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1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) is known to inhibit the proliferation and invasiveness of prostate cancer cells. However, 1alpha,25(OH)(2)D(3) can cause hypercalcemia and is not suitable as a therapeutic agent. 19-Nor-vitamin D derivatives are known to be less calcemic when administered systemically. In order to develop more potent anti-cancer agents with less calcemic side effect, we therefore utilized (3)H-thymidine incorporation as an index for cell proliferation and examined the antiproliferative activities of nine C-2-substituted 19-nor-1alpha,25(OH)(2)D(3) analogs in the immortalized PZ-HPV-7 normal prostate cell line. Among the nine analogs we observed that the substitution with 2alpha- or 2beta-hydroxypropyl group produced two analogs having antiproliferative potency that is approximately 500- to 1000-fold higher than 1alpha,25(OH)(2)D(3). The (3)H-thymidine incorporation data were supported by the cell counting data after cells were treated with 1alpha,25(OH)(2)D(3), 19-nor-2alpha-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) or 19-nor-2beta-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) for 7 days. 19-Nor-2alpha-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) and 19-nor-2beta-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) were also shown to be about 10-fold more active than 1alpha,25(OH)(2)D(3) in cell invasion studies using prostate cancer cells. In conclusion, a substitution at the C-2 position of 19-nor-1alpha,25(OH)(2)D(3) molecule with a hydroxypropyl group greatly increased the antiproliferative and anti-invasion potencies. Thus, these two analogs could be developed to be effective therapeutic agents for treating early and late stages of prostate cancer.

Background: Although topical 1,25-dihydroxyvitamin D3 (calcitriol, 1, 25(OH)2D3) and its analogues, calcipotriol and tacalcitol, are effective for patients with psoriasis, some patients show little or no response. There is a need to develop more potent analogues of 1, 25(OH)2D3. Hexafluoro-1,25(OH)2D3 (F6-1,25(OH)2D3) is at least 10 times more potent than 1,25(OH)2D3 on calcium metabolism., Objectives: We were interested in whether F6-1,25(OH)2D3 was also more potent than 1,25(OH)2D3 in inhibiting normal human and psoriatic keratinocyte proliferation., Methods: The antiproliferative activity of F6-1,25(OH)2D3 and 1,25(OH)2D3 was determined by 3H-thymidine incorporation into keratinocyte DNA and by counting basal cells., Results: F6-1,25(OH)2D3 was approximately 10-fold more active and had a longer lasting antiproliferative effect than 1,25(OH)2D3 on normal human keratinocytes, and was about 100-fold more potent than 1, 25(OH)2D3 on human psoriatic keratinocytes as determined by 3H-thymidine incorporation. F6-1,25(OH)2D3 also caused a dose-dependent decrease in the number of basal cells and was 100-fold more active than 1,25(OH)2D3., Conclusions: The increased potency and the long-lasting effects of F6-1,25(OH)2D3 suggest that F6-1,25(OH)2D3 may be a potent candidate agent for treating psoriasis.

Epidemiological and laboratory data support a role for vitamin D in the growth and differentiation of human prostatic cells. These findings prompted us to ask whether prostatic cells could convert 25-hydroxyvitamin D3 (25-OH-D3), the major circulating metabolite of vitamin D3, to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the hormonally active metabolite, in a manner similar to cultured human keratinocytes. Therefore, we investigated three well-characterized human prostate cancer cell lines, LNCaP, DU 145, and PC-3; two primary cultures of cells derived from noncancerous human prostates (one normal and one benign prostatic hyperplasia); and primary cultures of normal human keratinocytes for their ability to synthesize 1,25(OH)2D3. Assays were performed in the presence of 25-OH-D3 as the enzyme substrate and 1,2-dianilinoethane, an antioxidant and free radical scavenger, and in the presence and absence of clotrimazole, a cytochrome P450 inhibitor. DU 145 and PC-3 cells produced 0.31 +/- 0.06 and 0.07 +/- 0.01 pmol of 1,25(OH)2D3/mg protein/h, respectively. No measurable 1,25(OH)2D3 was detected in LNCaP cells. The normal and benign prostatic hyperplasia primary cultures and keratinocyte cultures produced 3.08 +/- 1.56, 1.05 +/- 0.31, and 2.1 +/- 0.1 pmol of 1,25(OH)2D3/mg protein/h, respectively, using a calf thymus receptor binding assay to measure 1,25(OH)2D3 in the presence of 1,2-dianilinoethane. The identity of the analyte as 1,25(OH)2D3 was supported by high performance liquid chromatography using [3H]25-OH-D3 as the enzyme substrate and a solvent system that is specific for 1,25(OH)2D3. The production of 1,25(OH)2D3 in the prostate cancer cell lines and in the primary cultures was completely inhibited in the presence of clotrimazole. This report demonstrates that two of three human prostate cancer cell lines, as well as primary cultures of noncancerous prostatic cells, possess 1alpha-hydroxylase activity and can synthesize 1,25(OH)2D3 from 25-OH-D3. Together with recent data indicating that 1,25(OH)2D3 inhibits the invasiveness of human prostate cancer cells (G. G. Schwartz et al., Cancer Epidemiol. Biomark. Prev., 6: 727-732, 1997), these data suggest a potential role for 25-OH-D3 in the chemoprevention of invasive prostate cancer.

