Acyl-coenzyme A: cholesterol O-acyltransferase 1 (ACAT1) [1].
EC₅₀: 9 nM (for human ACAT1) [1].
EC₅₀: 368 nM (for human ACAT2) [1].

When Nevanimibe (PD-132301; ATR101; 3 nM-30 μM) and cholesterol were co-incubated, the toxicity rose dramatically in a dose-dependent manner. After 24 hours, 3 nM of Nevanimibe reduced survival by 60% when 60 μg/mL of cholesterol was present. When cholesterol is present, all concentrations of Nevanimibe (3 nM–30 μM) cause cytotoxicity, however when cholesterol is present but not Nevanimibe-treated, cell viability is unaffected [1].

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In a cell-based fluorescence assay measuring cholesterol esterification, Nevanimibe potently and selectively inhibited human ACAT1 with an EC₅₀ of 9 nM, compared to ACAT2 with an EC₅₀ of 368 nM, demonstrating approximately 40-fold selectivity for ACAT1 [1].
In H295R human adrenocortical carcinoma cells, Nevanimibe (9 nM) treatment for 5 hours in the presence of exogenous cholesterol (45 μg/mL) increased intracellular free cholesterol levels by approximately 70% and decreased cholesteryl ester formation, consistent with ACAT1 inhibition. The free cholesterol to cholesteryl ester ratio shifted from 1.4:1 (cholesterol alone) to 5:1 (ATR-101 + cholesterol). Total intracellular cholesterol content was unchanged [1].
In H295R cells, Nevanimibe (30 nM) in the presence of cholesterol (45 μg/mL) induced apoptosis, as evidenced by increased caspase-3/7 activity (approximately 3-fold) and increased TUNEL-positive cells after 5-16 hours. In the absence of exogenous cholesterol, Nevanimibe at concentrations up to 3 μM showed no toxicity; only at 30 μM did it reduce viability by approximately 40% after 24 hours [1].
Co-incubation with exogenous cholesterol (45-60 μg/mL) markedly sensitized H295R cells to Nevanimibe, with 3 nM ATR-101 plus 60 μg/mL cholesterol reducing viability by approximately 60% after 24 hours [1].
The cholesterol transport inhibitor U18666A (100 nM) prevented Nevanimibe (30 nM)-induced caspase-3/7 activation, indicating that cholesterol trafficking to the endoplasmic reticulum is required for cytotoxicity [1].
Nevanimibe induced the unfolded protein response (UPR) in H295R cells. After 5 hours of treatment (30 nM ATR-101 + 45 μg/mL cholesterol), the following were observed: splicing of XBP-1 mRNA (XBP-1s), phosphorylation of PERK (indicated by a mobility shift on Western blot), and increased CHOP mRNA expression (approximately 2.5-fold) [1].
Inhibitors of endoplasmic reticulum calcium release (Xestospongin C, 10 μM; 2-APB, 100 μM), mitochondrial calcium uptake (Ruthenium Red, 15 μM), and mitochondrial membrane permeabilization (Cyclosporin A, 10 μM) all blocked Nevanimibe-induced caspase-3/7 activation in H295R cells [1].
Nevanimibe (30 nM + cholesterol) decreased mitochondrial membrane potential (TMRE fluorescence decreased by approximately 50% vs. control), an effect reversed by Cyclosporin A [1].
ACAT1 knockdown via shRNA in HAC15 adrenocortical cells (80% reduction in ACAT1 expression) mimicked the effects of Nevanimibe: increased free cholesterol (FC:CE ratio approximately 4:1 vs. 1:1 in control), decreased viability after 24 and 72 hours, and increased caspase-3/7 activity (approximately 3-4 fold) in the presence of exogenous cholesterol (60 μg/mL). These effects were blocked by U18666A, Ruthenium Red, and Cyclosporin A [1].

