Endogenous Metabolite; PPARβ/δ (Kd = 17 nM); PPARα (Kd = 103 nM); PPARγ (Kd = 178 nM); PPARα (IC50 = 14 nM); PPARγ (IC50 = 14 nM); RARβ (IC50 = 14 nM)

Retinoic acid, also known as all-trans retinoic acid, or ATRA, is a highly powerful derivative of vitamin A that is necessary for almost all vital physiological processes and functions. It plays a role in over 530 different genes' transcriptional control. The mechanism by which retinoic acid works is through its role as an activating ligand for the nuclear retinoic acid receptor (RARα-γ), which joins forces with the retinoic acid X receptor (RXRα-γ) to form a heterodimer[1]. With Kd values between 100 and 200 nM, retinoic acid (RA) binds to PPARα and PPARγ with low affinity. On the other hand, retinoic acid exhibits high affinity and isotype selectivity when it binds to PPARβ/δ, with a Kd of 17 nM [2]. The retinoic acid (RA) receptors RARα, RARβ, RARγ, and PPARβ/δ, as well as the retinoic acid-binding proteins CRABP-II and FABP5, are expressed by undifferentiated P19 cells. Induction of differentiation by treating cells with retinoic acid resulted in transitory overexpression of CRABP-II and downregulation of FABP5, which was detected at the relevant protein and mRNA levels. After an initial decline, FABP5 protein and mRNA levels climbed 2-2.5-fold in mature neurons compared with undifferentiated P19 cells. The levels of PPARβ/δ and RARα were not significantly impacted by the induction of differentiation. By day 4, RARγ mRNA levels declined nearly 5-fold and remained low in mature neurons [3]. Retinoic acid (RA) is a morphogen generated from retinol (vitamin A) that plays a crucial role in cell development, differentiation, and organogenesis. Retinoic acid interacts with retinoic acid receptors (RAR) and retinoic acid X receptors (RXR) to modulate the expression of target genes [4].

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UAB30 is an RXR selective agonist that has been shown to have potential cancer chemopreventive properties. Due to high efficacy and low toxicity, it is currently being evaluated in human Phase I clinical trials by the National Cancer Institute. While UAB30 shows promise as a low toxicity chemopreventive drug, the mechanism of its action is not well understood. In this study, we investigated the effects of UAB30 on gene expression in human organotypic skin raft cultures and mouse epidermis. The results of this study indicate that treatment with UAB30 results in upregulation of genes responsible for the uptake and metabolism of all-trans-retinol to all-trans-retinoic acid (ATRA), the natural agonist of RAR nuclear receptors. Consistent with the increased expression of these genes, the steady-state levels of ATRA are elevated in human skin rafts. In ultraviolet B (UVB) irradiated mouse skin, the expression of ATRA target genes is found to be reduced. A reduced expression of ATRA sensitive genes is also observed in epidermis of mouse models of UVB-induced squamous cell carcinoma and basal cell carcinomas. However, treatment of mouse skin with UAB30 prior to UVB irradiation prevents the UVB-induced decrease in expression of some of the ATRA-responsive genes. Considering its positive effects on ATRA signaling in the epidermis and its low toxicity, UAB30 could be used as a chemoprophylactic agent in the treatment of non-melanoma skin cancer, particularly in organ transplant recipients and other high risk populations. [1]

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retinoic acid (RA) modulates transcription of numerous target genes, thereby regulating a myriad of biological processes. It is well established that RA functions by activating retinoic acid receptors (RARs), which, in turn, control cell differentiation, proliferation, and apoptosis. However, perplexing reports of diverse and sometime opposing actions of RA have been published. Hence, while RA induces apoptosis and inhibits cell growth in some settings, it potentiates proliferation and acts as an anti-apoptotic agent in others. These observations raise the possibility that signaling pathways other than RAR may be involved in mediating RA activities. Here we show that RA is a high affinity ligand for another nuclear receptor, namely the orphan receptor peroxisome proliferator-activated receptor (PPAR) beta/delta. We demonstrate that while RA does not activate PPARalpha and PPARgamma, it binds to PPARbeta/delta with nanomolar affinity, modulates the conformation of the receptor, promotes interaction with the coactivator SRC-1, and efficiently activates PPARbeta/delta-mediated transcription. Transcriptional signaling by RA is thus exerted by a dual pathway, providing a rationale for understanding divergent cellular responses to this hormone. [2]

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retinoic acid (RA) regulates gene transcription by activating the nuclear receptors retinoic acid receptor (RAR) and peroxisome proliferator-activated receptor (PPAR) β/δ and their respective cognate lipid-binding proteins CRABP-II and FABP5. RA induces neuronal differentiation, but the contributions of the two transcriptional pathways of the hormone to the process are unknown. Here, we show that the RA-induced commitment of P19 stem cells to neuronal progenitors is mediated by the CRABP-II/RAR path and that the FABP5/PPARβ/δ path can inhibit the process through induction of the RAR repressors SIRT1 and Ajuba. In contrast with its inhibitory activity in the early steps of neurogenesis, the FABP5/PPARβ/δ path promotes differentiation of neuronal progenitors to mature neurons, an activity mediated in part by the PPARβ/δ target gene PDK1. Hence, RA-induced neuronal differentiation is mediated through RAR in the early stages and through PPARβ/δ in the late stages of the process. The switch in RA signaling is accomplished by a transient up-regulation of RARβ concomitantly with a transient increase in the CRABP-II/FABP5 ratio at early stages of differentiation. In accordance with these conclusions, hippocampi of FABP5-null mice display excess accumulation of neuronal progenitor cells and a deficit in mature neurons versus wild-type animals. [3]