Increasing evidence suggests involvement of integrins and CD44 isoforms in the pathogenesis of psoriasis, contributing to uncontrolled keratinocyte proliferation, neovascularization, and invasion of inflammatory cells. We have analyzed immunohistochemically in situ expression of integrins (CD29, CDw49b, CDw49c, CDw49e, CDw49f) and CD44 isoforms (CD44 standard, CD44 var/v6, CD44 v10) on frozen sections of normal and psoriatic skin (nonlesional skin, lesional skin before and along with topical calcitriol treatment). We did not observe visual changes of immunoreactivity in normal as compared to nonlesional psoriatic skin, while the staining pattern of CDw49c, CDw49f, and CD29 was severely altered in untreated lesional psoriatic skin. Most markedly, CDw49c, CDw49f, and CD29 were focally upregulated in suprapapillar epidermal compartments of lesional psoriatic skin, a staining pattern that is in accordance with the phenomenon that was described by Pinkus as "squirting papilla". Additionally, an increased proportion of inflammatory and endothelial cells revealed immunoreactivity for CD44(std.) in untreated lesional psoriatic as compared to nonlesional psoriatic or normal skin. After 8 weeks of topical calcitriol treatment (15 micrograms/g ointment), the staining pattern for CDw49c, CDw49f and CD29 was markedly changed in epidermis of lesional psoriatic skin, reverting to the staining pattern characteristic for the nonlesional psoriatic or normal human skin, although epidermal expression of CDw49f was still upregulated and CDw49e-, CDw49f-, CD29-, and CD44(std.)-immunoreactive inflammatory and endothelial cells were still to be found in the dermal compartment.

The potent calciotropic hormone calcitriol (1,25-dihydroxyvitamin D3, 1,25(OH)2D3) has been shown to be very effective and safe in the topical treatment of psoriasis. In vitro, 1,25(OH)2D3 inhibits proliferation and stimulates differentiation of human keratinocytes. Increasing evidence suggests an immunoregulatory function of this potent steroid hormone. To further characterize the biological effects of topical calcitriol treatment in psoriasis, we have analyzed immunohistochemically the expression of markers for epidermal proliferation (proliferating cell nuclear antigen=PCNA) and differentiation (transglutaminase K, involucrin, cytokeratin 16), as well as inflammation (CD1a, 55 kDa TNF-receptor, NAP-1/IL-8) in calcitriol-treated psoriatic skin in situ. Our findings strongly support the hypothesis that calcitriol modulates keratinocyte proliferation/differentiation as well as inflammation in human skin in vivo. The immunoreactivity of markers for epidermal proliferation and differentiation, as well as of CD1a and NAP-1/IL-8, changed after 8 weeks of calcitriol treatment almost completely to the pattern characteristic for non-lesional psoriatic skin, while a large number of 55 kDa TNF-receptor positive cells could be found in the dermal compartment.

Plaque-type psoriasis has been successfully treated with oral calcitriol, but there has been no long-term follow-up on the safety and efficacy of this calciotropic hormone for psoriasis. In a single centre study, patients were enrolled in an open trial to evaluate the efficacy and safety of oral calcitriol for psoriasis. Of the 85 patients who received oral calcitriol, 88.0% had some improvement in their disease; 26.5, 36.2 and 25.3%, had complete, moderate and slight improvement in their disease, respectively. The mean baseline psoriasis area severity index score (PASI) of 18.4 +/- 1.0 was reduced to 9.7 +/- 0.8 and 7.8 +/- 1.3 after 6 and 24 months on oral calcitriol therapy. Serum calcium concentrations and 24 h urinary calcium excretion increased by 3.9% and 148.2%, respectively, but were not outside the normal range. Bone mineral density remained unchanged. The clearance of creatinine decreased by 13.4% from baseline during the first 6 months of treatment, and thereafter, remained unchanged after 3 years of follow up. An evaluation of creatinine, inulin and paraaminohypurate (PAH) clearance was performed in eight patients. After 6 months on oral calcitriol, there was a 22.5% decline in creatinine clearance but no significant changes were observed in either inulin or PAH clearance, suggesting that calcitriol alters creatinine metabolism or secretion but does not affect renal function. Oral calcitriol is effective and safe for the treatment of psoriasis.

