生物活性

TTNPB is a synthetic stilbene analog of retinoic acid that acts as a potent retinoic acid receptor (RAR) agonist (EC50 = 21 nM, 4.0 nM, and 2.4 nM for RARα, RARβ, and RARγ, respectively). Extremely potent analog of retinoic acid, selective for the retinoic acid receptor (RAR) subtype. Enhances reprogramming efficiency in chemically induced pluripotent stem cells. TTNPB(Arotinoid Acid, Ro 13-7410), the third generation of synthetic retinoid, is ahighly potent pan-RAR-selective retinoid agonist that developed for thetreatment of psoriasis and other hyperkeratotic skin disorders.

TTNPB inhibits chondrogenesi with an IC50 of 0.14nMin mouse limb bud cells.[1]

Binding inhibition of [³H] tRA

体外研究

体内研究

激酶实验

Binding assays[1]

Binding assays were performed as follows.Briefly, labeled and unlabeled retinoids were added to nucleosol or cytosolicfractions in ethanol so that the total amount of ethanol added was constant inall tubes and did not exceed 2% of the incubation volume. The receptorpreparations were incubated with retinoids at 4^oC for 4–6 hr.Sephadex PD-10 desalting columns were used to separate bound radioligand fromfree radioligand after equilibrium was achieved. For competitive bindingassays, varying concentrations of unlabeled competing ligand were incubatedwith the appropriate nucleosol in the presence of a fixed concentration of [³H]tRA (sp. act. =49.3Ci/mmol) or [³H] 9-cis RA (sp. act. =24.0Ci/mmol). Finalconcentrations of [³H] tRA and [³H] 9-cis RA for nuclearreceptor binding assays were 5nM. Final concentrations of [³H]tRAfor CRABP binding assays was 30nM. The IC50s were calculated as described above.For saturation kinetics, increasing concentrations of radiolabeled ligand ([³H]tRAsp. act.=49.3Ci/mmol, [³H]TTNPB sp. act. =5.5Ci/mmol) were added to the nucleosol ofthe appropriate receptor subtype in the presence (nonspecific binding) orabsence (total binding) of a 100-fold molar excess of the correspondingunlabeled retinoid. Specific binding was defined as the total binding minusnonspecific binding. Saturation kinetics was calculated.

细胞实验

Analysis of NB4 cell differentiation[4]

NB4 cells were cultured in RPMI 1640supplemented with 10% FCS, glutamine (2mM), HEPES buffer (25mM), and gentamycin(40μg/mL) in a humidified incubator at 37^oC and 5% CO₂.Every 2 days the concentration of the cells was determined, the cells werediluted (to 1x10⁵cell/mL) and treated with the ligand (1μM finalconcentration of the corresponding ligand; total volume, 10mL). Thedifferentiation, apoptosis, and cell cycle analysis assays were performed onday 6, on a FACScan flow cytometer.

Briefly, NB4 cells (2.5x10⁵cells)were washed (PBS), centrifuged, and incubated (40min on ice, under exclusion oflight) in 20μL of the corresponding labeled BD Pharmingen antibodies (1/5 dilutedin 0.1% BSA in PBS solution): PE anti-human CD11c and FITC-labeled anti-humanCD14 in the test sample and PE mouse IgG1 and FITC mouse IgG2a antibodies,respectively, for the isotypic control to determine the non-specific backgroundstaining. After that the cells (on ice, under exclusion of light) were washed(2mL 0.1% BSA in PBS), centrifuged, and resuspended in 800μL0.1% BSA in PBS with 0.25μg/mL PI for the FACS analysis.

Analysisof apoptosis

For apoptosis assays recombinant human AnnexinV-FITC was used as a marker for apoptotic cells and PI (propidium iodide) fordiscrimination of the necrotic from the apoptotic cells. Aliquots of NB4 cells(2.5x10⁵ cells) were washed (PBS) and incubated (15 min, r.t., underexclusion of light) in 48μL HEPES containing buffer (10mM HEPES/NaOH pH 7.4, 150mM NaCl, 1mMMgCl₂, 5mM KCl, 1.8 mM CaCl₂) with 1μLAnnexin VFITC conjugate. After that HEPES buffer (800μL) with PI(0.25μg/mL) was added prior to the FACS analysis.

动物实验

Animals[5]

Outbred F/i-Albino rats were fed withground diet Nafag 850 as required. Tap water was freely available. Female ratswere mated overnight, and females which had a vaginal plug present thefollowing morning were considered to be at day 1 of gestation.

Dosingsolutions

The three arotinoids Ro 13-7410, Ro 13-6298and Ro 15-1570 were suspended in rapeseed oil and stored in the dark at 4 ^oCunder nitrogen. The arotinoids were administered orally by gavage using anadministration volume of 5 ml/kg. Controls received only rapeseed oil.

Embryotoxicityand teratogenicity studies

Inseminated females were treated once withthe indicated dose of the respective arotinoid on day 9 or 13 of gestation. Thesingle treatment with the various retinoids did not affect the body weightdevelopment and induced no toxic signs. Terminal necropsy was performed on day21 of gestation. The numbers of fetuses, implantations, and resorptions wererecorded. Fetuses were weighed and examined for external abnormalities andcleft palate. They were then prepared for skeletal examination using aNaOH-alizarin red S staining procedure.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Pignatello MA KF, Levin AA. Multiple factors contribute to the toxicity of the aromatic retinoid, TTNPB (Ro 13-7410): binding affinities and disposition. Toxicol Appl Pharmacol. . 1997;142(2):319-327.
[2] Anding AL, Nieves NJ, Abzianidze VV, Collins MD, Curley RW, Jr., Clagett-Dame M. 4-Hydroxybenzyl modification of the highly teratogenic retinoid, 4-[(1E)-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propen-1-yl]b enzoic acid (TTNPB), yields a compound that induces apoptosis in breast cancer cells and shows reduced teratogenicity. Chem Res Toxicol. 2011;24(11):1853-1861.
[3] Pignatello MA KF, Levin AA. Multiple factors contribute to the toxicity of the aromatic retinoid TTNPB (Ro 13-7410): interactions with the retinoic acid receptors. Toxicol Appl Pharmacol. 1999 159(2):109-116.
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体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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