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The identity of the endogenous RXR ligand has not been conclusively determined, even though several compounds of natural origin, including retinoids and fatty acids, have been postulated to fulfill this role. Filling this gap, 9-cis-13,14-dihydroretinoic acid (9CDHRA) was identified as an endogenous RXR ligand in mice. This review examines the physiological relevance of various potential endogenous RXR ligands, especially 9CDHRA. The elusive steps in the metabolic synthesis of 9CDHRA, as well as the nutritional/nutrimetabolic origin of 9CDHRA, are also explored, along with the suitability of the ligand to be the representative member of a novel vitamin A class (vitamin A5).

Dolichols are isoprenoid lipids of varying length that act as sugar carriers in glycosylation reactions in the endoplasmic reticulum. In Saccharomyces cerevisiae , there are two cis -prenyltransferases that synthesize polyprenol-an essential precursor to dolichol. These enzymes are heterodimers composed of Nus1 and either Rer2 or Srt1. Rer2-Nus1 and Srt1-Nus1 can both generate dolichol in vegetative cells, but srt1 ∆ cells grow normally while rer2 ∆ grows very slowly, indicating that Rer2-Nus1 is the primary enzyme used in mitotically dividing cells. In contrast, SRT1 performs an important function in sporulating cells, where the haploid genomes created by meiosis are packaged into spores. The spore wall is a multilaminar structure and SRT1 is required for the generation of the outer chitosan and dityrosine layers of the spore wall. Srt1 specifically localizes to lipid droplets associated with spore walls, and, during sporulation there is an SRT1 -dependent increase in long-chain polyprenols and dolichols in these lipid droplets. Synthesis of chitin by Chs3, the chitin synthase responsible for chitosan layer formation, is dependent on the cis -prenyltransferase activity of Srt1, indicating that polyprenols are necessary to coordinate assembly of the spore wall layers. This work shows that a developmentally regulated cis -prenyltransferase can produce polyprenols that function in cellular processes besides protein glycosylation., (Copyright © 2017 by the Genetics Society of America.)

Background: Dengue virus (DENV) is estimated to cause 390 million infections each year, but there is no licensed vaccine or therapeutic currently available., Methods: We describe a novel, high-throughput screen to identify compounds inhibiting the interaction between DENV nonstructural protein 5 and host nuclear transport proteins. We document the antiviral properties of a lead compound against all 4 serotypes of DENV, antibody-dependent enhanced (ADE) infection, and ex vivo and in vivo DENV infections. In addition, we use quantitative reverse-transcription polymerase chain reaction to examine cellular effects upon compound addition., Results: We identify N-(4-hydroxyphenyl) retinamide (4-HPR) as effective in protecting against DENV-1-4 and DENV-1 ADE infections, with 50% effective concentrations in the low micromolar range. 4-HPR but not the closely related N-(4-methoxyphenyl) retinamide (4-MPR) could reduce viral RNA levels and titers when applied to an established infection. 4-HPR but not 4-MPR was found to specifically upregulate the protein kinase R-like endoplasmic reticulum kinase arm of the unfolded protein response. Strikingly, 4-HPR but not 4-MPR restricted infection in peripheral blood mononuclear cells and in a lethal ADE-infection mouse model., Conclusions: 4-HPR is a novel antiviral that modulates the unfolded protein response, effective against DENV1-4 at concentrations achievable in the plasma in a clinical setting, and provides protection in a lethal mouse model., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: All-trans retinoic acid (ATRA), an active metabolite of vitamin A, is widely used in the treatment of acute promyelocytic leukaemia and myelodysplastic syndrome. However, its high lipophilicity is thought to be responsible for the slow dissolution and low bioavailability following oral administration. In order to obtain compounds with better solubility characteristics to improve the transportation and bioavailability of ATRA, derivatives of ATRA containing glutamic acid or its sodium salt were synthesised., Methods: The ATRA derivatives synthesised - all-trans retinoyl glutamate (RAE) and all-trans retinoyl sodium glutamate (RAENa(2)) - were characterised in terms of melting point, optical rotation, mass spectrometry, NMR and partition coefficient. A liposomal preparation formed from RAE was characterised by particle size and zeta potential. The anti-tumour activity of RAE and RAENa(2) was compared with that of ATRA in mice bearing S(180) tumours and their effects on the cell cycle were determined in human pro-myelocytic leukaemia HL-60 cells., Key Findings: RAE and RAENa(2) were more active than ATRA against tumour growth. Flow cytometry indicated that RAE and RAENa(2) induced HL-60 cell cycle arrest, similar to ATRA. DNA fragmentation studies suggested that apoptosis may be one of the mechanisms responsible for the anti-tumour activities., Conclusions: The two derivatives of ATRA, RAE and RAENa(2), exhibited improved aqueous solubility and were more effective in mice bearing S(180) tumours.