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retinoic acid (RA) is a morphogen derived from retinol (vitamin A) that plays important roles in cell growth, differentiation, and organogenesis. The production of RA from retinol requires two consecutive enzymatic reactions catalyzed by different sets of dehydrogenases. The retinol is first oxidized into retinal, which is then oxidized into RA. The RA interacts with retinoic acid receptor (RAR) and retinoic acid X receptor (RXR) which then regulate the target gene expression. In this review, we have discussed the metabolism of RA and the important components of RA signaling pathway, and highlighted current understanding of the functions of RA during early embryonic development. [4]

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retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs). Three subtypes of these receptors exist, RAR alpha, RAR beta, and RAR gamma. The receptors are differentially expressed in different cell types and stages of development, suggesting that they may regulate different sets of genes. We have identified a synthetic retinoid with the characteristics of a selective RAR alpha antagonist. This antagonist counteracts RA effects on HL-60 cell differentiation and on B-lymphocyte polyclonal activation. Beyond its potential practical relevance, this and other specific antagonists will be useful to dissect the RAR system and to assign to one given receptor each of the many RA-regulated functions. [5]

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Isothiocyanates and phenolic antioxidants can prevent cancer through activation of Nrf2 (NF-E2 p45-related factor 2), a transcription factor that controls expression of cytoprotective genes through the antioxidant response element (ARE) enhancer. Using a human mammary MCF7-derived AREc32 reporter cell line, we now report that all-trans retinoic acid (ATRA), and other retinoic acid receptor alpha (RARalpha) agonists, markedly reduces the ability of Nrf2 to mediate induction of ARE-driven genes by cancer chemopreventive agents including the metabolite of butylated hydroxyanisole, tert-butylhydroquinone (tBHQ). The basal and tBHQ-inducible expression of aldo-keto reductase (AKR) AKR1C1 and AKR1C2 genes, which are regulated by Nrf2, was also repressed by ATRA in AREc32 cells. Antagonists of RARalpha augmented induction of ARE-driven gene expression by tBHQ, as did knockdown of RARalpha by using RNAi [6].

After applying retinoic acid at a concentration of 0.3 μM to embryos submerged in retinoic acid-containing tank water, zebrafish exhibit faster rod differentiation after 24 and 48 hours[6].

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Repression of Basal ARE-Gene Battery Expression by RA in Mouse Small Intestine in Vivo. [6]
To investigate whether retinoic acid/RA inhibits the expression of ARE-regulated genes in vivo, both Nrf2−/− and Nrf2+/+ mice were placed on a vitamin A-deficient (VAD) diet. Western blotting of proteins known to be regulated through Nrf2 revealed a profound increase in the levels of Gstm5, GCLC, NQO1, and Gsta1/2 in the small intestine of WT mice on a VAD diet (Fig. 6). No increase was observed in the levels of these proteins in Nrf2−/− mice on the VAD diet. The administration of retinoic acid/ATRA (10 mg/kg, 2 weeks i.p.) to WT mice on the VAD diet almost completely blocked the increase in Gstm5, GCLC, NQO1, and Gsta1/2 proteins in the small intestine (Fig. 6, lane 5), demonstrating that retinoids inhibit Nrf2 function in vivo. Administration of ATRA to WT mice on a control diet did not affect the expression of Gstm5, GCLC, NQO1, or Gsta1/2 (data not shown).

Fluorescence Titrations [2]
Bacterially expressed mPPARα-LBD, mPPARβ/δ-LBD, and mPPARγ-LBD (0.2–1 μm) were titrated directly in a cuvette with retinoic acid/RA dissolved in ethanol. Ethanol concentration was usually below 1% and never exceeded 2%. To ensure equilibration between protein and ligand, the fluorescence was monitored until a constant value was reached. The progress of titrations was monitored by following the decrease in the intrinsic fluorescence of the protein (excitation, 280 nm; emission, 340 nm), which accompanies RA binding. Inner filtering by the ligand, reflected by linear slopes observed following saturation of the protein, was corrected for as described. Corrected data were analyzed to obtain equilibrium dissociation constants (Kd). Analyses were carried out by fitting the data to equation (1) derived from simple binding theory, (Eq. 1) where F is the observed fluorescence, F 0 and F ∞ are the fluorescence in the absence of ligand and at saturation, respectively, P T and R T are the total concentrations of protein and RA/retinoic acid, respectively, and Ka is the association constant (Ka = 1/Kd).

Detection of retinoic acid/ATRA in skin rafts [1]
Aliquots from a concentrated solution of UAB30 in DMSO (50 mM) were added to the culture medium to achieve a final concentration of 2 μM. The culture medium was replaced every other day, and supplemented with fresh UAB30. Medium for the control samples was supplemented only with DMSO. After harvesting, epidermis of raft cultures was peeled of underlying collagen beds. UAB30-treated or DMSO-treated cultures were pooled into three samples containing five rafts each, and retinoids were extracted essentially as described in [52]. Each sample was homogenized in 0.5 mL of ice-cold phosphate-buffered saline (PBS) in the dark, transferred to siliconized glass tube, and mixed with 0.5 mL of ethanol containing 0.025 M potassium hydroxide. Non-polar retinoids were extracted with 2 mL of hexane, organic phase was dried under the stream of nitrogen, reconstituted in 50 μL of hexane:acetonitrile (70:30), and analyzed by reverse phase HPLC as described before. The remaining aqueous phase was acidified by the addition of 45 μL of 4 M hydrochloric acid, and polar retinoids (including ATRA and UAB30) were extracted with another 2 mL of hexane. The extract was dried and reconstituted in 400 μL of acetonitrile.

