects in vitro and in vivo in multiple tumor models and enhances the activity of a variety of chemotherapeutic agents. We therefore investigated whether calcitriol could potentiate the cytotoxic activity of gemcitabine in the human pancreatic cancer Capan-1 model system. Isobologram analysis revealed that calcitriol and gemcitabine had synergistic antiproliferative effect over a wide range of drug concentrations. Calcitriol did not reduce the cytidine deaminase activity in Capan-1 tumors nor in the livers of Capan-1 tumor bearing mice. Calcitriol and gemcitabine combination promoted apoptosis in Capan-1 cells compared with either agent alone. The combination treatment also increased the activation of caspases-8, -9, -6 and -3 in Capan-1 cells. This result was confirmed by substrate-based caspase activity assay. Akt phosphorylation was reduced by calcitriol and gemcitabine combination treatment compared to single agent treatment. However, ERK1/2 phosphorylation was not modulated by either agent alone or by the combination. Tumor regrowth delay studies showed that calcitriol in combination with gemcitabine resulted in a significant reduction of Capan-1 tumor volume compared to single agent treatment. Our study suggests that calcitriol and gemcitabine in combination promotes caspase-dependent apoptosis, which may contribute to increased anti-tumor activity compared to either agent alone.

Epigenetic regulation of vitamin D 24-hydroxylase/CYP24A1 in human prostate cancer.

Calcitriol, a regulator of calcium homeostasis with antitumor properties, is degraded by the product of the CYP24A1 gene, which is downregulated in human prostate cancer by unknown mechanisms. We found that CYP24A1 expression is inversely correlated with promoter DNA methylation in prostate cancer cell lines. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) activates CYP24A1 expression in prostate cancer cells. In vitro methylation of the CYP24A1 promoter represses its promoter activity. Furthermore, inhibition of histone deacetylases by trichostatin A (TSA) enhances the expression of CYP24A1 in prostate cancer cells. Quantitative chromatin immunoprecipitation-PCR (ChIP-qPCR) reveals that specific histone modifications are associated with the CYP24A1 promoter region. Treatment with TSA increases H3K9ac and H3K4me2 and simultaneously decreases H3K9me2 at the CYP24A1 promoter. ChIP-qPCR assay reveals that treatment with DAC and TSA increases the recruitment of vitamin D receptor to the CYP24A1 promoter. Reverse transcriptase-PCR analysis of paired human prostate samples revealed that CYP24A1 expression is downregulated in prostate malignant lesions compared with adjacent histologically benign lesions. Bisulfite pyrosequencing shows that CYP24A1 gene is hypermethylated in malignant lesions compared with matched benign lesions. Our findings indicate that repression of CYP24A1 gene expression in human prostate cancer cells is mediated in part by promoter DNA methylation and repressive histone modifications.

CYP24A1 inhibition enhances the antitumor activity of calcitriol.

High systemic exposures to calcitriol are necessary for optimal antitumor effects. Human prostate cancer PC3 cells are insensitive to calcitriol treatment. Therefore, we investigated whether the inhibition of 24-hydroxylase (CYP24A1), the major calcitriol inactivating enzyme, by ketoconazole (KTZ) or RC2204 modulates calcitriol serum pharmacokinetics and biologic effects. Dexamethasone (Dex) was added to minimize calcitriol-induced hypercalcemia and as a steroid replacement for the KTZ inhibition of steroid biosynthesis cytochrome P450 enzymes. KTZ effectively inhibited time-dependent calcitriol-inducible CYP24A1 protein expression and enzyme activity in PC3 cells and C3H/HeJ mouse kidney tissues. Systemic calcitriol exposure area under the curve was higher in mice treated with a combination of calcitriol and KTZ than with calcitriol alone. KTZ and Dex synergistically potentiated calcitriol-mediated antiproliferative effects in PC3 cells in vitro; this effect was associated with enhanced apoptosis. After treatment with calcitriol and KTZ/Dex, although caspase-9 and caspase-3 were not activated and cytochrome c was not released by mitochondria, caspase-8 was activated and the truncated Bid protein level was increased. Translocation of apoptosis-inducing factor to the nucleus was observed, indicating a role of the apoptosis-inducing factor-mediated and caspase-independent apoptotic pathways. Calcitriol and KTZ/Dex combination suppressed the clonogenic survival and enhanced the growth inhibition observed with calcitriol alone in PC3 human prostate cancer xenograft mouse model. Our results show that the administration of calcitriol in combination with CYP24A1 inhibitor enhances antiproliferative effects, increases systemic calcitriol exposure, and promotes the activation of caspase-independent apoptosis pathway.

