Overexpression of cellular retinoic acid-binding protein type II (CRABP-II) and down-regulation of CRABP-I in psoriatic skin.

Cellular retinoic acid-binding proteins (CRABPs) might exert a physiological function by controlling the intracellular levels of free retinoic acid. The aim of this study was to analyze the relative expression of CRABP-I and CRABP-II in lesional (LPS) and nonlesional (NLPS) psoriatic skin. CRABP-I and -II proteins were analyzed by a PAGE-autoradioblotting technique, and their respective mRNA were studied by RNA blot and in situ hybridization. We found that CRABP-II levels were 6-fold higher in LPS and 2-fold in NLPS as compared to normal skin, whereas CRABP-I levels were decreased in NLPS and LPS. CRABP-II mRNA were grossly overexpressed in all LPS and some NLPS specimens. These results indicate a switch to the overexpression of CRABP-II mRNA in psoriasis which induces high levels of the protein mainly in LPS; these observations may be relevant to the pathophysiology and therapy of psoriasis as CRABP-I and -II have different ligand-binding affinities.     

Serum levels of Etretinate (Ro 10-9359) and its metabolite (Ro 10-1670)

Serum levels of Etretinate (ET, Ro 10-9359) and its metabolite (AM, Ro 10-1670) were monitored during and after cessation of ET treatment in 12 patients with acquired disorders of keratinization who underwent the ET therapy at initial doses of 30 to 60 mg/day. Studies were made of the possible correlations between ET and AM serum levels, the appearance and disappearance of side effects, and elimination time profiles. Side effects appeared 1 to 2 weeks after the initiation of ET administration in all cases, although serum ET and AM levels at this time varied considerably, indicating individual differences in ET and AM susceptibility in the appearance of side effects. Serum ET and AM levels at the time of disappearance of side effects were lower than those at the time of appearance, suggesting some correlation of side effects with serum levels of ET, AM, or both. Serum level curves for ET and AM after cessation of ET administration were diverse. The maximum ET and AM elimination times from serum were 3 and 6 months, respectively.     

Acitretin (Ro 10-1670) in the Treatment of Severe Psoriasis

A randomized double-blind parallel trial comparing acitretin and etretinate was performed in 20 patients with severe psoriasis during a treatment period of 12 weeks. The initial dose was 30 mg/day for 4 weeks, and the mean dose at the end of the study was slightly lower in the acitretin group when compared to the etretinate group (30.7 mg/day and 33.4 mg/day, respectively). Follow-up examinations were carried out every 2 weeks, and the efficacy of treatment was evaluated by using the PASI score. Percentage improvement in the PASI score was 50% at week 10 in both groups, and the difference between them at week 12 was insignificant. Both quantitatively and qualitatively, acitretin and etretinate do not significantly differ.     

Trimethylmethoxyphenyl-retinoic acid (Ro 10-1670) and lymphocyte DNA synthesis activity in vitro

Trimethylmethoxyphenyl-retinoic acid (TMMPRA), the main active therapeutic form of etretinate, was used for in vitro studies on human lymphocytes. Lymphocyte DNA synthesis remained unchanged when TMMPRA was added to the cultures at a concentration range of 1.25-25 micrograms/ml culture medium. No modulatory activity of the drug on DNA synthesis was seen on cultured lymphocytes stimulated by lectins (phytohemagglutinin, pokeweed mitogen, concanavalin A) or by a mitogen selective for a T lymphocyte subpopulation, phorbol myristate acetate. Concanavalin A induced T suppressor cell activity tested toward the proliferative response to allogeneic stimuli was lowered by TMMPRA. The results indicated that TMMPRA alone or in the presence of lectins or phorbol myristate acetate did not inhibit or stimulate DNA synthesis activity in vitro but that the drug could lower T suppressor cell activity.     

Overview of recent clinical pharmacokinetic studies with acitretin (Ro 10-1670, etretin)

Following oral administration of acitretin with food, peak plasma concentrations of unchanged drug (Ro 10-1670) are reached within 4 h. The mean absolute bioavailability of acitretin is 59% with high interpatient variability consistent with that of etretinate. Taking acitretin with food results in an increased and more consistent bioavailability. The drug appears to be extensively distributed throughout the body without unexpected accumulation and the elimination half-life is approximately 50 h. Acitretin has a profound pharmacokinetic advantage over etretinate because it is eliminated more rapidly from the body; etretinate is sequestered into fatty tissue due to its greater lipophilicity creating a deep compartment from which it is only slowly released. Acitretin is eliminated entirely by the metabolism and the resultant metabolites are excreted via the kidney and bile.     

