Calcitriol Analogs Research Articles

Synthesis of Spiro[4.5]decane CF-Ring Analogues of 1α,25-Dihydroxyvitamin D3

A novel series of analogues of calcitriol (1) is developed featuring a spirocyclic central core resulting from C18/C21-connection and C15/C16-deletion (2a, 2b). The synthesis of the key intermediate involves an Eschenmoser rearrangement of an enantiomerically pure bromo-substituted cyclohexenol.

The cytotoxicity of combined paricalcitol, docetaxel and radiation in head and neck squamous cell carcinoma

15526 Background: Paricalcitol is a calcitriol analog used in the treatment of secondary hyperparathyroidism. It produces less hypercalcemia compared to calcitriol and has low clinical toxicity. We have shown previously that paricalcitol is antiproliferative in head and neck squamous cell carcinomas (HNSCC) in vitro. However, its interaction in combination with docetaxel and radiation has not been elucidated. The purpose of this study is to evaluate the combined effects of paricalcitol, docetaxel, and radiation in vitro in SCC-15 cells, a human tongue squamous cell carcinoma cell line. Methods: The cytotoxic effects were assessed using MTT assays. SCC 15 cells were plated, allowed to attach for 24 hours, and then treated with paricalcitol at 0, 12.5, 25, or 50 nM. After 24 hours, docetaxel at 0, 0.05, 0.15, or 0.3 nM was applied, followed in 24 hours by single treatment with radiation at 0, 2, 4, or 6 Gy. MTT assays were performed after incubation for 5 days. CalcuSyn analysis was used to determine if the interaction between paricalcitol, docetaxel, and radiation is synergistic, additive, or antagonistic. Results: Paricalcitol alone at 12.5, 24, and 50 nM produced growth inhibition of 30, 38, and 40%, respectively. Docetaxel alone at 0.05, 0.15, and 0.3 nM resulted in 33, 61, and 72% growth inhibition, respectively. Radiation at 2, 4, and 6 Gy produced 44, 57, and 62% inhibition, respectively. Sequential paricalcitol and docetaxel at the concentrations tested followed by 2 Gy radiation produced additive to synergistic cytotoxicity. Antagonism was seen with 4 Gy and all combinations of paricalcitol and docetaxel. Antagonism was also seen with 6 Gy and 0.15 or 0.3 nM of docetaxel. Conclusions: Sequential application of paricalcitol, docetaxel, and then radiation at moderate doses produced additive to synergistic cytotoxicity in SCC-15 cells. The results of this study provide support for further investigations of combined paricalcitol, docetaxel and radiation therapy in the treatment of head and neck cancers. [Table: see text]

A phase I study of LR-103 (1,24α(OH)2D2) in patients with refractory malignancies

14608 Background: Preclinical studies of calcitriol and vitamin D analogs demonstrate potent growth inhibition of tumor cell lines. The clinical development of calcitriol and other analogs have been hindered by hypercalcemia. 1α (OH)D2 is a Vitamin D analog which has produced objective responses in advanced androgen-independent prostate cancer patients and can be administered daily at 10mcg. LR103 is an active metabolite of 1α (OH)D2 with equivalent anti-tumor activity and significantly less calcemic effects in preclinical studies. This phase I study explores the maximal tolerated dose and toxicity of LR-103. Methods: LR-103 was administered daily beginning at a dose of 5 μg/day with a cycle length of 14 to 28 days. Dose escalation proceeded in two stages. In step A, intra-patient dose escalation (starting dose of 5mcg daily) was allowed based on degree of toxicity regardless of attribution. Step A escalation continued until 2 patients had ≥ grade 2 toxicity at a dose level. Step B is a standard, inter-patient only, dose-escalation trial (starting dose based on Step A) with cohorts of three. Dose limiting toxicity is defined as ≥ grade 3 toxicity. Results: Nineteen patients have enrolled between May and December 2005, eight to step A and eleven to step B. During step A escalation, two patients at 15 μg/day experienced severe toxicity (grade 4 hyperuricemia and grade 3 alkaline phosphatase) and two patients at 15 μg/day experienced grade 1 hypercalcemia. Hypercalciuria has been common. No hypercalcemia ≥ grade 2 has been observed. Step B enrollment continues at 30 μg/day without hypercalcemia. Conclusions: Daily administration of LR-103 has not resulted in significant hypercalcemia to date. Dose escalation continues. No significant financial relationships to disclose.