LC-MS-MS analysis of retinoic acid/ATRA in tissue samples was carried out essentially as described before with several modifications. To quantify the concentration of ATRA or UAB30 present in the dried extracts, 50-μL aliquots were injected into a Shimadzu LC-10AD HPLC (including a degasser) coupled to an Applied Biosystems 4000 Q Trap mass spectrometer. The column used for all analyses was a SUPELCOSIL ABZ PLUS (10 cm x 2.1 mm, 3 μm). Mobile phase A containing 40% acetonitrile, 30% methanol, and 30% ultra-pure water was mixed with mobile phase B containing 55% acetonitrile, 30% methanol, and 15% ultra-pure water using a gradient program. Each mobile phase contained 0.01% v/v of formic acid. The gradient program used for mixing the mobile phases was: 0 to 5 min, 100% A to 100% B; 5 to 19 min, 100% B; 19 to 20 min, 100% B to 100% A; 20 to 30 min, 100% A. The flow rate of the mobile phase was kept constant at 200 μL/min. The mass spectrometer was operated with an atmospheric pressure chemical ionization (APCI) source in multiple reaction monitoring controlled by the Analyst 1.4.2 software. The dwell time for ATRA and UAB30 was 40 ms. The conditions for optimum positive APCI detection were: curtain gas 10, nebulizer gas 3, collision gas 6, ion source 70, and temperature 350°C.

Each sample was injected three times, and the average of three injections was used to estimate its retinoic acid/ATRA or UAB30 concentration. For ATRA, the 301 m/z was selected for in quadrupole 1 (Q1), and the 123 m/z ion fragment ions were quantified in quadrupole 3 (Q3). For UAB30, the 295 m/z was selected for Q1, and the 165 m/z was selected for Q3. Prior to analysis each peak was optimized for their declustering potential, entrance potential, collision energy, and collision cell exit potential using the optimization subroutine in Analyst.

To quantitate ATRA levels, a calibration curve was run 0.0–1.6 pmol/50 μL retinoic acid/ATRA injection (7 concentrations varying 2-fold) using 3 injections for each concentration. The total ion current area (TIC) of the 123 m/z peak was fit to a linear equation to establish the calibration curve. The TIC area of the 123 m/z peak was measured three times and averaged. The endogenous concentration of ATRA in the samples was determined using the averaged peak area and the linear calibration curve. The same method was used for UAB30 except the 165 m/z fragment peak was used for the calibration curve and quantitation and a different range of concentrations was used in construction of the calibration curve (0.0–1.0 pmol/50 μL UAB30 injection).

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Transient Transfection. [6]
Transfection of AREc32 cells with Nrf2 expression vectors was carried at 70–80% confluence by using Lipofectamine 2000 Reagent (Life Technologies). The culture medium was replaced 5 h after transfection with fresh DMEM containing 10 μM tBHQ in the presence or absence of 1 μM retinoic acid/ATRA. For control experiments, mock transfections (no plasmid DNA) and vehicle alone (0.1% vol/vol DMSO) was added to the medium. Cells were left for 24 h to respond to xenobiotics before being harvested and analyzed. In control experiments, the transfection reagent alone, without DNA, was added to the cells and treated with DMSO for 2 h.
For RAR knockdown experiments in AREc32 cells, two preannealed siRNA sequences 1 (5′-GGAAUUUGUGCUGUGUAUUtt-3′) and 2 (5′-GCUCACCACAUCUUCAUCAtt-3′), which target different regions of RARα mRNA, were purchased from Ambion. A prevalidated siRNA (5′-GGAAGCUGUGCGAAAUGACtt-′), specifically targeting human RARγ, was similarly used to transfect AREc32 cells. In these cases, the siRNA (200 pmol per well) and Lipofectamine 2000 reagent (10 μl per well) were diluted with 1 ml of Optimum in a six-well plate and incubated at 20°C for 20 min. Thereafter, 4 × 105 cells were diluted in 4 ml of growth medium without antibiotics and dispensed to each well directly. After 24 h incubation, the cells were treated for a further 24 h with 10 μM tBHQ, 1 μM retinoic acid/ATRA, or 10 μM tBHQ plus 1 μM ATRA in fresh DMEM.

