CINPA1 targets the constitutive androstane receptor (CAR; NR1I3) as an inverse agonist/inhibitor. In HepG2 cells transiently transfected with hCAR1 and CYP2B6-luc reporter, CINPA1 inhibits CAR-mediated transactivation with an IC50 of approximately 70 nM. [1] CINPA1 does not activate pregnane X receptor (PXR) at concentrations up to 40 μM. It is a weak antagonist of PXR with an estimated IC50 of 6.6 μM in HepG2-PXR stable cells. [1] CINPA1 does not modulate the activity of other nuclear receptors tested, including FXR, GR, LXRα, LXRβ, PPARγ, RXRα, RXRβ, or VDR, at 18 μM. [1]
Constitutive Androstane Receptor (CAR): CINPA1 is an inhibitor of CAR, reducing CAR-mediated transcription with an IC₅₀ of approximately 70 nM in HepG2-hCAR CYP2B6-luc reporter cells . It exhibits >90-fold selectivity for CAR over PXR . Pregnane X Receptor (PXR): CINPA1 does not activate PXR and shows only weak PXR antagonist potency (68% inhibition by 18 μM CINPA1 against 5 μM rifampicin) . Other Nuclear Receptors: CINPA1 has no agonist or antagonist activity toward FXR, GR, LXRα/β, PPARγ, RXRα/β, or VDR .

CINPA1 inhibits CAR-mediated transactivation in a dose-dependent manner with an IC50 of approximately 70 nM in HepG2 cells transfected with hCAR1 and CYP2B6-luc reporter. At 5 μM, CINPA1 completely inhibits CAR activity, comparable to the positive control PK11195 (50 μM). [1]
CINPA1 does not activate PXR-mediated gene expression at concentrations up to 40 μM, unlike PK11195 and clotrimazole. In HepG2-PXR stable cells, CINPA1 shows weak PXR antagonism with an IC50 of 6.6 μM. [1]
In HepG2-hCAR1 stable cells overexpressing hCAR1, CINPA1 (10 μM) significantly attenuates endogenous CYP2B6 and CYP3A4 mRNA levels (approximately 50-75% reduction). [1]
In LS174T cells (endogenously expressing both CAR and PXR), CINPA1 (10 μM) reduces CYP2B6 levels by approximately 50-60% without significantly affecting CYP3A4, whereas PK11195 increases CYP3A4 levels. [1]
In primary human hepatocytes from multiple donors, CINPA1 (0.3-5 μM) effectively inhibits CITCO-induced CYP2B6 expression (50-80% reduction). CINPA1 does not alter CAR protein levels or its nuclear localization. [1]
In a TR-FRET coactivator recruitment assay, CINPA1 inhibits PGC-1α binding to GST-hCAR-LBD with an IC50 of approximately 2-5 μM. [1]
In mammalian two-hybrid assays, CINPA1 (5 μM) reduces SRC-1 and TIF-2 coactivator binding to CAR-LBD and increases SMRTα and mNCoR corepressor binding. [1]
In chromatin immunoprecipitation assays using primary human hepatocytes, CINPA1 (5 μM) reduces CITCO-induced CAR recruitment to the CYP2B6 XREM (dNR3) region and reduces RNA polymerase II recruitment. [1]

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In HepG2 cells, CINPA1 (1 μM; 24 hours) transcribes PXR's CAR-mediated transactivation [1]. A potentiometric xenobiotic kit reagent is CINPA1 [1]. In primary human hepatocytes, where CAR is endogenously produced, CINPA1 transcribes CAR-mediated gene expression [1]. The subcellular location and protein levels of CAR are unaffected by CINPA1 [1]. In aided two-hybrid tests, CINPA1 enhances co-blocking, and in chromatin immunoprecipitation studies, CINPA1 interferes with CAR binding to target gene promoter regions [1]. The interaction between co-activating peptides and CAR-LBD is efficiently inhibited by CINPA1. The function demonstrates that CINPA1 is a CAR ligand [1].

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Inhibition of CAR-mediated Transcription: CINPA1 inhibits CAR-mediated transcription with an IC₅₀ of approximately 70 nM in HepG2-hCAR CYP2B6-luc reporter cells . In HepG2 cells, 1 μM CINPA1 treatment for 24 hours inhibits CAR-mediated transactivation without activating PXR . No Cytotoxicity: CINPA1 has no cytotoxic effects up to a concentration of 30 μM . Inhibition of Endogenous CAR Gene Expression: In primary human hepatocytes, where CAR is endogenously expressed, CINPA1 inhibits CAR-mediated gene expression . No Effect on CAR Protein Levels or Localization: CINPA1 does not alter the protein levels or subcellular localization of CAR . Alteration of Coregulator Interactions: In mammalian two-hybrid assays, CINPA1 increases corepressor interaction and reduces coactivator interaction with the CAR ligand-binding domain . Disruption of CAR Binding to DNA: In chromatin immunoprecipitation assays, 1 μM CINPA1 inhibits CITCO-induced CAR recruitment to CYP2B6 or CYP3A4 promoters by >85% . CINPA1 disrupts CAR binding to the promoter regions of target genes . Regulation of CFTR Chloride Transport: In T84 human colonic epithelial cells, CINPA1 acts as a CAR antagonist to reverse the inhibitory effect of CAR agonists (CITCO and phenytoin) on cAMP-dependent Cl⁻ secretion .

