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
Topical application of retinoic acid is expected to remain in the stratum corneum with minimal systemic absorption. One study showed that the total transdermal absorption rate of topically applied radiolabeled retinoic acid was 2% after 28 days. This study also examined the absorption of a once-daily combination of 1.9 g retinoic acid and benzoyl peroxide for 14 days. At steady state on day 14, the mean Cmax of retinoic acid was 0.15–0.19 ng/mL, while the mean Cmax of its metabolites, 4-keto-13-cis-retinoic acid and 13-cis-retinoic acid, was 0.27–0.34 ng/mL and 0.13–0.28 ng/mL, respectively. Cmax values varied across age groups (children, adolescents, and adults). The corresponding mean AUC0–24 ranges were 0.63–2.06, 2.39–2.89, and 0.96–1.99 ng·h/mL, respectively. The absolute bioavailability of retinoic acid after oral administration is approximately 50%. While the effect of food on retinoic acid is unclear, food increases the oral absorption of retinoids. When 22.5 mg/m² of retinoic acid was administered orally twice daily, the mean ± standard deviation (Cmax) after the first dose was 394 ± 89 ng/mL, and after one week of treatment it was 138 ± 139 ng/mL. The area under the curve (AUC) after the first dose was 537 ± 191 ng·h/mL, and after one week of treatment it was 249 ± 185 ng·h/mL. The time to peak concentration (Tmax) was 1 to 2 hours. Retinoic acid metabolites are excreted in bile and urine. Following administration of 2.75 mg and 50 mg of radiolabeled retinoic acid (0.53 and 9.6 times the approved recommended dose based on a body surface area of 1.7 m², respectively), approximately 63% of the radioactive material was recovered in urine within 72 hours, and 31% was recovered in feces within 6 days. Following oral administration, retinoic acid rapidly and extensively distributes to tissues but cannot cross the blood-brain barrier. The apparent volume of distribution (Vd) of intravenously administered retinoic acid is dose-dependent, with a significant increase at low doses. After a dose of 0.0125 mg/kg, Vd was 0.52 ± 0.12 L/kg; after a dose of 0.25 mg/kg, Vd was 0.21 ± 0.05 L/kg.
No further information available.
/Breast Milk/ It is unclear whether topically applied retinoic acid is excreted into human breast milk.
Studies with radiolabeled drugs have shown that after oral administration of 2.75 mg and 50 mg doses of retinoic acid, over 90% of the radioactive material is recovered in urine and feces. Based on data from three subjects, approximately 63% of the radioactive material was excreted in urine within 72 hours and 31% in feces within 6 days.
Following a single oral dose of 45 mg/m² (approximately 80 mg) in patients with acute promyelocytic leukemia (APL), the mean peak retinoic acid concentration was 347 ± 266 ng/mL. The time to peak concentration was 1 to 2 hours.
The apparent volume of distribution of retinoic acid has not been determined. Retinoic acid is bound to plasma at a rate exceeding 95%, primarily to albumin. Plasma protein binding remains constant across concentrations ranging from 10 to 500 ng/mL.
For more complete data on the absorption, distribution, and excretion of all-trans retinoic acids (12 in total), please visit the HSDB record page.