Homozygous Nrf2 KO mice were used. Two-month-old C57BL/6 Nrf2−/− and Nrf2+/+ male mice were used in this study. All animal procedures were carried out under a United Kingdom Home Office license and with local ethical approval. Nrf2−/− and Nrf2+/+ (n = 2–3) mice were placed on a VAD (Special Diet Service) or control diet for 6 weeks and then killed. Nrf2+/+ mice were also placed on a VAD diet for 6 weeks; during the last two weeks, they received either no treatment, retinoic acid/ATRA i.p. daily at 10 mg/kg, or the equivalent volume of corn oil. Mice were killed and the small intestine excised, washed, and frozen in liquid nitrogen. [6]

Absorption, Distribution and Excretion
Tretinoin applied topically is expected to remain on the stratum corneum and undergo minimal systemic absorption. In one study, the topical application of radiolabelled tretinoin for 28 days was associated with a total percutaneous absorption of 2%. The extent of absorption was examined after a once-daily application of 1.9 g of the combination product with [benzoyl peroxide] for 14 days. On Day 14, at steady-state, the mean Cmax was 0.15-0.19 ng/mL for tretinoin, 0.27-0.34 ng/mL for the metabolite 4-keto 13-cis retinoic acid, and 0.13-0.28 ng/mL for 13-cis retinoic acid, respectively. The Cmax varied across different age groups (children, adolescents, and adults). The corresponding ranges for the mean AUC0-24 were 0.63-2.06, 2.39-2.89, and 0.96-1.99 ng\*h/mL. Following oral administration, the absolute bioavailability of tretinoin was approximately 50%. While the effect of food on tretinoin is unclear, food increases the oral absorption of retinoids, as a class. When the oral dose of 22.5 mg/m2 tretinoin was administered twice daily, the mean ± SD Cmax was 394 ± 89 ng/mL after the first dose and 138 ± 139 ng/mL after one week of continuous treatment. The area under the curve (AUC) was 537 ± 191 ng·h/mL after the first dose and 249 ± 185 ng·h/mL after one week of continuous treatment. The Tmax was between one and two hours.
Tretinoin metabolites are excreted in bile and urine. Following administration of radiolabeled tretinoin at doses of 2.75 mg and 50 mg - which are 0.53 to 9.6 times the approved recommended dosage based on 1.7 m², respectively - approximately 63% of the radioactivity was recovered in the urine within 72 hours, and 31% appeared in the feces within six days.
Tretinoin is rapidly and extensively distributed to tissues following oral administration but does not cross the blood-brain barrier. The apparent volume of distribution (V_d) of intravenous tretinoin is dose-dependent and significantly greater at low doses. The V_d was 0.52 ± 0.12 L/kg after 0.0125 mg/kg and 0.21 ± 0.05 L/kg after 0.25 mg/kg.
No information is available.
/MILK/ It is not known whether topically applied tretinoin is excreted in human milk.
Studies with radiolabeled drug have demonstrated that after the oral administration of 2.75 and 50 mg doses of tretinoin, greater than 90% of the radioactivity was recovered in the urine and feces. Based upon data from 3 subjects, approximately 63% of radioactivity was recovered in the urine within 72 hours and 31% appeared in the feces within 6 days.
A single 45 mg/sq m (approximately 80 mg) oral dose to APL /acute promyelocytic leukemia/ patients resulted in a mean +/- SD peak tretinoin concentration of 347 +/- 266 ng/mL. Time to reach peak concentration was between 1 and 2 hours.
The apparent volume of distribution of tretinoin has not been determined. Tretinoin is greater than 95% bound in plasma, predominately to albumin. Plasma protein binding remains constant over the concentration range of 10 to 500 ng/mL.
For more Absorption, Distribution and Excretion (Complete) data for all-trans-Retinoic acid (12 total), please visit the HSDB record page.

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Metabolism / Metabolites
Tretinoin is rapidly metabolized to form various oxidized and conjugated metabolites. It forms several metabolites stereoisomerization derivatives (9-_cis_-retinoic acid or [alitretinoin] and 13-_cis_-retinoic acid or [isotretinoin]), oxidation derivatives (4-hydroxy-retinoic acid, 4-oxo-retinoic acid, 18-hydroxy-retinoic acid, 5,6-epoxy-retinoic acid, 3,4-didehydro-retinoic acid and retinotaurine), stereoisomerization and oxidation derivatives (13-_cis_-4-oxo-retinoic acid), glucuronidation derivatives (retinoyl beta-glucuronide, 13-_cis_-retinoyl beta-glucuronide, 4-oxo-retinoyl beta-glucuronide, 5,6-epoxyretinoyl beta-glucuronide and 13-_cis_-4-oxo-retinoyl beta-glucuronide), nonpolar metabolites of retinoic acid, and retinoic acid esters. Tretinoin is metabolized by several CYP enzymes, including CYP3A4, CYP2C8, and CYP2E. It also undergoes glucuronidation by UGT2B7. The metabolites 4-oxo retinoic acid and 4-oxo _trans_ retinoic acid glucuronide have one-third of the pharmacological activity of the parent compound. When the plasma concentrations decreased to one-third of their day-one concentrations after one week of continuous therapy, tretinoin induced its own metabolism.
Evidence suggests that tretinoin induces its own metabolism. In patients with APL receiving 45 mg/sq m tretinoin daily, urinary excretion of 4-oxo trans retinoic acid glucuronide increased approximately tenfold over the course of 2-6 weeks of continuous therapy, suggesting that increased metabolism of tretinoin may be the primary mechanism leading to the decreased plasma drug concentrations observed during continued administration. Possible mechanisms for the increased clearance of tretinoin with continuous daily dosing of the drug include induction of CYP enzymes or oxidative cofactors and increased expression of cellular retinoic acid binding proteins. Increasing the dosage of tretinoin to compensate for the apparent autoinduction has not been shown to increase therapeutic response. Reduced plasma retinoid concentrations have been associated with relapse and clinical resistance, and some investigators suggest that the clinical failure of tretinoin may be related to a lack of sustained effective concentrations of the drug during prolonged treatment.
Tretinoin metabolites have been identified in plasma and urine. Cytochrome P450 enzymes have been implicated in the oxidative metabolism of tretinoin. Metabolites include 13- cis retinoic acid, 4-oxo trans retinoic acid, 4-oxo cis retinoic acid, and 4-oxo trans retinoic acid glucuronide. In APL /acute promyelocytic leukemia/ patients, daily administration of a 45 mg/SQ m dose of tretinoin resulted in an approximately tenfold increase in the urinary excretion of 4-oxo trans retinoic acid glucuronide after 2 to 6 weeks of continuous dosing, when compared to baseline values.
Ethanol fed rats showed enhanced microsomal retinoic acid metabolism (50%) accompanied by increased microsomal cytochrome P450 content (34%). The increased hepatic microsomal cytochrome P450 dependent metabolism of retinoic acid after chronic ethanol consumption may contribute to the accelerated catabolism of retinoic acid in vivo.
Following ip administration of high doses of 15-(14)C- and 10,11-(3)H-labeled retinoic acid to rats, 3 major metabolites were isolated from feces in microgram amounts by column, thin-layer and high-pressure liquid chromatography. Mass spectrometry provided identification as all-trans-4-oxoretinoic acid, all-trans-5'-hydroxy-retinoic acid and 7-trans-9-cis-11-trans-13-trans-5'-hydroxyretinoic acid.
For more Metabolism/Metabolites (Complete) data for all-trans-Retinoic acid (8 total), please visit the HSDB record page.
Tretinoin has known human metabolites that include 5,6-Epoxy-retinoic acid, All-trans-retinoyl glucuronide, 18-Hydroxyretinoic acid, and 4-Hydroxyretinoic acid.
Tretinoin is a known human metabolite of retinal.
Hepatic
Half Life: 0.5-2 hours