Background: Isotretinoin is effective in the treatment of severe acne and rosacea. Both parent drug and its main metabolite 4-oxo-isotretinoin are potentially teratogenic compounds and contain a carboxylic acid moiety. In the presence of ethanol, naturally occurring as well as synthetic retinoids also containing a carboxylic acid moiety are capable of undergoing an ethyl esterification with the metabolic formation of more lipophilic compounds with a much longer terminal half-life., Objectives: To determine if isotretinoin (13-cis-RA), its main metabolite 4-oxo-isotretinoin (4-oxo-13-cis-RA), and other possible metabolites in the presence or absence of ethanol are converted to their corresponding ethyl derivatives in patients with severe acne or rosacea after multiple isotretinoin dosing. In addition, pharmacokinetic parameters of the parent drug and its 4-oxo metabolite were determined., Patients/methods: Eleven patients with severe acne or rosacea were treated with isotretinoin daily for 3 months and investigated pharmacokinetically during 24 h after 1 month of treatment and for up to 28 days after discontinuation of therapy. A possible influence of ethanol was evaluated using a simple self-administered questionnaire and by measuring serum ethanol levels during treatment. The concentrations of isotretinoin, 4-oxo-isotretinoin and possible ethylated and nonethylated metabolites were measured by reverse-phase high-performance liquid chromatography., Results: Although seven of 11 patients had a considerable weekly alcohol intake, no endogenous synthesis of ethyl derivatives of isotretinoin, the main 4-oxo metabolite or the all-trans compounds was chromatographically detectable in any of the patients' plasma samples during the treatment period. Multiple dose pharmacokinetic data for the parent drug and its main metabolite were comparable to previous studies., Conclusions: The metabolism and pharmacokinetics of isotretinoin and its main metabolites are not influenced by ethanol during long-term isotretinoin treatment. After ceasing long-term isotretinoin therapy the recommended period of 1 month for using anticonceptive measures in fertile women seems adequate.

Fast and sensitive liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) method for the specific determination of viaminate in 14 kinds of rat tissues pre-treated with simple procedure was developed and validated. Biological samples were prepared by direct precipitation to skin, stomach, intestine, and liver and extracted by liquid-liquid extraction to lung, kidney, muscle, spleen, brain, fat, testes (male and female), eye, and heart. After addition of menaquinon as internal standard to tissue homogenate, the supernatant was injected into the isocratic chromatographic system using a Waters Symmetry C8 column and methanol-water-formic acid (93:7:0.1) as the eluent. The eluate was completely led into an APCI interface with selected ion monitoring mode and the analytes were quantified using triple quadrupole MS. The assays were successfully validated in the ranges 0.02-20 ng/mL for lung, 0.02-10 ng/mL for kidney, spleen, muscle, brain, fat, eye, and heart, 0.05-10 ng/mL for testicle, 0.4-100 ng/mL for liver, skin and intestine, and 1.0-200 ng/mL for stomach. The accurate and precise studies showed good reproducibility with coefficients of variation below 8.5% and the recoveries range from 90 to 109%. The analytes were chemically stable under all relevant conditions and the assays were applied in tissue distribution study. The results showed that the viaminate concentration was high in skin, low in kidney, and almost undetectable in eye and brain.

Background: Patients with severe chronic hand eczema (CHE) refractory to topical corticosteroids currently have limited treatment options suited for chronic use, and few controlled clinical studies have investigated new therapies in this setting., Objectives: To assess the efficacy and safety of oral alitretinoin (9-cis retinoic acid) taken at 10 mg or 30 mg once daily for up to 24 weeks, compared with placebo control, in the treatment of severe CHE refractory to topical corticosteroids., Methods: A randomized, double-blind, placebo-controlled, prospective, multicentre trial was conducted in 111 dermatology outpatient clinics in Europe and Canada. A total of 1032 patients with severe refractory CHE were randomized in a 1 : 2 : 2 ratio to placebo, or 10 mg or 30 mg of oral alitretinoin once daily for up to 24 weeks. Safety was assessed for all patients during a follow-up period of 4 weeks, and responders were observed for relapse for 24 weeks after the end of therapy. The primary efficacy parameter was Physician Global Assessment of overall CHE severity, with response defined as clear or almost clear hands., Results: Responses, defined as clear or almost clear hands, were achieved in up to 48% of patients treated with alitretinoin, compared with 17% for placebo (P < 0.001), with up to 75% median reduction in disease signs and symptoms. Treatment was well tolerated, with dose-dependent adverse effects comprising headache, mucocutaneous events, hyperlipidaemia, and decreased free thyroxine and thyroid-stimulating hormone. The median time to relapse, defined as recurrence of 75% of initial signs and symptoms, was 5.5-6.2 months in the absence of anti-eczema medication., Conclusions: Alitretinoin given at well-tolerated doses induced clearing of CHE in a substantial proportion of patients with severe disease refractory to standard therapy.

Background: Chronic ultraviolet (UV) radiation from sunlight induces wrinkle formation. Retinoic acid (RA) can markedly improve wrinkles, although RA does have some side-effects, such as skin irritation. As the efficacy and cytotoxicity of RA has been traced to its free carboxylic acid, we synthesized a new molecule, N-retinoyl-D-glucosamine (GRA), in which a glucosamine has been attached to the polar end group of all-trans retinoic acid., Objectives: To analyse the effect of topical GRA in wrinkle repair and anti-irritation in photoaged mice compared with topical RA, as well as to determine retinoic acid receptor (RAR) and retinoid X receptor (RXR) transactivation activity in vitro., Methods: Hairless mice were irradiated with 60 mJ cm-2 of UVB for 10 weeks, and then topically treated with 0.05% GRA or 0.05% RA for 8 weeks. An in vitro transcriptional assay was performed and the activity of GRA in 293 cells transfected with RAR-alpha or RXR-alpha expression plasmid and luciferase reporter plasmid then determined., Results: Topical GRA and RA brought about almost complete disappearance of the wrinkles caused by UVB irradiation. The two ligands promoted both a wide repair zone histologically, and the expression of type 1 collagen in the skin. In contrast, topical GRA treatment did not produce irritation such as erythema or roughness, or alteration of transepidermal water loss values, compared with RA. In the in vitro luciferase assay, GRA resulted in significant dose-dependent RAR transactivation activity in a 100 times higher concentration range than RA. GRA did not mediate RXR transactivation activity at all., Conclusions: Topical GRA appears to be able to repair photoaged skin damage without any of the irritation caused by topical RA, probably via RAR transactivation activity.