Metabolism/Metabolites
Retinoic acid is rapidly metabolized to form various oxidative and conjugated metabolites. It forms a variety of metabolites, including stereoisomerized derivatives (9-cis-retinoic acid or [alvitamin] and 13-cis-retinoic acid or [isoretinoic acid]), oxidized derivatives (4-hydroxyretinoic acid, 4-oxoretinoic acid, 18-hydroxyretinoic acid, 5,6-epoxyretinoic acid, 3,4-didehydroretinoic acid, and retinoic acid taurine), stereoisomerized and oxidized derivatives (13-cis-4-oxoretinoic acid), and glucuronidated derivatives (retinoyl β-glucuronide, 13-cis-retinoyl β-glucuronide, 4-oxoretinoyl β-glucuronide, 5,6-epoxyretinoic acid β-glucuronide, and 13-cis-4-oxoretinoic acid). Retinoic acid metabolites include β-glucuronide, nonpolar metabolites of retinoic acid, and retinoic acid esters. Retinoic acid is metabolized by various CYP enzymes, including CYP3A4, CYP2C8, and CYP2E. It can also be glucuroninated via UGT2B7. The metabolites 4-oxoretinoic acid and 4-oxotrans-retinoic acid glucuronide have approximately one-third the pharmacological activity of the parent compound. After one week of continuous treatment, retinoic acid induces its own metabolism when plasma concentrations drop to one-third of the initial day concentration. There is evidence that retinoic acid induces its own metabolism. In patients with acute promyelocytic leukemia (APL) receiving 45 mg/m² retinoic acid daily for 2–6 weeks, urinary excretion of 4-oxotrans-retinoic acid glucuronide increased approximately tenfold, suggesting that increased retinoic acid metabolism may be the primary mechanism leading to decreased plasma drug concentrations during continuous administration. Possible mechanisms by which continuous daily administration increases retinoic acid clearance include induction by CYP enzymes or oxidative cofactors and increased expression of cellular retinoic acid-binding proteins. Increasing the dose of retinoic acid to compensate for significant self-induction did not show an improvement in treatment response. Decreased plasma retinoic acid concentrations are associated with relapse and clinical resistance, and some researchers believe that clinical treatment failure with retinoic acid may be related to insufficient maintenance of effective drug concentrations during long-term treatment. Retinoic acid metabolites have been detected in plasma and urine. Cytochrome P450 enzymes are involved in the oxidative metabolism of retinoic acid. Metabolites include 13-cis-retinoic acid, 4-oxotrans-retinoic acid, 4-oxocis-retinoic acid, and 4-oxotrans-retinoic acid glucuronide. In patients with acute promyelocytic leukemia (APL), daily subcutaneous injection of 45 mg/m² retinoic acid for 2 to 6 weeks resulted in approximately a tenfold increase in urinary excretion of 4-oxotrans-retinoic acid glucuronide compared to baseline. Ethanol-fed rats showed enhanced microsomal retinoic acid metabolism (50%), along with increased microsomal cytochrome P450 levels (34%). Long-term ethanol intake increases liver microsomal cytochrome P450-dependent retinoic acid metabolism, which may lead to accelerated retinoic acid catabolism in the body. After intraperitoneal injection of high doses of 15-(14)C- and 10,11-(3)H-labeled retinoic acid into rats, three major metabolites were isolated from feces by column chromatography, thin-layer chromatography, and high-performance liquid chromatography, with concentrations in the microgram range. Mass spectrometry analysis identified them as all-trans-4-oxoretinoic acid, all-trans-5'-hydroxyretinoic acid, and 7-trans-9-cis-11-trans-13-trans-5'-hydroxyretinoic acid. For more complete metabolite/metabolite data on all-trans retinoic acid (a total of 8 metabolites), please visit the HSDB record page.
Known metabolites of retinoic acid include 5,6-epoxyretinoic acid, all-trans retinoic acid glucuronide, 18-hydroxyretinoic acid, and 4-hydroxyretinoic acid.
Retinoic acid is a known metabolite of retinaldehyde.
Hepatic
Half-life: 0.5–2 hours

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Biological half-life
The terminal elimination half-life of retinoic acid after the first dose is 0.5 to 2 hours in patients with acute promyelocytic leukemia (APL).
In patients with acute promyelocytic leukemia (APL) treated with oral retinoic acid, the terminal elimination half-life after the first dose has been reported to be 0.5–2 hours.

