Immunomodulation by a novel, dissociated Vitamin D3 analogue

Abstract:  The biologically active metabolite of vitamin D₃, 1 α ,25-dihydroxyvitamin D₃, has potent immunomodulatory activity; however, its clinical use is limited because of its hypercalcaemic activity in anti-inflammatory active doses. Here, we present ZK203278, a novel, structurally different vitamin D₃ analogue with profound immunomodulatory activities. It potently inhibits lymphocyte proliferation in the mixed lymphocyte reaction, and release of cytokines that are central in inflammation, such as TNF α and IL-12 in the low nanomolar range. Similarly, expression of cell-surface molecules involved in cell adhesion and antigen presentation, e.g. to T cells, is down-regulated on human monocytes by low nanomolar concentrations of ZK203278. Potent anti-inflammatory activity has been demonstrated also in vivo in rodent disease models. ZK203278 inhibited allergic contact dermatitis in rodents after oral administration in doses approximately two orders of magnitude below the hypercalcaemic threshold dose. Moreover, ZK203278 significantly prolonged skin allograft survival in rats in well-tolerated doses. Altogether ZK203278, in contrast to 1 α ,25-dihydroxyvitamin D₃, exerts considerable immunomodulatory activity at non-hypercalcaemic dosages and may have therapeutic potential for immune disorders or transplant rejection.     

Clinical experience with topical calcitriol (1,25-dihydroxy-vitamin D3) in psoriasis

The use of vitamin D analogues for the treatment of chronic plaque psoriasis is well documented. Of importance now is their comparability and compatibility with other established treatments for psoriasis. This paper reviews five studies with calcitriol 3 μg g⁻¹ ointment (Silkis ointment®, Galderma Laboratories). Calcitriol applied twice daily was found to be as effective as short-contact dithranol in terms of global improvement and PASI scores. However, patients favoured calcitriol over dithranol when both quality of life and treatment acceptability were assessed. Two studies provide evidence of the benefit of combining calcitriol with other antipsoriatic therapies. Combination with ultraviolet (UV) B phototherapy proved as effective as UVB alone over an 8-week period; however, the combination had a radiation dose-sparing effect, thus reducing the risk of adverse events. Likewise, calcitriol combined with betamethasone valerate (each applied separately, once daily) was as efficacious as twice-daily betamethasone, thereby achieving a corticosteroid-sparing effect. Finally, two studies confirm that calcitriol 3 μg g⁻¹ ointment can be used safely in patients with psoriasis of the head and confirm the high level of clinical efficacy achieved with this compound.     

Effect of a topical corticosteroid, a retinoid and a vitamin D3 derivative on sodium dodecyl sulphate induced skin irritation

Exposure of the skin to sodium dodecyl sulfate (SDS) leads to disruption of barrier and skin irritation. We used repetitive short exposure to a low molarity SDS solution as an in vivo model to mimic the development of irritant contact dermatitis. In this model, we studied clinical (erythema), functional (transepidermal water loss(TEWL)) and cell biological changes, 24 healthy volunteers were patch tested with SDS (0.2%) for 4 h a the day for 5 consecutive days. After removal of the patches, the exposed sites were treated 1 × daily either with a topical corticosteroid (triamcinolon acetoide cream 0.05%). a retinoid (tretinoin cream 0.025%). or a vitamin D₃ derivative (calcipotriol ointment 50 microgram/g). Irritant reactions were assessed by erythema scoring and measurement of barrier function with TEWL up to 14 days after the first challenge. Skin biopsies were taken for cell biological changes at day 4. Vehicle-treated sites served as controls. Repetitive exposure of human skin to SDS resulted in a gradual increase in erythema scoring and TEWL associated with the upregulation of proliferative cells as measured by the expression of Ki-67-antigen and of differentiation markers, visualized by increased expression of involucrin and epidermal-fatty-acid binding protein (E-FABP). Skin irritation as assessed by erythema scoring and TEWL was not significantly suppressed by triamcinolone cream. However, a significant reduction of the number of cycling keratinocytes and a decrease in involucrin positive cell layers was observed in this group. Neither treatment with calcipotriol ointment nor with tretinoin cream induced improvement of skin irritation as judged by visual scoring and TEWL. In contrast to steroid treatment no significant elf eel of calcipotriol ointment or tretinoin cream treatment was observed with regard to the number of cycling cells and differentiation markers. Further studies are needed to assess whether treatment with topical corticosteroids is an effective modality in skin irritation and irritant contact dermatitis.     