We have shown previously that administration of acyclic retinoid to cirrhotic patients who had undergone curative treatment of preceding hepatocellular carcinoma (HCC) induced the disappearance of serum lectin-reactive alpha-fetoprotein (AFP-L3) and subsequently reduced the incidence of second liver cancers. AFP-L3 is a tumor marker that indicates the presence of occult tumors below the detection limit by diagnostic images. Therefore, we have proposed a new concept of 'clonal deletion' therapy with acyclic retinoid for the cancer chemoprevention against HCC. Such eradication of AFP-L3-producing latent malignant (or premalignant) cells from the liver suggested a new strategy to prevent HCC, which may be involved in the same category as cancer chemotherapy. In the present series of studies, we explored the molecular mechanism of 'clonal deletion' and found a novel mechanism of apoptosis induction by the retinoid. We have demonstrated a modification of a retinoid receptor, RXRalpha, by mitogen-activated protein (MAP) kinase-dependent phosphorylation, resulting in the loss of transactivating activity. This may lead HCC cells to be resistant to natural retinoic acid. However, acyclic retinoid restored the function of phosphorylated RXRalpha and induced its downstream pro-apoptotic genes including tissue transglutaminase, an enzyme that is implicated in apoptosis. Tissue transglutaminase-dependent apoptosis in HCC cells was independent of the activation of caspases. This novel mechanism of retinoid-induced apoptosis may give a clue to understand the molecular mechanism of clonal deletion.

Polyisoprenoid alcohols occurring in spinach leaves were analyzed by a two-plate TLC method. Z,E-mixed polyprenols (C(55-60)), glycinoprenols (C(50-55)), and solanesol (C(45)) were mainly found in chloroplasts, whereas dolichols (C(70-80)) were mainly found in microsomes. Analysis of enzymatic products derived from [1-(14)C]isopentenyl diphosphate and farnesyl diphosphate (FPP) with subcellular fractions revealed that chloroplasts and microsomes had the ability to synthesize Z,E-mixed polyprenyl (C(50-65)) and all E-polyprenyl (C(45-50)) diphosphates, and Z,E-mixed polyprenyl (C(70-85)) diphosphates, respectively. FPP and geranylgeranyl diphosphate (GGPP) were both accepted for these enzymatic reactions, the former being a better substrate than the latter. NMR analysis of naturally occurring spinach Z,E-mixed polyprenol (C(55)) and dolichol (C(75)) revealed that the number of internal trans isoprene residues in the former was three in comparison with two internal trans residues found for the latter. These results indicate that two kinds of polyprenyl diphosphate synthases occur in spinach: One is the chloroplast enzyme involved in the synthesis of the shorter-chain (C(50-65)) Z,E-mixed polyprenols and the other is the microsomal enzyme involved in the synthesis of longer-chain (C(70-85)) Z,E-mixed polyprenols, which is converted to dolichols.

Significant differences between the metabolism of retinoic acid by different tissues might be an important determinant of the effectiveness of a systemically administered inhibitor at a particular tissue site. Here the metabolism of retinoic acid has been studied in microsomal fractions from different tissues (liver, kidney, intestinal mucosa, lung, skin, brain) of the male rat to determine their relative metabolic activity. Kinetic analysis revealed major differences between the activity of different tissue microsomes. This is shown by the Vmax values for the metabolism of retinoic acid-liver (102+/-39.0 pmol (mg protein)(-1) min(-1)) was 100 times more active than the lung (1+/-0.03 pmol (mg protein)(-1) min(-1)), which was the least active. The range of Km values for microsomes from the different tissues was narrow (0.48-1.40 microM). Taking into account the mass of the tissue, the gross activity ranking for metabolism of retinoic acid was liver >> skin = kidney > brain > intestinal mucosa >> lung. It is concluded that metabolism of administered retinoic acid occurs mainly in the liver but that cellular retinoic acid levels in some other tissues (skin, kidney, brain) could be reduced (metabolized) to such an extent that higher levels might be observed after the use of inhibitors of retinoic acid metabolism.

Retinoyl beta-glucuronide is a naturally occurring, biologically active metabolite of vitamin A. Although retinoyl beta-glucuronide is regarded as a detoxification product of retinoic acid, it plays several roles in the functions of vitamin A. It can serve as a source of retinoic acid, and it may be a vehicle for transport of retinoic acid to target tissues. Topically applied retinoyl beta-glucuronide is comparable in efficacy to retinoic acid in the treatment of acne in humans, without the same side effects. Retinoyl beta-glucuronide may or may not be teratogenic, depending on the mode of administration and the species in which it is used. It may be a valuable therapeutic compound for the treatment of skin disorders and certain types of cancers.