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Metabolism of Vitamin A and Formation of All-Trans Retinoic Acid [4]
Vitamin A is a dietary vitamin essential for normal development and vision. As early as 1881, Nicolai Luning hinted at the importance of vitamin A, discovering that purified proteins, fats, and carbohydrates could not sustain normal growth in mice unless milk was added to their diet. In 1917, Elmer Verner McCollum determined that a key component of milk was actually a "fat-soluble factor A," contrasting with the previously discovered "water-soluble factor B" (i.e., vitamin B). These findings enabled Danish pediatrician Carl Edvard Bloch to determine that vitamin A deficiency was the cause of night blindness (or dry eye). While vitamin A is an essential dietary vitamin, it is not itself the primary bioactive mediator for its function. Key mediators of vitamin A function have been identified as all-trans retinoic acid (atRA) and 11-cis-retinal. atRA is a regulator of gene transcription, while 11-cis-retinal acts as a chromophore for visual function. In this section, we will review the metabolic process of vitamin A being converted into various retinoids, with a focus on the formation of atRA (Figure 1).

Toxicity Summary
Identification and Uses: All-trans retinoic acid (retinoic acid) is indicated for the topical treatment of acne vulgaris. Retinoic acid capsules are indicated for inducing remission in patients with acute promyelocytic leukemia. Human Studies: Heart failure occurred in 6% of patients receiving retinoic acid, and cardiac arrest, myocardial infarction, stroke, and pulmonary hypertension occurred in 3%. There is a risk of arterial or venous thrombosis involving any organ system during the first month of retinoic acid treatment. Thromboembolic events, including fatal pulmonary embolism, have been reported in patients receiving retinoic acid. One patient receiving retinoic acid experienced a fatal thromboembolism while concurrently receiving antifibrinolytic drugs. Bone marrow necrosis, sometimes fatal, has been reported in patients taking hydroxyurea while receiving retinoic acid. Thrombocytosis has been rarely reported in patients receiving retinoic acid. Rapidly progressive leukocytosis occurs in approximately 40% of patients receiving retinoic acid. Retinoic acid-acute promyelocytic leukemia (RA-APL) syndrome (also known as APL differentiation syndrome) is characterized by fever, dyspnea, acute respiratory distress, weight gain, pulmonary infiltration, pleural and pericardial effusion, edema, liver failure, kidney failure, and multiple organ failure. This syndrome occurs in approximately 25% of APL patients receiving retinoic acid treatment. RA-APL syndrome is sometimes accompanied by decreased myocardial contractility and paroxysmal hypotension, with or without leukocytosis. In severe cases, progressive hypoxemia may occur, requiring intubation and mechanical ventilation, and there have been reports of death due to progressive hypoxemia and multiple organ failure. Animal studies: No evidence of carcinogenicity was found when retinoic acid was administered topically to mice at a daily dose of 0.025 mg/kg. When 0.017% and 0.035% retinoic acid preparations were administered topically to mice, squamous cell carcinoma and papilloma of the skin were observed in some female mice, while dose-related liver tumors were observed in male mice. In vitro studies of cultured rat embryos have shown that retinoic acid is a direct-acting teratogenic agent. Major defects involve the gill arches and somites. Retinoid-induced malformations of the jaw, ears, face, skull, eyes, and heart in humans and rodents are well-known. In mice subjected to a single oral dose of 100 mg/kg retinoic acid on day 9 or 11 of gestation and sacrificed on day 17 of gestation, 90% of the fetuses exhibited skeletal defects (limbs) and cleft palate. Evidence suggests that daily topical application of retinoic acid exceeding 1 mg/kg in rats is teratogenic (tail shortening or curvature). Daily dermal application of 10 mg/kg retinoic acid in rats has also been reported to cause skeletal abnormalities. Topical application of retinoic acid cream is associated with an increased incidence of cleft palate and hydrocephalus in rabbits. In rabbits treated with topical retinoic acid, some fetuses developed dome-shaped heads and hydrocephalus, typical manifestations of retinoid-induced fetal malformations in this species. In mice, doses above 0.7 mg/kg/day resulted in significant external, soft tissue, and skeletal changes. In rats, doses above 2 mg/kg/day, 7 mg/kg/day, 10 mg/kg/day, and 10 mg/kg/day all