Psoriasis is a skin disorder characterized by hyperproliferation of epidermal keratinocytes. 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) and its analogs have been shown to inhibit keratinocyte proliferation in vitro and to be therapeutically effective for the treatment of psoriasis. Some patients with psoriasis, however, do not have a favorable response to 1 alpha,25 (OH)2D3 therapy. To evaluate the differential responsiveness to 1 alpha (OH)2D3 treatment, we examined the expression of vitamin D receptor mRNA in psoriatic lesions by reverse transcription-polymerase chain reaction using glyceraldehyde-3-phosphate dehydrogenase as an internal control. In this double-blind clinical trial, we recruited 18 patients who received topical treatment of 1 alpha,25(OH)2D3 (15 microgram/g Vaseline) or placebo on separated psoriatic lesions for 8 weeks. In patients who showed >90% clinical improvements of their psoriatic lesions with 1 alpha,25(OH)2D3 (n=9), an increase of 130+/-37% in vitamin D receptor mRNA level was observed in 1 alpha,25(OH)2D3-treated lesions when compared with the corresponding placebo controls. There was no increase in vitamin D receptor mRNA level in the lesions treated with this drug in patients who did not respond to the treatment. These data suggest that the antiproliferative activity of 1 alpha,25(OH)2D3 is closely associated with the expression of its cognate receptor.

Plaque-type psoriasis has been successfully treated with topical calcitriol, but there has been no long-term follow-up study of the safety and efficacy of this calciotropic hormone. In a single-centre study, patients with plaque or erythrodermic psoriasis were enrolled in a double-blind, right/left comparison, placebo-controlled study, and received 1.5 micrograms of calcitriol (15 micrograms/g of Vaseline) per day, or a placebo consisting of Vaseline alone. A subset of these patients (n = 22), with at least 25% involvement, applied 0.1 g of calcitriol ointment/50 cm2 on an area of from 2,500 to 5,000 cm2. Of the 84 patients enrolled in the double-blind control study, 96.5% responded to topical calcitriol therapy, compared with 15.5% whose lesions improved with Vaseline alone, after 2.4 months. After completion of the double-blind study, 22 patients applied calcitriol ointment (15 micrograms/g Vaseline) to all of their lesions (up to 10 g of calcitriol ointment; 150 micrograms calcitriol lesions showed either excellent or moderate clearing in 90.9% of all cases. The remaining 9.1% of cases showed slight improvement of their lesions. No abnormalities in calcium metabolism were noted in any of the patients using topical calcitriol. None of the patients experienced any local cutaneous side-effects, including six patients who applied calcitriol ointment to the face. Topical calcitriol is safe and effective for the treatment of psoriasis.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), has diverse effects in a variety of tissues and cell types, including skin. Since 1,25(OH)2D3 affects both fibroblast and keratinocytes, we evaluated the effect of 1,25(OH)2D3 on wound healing. We investigated the effect of the topically applied 1,25(OH)2D3 or vehicle on the healing of cutaneous wounds in rats in a blinded manner. Wound areas were measured by planimetry technique. Healing was expressed as the percentage of the original wound area that was healed. 1,25(OH)2D3 at concentrations between 5 and 50 ng/day caused a dose-dependent acceleration of healing. Time course and specificity studies indicated that 1,25(OH)2D3 specifically promoted healing between 1-5 days after wounding as compared with vitamin D (0.5 microgram/day), which showed no significant improvement over control. Our results suggest that 1,25(OH)2D3 and its analogues may be a new class of compounds that could be developed to enhance wound healing.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is a potent inhibitor of keratinocyte proliferation, as well as a stimulator of epidermal terminal differentiation. In the present studies, we investigated the influence of epidermal growth factor (EGF) and insulin on the antiproliferative and differentiation activities of 1,25(OH)2D3. Our results indicate the following: (1) EGF caused a dramatic potentiation of the 1,25(OH)2 D3-induced inhibition of 3H-thymidine incorporation into keratinocytes in a dose-dependent manner; (2) insulin acted antagonistically on the EGF-dependent potentiation of the 1,25(OH)2D3-induced antiproliferative activity; (3) transforming growth factor-alpha potentiated 1,25(OH)2D3-induced antiproliferative activity similar to EGF; (4) the EGF effect was not dependent upon 1,25(OH)2D3 receptor mRNA up-regulation; and (5) removal of insulin from medium supplemented with growth factors significantly potentiated the 1,25(OH)2D3-induced inhibition on the number of basal cells and the 1,25(OH)2D3-dependent cornified envelope formation. In conclusion, the antiproliferative activity of 1,25(OH)2D3 in cultured normal human keratinocytes is greatly enhanced by EGF or transforming growth factor-alpha and reduced by insulin. Insulin also inhibits 1,25(OH)2D3-induced terminal differentiation of cultured normal human keratinocytes.