Acitretin (Ro 10-1670, etretin): overall evaluation of clinical studies

Acitretin (Ro 10-1670, etretin) is the free acid derivative of etretinate. This compound possesses a much shorter half-life of elimination than etretinate and was therefore proposed for clinical development. A total of 635 patients with various dermatoses were studied in 12 main clinical trials performed in Europe and in the United States. It was shown that acitretin is effective in the treatment of psoriasis and other diseases of keratinization. Doses between 25 and 35 mg per day are recommended to initiate treatment since marked improvement or clearing was obtained in the majority of patients within this range. Hypervitaminosis A signs and symptoms have been observed in patients treated with acitretin. It is concluded that the efficacy and safety profile of acitretin resembles that of etretinate.     

连续外用维A酸皮肤刺激反应中的维A酸结合蛋白mRNA的表达

目的: 检测在连续外用维A酸引起的皮肤刺激反应中小鼠皮肤维A酸结合蛋白mRNA表达水平.方法: 连续外用0.1%维A酸和基质,用RT-PCR方法检测Balb/C小鼠皮肤中维A酸结合蛋白(CRABP I、CRABP II)mRNA的表达水平.结果: 外用0.1%维A酸霜,皮肤中CRABP II mRNA表达水平在用药第一天呈一过性升高.连续用药,CRABP II mRNA表达水平稳定升高而不受每天用药前后的影响,第6天达次高峰,第9天达高峰,第12天降至次高峰,一直到用药24天,均维持在次高峰水平.而CRABP I mRNA表达水平在用药期间未见改变.维A酸霜基质对这两种蛋白mRNA的表达没有影响.结论: 外用维A酸霜引起的一过性皮肤刺激反应可能是由CRABP II介导.     

Systemic treatment of psoriasis with an oral retinoic acid derivative (Ro 10-9359)

Ninety-seven patients with severe psoriasis took part in a 1-year study to evaluate the effect of a new oral synthetic retinoid (Ro 10-9359). The trial was performed in a double-blind cross-over fashion. The treatment started with either 100 mg daily of Ro 10-9359 or placebo and the maintenance dose was in most cases 50 mg. Follow-up examinations were performed monthly and the parameters erythema, desquamation, infiltration and extent of the lesions were followed. Throughout the study there was a significant to highly significant preference for Ro 10-9359 shown by all parameters. More patients were in complete remission after Ro 10-9359 periods than after placebo periods. The side-effects of Ro 10-9359 on uninvolved skin and mucous membranes seemed to be largely dose-dependent. Twenty-three patients interrupted the study, four of them because of side-effects, mainly alopecia. Laboratory examinations revealed no aberrations which could be attributed to the therapy. One patient developed hepatitis during a placebo period.     

Gene expression of retinoic acid receptors and cellular retinoic acid-binding proteins in rhino and hairless mouse skin

The rhino mouse comedolytic model and the hairless mouse photoaging model are established animal models for screening the in vivo activity of retinoids. However, the expression of the retinoic acid receptors (RARs) and cellular retinoic acid-binding proteins (CRABPs), known to regulate retinoid activity, is not completely understood in these mouse mutants. For this purpose, mRNA was isolated from rhino and hairless mouse skin and the gene expression of the RARs and CRABPs was measured by Northern blot hybridization. Results showed that RAR was the predominantly expressed RAR in both mouse strains. Two isoforms of RAR, RAR1 and RAR2, were detected with RAR1 being the more strongly expressed. RAR was also detected, but to a lesser degree than RAR. RAR expression was not detectable by our methodology. Additionally, topical treatment of these mice with 0.1% all-trans-retinoic acid (tRA) cream resulted in no significant alteration in the expression of the RAR genes. By contrast, CRABP-II was induced 2–4 fold by topical tRA treatment. CRABP-I, expressed to a lesser degree than CRABP-II, was not inducible. The relative expression of the RARs, CRABPs, and inducibility of CRABP-II by tRA in both rhino and hairless mouse skin paralleled that reported for human and mouse skin. These observations suggest that the altered phenotype observed in the rhino mouse most likely does not result from an altered expression level of these genes. The results also support these two animals as models for evaluating the therapeutic potential of retinoids.     