Inhibition of CAR Agonist-Induced Chloride Secretion: In in vivo mouse models, the CAR agonist TCPOBOP (3 mg/kg for 7 days) decreases CFTR mRNA and protein expression in intestinal tissues and inhibits cholera toxin-induced intestinal fluid accumulation, suggesting that CINPA1 as a CAR antagonist may have therapeutic potential for secretory diarrhea .

LanthaScreen TR-FRET coactivator recruitment assay: GST-tagged human CAR ligand-binding domain (GST-hCAR-LBD, 5 nM) is mixed with Tb-anti-GST antibody (5 nM) and fluorescein-labeled PGC-1α coactivator peptide (125 nM) in coregulator buffer G. Test compounds (CINPA1) are added at concentrations ranging from 70 μM to 3.5 nM (1:3 dilutions, 10 concentration levels). DMSO is used as negative control (0% inhibition), and clotrimazole (42 μM) is used as positive control (100% inhibition). After 1-hour incubation at room temperature, TR-FRET emissions at 490 nm and 520 nm are measured following 340 nm excitation. The 520:490 ratio is calculated, and IC50 values are derived from dose-response curves. CINPA1 inhibits PGC-1α binding to CAR-LBD with an IC50 of approximately 2-5 μM. [1]

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Coregulator Recruitment Assay: A mammalian two-hybrid assay is used to evaluate the effect of CINPA1 on CAR-coregulator interactions. The CAR ligand-binding domain is fused to the GAL4 DNA-binding domain as bait, and corepressor or coactivator proteins are fused to VP16 as prey. After transfection into cells and treatment with CINPA1, luciferase activity is measured to assess changes in CAR-coregulator interactions. Results show that CINPA1 increases corepressor interaction while decreasing coactivator interaction with the CAR-LBD . Molecular Docking Studies: Docking studies using the CAR ligand-binding domain structure are performed to analyze the binding modes of CINPA1 and its metabolites within the CAR ligand-binding pocket .

Luciferase reporter assay for CAR activity: HepG2 cells are transfected with FLAG-hCAR1 expression plasmid and CYP2B6-luc reporter plasmid. After 24 hours, cells are trypsinized and plated in 384-well plates (5000 cells/well). Cells are treated with test compounds for 24 hours before measuring firefly luciferase activity using SteadyLite reagent. Percentage of CAR inhibition is calculated by setting 50 μM PK11195 to 100% inhibition and DMSO to 0% inhibition. [1]
PXR activation assay: HepG2-PXR clone 1 cells (stably expressing hPXR and CYP3A4-luc) are treated with test compounds in phenol red-free DMEM with 5% charcoal-dextran-treated FBS for 24 hours. SteadyLite luciferase assay is performed. [1]
Gene expression analysis (qRT-PCR) : Cell lines or primary human hepatocytes are treated with chemicals for 24-48 hours. RNA is extracted and purified, and cDNA is prepared. Quantitative RT-PCR is performed using TaqMan probes with 18S as internal standard. [1]
Mammalian two-hybrid assay: HEK293T cells are cotransfected with pACT-hCAR1 (VP16AD-hCAR1 fusion), pBIND-coregulator (GAL4DBD-coregulator fusion), and pG5-Luc reporter. After 24-hour treatment, Dual-Glo luciferase assay is performed to measure firefly and Renilla luciferase activities. [1]
Chromatin immunoprecipitation (ChIP) assay: Primary human hepatocytes are treated with compounds for 45 minutes or overnight. Proteins are cross-linked with 1% formaldehyde, digested with micrococcal nuclease, and sonicated. Chromatin is immunoprecipitated with anti-CAR antibody, anti-RNA polymerase II antibody, or control IgG. Quantitative real-time PCR is performed to determine CAR occupancy at CYP2B6 PBREM and XREM regions. [1]

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CAR Reporter Gene Assay: HepG2 cells stably transfected with CYP2B6-luc reporter and hCAR1 (HepG2-hCAR1 CYP2B6-luc) are used to evaluate the inhibitory activity of CINPA1. Cells are seeded in 96-well plates and treated with various concentrations of CINPA1 in the presence of the CAR agonist CITCO to induce reporter gene expression. Luciferase activity is measured to calculate the inhibition rate, with an IC₅₀ of approximately 70 nM . Primary Human Hepatocyte Assay: Primary human hepatocytes are treated with CINPA1, and mRNA expression levels of CAR target genes (e.g., CYP2B6, CYP3A4) are detected by qPCR to validate the inhibitory effect of the compound on endogenous CAR activity . T84 Cell Chloride Secretion Assay: T84 human colonic epithelial cells are cultured as monolayers on Transwell inserts and co-treated with CINPA1 and CAR agonists (CITCO at 1 μM or phenytoin at 5 μM) for 24 hours. Short-circuit current measurements are used to assess changes in cAMP-dependent agonist-induced transepithelial Cl⁻ secretion, evaluating the effect of CINPA1 as a CAR antagonist on CFTR function .