It is well documented that 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25[OH]2D3), the most active vitamin D metabolite, inhibits epidermal keratinocyte proliferation and promotes differentiation. 1 alpha,25(OH)2D3 can be produced in keratinocytes from 25-hydroxyvitamin D3 by the enzyme 25-hydroxyvitamin D3-1 alpha-hydroxylase (1-OHase). Hydroxylation of 1 alpha,25(OH)2D3 by 25-hydroxyvitamin D3-24-hydroxylase (24-OHase), the first step in the catabolic pathway of 1 alpha,25(OH)2D3 could significantly reduce the intracellular concentration of 1 alpha,25(OH)2D3. Therefore, the expression of 24-OHase could have a critical regulatory role in 1 alpha,25(OH)2D3-dependent gene expression. As a first step to examine this possibility, the steady state level of 24-OHase mRNA in cultured human keratinocytes (CHK) was investigated. 24-OHase mRNA was not detected in control CHK. 1 alpha,25(OH)2D3 caused a dose- and time-dependent increase in 24-OHase mRNA level. The highest accumulation of 24-OHase mRNA was observed in CHK treated with 0.1-1 microM 1 alpha,25(OH)2D3. The level of 24-OHase mRNA reached a plateau 12-24 hr after 1 alpha,25(OH)2D3 treatment. 1 beta,25-dihydroxyvitamin D3, the stereoisomer of 1 alpha,25(OH)2D3, failed to induce 24-OHase mRNA expression significantly. In addition to 24-OHase mRNA, a 1.0-kb mRNA hybridized strongly with both rat and human 24-OHase cDNA probes. The origin of this 1.0-kb message is unknown at present, however, it was regulated by 1 alpha,25(OH)2D3. These results demonstrate that 1 alpha,25(OH)2D3 up-regulates the expression of 24-OHase mRNA, and this may be an important first step in the initiation of catabolism of 1 alpha,25(OH)2D3 in human keratinocytes.

It has been postulated that hyperparathyroidism in chronic renal failure results from hypocalcemia, occurring, in part, from phosphate retention and/or deficient 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] synthesis. However, many studies have failed to demonstrate hyperphosphatemia or low 1,25-(OH)2D levels in patients with mild renal failure. We measured creatinine clearance (CCr), fractional excretion of phosphorus (FEP), and serum phosphorus, ionized calcium, and plasma N-terminal PTH, and 1,25-(OH)2D concentrations in 21 normal subjects and 51 patients with renal failure. Patients with mild renal failure (Ccr, greater than 40 mL/min.1.73 m2) had normal mean serum phosphorus and ionized calcium and decreased mean 1,25-(OH)2D levels compared with those in normal subjects. In patients with moderate renal failure (CCr, 20-40), the mean ionized calcium level was normal, plasma PTH levels and FEP were elevated, and the decrement in 1,25-(OH)2D was more pronounced. The mean ionized calcium level was decreased only in the group of patients with severe renal failure (CCr, less than 20). The 1,25-(OH)2D values correlated positively with CCr and negatively with the log of plasma PTH and serum phosphorus concentrations. Log of plasma PTH correlated negatively with CCr and positively with FEP. The ionized calcium concentration correlated very weakly with CCr and the log of the plasma PTH level. These data demonstrate the presence of hyperparathyroidism, normocalcemia, and 1,25-(OH)2D deficiency in renal failure and are consistent with a role for 1,25-(OH)2D in the suppression of parathyroid activity through as yet unidentified mechanisms.