The testicular gene expression of the retinoic acid receptors, RAR alpha, -beta, and -gamma, was studied in normal mice and in vitamin A-deficient mice after the administration of all-trans-retinoic acid (ATRA). All three types of RARs were expressed in normal and/or vitamin A-deficient testes. Only the expression of RAR beta messenger RNA was transiently induced within 24 h after ATRA injection. ATRA-induced RAR beta expression was also found in purified Sertoli cells, suggesting that these cells mediate at least part of the effect of retinoids on germ cells. When an equimolar amount of retinol was administered instead of ATRA, no induction of RAR beta was seen at the point of maximal induction by ATRA, suggesting that the effect of retinol was delayed and probably less. The related nuclear receptors, RXR alpha, -beta, and, for the first time, gamma, were also shown to be present in the mouse testis. Upon administration of ATRA, messenger RNA expression of RXR alpha and -beta did not change significantly. The expression of RXR gamma was too low to allow quantification. Finally, the effect of the retinoid metabolism inhibitor liarozole on ATRA-induced proliferation of A spermatogonia was examined. The labeling index of A spermatogonia, 24 h after the administration of 0.25 mg ATRA, was significantly lowered by liarozole due to a shift of the maximal 5-bromo-deoxyuridine incorporation to an earlier point (20 h). This indicates that liarozole delays retinoid metabolism, thereby increasing the actual ATRA concentration, and more importantly, that ATRA by itself is an active retinoid in spermatogenesis. Apparently, ATRA does not need to be metabolized to 4-oxo-RA, which was previously shown to be a more potent inducer of spermatogonial proliferation than ATRA, to be effective.

Vitamin A deficiency leads to an arrest of spermatogenesis and a loss of advanced germ cells in male mice. In the present study, the effects of several retinoids and carotenoids on these mouse testis were investigated. First, the proliferative activity of the growth-arrested A spermatogonia in vitamin A-deficient (VAD) mice testis was determined, 20, 24, or 28 h after administration of 0.5 mg all-trans-retinoic acid (RA). The bromodeoxy-uridine (BrdU) labeling index of A spermatogonia in control VAD testis was 5 +/- 1% (n = 4, mean +/- SD). When RA was injected (ip), the highest labeling index was found 24 h after RA administration; 49 +/- 5%. When various concentrations of RA, all-trans-4-oxo-retinoic acid (4-oxo-RA) or all-trans-retinol acetate (ROAc), ranging from 0.13-1 mg, were injected, the labeling index of A spermatogonia always increased in comparison with the VAD situation. A maximum index at 24 h was found when 0.5 mg 4-oxo-RA was injected: 56 +/- 3%. This labeling index was even higher than those after injection of RA or ROAc, 49 +/- 5% and 34 +/- 6% respectively. The increase of the BrdU labeling index was dose dependent. After an initial increase of the labeling indices with increasing retinoid doses, the labeling indices decreased at a higher concentration. This decrease is likely due to a concentration dependent timeshift of the optimum of BrdU labeling to shorter time intervals after retinoid administration because a labeling index of 66 +/- 1% was found 20 h after injection of 1 mg RA. At 24 h, this labeling index was halved: 33 +/- 2%. These indices show that the degree of synchronization of spermatogenesis is also dependent on the retinoid dose. When the dimers of RA and 4-oxo-RA, respectively beta-carotene (beta C) and canthaxanthin, were given, 24 h after administration BrdU-labeling indices comparable with the VAD value were found. Repeated injection of beta C twice a week did induce a reinitiation of spermatogenesis, but compared with RA, the activity of beta C was lower and delayed. It is concluded that 4-oxo-RA is active in adult mammals in vivo. It is at least as potent as RA in the induction of the differentiation and subsequent proliferation of growth-arrested A spermatogonia in VAD mice testis. Furthermore, the degree of synchronization of spermatogenesis is influenced by the retinoid dose. Finally, carotenoids were shown to act in the induction of spermatogonial cell proliferation too but with a lower and delayed activity.