resulted in significant external, soft tissue, and skeletal changes. Subcutaneous injection in rabbits showed teratogenicity at a dose of 2 mg/kg/day, but not at 1 mg/kg/day. Neither in vivo nor in vitro (Ames assay) studies confirmed mutagenicity of retinoic acid. However, components in the microsphere formulation of this drug have shown potential genotoxicity and teratogenicity. Ecotoxicity studies: In Japanese flounder (Paralichthys olivaceus) 6–9 days after hatching, retinoic acid caused the most severe skeletal deformities among all tested retinoic acid isomers. Retinoic acid binds to α, β, and γ retinoic acid receptors (RARs). RAR-α and RAR-β are associated with the development and progression of acute promyelocytic leukemia and squamous cell carcinoma, respectively. RAR-γ is associated with the effects of retinoids on skin, mucous membranes, and bone. Although the exact mechanism of action of retinoic acid is not fully understood, existing evidence suggests that its effectiveness in treating acne is primarily attributed to its ability to improve abnormal follicular keratosis. Comedones form in hair follicles with an excess of keratinized epithelial cells. Retinoic acid promotes keratinocyte shedding and accelerates the shedding of follicular keratinocytes. By increasing the mitotic activity of follicular epithelial cells, retinoic acid also enhances the turnover rate of thin, loosely attached keratinocytes. Through these effects, comedone contents are expelled, and the formation of microcomedones (precursor lesions of acne vulgaris) is reduced. Retinoic acid is not a cell-dissolving agent, but it induces differentiation and inhibits the proliferation of acute promyelocytic leukemia (APL) cells in vitro and in vivo. When APL patients are treated systemically with retinoic acid, the treatment initially promotes the maturation of primitive promyelocytic cells derived from the leukemia clone, followed by the refilling of the bone marrow and peripheral blood with normal, polyclonal hematopoietic cells in patients achieving complete remission (CR). The exact mechanism of action of retinoic acid in acute promyelocytic leukemia (APL) remains unclear.

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Interactions
This study used mouse mammary organ culture technology to investigate the effects of retinoids, including trans-retinoic acid, on prolactin-induced mammary gland structural differentiation. The thymus of BALB/c mice pretreated with steroids differentiated into alveolar structures within 6 days in the presence of insulin and prolactin. Trans-retinoic acid inhibited prolactin-induced changes in glandular structure. To determine whether 2,3,7,8-tetrachlorodibenzo-dioxin and retinoic acid would enhance or antagonize the teratogenic effects of another compound, C57BL/6N female rats were orally administered 10 mL of corn oil per kg body weight containing 2,3,7,8-tetrachlorodibenzo-dioxin (0-18 μg/kg), retinoic acid (0-200 mg/kg), or a combination of both compounds on day 10 or 12 of gestation. The female rats were sacrificed on day 18 of gestation, and toxicity and teratogenicity were assessed. The combined administration of 2,3,7,8-tetrachlorodibenzo-dioxin and retinoic acid did not result in maternal or fetal toxicity exceeding the expectations of either compound alone. On day 10 of gestation, low doses of retinoic acid induced cleft palate, while on day 12, low doses of 2,3,7,8-tetrachlorodibenzo-dioxin induced cleft palate. The sensitivity to hydronephrosis induced by 2,3,7,8-tetrachlorodibenzo-dioxin was similar on both days 10 and 12 of gestation. Limb bud defects were observed only when retinoic acid was administered on day 10 of gestation, but not when administered on day 12. Other soft tissue or skeletal malformations were unrelated to administration of 2,3,7,8-tetrachlorodibenzo-dioxin or retinoic acid. No effect was observed on the incidence or severity of retinoic acid-induced limb bud defects, nor did retinoic acid affect the incidence or severity of 2,3,7,8-tetrachlorodibenzodioxin-induced hydronephrosis. However, the combined use of xenobiotics and vitamins significantly increased the incidence of cleft palate. The dose-response curves for cleft palate induction were parallel on days 10 and 12 of gestation, suggesting some similarities in the mechanisms of action of the two compounds. However, the combined treatment produced a synergistic effect that varied with developmental stage and was tissue-specific. Patients receiving retinoic acid had an increased risk of developing pseudotumor cerebri (intracranial hypertension). Concomitant use of other drugs known to