In Vitro Effects of Acitretin on Human Neuronal SH-SY5Y Cells.

Acitretin is an oral drug approved by the Food and Drug Administration that is commonly used to treat psoriasis. In recent years, acitretin has been identified as a candidate drug for the treatment of Alzheimer's disease, but its role in neuronal development is still unclear. In this study, the human neuroblastoma cell line SH-SY5Y was used as a model to study neuronal differentiation. We found that acitretin effectively promoted the differentiation of SH-SY5Y cells into neuronal cells and upregulated the expression of the neuronal marker ß-III tubulin and the mature neuronal marker NFH. Differentially expressed genes were identified by RNA sequencing and analyzed by bioinformatics approaches. The results showed that genes associated with neuron development-related pathways, such as SSPO and KCNT1, had significant changes in expression. Analysis showed that PRKCA and CAMK2B may play important roles in the process by which acitretin promotes neurodevelopment. Through whole-cell patch clamping and a microelectrode array assay, we found that acitretin-treated neurons generated electrical spikes similar to those generated by mature neurons. This study provided evidence to support an accessible and safe model of neuron-like cells and verified that acitretin can promote the differentiation of neurons and has the potential to treat brain tumors and neurodevelopmental and neurodegenerative diseases.

Regulatory Effect of Adipose-Derived Mesenchymal Stem Cells and/ or Acitretin on Adam10 Gene in Alzheimer's Disease Rat Model.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by progressive cognitive deterioration. All recent therapeutic strategies tend to inhibit the generation of the Aß peptide. These approaches tend to mediate both α - and γ -secretases to undergo the nonamyloidogenic pathway. ADAM10 is the main α-secretase that cleaves APP, and it is regulated by the metabolic product of vitamin A (retinoic acid), which is being widely used recently in AD research as a target for treatment. Mesenchymal stem cells (MSCs) are also used recently as a promising regenerative therapy for AD. OBJECTIVES: The present study aimed to: (1) study the effect of MSCs with/without acitretin on the regulation of Adam10 gene expression in AlCl3-induced AD rat model, and (2) validate the hypothesis that AD is a time-dependent progressive disease that spreads spontaneously even after the stopping of exposure to AlCl3. METHODS: The experimental work has been designed to include three successive phases; AlCl3 induction phase (I), AlCl3 withdrawal phase (W), and therapeutic phase (T). Forty-five male albino Wistar rats were randomly divided into 2 main groups: the control (C) group (15 rats) and AD group (30 rats). The therapeutic potential of MSCs with/without acitretin has been evaluated at behavioral, physiological, molecular, and histopathological levels. RESULTS: Among the three therapeutic groups, combined administration of both MSC and acitretin showed the best compensatory effects on most of the measured parameters. CONCLUSION: The present study approved that AD is a time-dependent progressive disease which spreads spontaneously without more AlCl3 exposure.

Ictiosis arlequín tratada con acitretino / Treatment of Harlequin Ichthyosis With Acitretin

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A novel blood-brain barrier co-culture system for drug targeting of Alzheimer's disease: establishment by using acitretin as a model drug.