BMS-181163 (4-acetamidophenyl retinoate, previously reported as BMY-30123), the acetamidophenyl ester of all-trans-retinoic acid (tRA), is topically active in various retinoid-sensitive animal models, but was recently shown to be ineffective for the treatment of acne in patients. To determine whether BMS-181163 functions as a prodrug of tRA in mice but not in man, the relative rates of ester hydrolysis in mouse and human skin homogenates were determined. In-vitro hydrolysis assays showed that BMS-181163 was substantially hydrolysed in mouse skin homogenates and minimally in human skin preparations. In addition, a series of phenyl esters of tRA and several known active synthetic retinoids (Ch-80: (E)-4-[3-oxo-3-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1 - propenyl] benzoic acid; CD-271: 6-[3-(1-adamantyl)-4-methyoxyphenyl]-2-naphthoic acid; and TTNPB: (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1- propenyl] benzoic acid) was prepared and hydrolysis rates and in-vivo (rhino mouse utriculi reduction) activities were compared. The hydrolysis rates of the six test retinoid phenyl esters, ranging from 0.06 to 2.0 h-1 were found to correlate with the in-vivo activity. Those esters (BMS-181163 and acetamidophenyl esters of Ch-80 and TTNPB) with a higher hydrolysis rate exhibited in-vivo activity only slightly lower than their parent free acid retinoids. In contrast, the three phenyl esters with a hydrolysis rate less than 0.3 h-1 were inactive in-vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Preclinical studies pertaining to the pharmacology and toxicology of BMY 30123 (4-acetamidophenyl retinoate) are reported. BMY 30123 is a novel compound which has topical retinoid activity. This compound exhibits lower toxicity, both local and systemic, than other clinically used topical retinoids such as tretinoin (all-trans retinoic acid) in animal models. BMY 30123 is effective in a number of retinoid sensitive skin models including the rhino mouse utriculi reduction assay, the mouse epidermal hyperplasia model and in the suppression of DNA synthesis in mouse skin stimulated with phorbol ester. BMY 30123 was equipotent with tretinoin in these topical models. In the rhino mouse model the ED30 values for BMY 30123 and tretinoin were 0.037 and 0.015 mM, respectively. In addition, BMY 30123 was active in the UVB-induced photodamaged mouse model, another retinoid sensitive model. One of the problems associated with topically applied tretinoin is local irritation. Therefore, for topical therapy to be optimal, it is important to reduce or minimize local irritation. Repeated applications of BMY 30123 to rabbit skin resulted in low skin irritation. The first perceptible signs of skin irritation produced by BMY 30123 occurred at a dose 10 times higher than that observed for tretinoin. BMY 30123 also exhibits low retinoid activity after oral or i.p. administration in mice and produced no signs of hypervitaminosis A-related toxicity at twenty times the no effect dose of tretinoin. Because retinoids are effective modulators of epidermal growth and differentiation, this compound should be useful for the treatment of cutaneous disorders that exhibit altered epidermal differentiation such as acne, psoriasis, ichthyosis and epithelial tumours.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of retinoids on the production of interleukin-1 (IL-1) by murine epidermal keratinocytes was investigated. Freshly isolated keratinocytes were cultured in the presence of etretinate, acitretin, all-trans retinoic acid or 13-cis retinoic acid at concentrations of 8 x 10(-9)-8 x 10(-6) mol/l. Exposure of keratinocytes to retinoids increased IL-1 bioactivity in culture supernatants and cell extracts at concentrations as low as 8 x 10(-9) mol/l, as assessed by T-cell proliferation. Prolongation of the culture period enhanced the augmentative effect of retinoids. All-trans retinoic acid and 13-cis retinoic acid had a greater ability to induce IL-1 production than the two aromatic retinoids, etretinate and acitretin. Treatment with 8-methoxypsoralen plus ultraviolet A radiation and treatment with triamcinolone acetonide both reduced the effect of retinoids on the production of bioactive IL-1.

Fenretinide [N-(4-hydroxyphenyl)retinamide, 4-HPR] is an effective agent for the inhibition of N-nitroso-N-methylurea-induced breast cancer in rats. This compound has been studied extensively and proved to be safer and less teratogenic than many other retinoids. A major characteristic of 4-HPR is its ability to concentrate in the granular and fat tissue of the breast instead of in the liver. Between January and June 1986, we carried out a phase I study on 101 patients divided into four randomized groups receiving placebo and 100, 200, and 300 mg/day of 4-HPR. Patients received the drug for 6 months without any major toxic effect. This finding was confirmed by another 6-month study in which patients received a common dose of 200 mg/day. In March 1987, a phase III study was started to evaluate the effectiveness of 4-HPR in preventing contralateral primary tumors in women who had already been treated for breast cancer. If 4-HPR succeeds in preventing second primaries in breast cancer patients, it may be useful for a wider group of subjects at high risk for breast cancer. This randomized study was designed with two arms: an intervention group versus a group receiving no treatment. Patients in the intervention group will be treated with 200 mg/day 4-HPR for 5 years. Patients in the control group will not be treated. A further 2 years of follow-up is planned for both groups. Currently, 2450 patients have been recruited. We expect a total accrual of 3500 patients by the end of 1992.

The aromatic synthetic retinoid acid derivative, acitretin, is efficacious in several cutaneous diseases. Its toxicological profile makes a topical form with no or reduced systemic adverse effects desirable. Direct application of a topical acitretin formulation might result in therapeutic skin concentrations at the site of the disease while minimizing systemic exposure. The present studies define the percutaneous absorption characteristics of acitretin from an isopropylmyristate formulation. We investigated, in-vitro, (1) the role of receptor solution variations, (2) the role of skin modifications, (3) the influence of skin from three different species on the absorption of topically applied acitretin and (4) the drug distribution within the skin. Addition of solubilizers (Polyethylenglycol-20 and albumin) to the receptor solutions improved the flux of acitretin through monkey skin, whereas the acitretin concentration in the skin was not affected by the various receptor solutions used. Acitretin flux through tape-stripped monkey skin and dermis was only slightly higher than through intact skin. Acitretin concentration in human skin was significantly higher than in rhesus monkey or guinea-pig skin. Topical application of acitretin can produce dermal concentrations in excess of those achieved by therapeutic oral doses.

Two patients with ichthyosis bullosa of Siemens (IBS) and one patient with bullous ichthyosiform erythroderma of Brocq (BIE) were treated with etretinate. Two additional patients with IBS received acitretin. All the patients had a marked improvement when on retinoids and the maintenance dose required was for IBS 10-25 mg per day. The patient with BIE was on a maintenance dose of 40-60 mg per day.