Keratin polypeptide profile in psoriatic epidermis normalized by treatment with etretinate (aromatic retinoid Ro 10-9359)

Summary The variations in the expression of epidermal keratins occurring during retinoid therapy were studied in patients with psoriasis before and during oral administration of etretinate (aromatic retinoid Ro 10-9359) and compared with normal epidermis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis densitometric readings were performed on keratins extracted from epidermal cells obtained through trypsinization of skin specimens. Epidermal cells were also tested by immunofluorescence (IF) for the presence of BMZ antigens as markers of basal cells and for the presence of TK and KP (67 K, 63 K, and 55 K) with sera with antibodies against BMZ antigens and specific antisera for TK and KP.In psoriasis-involved epidermis, SDS-PAGE analysis showed lower amounts of 67 K and increased amounts of 63 K and 55 K, as compared with normal epidermis. Low proportions of cells expressing by IF the 67 K and the 63 K were also noted, with a defective expression of these two KP by suprabasal keratinocytes in psoriatis-involved epidermis.During etretinate administration, a return toward the normal electrophoretic pattern and a correction of the defective cellular expression of these two KP were obtained parallel with clinical improvement. These findings indicate the presence in involved psoriatic epidermis of a population of suprabasal keratinocytes that do not express the high-molecular-weight KP and show a normalization of the relative proportions of the KP with etretinate.Abbreviations BMZ bazement zone - IIF indirect immunofluorescence - KP keratin polypeptides - MEM minimal essential medium - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TK total keratin - TUM TRIS-urea-mercaptoethanol - 67K, 63K, 55K keratin proteins with mol. wts., respectively, of 67,000, 63,000, and 55,000 daltons Supported in part by a grant INSERM CRL 81 2045 and a grant UER Grange Blanche (tranche C 1981)     

The antipsoriatic drug metabolite etretin (Ro 10-1670) alters the metabolism of fatty acids in human keratinocytes in culture

Summary We have studied the effect of etretin (Ro 10-1670), the active metabolite of the widely used antipsoriatic drug etretinate (Ro 10-9359), on the incorporation and release of arachidonic acid in human skin keratinocytes. During 24-h culture, radioactive ¹⁴C-arachidonic acid was avidly incorporated into the cellular lipids of the keratinocytes. When the cells were cultured for another 48 h in fresh medium, 8.8%±0.3% of the incorporated radioactivity was released from the cells. The presence of etretin (10^-8 M to 10^-5 M) in the medium stimulated the release of radiolabel. With 10^-5 M etretin in the culture medium, 13.0%±0.4% of the incorporated radioactivity was released, and this was accompanied by decreased labelling of phosphatidylethanolamine. This suggests that phosphatidylethanolamine may be an important source of the released arachidonic acid.Etretin pretreatment reduced the incorporation of ¹⁴C-arachidonic acid into diacylglycerols, triacylglycerols, and cholesteryl esters. Pretreatment for 48 h with 10^-5 M etretin reduced subsequent ¹⁴C-arachidonic acid incorporation into nonphosphorus lipids from a mean total of 8.2%±0.2% to 3.2%±0.1% (p<0.001). These findings suggest that etretin interferes with the esterification of arachidonic acid into nonphosphorus lipids. Etretin was also found to cause changes in the fatty acid composition of keratinocytes. Following 48 h culture with etretin, the percentage amount of the fatty acids belonging to the n3 series was increased whereas that of palmitic acid (16:0) and palmitoleic acid (16:1n7) was decreased. In conclusion, our study suggests that etretin in therapeutical concentrations affects fatty acid metabolism in human keratinocytes in culture.     

Treatment of severe psoriasis with etretin (RO 10-1670)

Eighty patients with severe psoriasis were treated in a double-blind fashion with either an initial dose of 10 mg, 25 mg or 50 mg of etretin daily or with placebo. Follow-up examinations were carried out monthly and the efficacy of treatment was evaluated by using the PASI score. Adverse effects of the treatment were recorded monthly; liver enzymes, cholesterol and triglycerides were measured. After 2 months of treatment the maintenance dose was reduced in some of the patients either because of complete remission or adverse effects. After 2 months treatment, groups receiving 25 mg/day and 50 mg/day showed significantly lower PASI scores than the placebo group. The 10 mg/day group showed a response intermediate between the 25 mg and 50 mg groups and the placebo group. Thus, the optimal initial dose seems to be approximately 25 mg/day and the maintenance dose somewhat lower. Six months after the start of treatment there were no significant differences between the four groups; the last follow-up examination took place during the summer and some of the patients probably experienced spontaneous improvement. Although clinical adverse effects were frequent in all groups, severe side effects, namely hair loss and paronychia, occurred frequently only among patients treated with an initial dose of 50 mg of etretin daily. The effect of treatment on liver enzymes, cholesterol and triglycerides was minimal.     