In the pathogenesis of Alzheimer's disease (AD) the homeostasis of amyloid precursor protein (APP) processing in the brain is impaired. The expression of the competing proteases ADAM10 (a disintegrin and metalloproteinase 10) and BACE-1 (beta site APP cleaving enzyme 1) is shifted in favor of the A-beta generating enzyme BACE-1. Acitretin--a synthetic retinoid-e.g., has been shown to increase ADAM10 gene expression, resulting in a decreased level of A-beta peptides within the brain of AD model mice and thus is of possible value for AD therapy. A striking challenge in evaluating novel therapeutically applicable drugs is the analysis of their potential to overcome the blood-brain barrier (BBB) for central nervous system targeting. In this study, we established a novel cell-based bio-assay model to test ADAM10-inducing drugs for their ability to cross the BBB. We therefore used primary porcine brain endothelial cells (PBECs) and human neuroblastoma cells (SH-SY5Y) transfected with an ADAM10-promoter luciferase reporter vector in an indirect co-culture system. Acitretin served as a model substance that crosses the BBB and induces ADAM10 expression. We ensured that ADAM10-dependent constitutive APP metabolism in the neuronal cells was unaffected under co-cultivation conditions. Barrier properties established by PBECs were augmented by co-cultivation with SH-SY5Y cells and they remained stable during the treatment with acitretin as demonstrated by electrical resistance measurement and permeability-coefficient determination. As a consequence of transcellular acitretin transport measured by HPLC, the activity of the ADAM10-promoter reporter gene was significantly increased in co-cultured neuronal cells as compared to vehicle-treated controls. In the present study, we provide a new bio-assay system relevant for the study of drug targeting of AD. This bio-assay can easily be adapted to analyze other Alzheimer- or CNS disease-relevant targets in neuronal cells, as their therapeutical potential also depends on the ability to penetrate the BBB.

The effects of alcohol on the metabolism and toxicology of anti-psoriasis drugs.

INTRODUCTION: Alcohol has long been suspected to be a triggering and precipitating factor of psoriasis. Alcohol misuse is common in patients with moderate-to-severe psoriasis and appears to impair treatment outcome. AREAS COVERED: In this article, the authors review the available data regarding the metabolic and toxicological interactions between anti-psoriasis systemic drugs and ethanol and/or alcoholic beverages. Special attention is given to the influence of alcohol consumption on the hepatotoxic risk of some anti-psoriasis drugs. The article was prepared using a MEDLINE literature search. EXPERT OPINION: The available knowledge highlights the existence of a few significant pharmacological interactions, such as the reduced exposure to cyclosporine by red wine, the possible increase of cyclosporine levels following a heavy acute alcohol intake, and, especially, the conversion of acitretin to etretinate, in the presence of ethanol, with important implications in females of child-bearing potential. There are limited data on the contributing role of alcohol in the hepatotoxicity induced by some anti-psoriasis drugs and the existing information on this topic is still controversial. However, further investigation is needed to assess the relevance of interactions between alcohol consumption and drug therapy for psoriasis, under both pharmacological and toxicological perspectives. Long-term prospective studies on large cohorts of patients are warranted to disclose the actual significance of such potential interactions in clinical practice.

Acitretin, an enhancer of alpha-secretase expression, crosses the blood-brain barrier and is not eliminated by P-glycoprotein.

BACKGROUND: ADAM10 (a disintegrin and metalloproteinase 10) has been demonstrated to act as the main physiological α-secretase. Enzymatic activity of the α-secretase on the one hand prevents the formation of toxic Aß peptides and on the other hand promotes the secretion of a neurotrophic and neuroprotective amyloid precursor protein fragment (APPs-α) by cleaving the amyloid precursor protein within its Aß sequence. Enhancement of ADAM10's gene expression may therefore present a valuable therapeutic approach for the treatment of Alzheimer's disease (AD), where Aß peptides are severely involved in the pathogenesis. OBJECTIVE: In cell culture and in a transgenic mouse model of AD, retinoids led to increased ADAM10 expression and activity. We therefore endeavor to develop a clinical application of synthetic retinoids such as acitretin in AD. METHODS: The effect of synthetic retinoids on ADAM10 gene expression was analyzed by reporter gene assays in human neuroblastoma cell line SH-SY5Y. Penetrance of acitretin into the murine brain was analyzed by high-performance liquid chromatography. P-glycoprotein (P-gp) double-knockout mice with a deficiency in both isoforms, mdr1a and 1b, were used to analyze a possible role of P-gp-dependent efflux on acitretin distribution. RESULTS: Acitretin and tamibarotene are both potent activators of ADAM10 promoter activity. Acitretin crosses the murine blood-brain barrier and its level in the mouse brain is not reduced by P-gp. CONCLUSION: Synthetic retinoids and especially acitretin seem to be ideal candidates to establish an ADAM10-based AD treatment, and therefore have already entered first clinical trials.