All-trans retinoic acid inhibits binding of 1,25-dihydroxy-vitamin D3 to the vitamin D receptor in cultured human keratinocytes

Abstract Psoriasis is characterized by hyperproliferation and impared differentiation of epidermal keratinocytes (KCs). Psoriasis can be treated with derivatives of relinoic acid (RA) and vitamin D₃. Analogues of vitamin D₃ are able to inhibit proliferation and stimulate differentiation of KCs. In contrast, RA inhibits terminal differentiation of KCs. Interactions are known to occur between RA and vitamin D₃ signalling pathways. The purpose of the present study was to determine the effect of all-trans RA on the binding of 1,25-dihydroxy-vitamin D₃ (1,25 (OH)₂D₃) to the vitamin D₃, receptor (VDR) of cultured human KCs. Cultured KCs from normal adults were incubated with or without RA (10^?9–10^?7 M) for 4-24 h. Cells were then harvested, homogenized and ultrasonicated. The extracted protein was incubated with 3H-1,25 (OH)₂D₃ (0.015-1.0 nM) with or without 250-fold excess nonradioactive 1,25 (OH)₂D₃ for 24 h and specific binding was determined by use of the dextran coated charcoal binding assay. Western blot analysis utilizing the monoclonal antibody 9A7γ to VDR was performed on protein extracted from the KCs. The bands resulting from Western blot analysis were visualized by enhanced chemiluminescence. From Scatchard analysis it was found that KCs bind 1,25 (OH)₂D₃ with high affinity (Kd = 0.175 nM). This binding was dose and time dependently inhibited by RA (60% inhibition at 10^?7 M after 24 h of incubation). By Western blot analysis, RA had no effect on the amount of protein extracted from KCs at any of the RA concentrations tested. In conclusion, these results show that binding of vitamin D₃ to its receptor of human KCs can be inhibited markedly by RA without effecting the amount of protein. These results are in contrast to results with other cell types in which RA upregulates binding of 1,25 (OH)₂D₃ to the VDR. Because interaction between retinoids and vitamin D₃ may occur at different levels during signal trans-duction, it is not possible to predict from our results whether RA will inhibit the effects of vitamin D₃, in vivo.     

Conversion of vitamin D3 to 1alpha,25-dihydroxyvitamin D3 in human skin equivalents

These results demonstrate for the first time that human keratinocytes under in vivo-like conditions have the capacity of the enzymatic hydroxylation of vitamin D3 to hormonally active calcitriol (1alpha,25(OH)2D3). Supplementation of the culture medium with bovine serum albumin (BSA) up to 1.5%) (w/v) amplifies the conversion of vitamin D3 to 1alpha,25(OH)2D3. The maximum turnover rate of this reaction at 780 nM vitamin D3 in presence of 1.0% (w/v) BSA amounts to approximately 3 pmol 1alpha,25(OH)2D3 per 10(6) cells after 6 h of incubation. The hydroxylation of vitamin D3 to 1alpha,25(OH)2D3 is inhibited by the P-450 oxidase inhibitor ketoconazole. The generation of 1alpha,25(OH)2D3 from vitamin D3 has an apparent Michaelis constant (Km) of 2.3x10(-6) M. The intrinsic conversion of vitamin D3 to biologically active 1alpha,25(OH)2D3 may be of importance for the regulation of proliferation and differentiation of keratinocytes.     