Highly antiproliferative, low-calcemic, side-chain amide and hydroxamate analogs of the hormone 1α,25-dihydroxyvitamin D 3

Four new side-chain amide ( 2 and 3) and hydroxamate ( 4 and 5) analogs of the hormone calcitriol ( 1) have been prepared. Even though lacking the 25-OH group characteristic of natural calcitriol ( 1), analogs 2– 4 are as antiproliferative in vitro as calcitriol ( 1) but are 20–40 times less calciuric in vivo than calcitriol ( 1).

Oral treatment with a vitamin D3 analogue (BXL628) has anti‐inflammatory effects in rodent model of interstitial cystitis

To investigate the effects of a vitamin D3 analogue (BXL628) in a model of chronic cystitis, as calcitriol analogues might be an interesting new therapeutic option for interstitial cystitis, for although the cause of the disease remains unclear, the increase in mast cells in the mucosa and detrusor muscle are significant. We devised a mouse model of allergen-induced allergic cystitis that is associated with the up-regulation of genes for interleukin-13, FcepsilonRIalpha and mast cells-derived proteases, a massive inflammatory reaction in the bladder tissue, and augmented levels of mast cell-derived protease 1 (MMCP1) detected in mouse sera. Oral administration of BXL628 significantly reduced the expression of interleukin-13, FcepsilonRIalpha and MMCP1 in the bladder. Furthermore, histological analysis showed a decrease in oedema and leukocyte infiltration in the bladder wall. BXL628 treatment reduced serum MMCP1 levels, indicating an effect on mast cell degranulation in vivo. Vitamin D3 analogues may successfully be used as anti-inflammatory agents in allergen-mediated inflammatory reactions. Moreover, the modulatory effect shown on mast cell activation by the BXL628 analogue strongly supports its potential therapeutic use in a possibly mast cell-dependent disease such as human interstitial cystitis.

Effects of Vitamin D analogs on gene expression profiling in human coronary artery smooth muscle cells

Vitamin D analogs provide survival benefit for chronic kidney disease patients with cardiovascular complications. Activation of smooth muscle cells plays a role in cardiovascular diseases. It is not known how Vitamin D analogs modulate gene expression in smooth muscle cells. In this study, DNA microarray technology was used to assess the gene expression profile in human coronary artery smooth muscle cells treated with 0.1 μM 1α,25-dihydroxyvitamin D 3 (calcitriol) or paricalcitol (an analog of calcitriol) for 30 h. The effects of calcitriol and paricalcitol were similar. A total of 176 target genes were identified with 115 up-regulated and 61 down-regulated genes in the paricalcitol group. Target genes fall into various categories including cell differentiation/proliferation. Real-time RT-PCR analysis demonstrated that paricalcitol dose- and time-dependently regulated the expression of IGF1, WT1 and TGFβ3, three genes known to modulate cell proliferation. Paricalcitol also down-regulated the expression of natriuretic peptide precursor B and thrombospondin 1. Both drugs inhibited cell proliferation in a dose-dependent manner. This study identified genes not previously known to be regulated by VDR, providing insight into understanding the role of VDR on regulating smooth muscle cell growth, thrombogenicity, fibrinolysis and endothelial regeneration.

Highly antiproliferative, low-calcemic, side-chain ketone analogs of the hormone 1α,25-dihydroxyvitamin D 3

A series 2a– 4b of seven new side-chain ketone analogs of calcitriol ( 1) have been prepared. Unexpectedly, several of these 24- and 25- tert-butyl ketones, even though lacking the classical side-chain tertiary hydroxyl group, are considerably more antiproliferative in vitro than the hormone calcitriol ( 1) even at physiologically relevant low nanomolar concentrations and are less calcemic than calcitriol ( 1) in vivo. In addition, ketone analog 19-nor- 2a is not significantly less calcemic in vivo than 19-methylene analog 2a.

Paricalcitol has a potent in vivo tumoricidal effect on head and neck squamous cell carcinoma