Vitamin A: a multifunctional tool for development.

Extensive research carried out over the last 100 years has established that the fat-soluble organic compound vitamin A plays crucial roles in early development, organogenesis, cell proliferation, differentiation and apoptosis as well as in tissue homeostasis. Given its importance during development, the delivery of vitamin A to the embryo is very tightly regulated with perturbations leading to severe malformations. This review discusses the roles of vitamin A during human development and the molecular mechanisms controlling its biological effects, hence bridging the gap between human development and molecular genetic work carried out in animal models. Vitamin A delivery during pregnancy and its developmental teratology in humans are thus discussed alongside work on model organisms, such as chicken or mice, revealing the molecular layout and functions of vitamin A metabolism and signaling. We conclude that, during development, vitamin A-derived signals are very tightly controlled in time and space and that this complex regulation is achieved by elaborate autoregulatory loops and by sophisticated interactions with other signaling cascades.

Up-regulation of the alpha-secretase ADAM10 by retinoic acid receptors and acitretin.

Late-onset Alzheimer's disease is often connected with nutritional misbalance, such as enhanced cholesterol intake, deficiency in polyunsaturated fatty acids, or hypovitaminosis. The alpha-secretase ADAM10 has been found to be regulated by retinoic acid, the bioreactive metabolite of vitamin A. Here we show that retinoids induce gene expression of ADAM10 and alpha-secretase activity by nonpermissive retinoid acid receptor/retinoid X receptor (RAR/RXR) heterodimers, whereby alpha- and beta-isotypes of RAR play a major role. However, ligands of other RXR binding partners, such as the vitamin D receptor, do not stimulate alpha-secretase activity. On the basis of these findings, we examined the effect of synthetic retinoids and found a strong enhancement of nonamyloidogenic processing of the amyloid precursor protein by the vitamin A analog acitretin: it stimulated ADAM10 promoter activity with an EC(50) of 1.5 microM and led to an increase of mature ADAM10 protein that resulted in a two- to three-fold increase of the ratio between alpha- and beta-secretase activity in neuroblastoma cells. The alpha-secretase stimulation by acitretin was completely inhibited by the ADAM10-specific inhibitor GI254023X. Intracerebral injection of acitretin in APP/PS1-21 transgenic mice led to a reduction of Abeta(40) and Abeta(42). The results of this study may have clinical relevance because acitretin has been approved for the treatment of psoriasis since 1997 and found generally safe for long-term use in humans.

Acitretin is converted to etretinate only during concomitant alcohol intake.

BACKGROUND: Acitretin has replaced etretinate in the treatment of various disorders of keratinization due to a considerably shorter terminal half-life. Possible esterification of acitretin to etretinate in the presence of ethanol has been reported. OBJECTIVES: To determine the plasma concentrations of etretinate as a metabolite in patients with various disorders of keratinization after multiple acitretin dosing, and to assess the influence of alcohol consumption using a questionnaire. In addition, to study the influence of alcohol consumption on the risk of metabolic formation of etretinate. PATIENTS/METHODS: Eighty-six acitretin (Neotigason(R), Roche)-treated outpatients from three centres provided pre-dose (trough) samples for determining plasma concentrations of acitretin and its metabolites 13-cis-acitretin and etretinate. Patients received acitretin doses of between 0.1 and 1.3 mg kg-1 daily. The concentrations of etretinate, acitretin and 13-cis-acitretin were determined by reverse-phase high-performance liquid chromatography. RESULTS: Of the 86 patients, 30 had detectable plasma etretinate levels. No etretinate was found in 20 patients who reported that they never drank alcohol, while etretinate was found in all 16 patients with an average weekly alcohol consumption of > 200 g ethanol, corresponding to about 15 U (1 U equals half a pint of standard beer or a wine glass of non-fortified wine). Etretinate was detected in 14 of 50 patients with a moderate weekly alcohol intake of up to 200 g ethanol. A trend linking higher alcohol intake with both higher risk of etretinate formation and higher etretinate levels was observed. The study also revealed that the ethylesterification only relates to acitretin (13-trans-) and not to the main metabolite 13-cis-acitretin, although the latter compound showed higher plasma trough concentration levels at steady state. CONCLUSIONS: Owing to the teratogenic potential and possible side-effects of oral retinoids, fertile women especially should be informed about the importance of strict alcohol abstinence during treatment and for at least 2 months after stopping therapy. In case of non-compliance with alcohol abstinence a post-therapy contraceptive period of 2-3 years should be recommended.