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Biological Half-Life
The terminal elimination half-life of tretinoin following initial dosing is 0.5 to 2 hours in patients with APL.
In patients with APL /acute promyelocytic leukemia/ receiving tretinoin orally, a terminal elimination half-life of 0.5-2 hours has been reported following initial dosing.

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Metabolism of vitamin A and the production of all-trans retinoic acid [4]
Vitamin A is a necessary dietary vitamin for the normal development and vision. The critical necessity of vitamin A was hinted as early as 1881 by Nikolai Lunin, who discovered that purified protein, fat, and carbohydrate did not sustain the normal growth of mice, unless the diet was supplemented with milk. Elmer Verner McCollum, then determined in 1917 that the critical component concerned in milk was actually a "fat-soluble factor A", named in contrast to the previously discovered "water-soluble factor B", or vitamin B. These discoveries allowed Carl Edvard Bloch, a Denmark paediatrician, to identify vitamin A deficiency as the cause of night blindness, or xerophthalmia. While vitamin A was a necessary dietary vitamin, vitamin A itself is not the main bioactive mediator of its function. The key mediators of vitamin A function were identified as atRA and 11-cis retinal. atRA is a regulator of gene transcription, while 11-cis retinal acts as a chromophore for visual functions. In this section, we will review the metabolic processes of converting vitamin A into various retinoids, with emphasis on the production of atRA (Figure 1).