A phase I, pharmacokinetic, and pharmacodynamic study of two schedules of vorinostat in combination with 5-fluorouracil and leucovorin in patients with refractory solid tumors.

PURPOSE: We conducted a phase I clinical trial to determine the maximum tolerated dose (MTD) of daily or twice daily vorinostat x 3 days when combined with fixed doses of 5-fluorouracil (FU) and leucovorin every 2 weeks. EXPERIMENTAL DESIGN: Vorinostat doses were escalated in a standard 3 x 3 phase I design. FU/leucovorin was started on day 2 of vorinostat and consisted of leucovorin 400 mg/m(2) i.v. over 2 hours followed by FU 400 mg/m(2) i.v. bolus and 2,400 mg/m(2) over 46 hours (sLV5FU2). RESULTS: Forty-three patients were enrolled. Grade 3 fatigue, and hand and foot syndrome were the dose-limiting toxicities (DLT) at the 2,000 mg vorinostat once-daily dose level. Grade 3 fatigue and mucositis were DLTs at the 800 mg vorinostat twice-daily dose level. None of six patients at the 1,700 mg once daily or six patients at the 600 mg twice daily dose levels had a DLT; those dose levels represent the MTD. Twenty-one of 38 patients with FU-refractory colorectal cancer had stable disease, and one had a partial response. Vorinostat maximum serum concentrations at the MTD exceeded concentrations associated with thymidylate synthase downregulation in vitro. No pharmacokinetic interactions were noted between vorinostat and FU. CONCLUSIONS: The MTD of vorinostat in combination with sLV5FU2 is 1,700 mg orally once daily x 3 or 600 mg orally twice daily x 3 days every 2 weeks. Clinical activity in refractory colorectal cancer supports further clinical development of this combination.

A phase I and pharmacokinetics study of intravenous calcitriol in combination with oral dexamethasone and gefitinib in patients with advanced solid tumors.

PURPOSE: The primary objective of this study was to determine the maximum tolerated dose (MTD) of intravenously (i.v.) calcitriol administered in combination with a fixed oral dose of dexamethasone and gefitinib in patients with refractory solid tumors. METHODS: A fixed oral dose of dexamethasone of 4 mg/day was given every 12 h x 3 doses starting 12 h prior to i.v. calcitriol administration. Calcitriol was administered i.v. over 1 h on weeks 1, 3, and weekly thereafter. The starting calcitriol dose level was 57 microg and escalation occurred in cohorts of three patients until the MTD was defined. Gefitinib was given at a fixed oral daily dose of 250 mg starting at week 2 (day 8). Serum calcitriol PK studies were performed on day 1 (calcitriol + dexamethasone) and on day 15 (calcitriol + dexamethasone + gefitinib). RESULTS: A total of 20 patients were treated. Dose-limiting hypercalcemia was observed in two out of the four patients receiving 163 mcg/week of calcitriol. Mean (+/-SE) peak serum calcitriol concentration (C (max)) at the MTD (125 microg/week calcitriol) was 11.17 +/- 2.62 ng/ml and the systemic exposure (AUC(0-72 h)) of 53.30 +/- 10.49 ng h/ml. The relationship between calcitriol dose and either C (max) or AUC was linear over the 57-163 microg dose range. CONCLUSIONS: The addition of a low dose of dexamethasone allowed the safe escalation of calcitriol to the MTD of 125 microg/week. This dose level resulted in serum calcitriol concentrations that are associated with pre-clinical antitumor activity. However, no antitumor activity was noted clinically in patients with solid tumors.