In vitro metabolism of acitretin by human liver microsomes: evidence of an acitretinoyl-coenzyme A thioester conjugate in the transesterification to etretinate.

The aromatic retinoid acitretin is the primary active metabolite of etretinate, and in this study we investigated the ethyl esterification of acitretin to etretinate using [(14)C]acitretin and human liver microsomes. Samples were analysed by TLC, HPLC, and LC-MS. Essential requirements for the transesterification reaction were identified and included viable microsomal protein, ATP, CoASH, and ethanol. Human liver microsomes catalysed formation of acitretinoyl-CoA at the rate of 0.08 +/- 0.02 nmol/min/mg (mean +/- SD, N = 10). Acitretinoyl-CoA was pivotal for the transesterification to etretinate and in the presence of methanol, ethanol, n-propanol, n-butanol, and hexanol, the corresponding esters, namely methyl-, ethyl (etretinate)-, propyl-, butyl-, and hexyl-acitretinate, were formed. On average, 1.7% of the acitretin present in the incubation was converted to etretinate in the presence of ethanol. In the absence of ethanol, transesterification did not proceed. Inhibition of the ester hydrolysis of etretinate by bis-p-nitrophenylphosphate (BNPP, 1 mM) prevented futile cycling of etretinate via acitretinoyl-CoA. An additional finding was that acitretin (15-30 microM) activated significantly human liver microsomal long-chain fatty acid-CoA ligase (E.C.6.2.1.3, LCL), resulting in enhanced formation of palmitoyl-CoA. This study demonstrated that in the presence of ethanol the ethyl esterification of acitretin to etretinate proceeds via formation of acitretinoyl-CoA. Predicting clearance of acitretin in vivo via this unique metabolic pathway will be a challenge, as the intracellular concentration of ethanol could never be predicted with any degree of accuracy in humans.

Acitretin in psoriasis: an overview of adverse effects.

Oral retinoids are among the drugs of choice for pustular and erythrodermic psoriasis. In addition, retinoids are effective in combination with other topical and systemic agents for the treatment of plaque-type psoriasis. Acitretin, the active retinoid metabolite, has replaced etretinate in retinoid therapy of psoriasis because of its more favorable pharmacokinetic profile, including a significantly shorter half-life. Retinoids, including acitretin, are potent teratogens, leading to strict requirements for pregnancy prevention during and after their use. Other retinoid side effects are generally preventable or manageable through proper patient selection, dose adjustments, and routine monitoring. Mucocutaneous side effects such as cheilitis and hair loss are the most common dose-dependent side effects, requiring dose reduction in some patients. Less common effects such as hepatotoxicity, serum lipid alterations, pancreatitis, and possible skeletal effects are also discussed.

Mechanistic studies on the ethyl-esterification of acitretin by human liver preparations in vitro.