Mouse Intestinal CFTR Expression Study: ICR mice receive intraperitoneal injections of the mouse-specific CAR agonist TCPOBOP (3 mg/kg body weight) for 7 days. After euthanasia, intestinal tissues are collected, and CFTR mRNA and protein expression levels are detected by qPCR and Western blot. CINPA1 can be used as a tool compound in such studies to validate CAR-mediated effects . Cholera Toxin-Induced Intestinal Fluid Accumulation Model: Mice are pretreated with TCPOBOP (3 mg/kg for 7 days), followed by cholera toxin injection using the intestinal loop ligation method to measure intestinal fluid accumulation. CAR agonists inhibit fluid accumulation, suggesting the potential application value of CINPA1 as a CAR antagonist .

Metabolic Profile: CINPA1 is converted to two main metabolites in human liver microsomes. CYP3A4 first converts CINPA1 to metabolite 1, which is then further metabolized by CYP2D6 to metabolite 2. Metabolite 1 exhibits very weak inhibitory activity on CAR function, while metabolite 2 is inactive. Docking studies indicate that while CINPA1 and metabolite 1 can bind in the CAR ligand-binding pocket, metabolite 2 may be incapable of the molecular interactions required for binding . Solubility: Solubility in DMSO is 150 mg/mL . Storage Stability: Powder is stable for 3 years at -20°C and for 2 years at 4°C; solutions in solvent are stable for 6 months at -80°C and 1 month at -20°C .

CINPA1 shows no cytotoxic effects up to 30 μM in HepG2 cells under conditions mimicking the luciferase screening assays (24-hour treatment). In extended 4-day cell viability assays, CINPA1 is not cytotoxic in HepG2, LS174T, or HEK293T cells up to 10 μM. At higher concentrations (30 μM and 60 μM), CINPA1, clotrimazole, and PK11195 are cytotoxic to varying degrees in different cell lines. [1]

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Cytotoxicity: CINPA1 has no cytotoxic effects on cells up to a concentration of 30 μM . Nuclear Receptor Selectivity: CINPA1 has no agonist or antagonist activity toward FXR, GR, LXRα/β, PPARγ, RXRα/β, or VDR, indicating good nuclear receptor selectivity . For Research Use Only: This product is for non-human scientific research use only and is not intended for therapeutic or veterinary use .

[1]. CINPA1 is an inhibitor of constitutive androstane receptor that does not activate pregnane X receptor. Mol Pharmacol. 2015 May;87(5):878-89.

Chemical structure: CINPA1 (chemical structure shown in Fig. 1B) was obtained from ChemDiv. [1]
Mechanism of action: CINPA1 binds to the CAR ligand-binding domain, reducing coactivator recruitment and increasing corepressor interaction, leading to reduced CAR binding to target gene promoters and decreased transcription of CAR-regulated genes (CYP2B6, CYP3A4). [1]
Species selectivity: CINPA1 does not inhibit mouse CAR activity. In mouse primary hepatocytes, CINPA1 does not inhibit Cyp2b10 expression, and in HepG2 cells transfected with mouse CAR, CINPA1 does not inhibit luciferase reporter activity. [1]
Specificity: CINPA1 does not activate any of the nuclear receptors tested (FXR, GR, LXRα, LXRβ, PPARγ, RXRα, RXRβ, VDR) and does not inhibit their agonist-induced activation at 18 μM. [1]

C23H29N3O3

395.49466586113

395.22

C, 69.85; H, 7.39; N, 10.62; O, 12.14

102636-74-8

102636-74-8;

969470

White to off-white solid powder

3.9

1

4

7

29

552

0

C(OCC)(=O)NC1C=C2C(=CC=1)CCC1=CC=CC=C1N2C(CN(CC)CC)=O

AYQBYSPEGRYKFA-UHFFFAOYSA-N

InChI=1S/C23H29N3O3/c1-4-25(5-2)16-22(27)26-20-10-8-7-9-17(20)11-12-18-13-14-19(15-21(18)26)24-23(28)29-6-3/h7-10,13-15H,4-6,11-12,16H2,1-3H3,(H,24,28)

Ethyl [5-[(diethylamino)acetyl]-10,11-dihydro-5H-dibenz[b,f]azepin-3-yl]carbamate

CINPA1 CINPA-1 CINPA 1

2934.99.03.00

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)

DMSO : ~250 mg/mL (~632.13 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.26 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 20.8 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: ≥ 2.08 mg/mL (5.26 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 20.8 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: ≥ 2.08 mg/mL (5.26 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 20.8 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.)