The activities of the research group on cancer chemoprevention supported by the Ministry of Health and Welfare of Japan are summarized. Since the research on cancer chemoprevention is quite new in Japan, this paper describes the present status of the development of possible cancer preventive agents. Acyclic retinoid (E-5166) and sarcophytol A encourage further studies. New inhibitory compounds of experimental carcinogenesis, such as cryptoporic acid, oleanolic acid, mokko lactone and ursolic acid were isolated from plants. The importance of the keen collaboration between natural product chemists and clinicians for further meaningful progress in this research field has been emphasized.

This is a review of the efficacy of etretinate/acitretin in the treatment of psoriasis and of the currently reported side-effects. The data indicate that retinoids bring significant improvement (if not total clearing) with frequent low-morbidity but rarely serious side-effects. The most serious side-effect of etretinate/acitretin is teratogenicity.

Thirty-four patients with extensive psoriasis were treated in a double-blind parallel fashion with either acitretin plus bath PUVA (trimethylpsoralen bath + UVA) or etretinate plus bath PUVA. Each group consisted of 17 patients. The dose of retinoid was 40 mg/day during the 2-week monotherapy phase and subsequently 20 mg/day during the combination treatment. Bath PUVA was given three times a week starting with a UVA dose of 0.06 J/cm2. Remission (greater than 90% improvement) was achieved in all patients in 6-10 weeks. There were no significant differences in clinical response between the two groups; the mean +/- SD PASI score (psoriasis area and severity index) before treatment was 22.6 +/- 7.1 in the acitretin-PUVA group and 19.4 +/- 7.8 in the etretinate-PUVA group. The corresponding figures after treatment were 0.6 +/- 0.6 and 1.0 +/- 0.5, respectively. Side-effects related to retinoid treatment were frequent in both groups but they were usually mild and well-tolerated. There was only one case of diffuse alopecia after 8 weeks in the etretinate-PUVA group. Scaling of the palms and soles was seen in six patients in the acitretin-group but only in two patients in the etretinate-group. Triglycerides were elevated in about half of the patients in both groups. The present study shows that acitretin is as effective as etretinate in the combination with bath PUVA.

Treatment with an oral aromatic retinoid (etretinate) and with PUVA significantly reduced the elevated levels of putrescine, spermidine and spermine in psoriatic lesions. Both treatments also significantly reduced the spermidine/spermine ratio, which is considered to be an indicator of proliferation activity. Although both regimens produced a roughly parallel reduction of epidermal polyamines the initial fall of putrescine was much more rapid in patients receiving retinoid. This may indicate that one of the primary targets of retinoids could be ornithine decarboxylase.

The light and electron microscopic structure of pityriasis rubra pilaris (PRP) is described in five patients. Hyperkeratosis, hypergranulosis, keratotic plugs in the follicular openings, acanthosis and focal parakeratosis were observed. A moderate perivascular infiltrate was seen in the upper dermis. Electron microscopy revealed moderately activated keratinocytes, a decreased number of tonofilaments and desmosomes, enlarged intercellular spaces, parakeratosis with lipid-like vacuoles and a large number of keratinosomes. Lymphoid cells were present in the epidermis in moderate numbers. At the dermo-epidermal junction, the basal lamina was focally split, containing gaps. Etretinate therapy produced moderate to marked clinical improvement. The histological picture improved but the typical signs of PRP, including follicular plugging, persisted. Ultrastructurally the cellular activity and the amount of hyperkeratosis and parakeratosis decreased, while increases in keratinosomes, intercellular substance, microvilli and desmosomes were observed during treatment.

Oral retinoids obviously influence dermal components such as cutaneous capillaries and dermal inflammatory cells in addition to their well-known action on keratinizing epithelia. On this basis, they act as an anti-inflammatory drug. In particular, they reduce the elevated skin temperature, inhibit the motility of neutrophils and eosinophils and their migration into the epidermis, decrease DNA synthesis of human lymphocytes by blocking their response to lectins and stimulate Langerhans cells, monocytes and macrophages in various in vitro and in vivo models. These data indicate that oral retinoids may not only normalize disorders of keratinization but also exert distinct therapeutic effects on various skin diseases with dermal inflammatory involvement regardless of their particular aetiology. In some respects, retinoids resemble corticosteroids, acting as a modified hormone. Preliminary clinical experiences with oral retinoid treatment in skin diseases such as cutaneous disseminated LE, bullous pemphigoid, Duhring's disease, pemphigus, Behçet's disease and necrotizing vasculitis with eosinophilia support these data. Monotherapy or combined administration of oral retinoids with corticosteroids in low doses seems therapeutically beneficial in these disorders.

Synthetic and natural retinoids were analysed in epidermis, dermis, subcutis and serum of twenty-seven patients treated with etretinate (0.6-1.0 mg/kg/day) for 1-36 months. The concentrations of etretinate (including its major metabolite) in serum and normal-appearing epidermis were 150-600 ng/ml and 50-350 ng/g, respectively. The serum and epidermal values were significantly correlated (P less than 0.05). The drug progressively accumulated in the subcutis attaining a maximum value of 15,500 ng/g. After the treatment, etretinate disappeared from the epidermis within 1 week. By contrast, the drug remained in the subcutis for several months after cessation of treatment. The epidermal composition of endogenous retinoids changed during etretinate therapy as reflected in an increased ratio of 3-dehydroretinol to all-trans retinol.