Toxicity Summary
IDENTIFICATION AND USE: All-trans-Retinoic acid (tretinoin) indicated for topical application in the treatment of acne vulgari. Tretinoin capsules are indicated for the induction of remission in patients with acute promyelocytic leukemia. HUMAN STUDIES: Cardiac failure occurred in 6% of patients receiving tretinoin, and cardiac arrest, myocardial infarction, stroke, and pulmonary hypertension each occurred in 3% of patients. There is a risk of arterial or venous thrombosis, involving any organ system, during the first month of tretinoin therapy. Thromboembolic events, including fatal pulmonary embolism, have been reported in patients receiving tretinoin. In one patient receiving tretinoin, fatal thromboembolism occurred during concomitant therapy with an antifibrinolytic agent. Bone marrow necrosis, sometimes fatal, has been reported in several patients receiving hydroxyurea during tretinoin therapy. Thrombocytosis has been reported rarely in patients receiving tretinoin. Rapidly evolving leukocytosis occurs in approximately 40% of patients receiving tretinoin. Retinoic acid-acute promyelocytic leukemia (RA-APL) syndrome (also known as APL differentiation syndrome), characterized by fever, dyspnea, acute respiratory distress, weight gain, pulmonary infiltrates, pleural and pericardial effusions, edema, hepatic failure, renal failure, and multiorgan failure, occurs in approximately 25% of patients receiving tretinoin for the treatment of APL. RA-APL syndrome occasionally has been accompanied by impaired myocardial contractility and episodic hypotension and can occur with or without concomitant leukocytosis. In severe cases, progressive hypoxemia requiring endotracheal intubation and mechanical ventilation may occur, and deaths secondary to progressive hypoxemia and multiorgan failure have been reported. ANIMAL STUDIES: There was no evidence of carcinogenic potential when tretinoin dosages of 0.025 mg/kg daily were administered topically to mice. When mice received 0.017 and 0.035% formulations of tretinoin applied topically, cutaneous squamous cell carcinomas and papillomas in the treatment area were observed in some female mice and dose-related hepatic tumors were observed in male mice. Experiments in vitro with cultured rat conceptuses have shown that tretinoin is a direct-acting dysmorphogen. Major defects involved the branchial arches and somites. Retinoid-induced malformations of the jaw, ears, face, skull, eyes, and heart in humans and rodents are well known. In mice that were administered a single oral dose of 100 mg/kg tretinoin on gestation days 9 or 11 and were killed on gestation day 17 skeletal defects (limbs) and cleft palate were present in 90% of the fetuses. There is evidence for teratogenicity (shortened or kinked tail) of topical tretinoin in rats at dosages exceeding 1 mg/kg daily. Bone anomalies also have been reported in rats when tretinoin 10 mg/kg daily was applied dermally. Topical tretinoin cream was associated with an increased incidence of cleft palate and hydrocephaly in rabbits. In rabbits treated with topical tretinoin an increased incidence of domed head and hydrocephaly was noted in some of the fetuses, typical of retinoid-induced fetal malformations in this species. Gross external, soft tissue and skeletal alterations occurred at doses higher than 0.7 mg/kg/day in mice, 2 mg/kg/day in rats, 7 mg/kg/day in hamsters, and at a dose of 10 mg/kg/day, the only dose tested, in pigtail monkey. When given subcutaneously to rabbits, tretinoin was teratogenic at a dosage of 2 mg/kg daily but not at 1 mg/kg daily. In vivo and in vitro (Ames) tests have not demonstrated that tretinoin is mutagenic. However, ingredients in the microsphere formulation of the drug have shown potential for genetic toxicity and teratogenesis. ECOTOXICITY STUDIES: In Japanese flounder, Paralichthys olivaceus, at 6-9 days post-hatching tretinoin induced the most severe deformity in all skeletons examined among retinoic acid isomers.
Tretinoin binds to alpha, beta, and gamma retinoic acid receptors (RARs). RAR-alpha and RAR-beta have been associated with the development of acute promyelocytic leukemia and squamous cell cancers, respectively. RAR-gamma is associated with retinoid effects on mucocutaneous tissues and bone. Although the exact mechanism of action of tretinoin is unknown, current evidence suggests that the effectiveness of tretinoin in acne is due primarily to its ability to modify abnormal follicular keratinization. Comedones form in follicles with an excess of keratinized epithelial cells. Tretinoin promotes detachment of cornified cells and the enhanced shedding of corneocytes from the follicle. By increasing the mitotic activity of follicular epithelia, tretinoin also increases the turnover rate of thin, loosely-adherent corneocytes. Through these actions, the comedo contents are extruded and the formation of the microcomedo, the precursor lesion of acne vulgaris, is reduced. Tretinoin is not a cytolytic agent but instead induces cytodifferentiation and decreased proliferation of APL cells in culture and in vivo. When Tretinoin is given systemically to APL patients, tretinoin treatment produces an initial maturation of the primitive promyelocytes derived from the leukemic clone, followed by a repopulation of the bone marrow and peripheral blood by normal, polyclonal hematopoietic cells in patients achieving complete remission (CR). The exact mechanism of action of tretinoin in APL is unknown.

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Interactions
Mouse mammary gland organ culture technique was utilized to determine the effects of retinoids, including trans-retinoic acid, on the prolactin-induced structural differentiation of the mammary gland. Thoracic glands from BALB/C mice pretreated with steroids differentiate in 6 days into alveolar structures in presence of insulin and prolactin. Trans-retinoic acid inhibited prolactin-induced structural changes in the glands.
To determine whether 2,3,7,8-tetrachlorodibenzo-p-dioxin and retinoic acid would enhance or antagonize the teratogenic effects of the other compound, C57BL/6N dams were treated orally on gestation days 10 or 12 with 10 ml corn oil/kg containing 2,3,7,8-tetrachlorodibenzo-p-dioxin (0-18 ug/kg), retinoic acid (0-200 mg/kg), or combinations of the two chemicals. Dams were killed on gestation day 18 and toxicity and teratogenicity assessed. Coadministration of 2,3,7,8-tetrachlorodibenzo-p-dioxin and retinoic acid had no effect on maternal or fetal toxicity beyond what would be expected by either compound alone. Cleft palate was induced by retinoic acid at lower doses on gestation day 10 than on gestation day 12, but by 2,3,7,8-tetrachlorodibenzo-p-dioxin at lower doses on gestation day 12 than on gestation day 10. Sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin induced hydronephrosis was similar on both gestation days 10 and 12. The limb bud defects were only observed when retinoic acid was administered on gestation day 10, not when given on gestation day 12. No other soft tissue or skeletal malformations were related to administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin or retinoic acid. No effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin was observed on the incidence or severity of limb bud defects induced by retinoic acid, nor did retinoic acid influence the incidence or severity of hydronephrosis induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, the incidence of cleft palate was dramatically enhanced by coadministration of the xenobiotic and vitamin. On both gestation day 10 and 12, the dose-response curves for cleft palate induction were parallel, suggesting some similarities in mechanism between the two compounds. However, combination treatment resulted in a synergistic response that varied with the stage of development and was tissue specific.
Risk of pseudotumor cerebri (intracranial hypertension) is increased in patients receiving tretinoin. Concomitant use of other agents known to cause pseudotumor cerebri or intracranial hypertension, such as tetracyclines, may increase the risk of this condition in patients receiving tretinoin.
Concurrent use of hydroxyurea, which is cytotoxic to cells in S phase, and tretinoin, which induces cells to enter the S phase, may cause a synergistic effect leading to massive cell lysis. Bone marrow necrosis, sometimes fatal, has been reported in patients receiving hydroxyurea during tretinoin therapy. Although some clinicians have administered hydroxyurea in conjunction with tretinoin therapy to reduce leukocytosis, the safety and efficacy of this practice have not been established, and caution is recommended in the use of hydroxyurea in patients receiving tretinoin.
For more Interactions (Complete) data for all-trans-Retinoic acid (14 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Rat oral 1960 mg/kg
LD50 Rat ip 96 mg/kg
LD50 Rat sc 53 mg/kg
LD50 Rat iv 78 mg/kg
For more Non-Human Toxicity Values (Complete) data for all-trans-Retinoic acid (12 total), please visit the HSDB record page.

