Retinoic acid receptor α (RARα)

In T-cell lymphoma cells, tamibarotene (20, 40 μM) suppresses the expression of genes related to the cell cycle. When tamibarotene (5 μM) is added to RARA-overexpressing cells, it significantly boosts RARE activity compared to RARAlow cells. Moreover, treatment with tamibarotene enhances the degree of CDK2, CDK4, and CDK6 inhibition due to RARAwt overexpression [1]. Tamibarotene directly counteracts the profibrotic phenotype of dermal fibroblasts treated with transforming growth factor-β1, suppresses ICAM-1 expression in endothelial cells, and stimulates the development of M1 macrophages in vitro [2]. In VSMC, tamibarotene (4 μM) dose-dependently increases apelin mRNA and protein levels. As a result of interacting with KLF5 and Sp1, which are pre-bound to the apelin promoter's TCE site, RARα (retinoic acid receptor α) is recruited to the apelin promoter during tamibarotene stimulation. This transcriptional activation complex then triggers apelin expression. elevated in the VSMC. By directly binding to TCE on the apelin promoter, KLF5 and Sp1 work together to mediate the expression of apelin induced by tamibarotene [4].

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Am80 inhibits the production of IL-4, IL-17A, and IFN-γ in CD4+ T cells. Am80 suppresses M2 polarization of macrophages. Am80 reduces ICAM-1 expression in dermal microvascular endothelial cells. Am80 directly ameliorates the TGF-β-induced profibrotic phenotype of human dermal fibroblasts [2].

In mice treated with bleomycin (BLM) and skin-tightening 1 mice, Tamibarotene (Am80) (1 mg/kg/day) significantly decreased dermal and subdermal fibrosis, respectively. Tamibarotene consistently and significantly inhibits the expression of several molecules linked to tissue fibrosis in the lesional skin of BLM-treated mice, including transforming growth factor-β1, connective tissue growth factor, IL-4, IL-10, IL-13, IL-17A, tumor necrosis factor-α, IFN-γ, and monocyte chemoattractant protein 1. Furthermore, in the draining lymph nodes of mice treated with BLM, tamibarotene increased the proportion of naive T cells and decreased the proportion of effector T cells among CD4+ T cells [2]. When compared to mice not receiving treatment, mice with periodontitis did not exhibit any appreciable changes in serum levels of AST, ALT, or ALP when given tamibarotene (2.5 mg/kg, po). Tamibarotene decreases the quantity of P. gingivalis-induced mouse osteoclasts and enhances alveolar bone resorption. When compared to EPD mice, tamibarotene significantly raised the percentage of CD4+ Foxp3+ Treg cells. Additionally, tamibarotene successfully lowers the expression of Th17 (CD4+ROR-γt+) cells in P. CLNs and gingival tissue were infected by gingivalis[3]. Rats with balloon-injured arteries express more apelin when given tamibarotene (1 mg/kg, po), which is in line with findings from cultured VSMCs [4]. The hippocampal ADAM10 levels in aged SAMP8 mice showed an improvement following the administration of Tamibarotene (1 mg/kg/day). After taking tamibarotene, Hes5 and Ki67 were recovered, and spatial working memory was enhanced [5].

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Tamibarotene (Am80) is a synthetic retinoid that modulates the pathologic processes of various autoimmune and inflammatory diseases and their animal models. We here investigated the therapeutic potential of Tamibarotene (Am80) against systemic sclerosis using its animal models. Am80 significantly attenuated dermal and hypodermal fibrosis in bleomycin (BLM)-treated mice and tight skin 1 mice, respectively. Consistently, Tamibarotene (Am80) significantly suppressed the expression of various molecules related to tissue fibrosis, including transforming growth factor-β1, connective tissue growth factor, IL-4, IL-10, IL-13, IL-17A, tumor necrosis factor-α, IFN-γ, and monocyte chemotactic protein 1 in the lesional skin of BLM-treated mice. Furthermore, Am80 decreased the proportion of effector T cells, while increasing that of naïve T cells among CD4+ T cells in the draining lymph nodes of BLM-treated mice. [2]