Twenty patients with disabling disorders of keratinization were treated with Tigason (etretinate, Ro 10-9359), an oral aromatic retinoid, and the clinical responses and the effects on the skin were monitored. Most patients showed a considerable clinical improvement within 4 weeks. Side effects, such as cheilitis, were common but mostly transient or minor. In the skin there was a decrease in glucose-6-phosphate dehydrogenase activity within the granular cell layer of the epidermis, and an increase in mean epidermal thickness and mean corneocyte area. However, there was little apparent effect on epidermal proliferation or on histological and ultrastructural appearances. These findings suggest that the drug acts at a late stage of epidermal differentiation.

The administration of UVB phototherapy and low-dose acitretin (0.34-0.44 mg/kg body weight) was compared with the effect of acitretin alone and UVB phototherapy alone in 41 patients with plaque-type psoriasis. Of these patients, 32 received standard UVB phototherapy without acitretin. The other nine were treated with acitretin and the effect of UVB irradiation (Sylvania UV 21-tubes), applied to one half of the body, was assessed. Clearance was defined as 80-100% improvement and this occurred in eight out of the nine patients (89%) treated with acitretin-UVB (ReUVB) and, in two of them (23%), on the untreated side. Clearance occurred in 20 of the 32 (62.5%) patients given UVB alone. The improvement score was significantly higher for the ReUVB side than the acitretin side. Patients treated with ReUVB showed a statistically higher therapeutic score (95-100% clearance) than those receiving UVB alone. However, taking 80-100% improvement as the criterion, no significant difference was found. The number of treatments to clearance was significantly less for the patients treated with ReUVB than for the UVB (19.3 as compared with 24.9). The total UVB dose and the number of minimal erythema doses (MEDs) could be reduced by approximately 20% in the ReUVB group relative to the UVB group.

The mode of action of an aromatic analogue of retinoic acid, ethyl all-trans-9-(4-methoxy-2,3,6-trimethyl-phenyl)-3,7-dimethyl-2,4,6,8-nonatetraenoate (Ro 10-9359), a compound known to possess a considerable prophylactic and therapeutic effect on skin papillomas and carcinomas, was investigated with autoradiographic and histopathologic methods. The ip application of a single dose of 1,000 mg Ro 10-9359/kg to female Swiss mice with chemically induced skin papillomas caused a 29% regression of the mean tumor diameter after 3 days and a 51% regression after 7 days. In the tumors, the number of DNA-synthesizing cells [measured by the labeling index (LI)] and the length of the cell cycle were not affected by the retinoid; thus a mode of action at the level of cell proliferation can be excluded. In the normal skin, an increase in the LI of about 30% was observed. A small effect on the cell loss was observed; however, it was not sufficient to explain quantitatively the regression of the tumors. When measured histometrically, it appeared that the loss of the horn and the formation of necroses, 3-10 times larger than in the placebo groups, were mainly responsible for the tumor regressions caused by the retinoid. After 7 days, the proportion of stroma in the tumors was increased, and dilation of the vessels and edema in the stroma proximal to the necroses were frequent.

Male Syrian golden hamsters received 12 weekly intratracheal exposures to 0.5% N-methyl-N-nitrosourea with a special catheter. Following exposures, animals were randomized into 4 groups of 63 hamsters and placed on diets of lab meal or meal with 120 mg 13-cis-retinoid acid (CRA)/kg, 327 mg ethyl retinamide (ER)/kg, or 343 mg N-(2-hydroxyethyl)retinamide (HR)/kg for 6 months at which time all hamsters were killed. The observed incidences of tracheal epithelial neoplasms (No. of animals with tumors/total No. of animals) were 10/63 (lab meal), 22/61 (CRA), 24/63 (ER), and 17/62 (HR). The incidence of carcinomas (No. of animals with tumors/total No. of animals) were 4/63 (lab meal), 12/61 (CRA), 12/63 (ER), and 11/62 (HR). The weight loss and mortality relative to those in the group fed the lab meal were significantly in the group fed HR but not in the other retinoid-treated groups.

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.

An oral dose of all-trans-retinoic acid or 13-cis-retinoic acid in the pregnant [Lak:LVG(SYR)] hamster caused a dose-dependent increase in malformations in the offspring, but an equivalent dose of all-trans-N-ethyl retinamide or 13-cis-N-ethyl retinamide failed to result in embryotoxicity. The present results show that structural modification of the retinoid skeleton can produce compounds that retain cancer chemopreventive activity but that lack the teratogenic activity common to many synthetic and naturally occurring forms of vitamin A. The results indicate that in the case of the retinoids the two kinds of activity--interference with the process of carcinogenesis and interference with embryonic development--may be divorced.

Eight patients with psoriasis or Darier's disease were studied during 1-3 months of 'Tigason' treatment (0.7-0.8 mg/kg body weight). The levels of triglycerides and cholesterol in whole serum and in different lipoprotein fractions were repeatedly determined. Before treatment all lipid values were within the normal range. During treatment the values changed gradually and five of the eight patients developed pathological VLDL triglyceride concentrations. A significant increase of VLDL cholesterol and a decrease in the HDL cholesterol was also noted. Until more information is available, prolonged treatment periods with 'Tigason' should perhaps be avoided, at least in high risk patients.