Evaluation of retinoids as inhibitors of [3H] all-trans retinoic acid binding to cellular retinoic acid-binding protein in rat skin and testes

Summary These experiments were designed to test the ability of certain analogs and metabolites of all-transretinoic acid (RA), 13-cis-retinoic acid, 4-hydroxy-alltrans-retinoic acid, 4-keto-all-trans-retinoic acid, trimethylmethoxyphenol (TMMP) analog of retinoic acid, and TMMP analog of ethyl retinoate (etretinate) to compete for cellular retinoic acid-binding protein (CRABP) in skin and testes of rats. All retinoids, except etretinate, bind to CRABP in a competitive manner with a similar affinity (approximately 5×10^-9 M for skin and 3×10^-9 M for testes). In contrast, etretinate binds in a noncompetitive manner with a much lower affinity (7.7×10^-5 M for skin and 7.5×10^-5 M for testes). The values (M) of IC₅₀ for CRABP from rat skin are 0.43, 0.41, 0.95, 0.83, and 77.4 and those from rat testes are 0.59, 1.29, 2.25, 2.30, and 75.25 for all-trans-RA, 13-cis-RA, 4-hydroxy-all-trans RA, 4-keto-all-trans-RA, TMMP analog of RA, and etretinate, respectively. Etretinate is a potent retinoid that is used in the treatment of psoriasis. The lack of quantitative correlation between IC₅₀ and the biological activity of etretinate may be explained in that the active form of etretinate in the body may be the carboxylic acid form (TMMP analog of RA) which binds to CRABP with higher affinity.Abstract of this paper was presented at the Joint Meetings of the Western Section, American Federation for Pediatric Research, and Western Region, the Society for Investigative Dermatology, February 1985, Carmell, California     

Trimethylmethoxyphenyl-retinoic acid (Ro 10–1670) inhibits mitogen-induced DNA-synthesis in peripheral blood lymphocytes in vitro

Trimethylmethoxyphenyl-retinoic acid (TMMP-retinoic acid, Ro 10-1670) is the main metabolite of the aromatic retinoid Ro 10-9359 (Tigason, etretinate), which is now in clinical use. Therefore, we selected this metabolite for our in vitro studies on human lymphocytes. The following findings were obtained: TMMP-retinoic acid is practically insoluble in aqueous solution. It remains partly in solution only if bound to protein. Under these conditions it had no influence on DNA-synthesis of human peripheral blood lymphocytes added in vitro without lectins (5--12.5 ng/ml culture medium). However, if human peripheral blood lymphocytes were cultured in vitro with lectins (ConA, PHA-M, PHA-P and PWM) and in the presence of TMMP-retinoic acid (25 microgram/ml culture medium) no induction of DNA-synthesis was seen. The altered lymphocytic response in presence of TMMP-retinoid in vitro was clearly dose-dependent at these levels. Trypan blue exclusion tests showed no evidence of cytotoxicity. Our studies clearly indicate that TMMP-retinoic acid inhibits the biological response of human blood lymphocytes to lectins in vitro, at dosages close to therapeutic levels in vivo. They confirm that retinoids may exert distinct effects on dermal cells in addition to their influence on keratinocytes, and suggest that the therapeutic action of the drug may be also related to the altered lymphocytic response.     

Cellular retinoic acid- but not cellular retinol-binding protein is elevated in psoriatic plaques

Cellular retinoid binding proteins are thought to be involved in the molecular action of retinoids, a family of compounds successfully used in the treatment of psoriasis. Therefore, both cellular retinol (CRBP)- and retinoic acid (CRABP)-binding proteins were analyzed in psoriatic skin. Three facts emerged from our study: both CRABP and CRBP are detectable in the skin of psoriatic patients; qualitatively, they both appear similar to the corresponding proteins of normal human skin, in terms of their elution profile and apparent Kd; and quantitatively, only CRABP was found to be 3 times higher in psoriatic plaques as compared to either nonlesional skin of psoriatic patients or the skin of normal subjects. Since psoriatic plaques are particularly responsive to systemic retinoids, specifically to retinoic acid analogues, our results suggest for the first time a link between the levels of CRABP and the responsiveness of a nonneoplastic hyperproliferative tissue to systemic administration of retinoids in the human.