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Tamibarotene (Am80), a synthetic retinoic acid receptor (RAR), is an agonist with high specificity for RARα and RARβ. Retinoid agonists have been shown to inhibit Th17 cell polarization and to enhance forkhead box P3 (Foxp3) expression during the course of inflammatory diseases. The aim of this study was to evaluate the previously unrecognized role of Am80 in regulating the immune responses of periodontitis within the oral microenvironment. The experimental model of periodontitis in mice was induced by oral infection with Porphyromonas gingivalis (P. gingivalis) W83. Our results indicated that Am80 effectively suppressed alveolar bone resorption induced by P. gingivalis W83 and decreased the number of osteoclasts. We clarified that these effects were closely associated with the reduced percentage of CD4(+) retinoid-related orphan receptor (ROR)γt(+) cells and increased the percentage of CD4(+) Foxp3(+) cells in the gingival tissues, cervical lymph nodes (CLNs), and spleen. Furthermore, in P. gingivalis-infected mice, Am80 down-regulated mRNA expression levels of interleukin-17A (IL-17A), receptor activator of nuclear factor-kappa beta ligand (RANKL), monocyte chemotactic protein-1 (MCP-1), IL-6, and IL-1β. Simultaneously, Am80 up-regulated expression levels of IL-10 and transforming growth factor-β1 (TGF-β1) in gingival tissues and the CLNs. Our results suggest that Am80 could protect against periodontal bone resorption, primarily through the modulation of immune responses in the oral microenvironment, and demonstrate the potential of Am80 as a novel clinical strategy for preventing periodontitis.[3]

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An in vivo study indicated that Tamibarotene (Am80) increased apelin expression in balloon-injured arteries of rats, consistent with the results from the cultured VSMCs. [4]

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The retinoic acid (RA, a vitamin A metabolite) receptor (RAR) is a transcription factor. Vitamin A/RA administration improves the Alzheimer's disease (AD)- and age-related attenuation of memory/learning in mouse models. Recently, a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was identified as a key molecule in RA-mediated anti-AD mechanisms. We investigated the effect of chronic administration of the RAR agonist Tamibarotene (Am80) on ADAM10 expression in senescence-accelerated mice (SAMP8). Moreover, we estimated changes in the expression of the amyloid precursor protein (APP), amyloid beta (Aβ), and hairy/enhancer of split (Hes), which are mediated by ADAM10. Spatial working memory and the levels of a hippocampal proliferation marker (Ki67) were also assessed in these mice. ADAM10 mRNA and protein expression was significantly reduced in the hippocampus of 13-month-old SAMP8 mice; their expression improved significantly after Am80 administration. Further, after Am80 administration, the expression levels of Hes5 and Ki67 were restored and the deterioration of working memory was suppressed, whereas APP and Aβ levels remained unchanged. Our results suggest that Am80 administration effectively improves dementia by activating the hippocampal ADAM10-Notch-Hes5 proliferative pathway [5].

siRNA transfection [4]
The siRNAs against the rat RARα and KLF5 sequences were designed and synthesized by Sigma–Aldrich, and the siRNAs against the rat apelin sequences were designed and synthesized by Invitrogen. si-NS (non-specific siRNA) and siRNAs specific for Sp1 were used. The siRNA sequences against RARα (si-RARα) were 5′-CUCAGAACAACGUGUCUCU-3′ and 5′-AGAGACACGUUGUUCUGAG-3′. The siRNA sequences against apelin (si-apelin) were 5′-GGCUAGAAGAAGGCAACAUTT-3′ and 5′-AUGUUGCCUUCUUCUAGCCTT-3′. Transfection was performed using Lipofectamine™ reagent following the manufacturer's instructions. At 24 h following transfection, VSMCs were treated with or without Tamibarotene (Am80) (4 μM). Cells were then harvested and used for PCR, qRT-PCR and Western blotting assays.