5584 Background: Paricalcitol is a calcitriol analog that has low clinical toxicity and produces significantly less hypercalcemia compared to calcitriol. Previous in vitro study from our lab has shown that paricalcitol is antiproliferative in 3 human head and neck squamous cell carcinoma cell lines, SCC 9, 15, and 25. The purpose of this study is to evaluate the in vivo activity of paricalcitol on SCC 15 cells grown in athymic nude mice. The effect of paricalcitol on serum calcium was also assessed. Methods: Each flank of nude mice was injected subcutaneously with 2.5 million SCC 15 cells. After the xenografts reached at least 50 mm3 in size, paricalcitol at 1.25 mcg/kg (treatment group with 4 mice) or drug carrier (control group with 4 mice) was administered by intraperitoneal (i.p.) injection every other day, three times a week, for 2 weeks. Serum calcium was obtained on days 0 and 14 of treatment. Results: In the control group, the xenografts reached a maximum mean size of 336 mm3 (S.E.193 mm3) at day 16, and all mice had to be euthanized due to tumor burden by day 43. In the treatment group, the xenografts grew to a maximum mean size of 252 mm3 (S.E. 195 mm3) at day 13. One mouse in the treatment group was euthanized due to tumor burden at day 7, but three mice remained alive and healthy at day 93. At day 93, two treated mice had no palpable residual tumors and one treated mouse had stable tumors measuring 38 and 27 mm3. In the control and treatment groups, calcium levels at days 0 and 14 were within normal limits. Conclusions: Paricalcitol, given at 1.25 mcg/kg i.p. every other day for 2 weeks, produced a potent tumoricidal effect on SCC 15 xenografts without hypercalcemia in nude mice. The results of this study provide support for further investigations of paricalcitol in the treatment of head and neck cancers. No significant financial relationships to disclose.

A-ring hydroxymethyl 19-nor analogs of the natural hormone 1α,25-dihydroxyvitamin D 3: synthesis and preliminary biological evaluation

A series 5– 8 of 1- and 3-CH 2OH 19-nor analogs of the hormone calcitriol ( 1) has been prepared. Surprisingly, 19-nor 1α-CH 2OH analog 5a is more antiproliferative at 100 nM concentration than the corresponding regioisomeric analog 6a with the natural 1α-OH group, and 1α-CH 2OH hybrid analog 7a is similar in antiproliferative potency to calcitriol ( 1) even at low nanomolar concentrations.

Modulation of Vitamin D‐induced growth inhibition in melanoma cell lines: implications for an important function of vitamin D receptor (VDR) and 1,25‐dihydroxyvitamin D3‐24‐hydroxylase (24‐OHase) expression, histonedeacetylation, and calpain activity

Increasing evidence indicates an important role of the skin vitamin D system for tumorigenesis and progression of malignant melanoma. We have now characterized the vitamin D system in melanoma cell lines, detecting strong RNA expression of vitamin D receptor (VDR), 25‐hydroxyvitamin D‐1α‐hydroxylase (1αOHase), vitamin D‐25‐hydroxylase (25OHase) and 1,25‐dihydroxyvitamin D‐24‐hydroxylase (24OHase) in various melanoma cell lines (MEWO, SKMEL28, BU47HOM) using real time PCR (LightCycler) and specific hybridisation probes. We then have analyzed effects of 1,25‐dihydroxyvitamin D3 and analogs (EB 1089, MC 1288) on proliferation and apoptosis as well as on expression of VDR, 1αOHase), 25OHase, and 24OHase in various melanoma cell lines (BUHOM, MEWO, SKMEL)in vitro. Using a tetrazolium salt (WST‐1) based colorimetric assay, we detected dose‐dependent inhibition of cell growth (up to 40%) induced by calcitriol or its analogs. Treatment of melanoma cells with calcitriol resulted in decreased immunoreactivity of proliferation markers including Ki‐67 and PCNA. Flow cytometry experiments (bcl‐2, bcl‐xl, bcl‐xs, bcl‐x, bax, CD95) and analysis of annexin Pi expression revealed no induction of apoptosis by calcitriol (10−7 M) or its analogs, while increased levels of bcl‐2 protein were detected. Additionally, we show modulation of vitamin D‐induced inhibition of cell proliferation by calpain inhibitors I and II (Calbiochem) as well as by inhibitors of histonedeacetylation (sodium butyrate, trichostatin A). In conclusion our findings indicate that (i) vitamin D analogs suppress proliferation but do not induce apoptosis in melanoma cell lines, (ii) induction of VDR and 24‐OHase expression as well as histonedeacetylation and calpain activity modulate vitamin D‐induced growth inhibition, (iii) new calcitriol analogs exerting less systemic side effects may be interesting candidates for the treatment of metastasizing malignant melanoma.

Design and Synthesis of Novel 20-epiAnalogues of Calcitriol with Restricted Side Chain Conformation

The design and efficient preparation of two 20-epi analogues of calcitriol with restricted side chain conformation is described. The formation of the tetrahydropyran ring was achieved via zinc chloride mediated etherification of alcohols. Docking experiments show that these novel analogues could be interesting synthetic targets.