[1]. Retinoid X Receptor Agonists Upregulate Genes Responsible for the Biosynthesis of All-Trans-Retinoic Acid in Human Epidermis. PLoS One. 2016 Apr 14;11(4):e0153556.
[2]. Retinoic acid is a high affinity selective ligand for the peroxisome proliferator-activated receptor beta/delta. J Biol Chem. 2003 Oct 24;278(43):41589-92.
[3]. Retinoic acid induces neurogenesis by activating both retinoic acid receptors (RARs) and peroxisomeproliferator-activated receptor β/δ (PPARβ/δ). J Biol Chem. 2012 Dec 7;287(50):42195-205.
[4]. Retinoic acid synthesis and functions in early embryonic development. Cell Biosci. 2012 Mar 22;2(1):11.
[5]. A retinoic acid receptor alpha antagonist selectively counteracts retinoic acid effects. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7129-33.
[6]. Identification of retinoic acid as an inhibitor of transcription factor Nrf2 through activation of retinoic acid receptor alpha. Proc Natl Acad Sci U S A. 2007 Dec 4;104(49):19589-94

Therapeutic Uses
Antineoplastic Agents Keratolytic Agents
/CLINICAL TRIALS/ ClinicalTrials.gov is a registry and results database of publicly and privately supported clinical studies of human participants conducted around the world. The Web site is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each ClinicalTrials.gov record presents summary information about a study protocol and includes the following: Disease or condition; Intervention (for example, the medical product, behavior, or procedure being studied); Title, description, and design of the study; Requirements for participation (eligibility criteria); Locations where the study is being conducted; Contact information for the study locations; and Links to relevant information on other health Web sites, such as NLM's MedlinePlus for patient health information and PubMed for citations and abstracts for scholarly articles in the field of medicine. trans-Retinoic acid is included in the database.
Tretinoin gel and cream are indicated for topical application in the treatment of acne vulgaris. The safety and efficacy of the long-term use of this product in the treatment of other disorders have not been established. /Included in US product labeling; Tretinoin, topical/
Tretinoin is used topically as a 0.05 or 0.1% cream for palliative therapy to improve dermatologic changes (e.g., fine wrinkling, mottled hyperpigmentation, roughness) associated with photodamage. /NOT included in US product labeling; Tretinoin, topical/
For more Therapeutic Uses (Complete) data for all-trans-Retinoic acid (9 total), please visit the HSDB record page.

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Drug Warnings
/BOXED WARNING/ Experienced Physician and Institution. Patients with acute promyelocytic leukemia (APL) are at high risk in general and can have severe adverse reactions to tretinoin capsules. Tretinoin capsules should therefore be administered only to patients with APL under the strict supervision of a physician who is experienced in the management of patients with acute leukemia and in a facility with laboratory and supportive services sufficient to monitor drug tolerance and protect and maintain a patient compromised by drug toxicity, including respiratory compromise. Use of tretinoin capsules requires that the physician concludes that the possible benefit to the patient outweighs the following known adverse effects of the therapy. /Tretinoin, systemic/
/BOXED WARNING/ Retinoic Acid-APL Syndrome. About 25% of patients with APL treated with tretinoin capsules have experienced a syndrome called the retinoic acid-APL (RA-APL) syndrome characterized by fever, dyspnea, acute respiratory distress, weight gain, radiographic pulmonary infiltrates, pleural and pericardial effusions, edema, and hepatic, renal, and multi-organ failure. This syndrome has occasionally been accompanied by impaired myocardial contractility and episodic hypotension. It has been observed with or without concomitant leukocytosis. Endotracheal intubation and mechanical ventilation have been required in some cases due to progressive hypoxemia, and several patients have expired with multi-organ failure. The syndrome generally occurs during the first month of treatment, with some cases reported following the first dose of tretinoin capsules. The management of the syndrome has not been defined rigorously, but high-dose steroids given at the first suspicion of the RA-APL syndrome appear to reduce morbidity and mortality. At the first signs suggestive of the syndrome (unexplained fever, dyspnea and/or weight gain, abnormal chest auscultatory findings or radiographic abnormalities), high-dose steroids (dexamethasone 10 mg intravenously administered every 12 hours for 3 days or until the resolution of symptoms) should be immediately initiated, irrespective of the leukocyte count. The majority of patients do not require termination of tretinoin capsules therapy during treatment of the RA-APL syndrome. However, in cases of moderate and severe RA-APL syndrome, temporary interruption of tretinoin capsules therapy should be considered. /Tretinoin, systemic/
/BOXED WARNING/ During tretinoin capsules treatment about 40% of patients will develop rapidly evolving leukocytosis. Patients who present with high WBC at diagnosis (>5x10 9/L) have an increased risk of a further rapid increase in WBC counts. Rapidly evolving leukocytosis is associated with a higher risk of life-threatening complications. If signs and symptoms of the RA-APL syndrome are present together with leukocytosis, treatment with high-dose steroids should be initiated immediately. Some investigators routinely add chemotherapy to tretinoin capsules treatment in the case of patients presenting with a WBC count of >5x10 9/L or in the case of a rapid increase in WBC count for patients leukopenic at start of treatment, and have reported a lower incidence of the RA-APL syndrome. Consideration could be given to adding full-dose chemotherapy (including an anthracycline if not contraindicated) to the tretinoin capsules therapy on day 1 or 2 for patients presenting with a WBC count of >5x10 9/L, or immediately, for patients presenting with a WBC count of <5x10 9/L, if the WBC count reaches >/= 6x10(9)/L by day 5, or >/= 10x10(9)/L by day 10, or >/=15x10(9)/L by day 28. /Tretinoin, systemic/
/BOXED WARNING/ Teratogenic Effects. Pregnancy Category D. There is a high risk that a severely deformed infant will result if tretinoin capsules are administered during pregnancy. If, nonetheless, it is determined that tretinoin capsules represent the best available treatment for a pregnant woman or a woman of childbearing potential, it must be assured that the patient has received full information and warnings of the risk to the fetus if she were to be pregnant and of the risk of possible contraception failure and has been instructed in the need to use two reliable forms of contraception simultaneously during therapy and for 1 month following discontinuation of therapy, and has acknowledged her understanding of the need for using dual contraception, unless abstinence is the chosen method. Within 1 week prior to the institution of tretinoin capsules therapy, the patient should have blood or urine collected for a serum or urine pregnancy test with a sensitivity of at least 50 mIU/mL. When possible, tretinoin capsules therapy should be delayed until a negative result from this test is obtained. When a delay is not possible, the patient should be placed on two reliable forms of contraception. Pregnancy testing and contraception counseling should be repeated monthly throughout the period of tretinoin capsules treatment. /Tretinoin, systemic/
For more Drug Warnings (Complete) data for all-trans-Retinoic acid (44 total), please visit the HSDB record page.