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EMSAs [4]
EMSAs were performed using the LightShift Chemiluminscent EMSA Kit, according to the manufacturer's recommendations. Briefly, a biotin-labelled probe containing TCE-1 site for the apelin promoter was synthesized by Sangon. The sequence is as follows: 5′-CTTCCTGCCTCCCCCCCCCCCACCCCCGCCTCCTAATGACCAC-3′. A total of 5 μg of nuclear protein from VSMCs treated with Tamibarotene (Am80) for 24 h was incubated with the biotin-labelled probe for 30 min at room temperature, loaded on to a 6% non-denaturating polyacrylamide gel and run at 100 V for 40 min, then electrotransferred on to a nylon membrane. The probe was cross-linked to the membrane by exposure to 254 nm UV radiation for 15 min. Finally, the biotin-labelled probe was detected by chemiluminescence, according to the manufacturer's instructions. To specifically identify KLF5, RARα and Sp1 protein in binding complexes, 2 mg of anti-KLF5, anti-RARα and anti-Sp1 antibodies were added to the binding reaction mix and incubated for 30 min at room temperature before adding the probe.

Luciferase reporter assays [1]
RARE reporter and pCI empty vector or pCI-RARA (wild-type) were co-transfected into HuT78 cells by electroporation. After recovery for 24 hours, the transfected cells were treated with Tamibarotene (Am80) or vehicle (dimethyl sulfoxide) in RPMI containing 2% charcoal-stripped FBS for 24 hours. Luciferase expression was measured using the Dual-Luciferase Reporter Assay System following manufacturer's protocol using the Centro XS3 LB 960 microplate luminometer as previously described. Total protein concentrations of each lysate were used for normalization.

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Transcriptome analysis [1]
Mac-1, Karpas 299 and HuT78 cells were treated with Tamibarotene (Am80) (0 μM, 20 μM, or 40 μM), ATRA (0 μM, 10 μM, or 20 μM), or bexarotene (0 μM, 10 μM, or 20 μM) in medium containing 2% charcoal-stripped FBS. Cell pellets were collected at 24 hours and immediately stored at −80°C pending RNA extraction. RNA sequencing was performed as previously described. Briefly, following extraction of total RNA, mRNA libraries were constructed and 100 base-pair paired-end sequencing was performed on an Illumina HiSeq instrument. The Sequencing data were mapped to hg19 and genes with a minimum of 100 reads in at least one sample were retained for analysis. Dose-dependent effects of each drug were determined using paired t-tests (EdgeR) for medium-versus-low and high-versus-medium comparisons. Genes were considered differentially expressed if they met fold-change and P-value criteria (log2 fold change ≥ 0.5 or ≤ −0.5 and P-value ≤ 0.05) for both comparisons. GSEA was performed on a ranked dataset generated by the product of the fold-change sign and –log(P-value) for the 0 μM versus 40 μM AM80 compariso. Pathway analysis was performed using Ingenuity Pathway Analysis software on differentially expressed genes.

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Macrophage polarization [2]
Thioglycollate-elicited peritoneal macrophages were prepared from wild-type mice as described previously (Matsumoto et al., 2007). Peritoneal macrophages, cultured overnight, were polarized to M2 macrophages by stimulating with 20 ng/ml of IL-4 in the presence or absence of Tamibarotene (Am80) for 24 hours. Total RNA was isolated from cultured cells and analyzed by quantitative real-time reverse transcription PCR. Additionally, macrophages were stained with anti-CD204 and anti-CD206 antibodies and quantified using flow cytometry.

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Cell culture of THP-1 cells [2]
THP-1 cells were purchased from the American Type Culture Collection. THP-1 cells in log phase growth were primed with 100 ng/ml of IFN-γ for 6 hours and stimulated with 100 ng/ml of lipopolysaccharides in the presence or absence of Tamibarotene (Am80) for 24 hours. Total RNA was isolated from cell lysates, as described above.

