kh-1060 and Hyperplasia

KH 1060 is the 20-epi-22-oxa-24a-homo-26,27-dimethyl analogue of the natural hormone, 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). We have previously shown that after topical application in hairless mice both KH 1060 and 1 alpha,25(OH)2D3 cause epidermal hyperproliferation. MC 1582 differs from KH 1060 by the lack of hydroxyl group in the side chain which is required for receptor binding. We found that MC 1582 strongly stimulates epidermal hyperplasia in hairless mice after topical application in vivo, approaching in potency KH 1060. A similar, although much weaker response was also obtained with 1 alpha-hydroxyvitamin D3 (1 alpha(OH)D3). Since only the vitamin D compounds which possess hydroxyl groups both in the position 1 alpha and in the side chain, bind to the vitamin D receptor, we suggest that a local metabolism of MC 1582 and 1 alpha(OH)D3 takes place in the skin to the active, side-chain-hydroxylated species, probably to KH 1060 and 1 alpha,25(OH)2D3. This study suggests that 1 alpha hydroxylated prodrugs may be of use in the dermatological treatment of the future.

Topics: Analysis of Variance; Animals; Antineoplastic Agents; Calcitriol; Female; Hydroxylation; Hyperplasia; Mice; Mice, Hairless; Mice, Inbred C3H; Prodrugs; Receptors, Calcitriol; Skin; Vitamin D

The hormonal forms of vitamin D3 (1,25-dihydroxyvitamin D3 and synthetic vitamin D3 analogues) are potent regulators of keratinocyte growth and stimulators of keratinocyte differentiation. Recent experiments in vitro on cultured keratinocytes indicate that Ca2+ may be a second messenger mediating the effects related to the induction of keratinocyte differentiation by the hormonal forms of vitamin D3. In this study we employed the technique of ion capture cytochemistry to investigate the effects of a potent vitamin D3 analogue, KH 1060 (20-epi-22-oxa-24 alpha-homo-26,27-dimethyl-1,25-dihydroxyvitamin D3), on the distribution of bound intracellular and extracellular calcium in murine epidermis in vitro. Topical treatment of the skin with KH 1060 (0.4 mmol/l) resulted in the development of epidermal hyperplasia and hyperkeratosis. We observed that epidermis treated with KH 1060 contained fewer calcium deposits in the upper epidermal strata (both intra- and extracellularly) than the control skin. This phenomenon was rapid and occurred after only a single application of KH 1060. Calcium distribution in the basal cell layer was not affected. We propose that observed reduction in the quantity of calcium deposits was caused by the release of sequestered calcium from the intracellular stores and cellular Ca2+ uptake, leading eventually to the increase in the intra-cellular concentration of ionized calcium. The ability of the active vitamin D3 compounds to release Ca2+ may be important for their activity in psoriasis.

Topics: Administration, Cutaneous; Animals; Body Water; Calcitriol; Calcium; Cell Membrane Permeability; Epidermal Cells; Epidermis; Female; Histocytochemistry; Hyperplasia; Immunosuppressive Agents; Keratinocytes; Keratosis; Mice; Mice, Hairless; Mice, Inbred C3H; Microscopy, Electron; Skin; Skin Diseases; Water Loss, Insensible

Dermal effects of KH 1060, a novel, highly potent 20-epi analogue of 1 alpha,25-dihyroxyvitamin D3, were investigated in a hairless mouse model. During daily topical applications of a 0.4 microM solution of KH 1060 for 4 weeks, epidermal hyperplasia and an increase of dermal thickness and mass were observed. KH 1060 upregulated glycosaminoglycan and collagen synthesis in the skin, and increased glycosaminoglycan deposition in the subepidermal region. Reverse transcription-polymerase chain reaction amplification of the transforming growth factor (TGF) beta 1-specific mRNA revealed that KH 1060 stimulated expression of this growth factor in the epidermis, but not in the dermis. Changes observed after application of 1 alpha,25-dihydroxyvitamin D3 were much less pronounced but qualitatively similar to the effects of KH 1060, whereas structurally related but receptor inactive compounds, vitamin D3 and 1 beta,25-dihydroxyvitamin D3, did not produce any effects. Furthermore, we were unable to demonstrate the involvement of the non-genomic, receptor-independent vitamin D signalling in the skin, using a specific stimulator (Ro 24-2090) and a blocker (1 beta,25-dihydroxyvitamin D3) of this pathway. Our findings provide the first evidence that a strong vitamin D3 analogue triggers synthesis of skin connective tissue, possibly via vitamin D receptor activation and the paracrine action of epidermis-derived TGF-beta 1.

Topics: Animals; Calcitriol; Collagen; Epidermis; Female; Glycosaminoglycans; Hyperplasia; Immunosuppressive Agents; Mice; Mice, Hairless; Mice, Inbred C3H; Skin; Transforming Growth Factor beta

1,25-dihydroxyvitamin D3 (calcitriol) affects differentiation and proliferation of epidermal keratinocytes in vitro and in vivo. We have studied the topical effects of calcitriol (0.08-2.0 micrograms/ml) and of a new vitamin D analogue, the epi-20-analogue KH1060 (0.4-2.0 micrograms/ml) on epidermal proliferation in normal hairless mice. Epidermis was examined at intervals from 4 h to 8 days after a single-dose application. The mitotic rate was assessed by the stathmokinetic method and hyperplasia was scored in histological sections. Cell cycle parameters were measured by bivariate bromodeoxyuridine (BrdUrd)/DNA flow cytometry on isolated epidermal basal cells after pulse-labelling with BrdUrd. Both calcitriol and KH1060 induced a dose- and time-dependent increase in the mitotic rate and in hyperplasia, the latter drug being the most effective. Calcitriol and KH1060 induced changes in the cell cycle traverse compatible with the regenerative reaction seen after other hyperplasiogens, but with an additionally increased accumulation of cells in the G2 phase. This is similar to that seen after topical application of retinoic acid to mouse skin. Our results are thus in contrast to the anti-proliferative effects of calcitriol observed in vitro and following treatment of the hyperproliferative disease psoriasis with calcitriol as well as other vitamin D analogues.

Topics: Animals; Bromodeoxyuridine; Calcitriol; Calcium; Cell Cycle; Cell Division; DNA; Epidermis; Flow Cytometry; Hyperplasia; Mice; Mice, Hairless