Retinoic acid 5,6-epoxidase activity was found in several hemoproteins such as human oxy- and methemoglobin (HbO2 and MetHb), equine skeletal muscle oxy- and metmyoglobin (MbO2 and MetMb), bovine liver catalase, and horseradish peroxidase. Hematin also catalyzed retinoic acid 5,6-epoxidation. The results suggest that the heme moiety participates in the epoxidation. However, neither horse heart cytochrome c, nor free ferrous ion nor free ferric ion exhibited the epoxidase activity. Some hemoproteins (HbO2, MetHb, MbO2, MetMb, catalase, peroxidase, and hematin) exhibited characteristic individual pH dependences of the activity, suggesting that the epoxidase activities of the hemoproteins are influenced by the apoenzymes to some degree. This view is also supported by the finding that preincubation of an HbO2 preparation at various temperatures (37-70 degrees C) reduced its epoxidase activity with increasing temperature, whereas the activity of hematin was unaffected. Active oxygen scavengers such as mannitol, catalase, and superoxide dismutase exhibited no effect on the epoxidase activities of HbO2, MetHb, MbO2, and MetMb. A ligand of heme, CN- (100 mM), inhibited the epoxidase activities but N3- (100 mM) did not. The epoxidase activities were completely inhibited by NADPH, NADH, and/or 2-mercaptoethanol but not by NADP+ and/or NAD+. An intermediate in the epoxidation may be reduced by NADPH, NADH and/or 2-mercaptoethanol. Radical species can be considered as plausible candidates for the intermediate.

In five patients with Darier's disease, lymphocyte subpopulations, lymphocyte responsiveness to phytohaemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and purified protein derivative of tuberculin (PPD), leukocyte migration inhibitory factor (LMIF) production and suppressor cell activities were studied before and during oral etretinate treatment. Pre-therapy investigations of cell-mediated immunity showed no severe immunological dysfunction, although high responses to supra-optimal Con A concentrations suggested abnormalities in immunoregulatory lymphocyte subpopulations. In addition, two patients showed enhanced LMIF production upon stimulation with Con A, PWM and PPD. Retinoid therapy decreased the number of peripheral blood total leukocytes, lymphocytes and T-cells, normalized the LMIF production, and decreased the lymphocyte responses to mitogens. Furthermore, the dose-response curve to Con A changed toward normal and the suppressor cell activity regulating Con A responses tended to increase.

When treated with a retinoic acid analog (Ro 11-1430), the Swarm rat chondrosarcoma regressed (t 1/2 = 11-12 days) with a rapid removal of tumor proteoglycan, histologic evidence of mineralization, and cartilage proteoglycan synthesis was suppressed down to a value of 1% of the control. During the first 3 weeks of treatment, the newly synthesized proteoglycan was similar both in aggregation and size to the proteoglycan present in the control. However, after 5 weeks of treatment synthesis shifted to a small nonaggregating proteoglycan with longer glycosaminoglycan chains now containing dermatan sulfate, possibly representing a switch in proteoglycan synthesized. Heparan sulfate was also detected. Unlabeled proteoglycan released from the tissue during Ro 11-1430 treatment was large (Kav = 0.25 on Sepharose CL-2B) but incapable of aggregation, suggesting the initial proteolytic cleavage was in or near the hyaluronic acid-binding region of the proteoglycan. Degradative enzyme activity varied during the period of treatment. Since other tissues remained histologically normal during the treatment with Ro 11-1430, this drug may have possible therapeutic value.

A double-blind study of twenty-eight patients with severe oral lichen planus treated with etretinate (75 mg daily) or a placebo for 2 months, showed that the oral retinoid had a marked beneficial effect. Nine non-responders who had received only placebo then entered an open cross-over study and they responded well to etretinate. Etretinate thus provided effective symptomatic relief for severe oral lichen planus, but side-effects were common, and six patients stopped treatment because of them.

The usefulness of a short-term azaserine [CAS: 115-02-6; diazoacetate serine (ester)]-rat model for the screening of retinoids (known chemopreventive agents) and the effect of two retinoids on the growth of azaserine-induced, presumptive preneoplastic foci of acinar cells were examined. At 14 days of age, male Lewis rats were each given injections of a single dose of 30 mg azaserine/kg body weight. These rats were weaned to test diets to which retinoids were added. At 4 months post initiation, pancreata were examined by quantitative stereologic methods to determine number and mean size of foci. Two phenotypically different populations of foci were observed and characterized as acidophilic or basophilic. Retinylidene dimedone and N-2-hydroxyethylretinamide decreased the number and size of the acidophilic foci but not the basophilic foci. The inhibition of growth of the acidophilic foci correlates well with the known effects of these retinoids in long-term carcinogenicity studies.

A patient with subcorneal pustular dermatosis with a fatal outcome is presented. Treatment with corticosteroids, vitamin E, dapsone, sulphapyridine and levamisole was ineffective. Only systemic treatment with retinoic acid and a new aromatic retinoic acid derivative (Ro 10-9359) produced a satisfactory clinical response, but a complete remission was not obtained.