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Cell culture of human dermal microvascular endothelial cells [2]
Human dermal microvascular endothelial cells were used. Human dermal microvascular endothelial cells were treated with Tamibarotene (Am80) at the indicated concentrations or an equal amount of DMSO, 1 hour before the stimulation with 0.5 ng/ml of TNF-α. Cells were cultured for an additional 24 hours. Total RNA was isolated from cell lysates as described above.

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Immunoblotting [2]
Human dermal fibroblasts were stimulated with TGF-β1 in the presence or absence of Tamibarotene (Am80) for 24 hours. Cell lysates were prepared and subjected to immunoblotting, as described previously (Noda et al., 2014).

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Cells, cell culture and treatment [4]
Male Sprague–Dawley rats (80–100 g) were anaesthetized with ketamine (60 mg/kg of body weight) and xylazine (5 mg/kg of body weight) intraperitoneally, and VSMCs were extracted from the thoracic aorta as described previously. Next, the rats were killed by exsanguination under anaesthesia. VSMCs were maintained in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS in a humidified atmosphere with 5% CO2 at 37°C. Cells used in this study were passaged for three to six generations. Prior to Tamibarotene (Am80) stimulation, VSMCs were maintained in serum-free DMEM for 24 h. They were then cultured in DMEM containing 2% FBS and 4 μM Tamibarotene (Am80) for the indicated times.

Mice:For the infection, mice are given sulfamethoxazole and trimethoprim in an oral suspension at 10 mL of deionized water ad libitum for 10 days to reduce the native flora and to support colonization of P. gingivalis W83. Four days after the antibiotic therapy finishes, periodontal infection is established through oral inoculation using 1010 colony-forming units of P. gingivalis suspended in 100 μL 4% carboxymethyl cellulose (CMC) for 7 days. The mice are euthanized 4 weeks after the first oral inoculation. Tamibarotene (2.5 mg/kg) is suspended in a 0.5% carboxymethyl cellulose solution. The drug is orally gavaged into the esophagus daily in a volume of 0.1 mL/10 g body weight. Tamibarotene is administered 1 h before the induction of periodontitis and then given daily per the protocol until day 28. Control mice with periodontal disease receive the same volume of 0.5% carboxymethyl cellulose solution. The body weight of each mouse is measured every 3 days.

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TSK1 mice [2]
TSK1 mice were used. Genotyping of TSK1 mice was performed by PCR with the following primers (forward 5′-GTTGGCAACTATACCTGCAT-3′ and reverse 5′-CCTTTCCTGGTAACATAGGA-3′). Tamibarotene (Am80) treatment to TSK1 mice was began at the age of 3 weeks and finished at the age of 7 weeks.

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Tamibarotene (Am80) treatment [2]
Feeding pellets were made by mixing Am80 with a commercial rodent diet. Approximately 1 mg/kg/day Am80 were administered to mice, according to the procedure of Miwako and Shudo (2009). Administration was started 1 week before BLM or PBS subcutaneous injection. Mouse model of periodontitis [3]
Periodontal infection was induced by P. gingivalis W83 as described previously, with minor modifications. P. gingivalis W83 was cultivated on a brain–heart infusion (BHI) agar, supplemented with Vitamin K1 (10 μg/mL), hemin (0.25%), and sterile defibrinated sheep blood (5%). The bacteria were incubated in an anaerobic chamber at 37 °C for 7 days in a 10% CO2, 5% H2, and 85% N2 atmosphere. The bacteria were collected and cultured in complete-BHI liquid at 37 °C for 24 h and then used during the logarithmic growth phase for oral infection.
For the infection, mice were given sulfamethoxazole and trimethoprim in an oral suspension at 10 mL of deionized water ad libitum for 10 days to reduce the native flora and to support colonization of P. gingivalis W83. Four days after the antibiotic therapy finished, periodontal infection was established through oral inoculation using 1010 colony-forming units of P. gingivalis suspended in 100 μL 4% carboxymethyl cellulose (CMC) for 7 days. The mice were euthanized 4 weeks after the first oral inoculation.