cause pseudotumor cerebri or intracranial hypertension, such as tetracyclines, may increase this risk in patients receiving retinoic acid. Concomitant use of hydroxyurea, which is cytotoxic to S-phase cells, and retinoic acid, which induces cell entry into S phase, may produce a synergistic effect, leading to extensive cell lysis. There have been reports of bone marrow necrosis, sometimes even fatal, in patients taking hydroxyurea while receiving retinoic acid treatment. Although some clinicians have combined hydroxyurea with retinoic acid to reduce leukocytosis, the safety and efficacy of this practice have not been established, and therefore caution is advised when using hydroxyurea in patients receiving retinoic acid treatment. For more complete data on interactions of all-trans retinoic acids (14 in total), please visit the HSDB record page. Non-human toxicity values: Oral LD50 in rats: 1960 mg/kg Intraperitoneal LD50 in rats: 96 mg/kg Subcutaneous LD50 in rats: 53 mg/kg Intravenous LD50 in rats: 78 mg/kg For more complete data on non-human toxicity values of all-trans retinoic acids (12 in 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
Anti-tumor drugs, keratolytic agents
/Clinical Trials/ ClinicalTrials.gov is a registry and results database that lists human clinical studies funded by public and private institutions worldwide. The website is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each record on ClinicalTrials.gov includes a summary of the study protocol, including: the disease or condition; the intervention (e.g., the medical product, behavior, or procedure being studied); the title, description, and design of the study; participation requirements (eligibility criteria); the location of the study; contact information for the study location; and links to relevant information from other health websites, such as the NLM's MedlinePlus (for providing patient health information) and PubMed (for providing citations and abstracts of academic articles in the medical field). Trans-retinoic acid is listed in the database.
Retinoic acid gels and creams are indicated for topical treatment of acne vulgaris. The safety and efficacy of this product for the long-term treatment of other conditions have not been established. /Included on US product label; Retinoic acid, for topical use/
Retinoic acid is used topically as a 0.05% or 0.1% cream to relieve photodamage-related skin changes (e.g., fine lines, patchy pigmentation, roughness). /Not included on US product label; Retinoic acid, for topical use/
For more complete data on the therapeutic uses of all-trans retinoic acid (9 types), please visit the HSDB record page.

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Drug Warning
/Black box warning/ Experienced physicians and institutions. Patients with acute promyelocytic leukemia (APL) are at high risk and taking retinoic acid capsules may cause serious adverse reactions. Therefore, retinoic acid capsules should only be used in APL patients under the strict supervision of an experienced physician treating acute leukemia in a healthcare facility with adequate laboratory and support services to monitor drug tolerance and protect and maintain patients impaired by drug toxicity, including respiratory impairment. Retinoic acid capsules should only be used if the physician believes the potential benefit to the patient outweighs the following known adverse treatment reactions. /Retinoic acid, systemic medication/
/Black box warning/ Retinoic acid-APL syndrome. Approximately 25% of patients with acute promyelocytic leukemia (APL) receiving retinoic acid capsules develop a syndrome called retinoic acid-APL (RA-APL), characterized by fever, dyspnea, acute respiratory distress, weight gain, chest infiltrates on X-ray, pleural and pericardial effusions, edema, and liver, kidney, and multiple organ failure. This syndrome is sometimes accompanied by decreased myocardial contractility and paroxysmal hypotension. Leukocytosis may or may not be present. Some cases require intubation and mechanical ventilation due to progressive hypoxemia, and some patients die from multiple organ failure. This syndrome usually occurs within the first month of treatment, but there are also reports of it occurring immediately after the first dose of retinoic acid capsules. Treatment for this syndrome is not well-established, but immediate administration of high-dose glucocorticoids upon suspicion of rheumatoid arthritis-associated acute promyelocytic leukemia (RA-APL) syndrome appears to reduce morbidity and mortality. Once early symptoms suggestive of the syndrome appear (unexplained fever, dyspnea and/or weight gain, abnormal chest auscultation or imaging findings), regardless of white blood cell count, high-dose glucocorticoid therapy (dexamethasone 10 mg, intravenously, every 12 hours for 3 days or until symptom relief) should be initiated immediately. Most patients do not need to discontinue tretinoin capsules during treatment for RA-APL syndrome. However, for patients with moderate to severe RA-APL syndrome, temporary discontinuation of tretinoin capsule therapy should be considered. /Tretinoin, systemic medication/