Side-chain-modified analogs of calcitriol cause resistance of human HL-60 promyelocytic leukemia cells to drug-induced apoptosis

Calcitriol and some of its analogs have antiproliferative activity, but at the same time, can cause resistance to apoptosis induced by known cytostatic drugs. In this paper, we examined the effects of treatment with calcitriol or its side-chain-modified analogs, analog of Vitamin D 2, coded PRI-1906, with monohomologated and unsaturated side-chain and the analog of Vitamin D 3, coded PRI-2191, with (24 R) hydroxyl group, and those of known cytostatics (genistein, etoposide, doxorubicin, cisplatin, and taxol) on the apoptosis of HL-60 promyelocytic leukemia cells. HL-60 cells were incubated in three different sequences: (1) pre-treatment with calcitriol or its analogs and then treatment with cytostatics; (2) pre-treatment with cytostatics and then treatment with calcitriol; (3) simultaneous treatment with calcitriol and cytostatics. Apoptosis was examined either by DNA fragmentation in agarose gel electrophoresis or by cell-cycle analysis in a FACS Calibur flow cytometer. We showed that pre-treatment with calcitriol or one of its side-chain-modified analogs PRI-1906 or PRI-2191 caused resistance of HL-60 promyelocytic leukemia cells to genistein-, doxorubicin-, cisplatin-, and taxol-induced apoptosis. Simultaneous exposure of HL-60 cells to calcitriol and drug caused a significant decrease in the apoptotic level of HL-60 cells compared with cells treated with drug alone. The pre-treatment of HL-60 cells with drug and then treatment with calcitriol did not increase the level of apoptosis compared with the drug effect alone. These results indicate the potential limitations of calcitriol analogs for treatment of leukemia.

The cytotoxicity of combined paricalcitol and docetaxel in head and neck squamous cell carcinomas

5609 Background: Paricalcitol is a calcitriol analog used in the treatment of secondary hyperparathyroidism. It produces less hypercalcemia compared to calcitriol and has low clinical toxicity. We ...

Anti-endothelial properties of 1,25-dihydroxy-3-epi-vitamin D 3, a natural metabolite of calcitriol

Background: Calcitriol [1,25-(OH) 2D 3] is a strong anti-proliferative agent both in vitro and in vivo. Earlier studies have established that calcitriol inhibits the growth factor-stimulated proliferation of endothelial cells (EC) and angiogenesis. However, the lethal calcemic side effects of calcitriol prohibit its use as a therapeutic agent. Several analogs of vitamin D have been developed to minimize these calcemic side effects. 1,25-dihydroxy–3–epi–vitamin D 3 (3-epiD 3), a naturally formed vitamin D metabolite is one such analog. Objective: To demonstrate that 3-epiD 3, a calcitriol analog, inhibits endothelial cell proliferation and induces apoptosis. Results: Treatment of EC with 3-epiD 3 showed 60% inhibition ( P<0.006) of proliferation. Cell viability assays corroborated these results. Pro-apoptotic caspase-3 activity was increased fourfold ( P<0.01) in 3-epiD 3-treated cells over controls. 3-epiD 3 induced apoptosis in EC as shown by genomic DNA fragmentation. Cell cycle analysis of 3-epiD 3-treated EC revealed a G0/G1 arrest. Conclusions: 3-epiD 3, a low-calcemic, natural analog of calcitriol, inhibits EC proliferation by causing a G0/G1 arrest and induces apoptosis more effectively than 1,25-(OH) 2D 3. These results suggest that 3-epiD 3 is a potent inhibitor of EC growth.

Anti-endothelial properties of 1,25-dihydroxy-3-epi-vitamin D3, a natural metabolite of calcitriol*1

Background: Calcitriol [1,25-(OH) 2 D 3 ] is a strong anti-proliferative agent both in vitro and in vivo. Earlier studies have established that calcitriol inhibits the growth factor-stimulated proliferation of endothelial cells (EC) and angiogenesis. However, the lethal calcemic side effects of calcitriol prohibit its use as a therapeutic agent. Several analogs of vitamin D have been developed to minimize these calcemic side effects. 1,25-dihydroxy–3–epi–vitamin D 3 (3-epiD 3 ), a naturally formed vitamin D metabolite is one such analog. Objective: To demonstrate that 3-epiD 3 , a calcitriol analog, inhibits endothelial cell proliferation and induces apoptosis. Results: Treatment of EC with 3-epiD 3 showed 60% inhibition ( P <0.006) of proliferation. Cell viability assays corroborated these results. Pro-apoptotic caspase-3 activity was increased fourfold ( P <0.01) in 3-epiD 3 -treated cells over controls. 3-epiD 3 induced apoptosis in EC as shown by genomic DNA fragmentation. Cell cycle analysis of 3-epiD 3 -treated EC revealed a G0/G1 arrest. Conclusions: 3-epiD 3 , a low-calcemic, natural analog of calcitriol, inhibits EC proliferation by causing a G0/G1 arrest and induces apoptosis more effectively than 1,25-(OH) 2 D 3 . These results suggest that 3-epiD 3 is a potent inhibitor of EC growth.