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Drug treatments [3]
Tamibarotene (Am80) (2.5 mg/kg) was suspended in a 0.5% carboxymethyl cellulose solution. The drug was orally gavaged into the esophagus daily in a volume of 0.1 mL/10 g body weight. Am80 was administered 1 h before the induction of periodontitis and then given daily per the protocol until day 28. Control mice with periodontal disease received the same volume of 0.5% carboxymethyl cellulose solution. The body weight of each mouse was measured every 3 days.

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Balloon injury model [4]
Male Sprague–Dawley rats were anaesthetized with ketamine (60 mg/kg of body weight) and xylazine (5 mg/kg of body weight) intraperitoneally. Briefly, after a median incision on the anterior neck, the carotid arteries of the left side were isolated and a distal incision was made. After the blood was removed, a 0.13-mm-diameter balloon catheter was advanced gently into the left common carotid artery. The balloon was inflated with saline to distend the common carotid artery and then pulled back to the external carotid artery. The catheter was withdrawn and the proximal end of the external carotid artery was ligated, and blood flow was re-established after removing the clamps on the arteries. All procedures were performed by a single operator. Tamibarotene (Am80) was administrated orally at a dose of 1 mg/kg of body weight per day beginning 1 day before balloon injuring and continuing for 14 days thereafter. On day 14 after injury and administration of Am80 orally, the rats were killed by exsanguination under anaesthesia, and the carotid artery was collected for qRT-PCR or immunohistochemistry.

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The animals used were male SAMP8 and SAMR1 mice (Japan SLC, Shizuoka, Japan) aged 2–13 months at the beginning of the experiment. The animals were maintained on a 12:12 h light:dark cycle (lights on at 09:00) at an ambient temperature of 23 ± 1 °C and had free access to food and water. An Tamibarotene (Am80)-containing diet was prepared to a concentration of 0.001% (w/w) by mixing the drug with the control diet (MF, 360 kcal/100 g; Oriental Yeast, Tokyo, Japan). The concentration of Tamibarotene (Am80) was equivalent to 1 mg/kg/day for a mouse weight of 40 g and a food consumption of 4 g/day. The Am80-containing and control diets were fed for 4 weeks to each group before radial maze test setting. Obvious adverse effects were not observed in the Am80-treated group.[5]

Protein Binding
Over 99%, predominantly to serum albumin.

[1]. Retinoic acid receptor alpha drives cell cycle progression and is associated with increased sensitivity to retinoids in T-cell lymphoma. Oncotarget. 2017 Apr 18;8(16):26245-26255.

[2]. Tamibarotene Ameliorates Bleomycin-Induced Dermal Fibrosis by Modulating Phenotypes of Fibroblasts, Endothelial Cells, and Immune Cells. J Invest Dermatol. 2016 Feb;136(2):387-98.

[3]. Tamibarotene modulates the local immune response in experimental periodontitis. Int Immunopharmacol. 2014 Dec;23(2):537-45.

[4]. Synthetic retinoid Am80 up-regulates apelin expression by promoting interaction of RARα with KLF5 and Sp1 in vascular smooth muscle cells. Biochem J. 2013 Nov 15;456(1):35-46.

[5]. The retinoic acid receptor agonist Am80 increases hippocampal ADAM10 in aged SAMP8 mice. Neuropharmacology. 2013 Sep;72:58-65.

Tamibarotene is a dicarboxylic acid monoamide resulting from the condensation of one of the carboxy groups of terephthalic acid with the amino group of 5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-amine. It has a role as an antineoplastic agent and a retinoic acid receptor alpha/beta agonist. It is a member of tetralins, a retinoid and a dicarboxylic acid monoamide. It is functionally related to a 4-carbamoylbenzoic acid and a terephthalic acid.
Tamibarotene is a novel synthetic retinoid for acute promyelocytic leukaemia (APL). Tamibarotene is currently approved in Japan for treatment of recurrent APL, and is undergoing clinical trials in the United States.
Tamibarotene is an orally active, synthetic retinoid, developed to overcome all-trans retinoic acid (ATRA) resistance, with potential antineoplastic activity. As a specific retinoic acid receptor (RAR) alpha/beta agonist, tamibarotene is approximately ten times more potent than ATRA in inducing cell differentiation and apoptosis in HL-60 (human promyelocytic leukemia) cell lines in vitro. Due to a lower affinity for cellular retinoic acid binding protein (CRABP), tamibarotene may show sustained plasma levels compared to ATRA. In addition, this agent may exhibit a lower toxicity profile than ATRA, in part, due to the lack of affinity for the RAR-gamma receptor, the major retinoic acid receptor in the dermal epithelium.