/Black box warning/ Approximately 40% of patients develop rapidly progressive leukocytosis during tretinoin capsule therapy. Patients with elevated white blood cell counts at diagnosis (>5 × 10⁹/L) are at increased risk of further rapid increases in white blood cell count. Rapidly progressive leukocytosis is associated with an increased risk of life-threatening complications. If signs and symptoms of rheumatoid arthritis complicated with acute promyelocytic leukemia (RA-APL) syndrome appear, accompanied by leukocytosis, high-dose glucocorticoid therapy should be initiated immediately. Some researchers routinely add chemotherapy to retinoic acid capsule therapy when patients have a white blood cell count >5×10⁹/L at initial diagnosis, or when leukopenia is present at the start of treatment and the white blood cell count rapidly increases, and report a low incidence of RA-APL syndrome. For patients with a white blood cell count >5×10⁹/L, full-dose chemotherapy can be considered on day 1 or 2 to add to retinoic acid capsule therapy (anthracyclines can be added if there are no contraindications); for patients with a white blood cell count <5×10⁹/L, if the white blood cell count reaches ≥6×10⁹/L on day 5, or ≥10×10⁹/L on day 10, or ≥15×10⁹/L on day 28, full-dose chemotherapy can be added immediately. /Retinoic acid, systemic medication/
/Black box warning/ Teratogenic effects. Pregnancy category D. Taking retinoic acid capsules during pregnancy carries a high risk of serious birth defects. However, if retinoic acid capsules are determined to be the optimal treatment option for pregnant women or women of childbearing age, it must be ensured that the patient is fully informed of the potential risks to the fetus if pregnancy occurs, as well as the risk of contraceptive failure, and is informed of the need to use two reliable methods of contraception simultaneously during treatment and for one month after discontinuation of the medication. The patient must also confirm that they understand the necessity of dual contraception unless abstinence is chosen. A serum or urine pregnancy test with a sensitivity of at least 50 mIU/mL should be performed on a blood or urine sample within one week before starting retinoic acid capsule treatment. If possible, retinoic acid capsule treatment should be postponed until a negative result is obtained. If treatment cannot be postponed, two reliable methods of contraception should be started immediately. Pregnancy tests and contraceptive counseling should be repeated monthly throughout the entire retinoic acid capsule treatment period. /Retinoic Acid, Systemic Use/
For more complete data on all-trans retinoic acid (44 items), please visit the HSDB record page.

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Pharmacodynamics
Retinoic acid is a vitamin A derivative that promotes cell generation, proliferation, and differentiation. When applied topically, retinoic acid regulates epidermal cell renewal and collagen production. It also prevents collagen loss, reduces inflammation, and inhibits the induction of matrix metalloproteinases (MMPs), enzymes that destroy collagen and elastin fibers. In short-term and long-term studies, topical application of retinoic acid at concentrations of 0.001% to 0.1% was associated with improved clinical symptoms of photoaging and fine lines, epidermal thickening, stratum corneum densification, and reduced melanin content. It also improves melanocyte differentiation and distribution, and promotes epidermal proliferation and angiogenesis. Oral retinoic acid has antitumor activity. Studies have shown that retinoic acid can induce tumor cell differentiation. It can induce cell differentiation and reduce the proliferation of acute promyelocytic leukemia (APL) cells in vitro and in vivo. In APL patients, retinoic acid promotes the initial maturation of primitive promyelocytes derived from leukemia clones, and subsequently, in patients who achieve complete remission, the bone marrow and peripheral blood are refilled with normal, polyclonal hematopoietic cells.

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.)