Canine Study (Pharmacodynamics and Toxicity): Three male beagle dogs were administered Nevanimibe orally once daily at 3 mg/kg/day for 7 days followed by 30 mg/kg/day for an additional 7 days. After 14 days of treatment, histologic examination of adrenal glands revealed cortical atrophy, vacuolation, degeneration/necrosis, and mononuclear cell infiltration primarily affecting the zona fasciculata. Approximately 60% of cortical cells in each representative field stained positive for TUNEL, indicating ongoing apoptosis. No abnormalities were observed in the adrenal medulla [1].
Steroidogenesis Inhibition: In the same dogs, ACTH-stimulated cortisol levels decreased by 62% after 7 days of 3 mg/kg/day and by 71% after 14 days of 30 mg/kg/day compared to baseline. ACTH-stimulated levels of corticosterone, 17-hydroxyprogesterone, 11-deoxycortisol, and 11-deoxycorticosterone were also significantly reduced. Pre-ACTH levels of adrenal androgens (androstenedione, DHEA-S) were significantly lower after 14 days of treatment [1].
Adrenal Cholesterol Content: Adrenal glands of treated dogs showed a marked decrease in cholesteryl ester content (191 μg/mg protein) compared to an untreated dog (606 μg/mg protein), consistent with ACAT1 inhibition. Free cholesterol levels were unchanged (37 vs. 38 μg/mg protein) [1].

ACAT1/ACAT2 Fluorescent Cell-Based Assay: ACAT-deficient Chinese hamster ovary (AC29) cells were transfected with constructs expressing human ACAT1 or ACAT2. Esterification of a fluorescent cholesterol analog (22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-23,24-bisnor-5-cholen-3-ol) was measured. Cells were incubated with increasing concentrations of Nevanimibe, and esterification was determined to calculate EC₅₀ values [1].

Cytotoxicity Assay[1]
Cell Types: H295R and HAC Clone 15 (HAC15) Human ACC Cell Line
Tested Concentrations: 3 nM-30 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: 3 nM-3 μM non-toxic, while 30 μM treatment diminished 24 hrs (hours) Memory survival rate increases by approximately 40%.

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Cytotoxicity Assay (MTT): H295R cells were plated in 96-well plates and treated with Nevanimibe in the presence or absence of water-soluble cholesterol (cholesterol-methyl-β-cyclodextrin complex) for 24 hours. MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) was added at 0.5 mg/mL for 2 hours. Formazan dye was extracted with DMSO, and absorbance was read at 570 nm [1].
Crystal Violet Staining: Cells were treated similarly, fixed with 4% paraformaldehyde, stained with 0.4% crystal violet in 10% ethanol, and after solubilization with 1% SDS, absorbance was measured at 570 nm [1].
Caspase-3/7 Assay: Cells were treated for 5 hours, and caspase-3/7 activity was measured using a luminescence-based kit. Caspase detection reagent was added, and luminescence was measured with a luminometer [1].
TUNEL Staining: Cells on chamber slides were treated for 16 hours, and TUNEL staining was performed using a fluorometric TUNEL system. Cells were counterstained with DAPI, and images were captured [1].
Mitochondrial Membrane Potential Measurement: Cells were treated for 5 hours, then TMRE (tetramethyl rhodamine) was added at 2 μM for 15 minutes. Live-cell TMRE fluorescence was measured on a plate reader [1].
Free and Esterified Cholesterol Measurement: Cells were treated for 5 hours, lipids extracted with hexane/isopropanol (3:2), and free and total cholesterol were measured using a fluorometric assay. Esterified cholesterol was calculated by subtracting free cholesterol from total cholesterol [1].
Quantitative RT-PCR: RNA was extracted, reverse transcribed, and CHOP mRNA expression was quantified using SYBR Green PCR and normalized to GAPDH. XBP-1 splicing was detected by PCR with primers spanning the splice site [1].
Western Blotting: PERK phosphorylation was detected by immunoblotting using an anti-PERK antibody. Activated PERK was identified by an upward mobility shift [1].
ACAT1 Knockdown: HAC15 cells were transduced with lentiviral shRNA targeting SOAT1 (human ACAT1). Stable knockdown cells were selected with puromycin, and ACAT1 expression was confirmed by qPCR (80% reduction) [1].