Epigenetic silencing of CYP24 in tumor-derived endothelial cells contributes to selective growth inhibition by calcitriol.

Calcitriol (1,25-dihydroxycholecalciferol), the most active form of vitamin D, has selective anti-proliferative effects on tumor-derived endothelial cells (TDEC) compared with Matrigel-derived endothelial cells (MDEC). Although both cell types have an intact vitamin D receptor-signaling axis, this study demonstrates that upon treatment with calcitriol, 24-hydroxylase (CYP24) mRNA, protein and enzymatic activity were markedly induced in MDEC in a time-dependent manner but not in TDEC. Furthermore, treatment of MDEC with a CYP24 small interfering RNA restored sensitivity to calcitriol. To investigate the lack of CYP24 induction in TDEC, we examined methylation patterns in the promoter regions of the CYP24 gene in these two cell types. We identified two putative CpG island regions located at the 5' end. Using methylation-specific PCR and bisulfite sequencing, we determined that these CpG islands were hypermethylated in TDEC but not in MDEC. These data may explain the recruitment of vitamin D receptor to the promoter region in MDEC but not TDEC, as revealed by chromatin immunoprecipitation analyses. Treatment of TDEC with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine restored calcitriol-mediated induction of CYP24, which led to loss of sensitivity to calcitriol growth inhibitory effects. CYP24 promoter hypermethylation was also observed in endothelial cells isolated from other tumors but not in endothelial cells isolated from normal mouse tissues. These observations indicate that the methylation status of the CYP24 promoter differs in endothelial cells isolated from different microenvironments (tumor versus normal) and that methylation silencing of CYP24 contributes to selective calcitriol-mediated growth inhibition in endothelial cells.

Pharmacokinetics of 1alpha,25-dihydroxyvitamin D3 in normal mice after systemic exposure to effective and safe antitumor doses.

OBJECTIVES: Calcitriol, 1alpha,25-dihydroxyvitamin D(3) (1,25-D(3)) has potent antiproliferative effects and potentiates the antitumor activity of many other cytotoxic drugs. 1,25-D(3) plasma pharmacokinetic (PK) parameters associated with antitumor activity in experimental animal models are unknown. The objective of this study was to determine plasma calcitriol PK in normal mice at doses of calcitriol which are active in suppressing tumor growth. METHODS: Plasma 1,25-D(3) PK were examined in normal C3H/HeJ mice after a single intraperitoneal dose of 0.125 or 0.5 microg 1,25-D(3)/mouse. PK blood samples were collected from groups of 5-9 mice at each time point up to 24 h after 1,25-D(3) administration. Plasma 1,25-D(3) concentrations were measured by radioimmunoassay. Plasma 1,25-D(3) concentration diurnal variation was determined in blood samples from untreated animals collected in the morning (9:00-11:00 a.m.) and in the evening (4:00-9:00 p.m.). RESULTS: Median baseline plasma 1,25-D(3) concentration measured in the morning and in the evening were 0.082 ng/ml (CI 95%, 0.076-0.099) and 0.067 ng/ml (CI 95%, 0.058-0.075), respectively (p = 0.004). After 0.125 and 0.5 microg dosing, peak plasma 1,25-D(3) concentrations (Cp(max)) were 12.0 ng/ml (CI 95%, 10.8-12.6) and 41.6 ng/ml (CI 95%, 40.8-53.6), respectively. The corresponding areas under the curve (AUC(0->24 h)) were 47.0 (CI 95%, 43.2-51.1) and 128.0 (CI 95%, 127.0-130.0) ng.h/ml. No dose-related changes in time to Cp(max) and apparent total plasma clearance were observed. CONCLUSIONS: These results demonstrate diurnal variation in baseline plasma 1,25-D(3) concentrations in mice. Plasma 1,25-D(3) PK in mice receiving doses that are effective in slowing tumor growth, inducing cell cycle arrest and apoptosis, and potentiating taxanes and platinum analogue antitumor activity are at least 5-10 times higher than those easily achieved and nontoxic in patients receiving high-dose intermittent oral therapy.