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Drug Indication
Investigated for use/treatment in leukemia (unspecified).

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Mechanism of Action
Tamibarotene is a specific agonist for retinoic acid receptor alpha/beta with possible binding to retinoid X receptors (RXR).

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Pharmacodynamics
Tamibarotene is a new synthetic retinoid drug recently approved for relapsed or refractory acute promyelocytic leukemia (APL) in Japan. It is a specific agonist for retinoic acid receptor alpha/beta. Compared to all-trans retinoic acid (ATRA), a natural retinoid indicated for a first-line treatment of APL, tamibarotene is chemically more stable and several times more potent as an inducer of differentiation in promyelocytic leukemia cells. In contrast to ATRA, whose plasma concentration declines considerably during daily administration, tamibarotene sustains plasma level probably due to a lower affinity for cellular retinoic acid binding protein. Furthermore, adverse side effects were milder than those of ATRA in clinical trials.

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Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with generally poor outcomes following standard therapy. Few candidate therapeutic targets have been identified to date. Retinoic acid receptor alpha (RARA) is a transcription factor that modulates cell growth and differentiation in response to retinoids. While retinoids have been used to treat some cutaneous T-cell lymphomas (CTCLs), their mechanism of action and the role of RARA in CTCL and other mature T-cell lymphomas remain poorly understood. After identifying a PTCL with a RARAR394Q mutation, we sought to characterize the role of RARA in T-cell lymphoma cells. Overexpressing wild-type RARA or RARAR394Q significantly increased cell growth in RARAlow cell lines, while RARA knockdown induced G1 arrest and decreased expression of cyclin-dependent kinases CDK2/4/6 in RARAhigh cells. The retinoids, AM80 (tamibarotene) and all-trans retinoic acid, caused dose-dependent growth inhibition, G1 arrest, and CDK2/4/6 down-regulation. Genes down-regulated in transcriptome data were enriched for cell cycle and G1-S transition. Finally, RARA overexpression augmented chemosensitivity to retinoids. In conclusion, RARA drives cyclin-dependent kinase expression, G1-S transition, and cell growth in T-cell lymphoma. Synthetic retinoids inhibit these functions in a dose-dependent fashion and are most effective in cells with high RARA expression, indicating RARA may represent a therapeutic target in some PTCLs. [1]

C22H25NO3

351.44

351.183

C, 75.19; H, 7.17; N, 3.99; O, 13.66

94497-51-5

108143

White to off-white solid powder

1.2±0.1 g/cm3

449.6±45.0 °C at 760 mmHg

231-232ºC

225.7±28.7 °C

0.0±1.2 mmHg at 25°C

1.593

6.48

2

3

3

26

546

0

O=C(O)C1=CC=C(C(NC2=CC=C3C(C)(C)CCC(C)(C)C3=C2)=O)C=C1

MUTNCGKQJGXKEM-UHFFFAOYSA-N

InChI=1S/C22H25NO3/c1-21(2)11-12-22(3,4)18-13-16(9-10-17(18)21)23-19(24)14-5-7-15(8-6-14)20(25)26/h5-10,13H,11-12H2,1-4H3,(H,23,24)(H,25,26)

4-[(5,5,8,8-tetramethyl-6,7-dihydronaphthalen-2-yl)carbamoyl]benzoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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 (7.11 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 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 (7.11 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), 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 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 (7.11 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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 (Please use freshly prepared in vivo formulations for optimal results.)