Canine Toxicology and Pharmacodynamics Study: Three male beagle dogs (approximately 10-12 kg) were administered Nevanimibe orally once daily at 3 mg/kg/day for 7 days followed by 30 mg/kg/day for an additional 7 days. The compound was formulated in 0.5% hydroxypropylmethylcellulose at a dose volume of 5 mL/kg. Blood samples were collected for serum cortisol and steroid analysis before and 1 hour after ACTH stimulation (5 μg/kg, IV) on days -3, 1, 3, 7, 8, 10, and 14. On day 14, animals were euthanized, and adrenal glands were collected for histology (H&E and TUNEL staining), and for free and esterified cholesterol analysis [1].
Canine Tissue Distribution Study: Three female beagle dogs (approximately 19 months old, 7-10 kg) were administered Nevanimibe orally once daily at 3 mg/kg/day for 7 days in 0.5% hydroxypropylmethylcellulose (5 mL/kg). On the last day, blood was collected 4 hours post-dose, and animals were euthanized. Tissues (adrenal glands, kidney, liver, skeletal muscle, sc fat, ovaries, and CSF) were collected for determination of ATR-101 content by LC-MS/MS [1].

Tissue Distribution in Dogs: After 7 days of oral dosing at 3 mg/kg/day, Nevanimibe showed preferential distribution to the adrenal glands. When normalized to plasma concentration, only the adrenal glands had concentrations approaching equivalence to plasma. No other tissues had concentrations exceeding 30% of plasma concentration. ATR-101 was not detectable in cerebrospinal fluid (CSF) [1].
Plasma Concentrations: Plasma concentrations of Nevanimibe after dosing at 3 mg/kg/day and 30 mg/kg/day were reported to approximate those previously seen in toxicology studies (data shown in Supplemental Figure 1) [1].

Adrenal-Specific Toxicity in Dogs: After 14 days of oral Nevanimibe treatment (3 mg/kg/day for 7 days, then 30 mg/kg/day for 7 days), histologic examination revealed adrenal cortical atrophy, vacuolation, degeneration/necrosis, and mononuclear cell infiltration, primarily in the zona fasciculata. Approximately 60% of cortical cells stained TUNEL-positive. No abnormalities were observed in the adrenal medulla [1].
No Effects on Other Tissues: A separate 28-day toxicity study in male dogs showed no effects on testes or macrophages (data not shown) [1].
Steroidogenesis Impairment: Nevanimibe treatment resulted in progressive reductions in both pre- and post-ACTH-stimulated levels of glucocorticoids, mineralocorticoids, and adrenal androgens, consistent with reduced adrenal function and cell death [1].
In vitro Toxicity: In H295R cells, Nevanimibe was non-toxic up to 3 μM in the absence of exogenous cholesterol; only at 30 μM did it reduce viability by ~40% after 24 hours [1].

[1]. ATR-101, a Selective and Potent Inhibitor of Acyl-CoA Acyltransferase 1, Induces Apoptosis in H295R Adrenocortical Cells and in the Adrenal Cortex of Dogs. Endocrinology. 2016 May;157(5):1775-88.

Nevanimibe is being studied in the clinical trial NCT03053271 (an investigation of ATR-101 for the treatment of endogenous Cushing's syndrome).

C28H41N3O

435.64464

435.325

C, 76.92; H, 9.32; N, 9.97; O, 3.79

133825-80-6

Nevanimibe hydrochloride;133825-81-7

131679

Typically exists as White to off-white solid at room temperature

1.062g/cm3

528.1ºC at 760mmHg

273.2ºC

3.05E-11mmHg at 25°C

1.58

6.905

2

2

7

31

543

0

O=C(NCC1(C2=CC=C(N(C)C)C=C2)CCCC1)NC3=C(C(C)C)C=CC=C3C(C)C

PKKNCEXEVUFFFI-UHFFFAOYSA-N

InChI=1S/C27H39N3O/c1-19(2)23-10-9-11-24(20(3)4)25(23)29-26(31)28-18-27(16-7-8-17-27)21-12-14-22(15-13-21)30(5)6/h9-15,19-20H,7-8,16-18H2,1-6H3,(H2,28,29,31)

1-[[1-[4-(dimethylamino)phenyl]cyclopentyl]methyl]-3-[2,6-di(propan-2-yl)phenyl]urea

Nevanimibe; 133825-80-6; ATR-101 free base; PD-132301; nevanimiba;

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)

DMSO: 25 mg/mL (59.3 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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