Studies have been performed with human liver microsome preparations in vitro, to investigate the reaction mechanisms involved in the conversion of acitretin to the corresponding ethyl ester, etretinate. The results indicate that: Three fresh samples of human liver, which had been stored in liquid nitrogen for up to 8 months, all produced traces of etretinate (5.8 +/- 0.8 ng/ml) in the presence of ethanol but not when the acitretin was added in acetone, or when the sample was denatured by preheating. Studies with pooled human liver microsomes, to identify the cellular location of the enzymes and the co-factors involved in this esterification, indicate a primary requirement for both ethanol and CoA + ATP with a secondary potentiation in the presence of an NADPH regenerating system. A possible explanation for these finding is that the microsomal ligase enzymes form an intermediate ester between CoA and acitretin, which is then trans-esterified by the ethanol. The low formation with CoA + ATP may indicate that second stage of this process occurs spontaneously, with the NADPH potentiation suggesting that it could also be mediated enzymically.

Simultaneous determination of etretinate, acitretin and their metabolites in perfusate, perfusate plasma, bile or hepatic tissue with reversed-phase high-performance liquid chromatography.

Etretinate is a synthetic aromatic retinoid used in the treatment of psoriasis and other disorders affecting the skin. Acitretin is the primary active metabolite of etretinate. The in situ perfused rat liver model was used to study the first-pass hepatic metabolism of etretinate and acitretin and a reliable method of quantifying etretinate and its metabolites was needed. Previously published assays allow for the simultaneous quantitation of etretinate and acitretin in blood or plasma. This paper describes an accurate and reliable reversed-phase HPLC method for the determination of etretinate, acitretin and their metabolites in whole perfusate, plasma, bile and hepatic tissue.

Measurement of subnanomolar retinoic acid binding affinities for cellular retinoic acid binding proteins by fluorometric titration.

Cellular retinoic acid binding protein I (CRABP-I) and cellular retinoic acid binding protein II (CRABP-II) are small, cytoplasmic proteins which bind all-trans-retinoic acid with high affinity. Both of these proteins belong to a family of intracellular proteins which bind amphiphilic lipids, including fatty acids, bile salts, and retinoids. Because CRABP-I and -II exhibit different tissue distributions and differential transcriptional regulation, they are proposed to serve different functions. The binding properties of mouse CRABP-I and -II purified from Escherichia coli were examined to further understand their role in intracellular retinoic acid processing. Fluorescence titrations were performed using nanomolar protein concentrations, near the obtained dissociation constants, and analyzed by direct mathematical fitting to raw data, in order to extend the range and accuracy of binding constant determination. The apparent dissociation constants, K'd, of mouse CRABP-I and CRABP-II binding all-trans-retinoic acid were determined to be 0.4 +/- 0.3 nM and 2 +/- 1 nM respectively, stronger binding than previously reported. The K'd of mCRABP-I and mCRABP-II complexing with acitretin, a pharmacologically active synthetic retinoid used in the treatment of psoriasis, was 3 +/- 1 nM and 15 +/- 11 nM. Both CRABPs bound 9-cis-retinoic acid with a K'd of roughly 200 nM, and neither exhibited significant binding of 13-cis-retinoic acid.

Inability to detect plasma etretinate and acitretin is a poor predictor of the absence of these teratogens in tissue after stopping acitretin treatment.

1. Concentrations of etretinate, acitretin and its main metabolite 13-cis-acitretin were measured in plasma and subcutaneous fat samples from 37 women of childbearing age exposed to acitretin before November 1990. Twenty of the women still used acitretin and 17 had stopped therapy for a period ranging from 1 to 29 months. 2. The prevalences of detectable etretinate concentrations were 45% and 83% in plasma and subcutaneous tissue, respectively, among current acitretin users and 18% and 86% among those who had stopped acitretin therapy. Thus, inability to detect plasma etretinate is a poor predictor of the absence of etretinate in fat. 3. Acitretin and/or etretinate were detectable in fat and in some cases in plasma from women who had ceased acitretin therapy for up to 29 months. 4. We suggest that (cis)-acitretin and etretinate should be monitored in subcutaneous tissue when plasma measurements are negative. The recommended contraception period of 2 years after cessation of acitretin therapy should be reconsidered to avoid the risk of teratogenicity.