Inhibition of prostate cell growth by BXL-628, a calcitriol analogue selected for a phase II clinical trial in patients with benign prostate hyperplasia.

Calcitriol analogues might represent an interesting new therapy for benign prostate hyperplasia (BPH). We here report the preclinical characterization of BXL-628, an analogue selected for an ongoing double-blind, randomized, placebo-controlled phase II trial in BPH. Experiments with BXL-628 were carried out in human BPH cells and in the ventral prostate of intact and castrated rats. BPH cell and rat prostate growth were evaluated along with morphological and biochemical hallmarks of apoptosis. BXL-628 inhibited human BPH cell proliferation and induced apoptosis even in the presence of androgens or growth factors. It also decreased prostate growth to an extent similar to finasteride, inducing DNA fragmentation and apoptosis, both in intact and in testosterone-supplemented castrated rats. Accordingly, BXL-628, like finasteride, increased the expression of clusterin, a prostatic atrophy marker. However, BXL-628 did not inhibit 5 alpha-reductase 1 and 2, did not bind to the androgen receptor (AR) in BPH homogenates and did not affect AR-coupled luciferase activity. In addition, BXL-628 did not affect rat pituitary and testis activity or calcemia. BXL-628 inhibited in vitro and in vivo prostate cell proliferation, and therefore might represent a novel, interesting option for the treatment of BPH.

1349: Inhibition of Spontaneous and Androgen-Induced Prostate Growth by a Nonhypercalcemic Calcitriol Analogue

1349: Inhibition of Spontaneous and Androgen-Induced Prostate Growth by a Nonhypercalcemic Calcitriol Analogue

65 Inhibition of spontaneous and androgen-induced prostate growth by a non-hypercalcemic calcitriol analogue

65 Inhibition of spontaneous and androgen-induced prostate growth by a non-hypercalcemic calcitriol analogue

Vitamin D receptor and analogs

In chronic kidney disease (CKD), high circulating levels of parathyroid hormone (PTH) cause osteitis fibrosa, bone loss, and cardiovascular complications that increase morbidity and mortality. Impaired production of 1,25-dihydroxyvitamin D (calcitriol), the hormonal form of vitamin D, is a major contributor to the generation and maintenance of parathyroid hyperplasia and increased synthesis and secretion of PTH. Calcitriol inhibits PTH gene transcription and ameliorates parathyroid hyperplasia by suppressing the expression of and growth signals from the autocrine transforming growth factor α (TGFα)/epidermal growth factor receptor (EGFR)-growth loop, a main determinant of parathyroid cell proliferation. Calcitriol reduction of parathyroid hyperplasia and serum PTH levels demands a functional vitamin D receptor (VDR). Although VDR is normal in CKD, parathyroid VDR content is reduced markedly. Furthermore, VDR function, as a transcriptional regulator of vitamin D responsive genes, is impaired by several factors including hypocalcemia, hyperphosphatemia, accumulation of uremic toxins, and reduction in cellular levels of the VDR partner, retinoid X receptor. Therapy with calcitriol analogs can overcome the antagonism on calcitriol-VDR actions induced by CKD. Although not all analog formulations are equally effective, they offer a wider therapeutic window in counteracting vitamin D resistance and survival advantage over exclusive calcitriol therapy.

Prediction of in vitro metabolic stability of calcitriol analogs by QSAR.

The metabolic stability of a drug is an important property for potential drug candidates. Measuring this property, however, can be costly and time-consuming. The use of quantitative structure-activity relationships (QSAR) to estimate the in vitro stability is an attractive alternative to experimental measurements. A data set of 130 calcitriol analogs with known values of in vitro metabolic stability was used to develop QSAR models. The analogs were encoded with molecular structure descriptors computed mainly with the commercial software QikProp and DiverseSolutions. Variable selection was carried out by five different variable selection techniques and Partial Least Squares Regression (PLS) models were generated from the 130 analogs. The models were used for prediction of the metabolic stability of 244 virtual calcitriol analogs. Twenty of the 244 analogs were selected and the in vitro metabolic stability was determined experimentally. The PLS models were able to predict the correct metabolic stability for 17 of the 20 selected analogs, corresponding to a prediction performance of 85%. The results clearly demonstrate the utility of QSAR models in predicting the in vitro metabolic stability of calcitriol analogs.