1α,25(OH)2 vitamin D3 increases intracellular calcium in human keratinocytes

Vitamin D3 metabolites have been found to improve psoriasis but their mechanism of action is not clear. Keratinocyte proliferation and differentiation are known to be dependent on calcium concentrations in vitro. The aim of this study was to examine whether 1 alpha,25(OH)2 vitamin D3 had any direct effect on intracellular free calcium concentrations in cultured keratinocytes. A response to 1 alpha,25(OH)2 vitamin D3 was seen in 88% of monolayers of normal human keratinocytes attached to glass coverslips. An increase in intracellular free calcium was seen in 80% of the reactive cultures, with over half the responses occurring within 30 s of exposure to 1 alpha,25(OH)2 vitamin D3 and the remainder occurring within minutes. Responses could be seen at physiological concentrations of 1 alpha,25(OH)2 vitamin D3 and were not blocked by the protein synthesis inhibitor cycloheximide. The response to 1 alpha,25(OH)2 vitamin D3 took the form of rapid transient increases in intracellular free calcium in 29 out of 59 coverslips. The basal intracellular free calcium was calculated to be 245 +/- 47 nM rising to a maximum of 834 +/- 267 nM (mean +/- SEM; n = 20) following exposure to 1 alpha,25(OH)2 vitamin D3. We conclude that 1 alpha,25(OH)2 vitamin D3 acts directly on keratinocytes to increase intracellular free calcium and that this may be relevant to its mechanism of action in psoriasis.     

Effects of lithium carbonate (Li2CO3) on in-vitro-cultured normal human skin explants

The early morphological changes induced by lithium carbonate, a well-known psoriasis-provoking drug, were studied on cultured skin. Normal human skin from patients undergoing mastectomy was cultured in the presence of 3 mM, 6 mM and 10 mM of Li2CO3 for 4 days. The morphological changes were then evaluated by three observers in a blind manner and their reports were matched and collated. The cultured skin in the presence of Li2CO3 showed cell crowding of keratinocytes in the lower part of the epidermis, indicating epidermal hyperplasia. Another striking finding was intercellular oedema and vacuolar alteration with formation of small cavities in the upper dermis. There was no evidence of parakeratosis or any other histological characteristic of psoriasis, except hyperproliferation of the epidermis. Based on our knowledge of mechanisms of lithium action, we proposed two competitive explanations for its action on the epidermis: i) that lithium acts directly on dividing cells of the epidermis; and ii) that it acts indirectly by altering epidermal barrier function. Although we lack definite proof, we suggest that the observed morphological changes, in particular the non-specific stimulus to epidermal proliferation, are the primary events which initiate the process that will ultimately lead to the development of psoriasis in a predisposed patient.     

DNA content and Ks8.12 binding of the psoriatic lesion during treatment with the vitamin D3 nalogue MC903 and betamethasone

Twenty patients with psoriasis were treated with the vitamin D3 analogue MC903 and betamethasone ointment in a double-blind trial with a left-right comparison. In addition to the clinical severity scores, Ks8.12 binding which detects keratin 16 expression and the DNA synthesis were quantified using flow cytometry. Both markers decreased significantly with treatment, but remained above the normal range even in those who had total clearance of the lesions. Treatment with MC903 with regard to Ks8.12 binding was significantly better than with betamethasone.     

RANTES expression in psoriatic skin, and regulation of RANTES and IL-8 production in cultured epidermal keratinocytes by active vitamin D3 (tacalcitol)

The chemokine RANTES is a chemoattractant for eosinophils, T lymphocytes of memory phenotype and monocytes, suggesting that it plays an important part in chronic inflammatory and allergic diseases. In various types of cells, RANTES production is markedly induced by tumour necrosis factor (TNF)-α and interferon (IFN)-γ in combination. Psoriasis vulgaris is a chronic cutaneous inflammatory disease. Cytokines and chemokines produced by T cells and epidermal keratinocytes, such as interleukin (IL) 8, are involved in the pathogenesis of psoriasis. T-cell clones obtained from psoriatic skin have been shown to produce the Th1 cytokine IFN-γ. In addition, abnormal expression of proinflammatory cytokines including TNF-α has been observed in psoriatic lesions. These reports led us to hypothesis that psoriatic skin could provide epidermal keratinocytes with TNF-α and IFN-γ, so that keratinocytes could produce RANTES. In this study, we addressed the question as to whether RANTES was involved in psoriasis vulgaris. Immunohistochemistry of skin biopsies showed RANTES was present in the intercellular spaces between epidermal keratinocytes, in the fully developed lesions from the middle to the edge of psoriatic plaques, but not in the perilesional uninvolved and healthy control skin. Further, we confirmed the production of RANTES, together with IL-8, by cultured normal human epidermal keratinocytes, using an enzyme-linked immunosorbent assay. Stimulation with TNF-α and IFN-γ in combination synergistically increased the RANTES production in this system. These results clearly demonstrate the expression of RANTES in psoriatic lesions and suggest the involvement of this chemokine in the outcome of cutaneous inflammatory diseases. Tacalcitol (1α,24(R)-dihydroxyvitamin D₃), an active vitamin D₃ analogue, inhibited RANTES and IL-8 production in cultured normal epidermal keratinocytes. This result indicates that active vitamin D₃ is effective in the regulation of chemokine production by epidermal keratinocytes, which may partly account for its action as an antipsoriatic drug.     