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Pharmacodynamics
Tretinoin is a vitamin A derivative that promotes cell production, proliferation, and differentiation. When used topically, tretinoin regulates epidermal cell turnover and collagen production. It also prevents collagen loss, reduces inflammation, and blocks the induction of matrix metalloproteinase (MMP), which are enzymes that disrupt collagen and elastic fibres. In short-term and long-term studies, topical application of tretinoin at doses ranging from 0.001% to 0.1% was associated with improvements in clinical signs of photoaging and fine wrinkles, increased epidermal thickness, compaction of the stratum corneum, and decreased melanin content. It also improved melanocyte differentiation and distribution, promotion of epidermal hyperplasia, and angiogenesis. Tretinoin exhibits antineoplastic activities when given orally. Tretinoin was shown to induce differentiation in tumour cells. It induced cytodifferentiation and decreased acute promyelocytic leukemia (APL) cell proliferation in culture and _in vivo_. In patients with APL, tretinoin promoted the initial maturation of the primitive promyelocytes derived from the leukemic clone, followed by a repopulation of the bone marrow and peripheral blood by normal, polyclonal hematopoietic cells in patients achieving complete remission.

C20H28O2

300.4

300.208

C, 79.96; H, 9.39; O, 10.65

302-79-4

Retinoic acid-d5;78996-15-3;Retinoic acid;302-79-4;11-cis-Retinoic Acid-d5;Retinoic acid-d6;2483831-72-5

444795

Yellow to light-orange crystalline powder
Crystals from ethanol

1.0±0.1 g/cm3

462.8±14.0 °C at 760 mmHg

179-184ºC

350.6±11.0 °C

0.0±2.5 mmHg at 25°C

1.556

6.83

1

2

5

22

567

0

CC1(C)C(/C=C/C(C)=C/C=C/C(C)=C/C(O)=O)=C(C)CCC1

SHGAZHPCJJPHSC-YCNIQYBTSA-N

InChI=1S/C20H28O2/c1-15(8-6-9-16(2)14-19(21)22)11-12-18-17(3)10-7-13-20(18,4)5/h6,8-9,11-12,14H,7,10,13H2,1-5H3,(H,21,22)/b9-6+,12-11+,15-8+,16-14+

(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid

All-trans Retinoic Acid; Ro 5488; Ro-5488; tretinoin; ATRA; Renova; Aknefug; Atralin; Retin-A Micro; Tretinoina; ...; 302-79-4; Vitamin A acid; ATRA; TRA; Ro5488; alltrans vitamin A acid; betaretinoic acid; retinoic acid; TRA; trans retinoic acid; trans vitamin A acid; tretinoinum; Trade names: Avita; Renova; Aberel; Aknoten; RetinA; RetinA MICRO; Vesanoid. Foreign brand names: Airol; Eudyna; RetisolA; StievaA; Cordes Vas; Dermairol; EpiAberel; StievaA Forte; Vitinoin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2.5 mg/mL (8.32 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.32 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2.5 mg/mL (8.32 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: 2.5 mg/mL (8.32 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 6: 5 mg/mL (16.64 mM) in 50% PEG300 50% PBS (add these co-solvents sequentially from left to right, and one by one), suspension solution; Need ultrasonic and warming and heat to 40°C.

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 (Please use freshly prepared in vivo formulations for optimal results.)