Kinetics of tissue distribution and elimination of retinoid drugs in the rat. II. Etretinate.

Rats were injected with single intravenous doses of etretinate (6 mg/kg), and concentrations of the drug and its metabolites, acitretin and 13-cis-acitretin, were determined in plasma and nine tissues up to 96 hr. A newly developed sensitive method for the determination by HPLC of the three retinoids in tissues was used. Etretinate rapidly appeared in most tissues and underwent a redistribution from highly perfused organs into muscle, skin, and ultimately, adipose tissue. Tissue/plasma concentration ratios ranged from 14 to 1, with the highest value in adipose tissue. In this tissue, maximum concentration was reached after 1.5 hr and remained practically constant up to 96 hr. Etretinate was rapidly hydrolyzed to form acitretin at concentrations that surpassed those of the parent drug in plasma, liver, kidney, and brain. After 6 hr, approximately 45% of etretinate had been metabolized to acitretin and approximately 40% to unidentified metabolites. These metabolites were not observed in tissues after 6 hr postdose. The parent drug was not observed 12 hr postdose, except for 6% of the dose remaining in adipose tissue. Etretinate elimination, in most tissues, was biphasic with terminal half-lives of 41 hr in skin, 1-6 hr in other lean tissues, and 1.7 hr in plasma. A volume of distribution of 1.7 liters/kg was determined, and a clearance of 12 ml.min-1.kg-1. Etretinate is characterized by rapid metabolism, transient storage in skin, and prolonged storage at a low level in adipose tissue as a deep compartment. A comparison of the pharmacokinetics of the closely related retinoids, etretinate and acitretin, disclose very pronounced differences.

Conversion of acitretin to etretinate in psoriatic patients is influenced by ethanol.

Acitretin has recently been introduced to replace etretinate in the treatment of severe psoriasis due to a considerable shorter terminal half-life. The previously recommended 2-month anticonceptive period after acitretin treatment has been extended to 2 years after the detection of etretinate in certain acitretin recipients. In the present study, 10 patients with severe psoriasis were treated with 30 mg acitretin daily for 3 months. Seven patients had detectable mean steady-state plasma etretinate concentrations in the range of 2.5 to 56.7 ng/ml. Four of the patients showed teratogenic levels of plasma etretinate. Consumption of alcohol appeared to be an important contributing factor for the formation of etretinate. As judged from the dose- and body-weight-normalized AUC values (AUCcor) there was a great inter-individual variation (sixfold) in the systemic availability of acitretin. After discontinuation of therapy, the rate of elimination of both acitretin (t1/2 range 1.0 to 25.4 d) and 13-cis-acitretin (t1/2 range 1.5 to 25.7 d) was found to be related to the observed mean steady-state level of etretinate as evidenced by a longer terminal t1/2 of patients with high levels of etretinate in plasma. A mean terminal elimination half-life of etretinate was found to be 45.7 d +/- 10.6 (mean +/- SD; range 27.0 to 59.3 d). The risk of metabolic formation of etretinate in acitretin recipients makes it impossible to draw any definite conclusion with regard to recommendation of length of anticonceptive period following acitretin therapy in psoriatics. Monitoring of plasma etretinate levels in acitretin-treated fertile women is advisable.

The metabolism of acitretin and isoacitretin in the in situ isolated perfused rat liver.

1. The metabolism of acitretin and its 13-cis isomer, isoacitretin, has been investigated in the in situ isolated perfused rat liver in order to differentiate the action of the liver from that of the gut on the metabolism of these isomers. 2. Acitretin undergoes alpha-oxidation, chain shortening O-demethylation, and glucuronidation in the perfused rat liver. 3. Isoacitretin undergoes glucuronidation as the major, almost exclusive, route of metabolism in the perfused rat liver. 4. The difference in the hepatic metabolism of the cis and trans isomers of this retinoid may explain the differences in their pharmacokinetics, and may help in understanding the pharmacokinetics of related retinoids.