A double-blind study of topical 1α,25-dihydroxyvitamin D3 in psoriasis

Several open studies with active forms of vitamin D3 have been reported to be effective in the treatment of psoriasis. We report a double-blind study of 1 alpha,25-dihydroxycholecalciferol in psoriasis using 0.5 microgram of active substance applied topically twice daily. In contrast to previous studies, the present investigation does not provide evidence of clinical efficacy for the drug at that dosage and with the vehicle that was used.     

The epidermis of chronic idiopathic lichen planus during topical treatment with the vitamin D3 analogue KH 1060

A parallel-group, double-blind, randomised study was performed to establish the effect of the vitamin D₃ analogue KH 1060, in an ointment versus vehicle only, on the epidermal cell characteristics of chronic idiopathic lichen planus; KH 1060 also has marked immunosuppressive activity.   A group of 10 patients were treated for 8 weeks with either KH 1060 ointment or vehicle only. In addition to the assessment of clinical scores, keratotome biopsies were taken before and after 8 weeks' treatment. Epidermal cell suspensions were prepared with trypsin and the suspensions incubated with TO-PRO-3 (DNA marker), RKSE 60 (marker for keratin 10-positive cells) and antivimentin (marker for all non-keratinocytes). In nine of the 10 patients, keratotome biopsies were obtained both before and after 8 weeks treatment. The vehicle alone had no significant effect on the clinical severity scores or epidermal cell characteristics. In contrast, the KH 1060 ointment resulted in a statistically significant reduction in the percentage of cells in S- and G₂M phase and the percentage of vimentin-positive cells, but it did not affect the percentage of keratin 10-positive cells.     

Improvement of psoriasis by a topical vitamin D3 analogue (MC 903) in a double-blind study

The effect of the non-calciotropic vitamin D3 analogue MC 903 on psoriasis vulgaris was assessed in a double-blind, placebo controlled trial in 30 patients. Lesions on one side of the body were treated for 6 weeks with a cream containing 10 μ/g, 33 μg//g or 100 μg/g MC 903 and lesions on the other side were treated with the cream base alone, according to a randomized design. Nine of the 10 patients in each treatment group completed the study. MC 903 cream gave a statistically significant decrease in erythema, thickness and scaling of the lesions, compared with the control. Overall assessment of psoriasis after 6 weeks showed moderate or excellent improvement in two of nine patients treated with 10 μg/g, in five of nine patients treated with 33 μ/g, and in seven of nine patients treated with 100 μ/g MC 903. Placebo treatment showed a moderate improvement in only one of the 27 patients. The histopathological picture of the psoriatic lesions corresponded with the clinical changes. The patients reported no adverse reactions, and laboratory tests did not show any significant changes; in particular there was no change in serum calcium levels. These results suggest that the vitamin D₃ analogue MC 903 is an effective and safe topical treattnent for psoriasis.     

SkinChip®, a new tool for investigating the skin surface in vivo

Background/aims: Non-invasive methods used for characterizing skin micro-relief and skin surface hydration were developed in the 1980s. Although they allowed some progress in the knowledge of skin properties, they are not completely satisfactory in many aspects. Today, new technologies are emerging that may address such issues.
Methods: We adapted the technology produced by the ST Microelectronics Company for sensing fingerprint for the measurement of skin surface properties. Accordingly, we developed acquisition software for obtaining routinely the distribution of skin surface capacitance along different body sites. Image analysis softwares were also processed for collecting both the main orientations of the micro-relief lines and their density. The average value of skin capacitance is also obtained.
Results: The images allow a highly precise observation of the skin topography that can be easily quantified in terms of line density and line orientation. The mean gray levels of the images appear much closely correlated to the Corneometer values.
Conclusion: This new device appears to be a very convenient way for characterizing the properties of the skin surface. With regard to hydration, it usefully provides both the average value and the hydration chart of the investigated skin zones.