ACAT1 (IC50 = 24 μM); ACAT2 (IC50 = 9.2 μM)

Prostate cancer (PCa) cells proliferate less when avasimibe (0, 0.25, 5, 10, 20, 40, and 80 μM; 1, 2 and 3 days) is administered [2]. The expression of β-catenin, Vimentin, N-cadherin, Snail, and MMP9—all of which are closely linked to the epithelial-mesenchymal transition (EMT)—is reduced by avasimibe (10 and 20 μM; 48 hours) [2]. In prostate cancer, avasimibe (10 and 20 μM) induces cell cycle arrest via the E2F-1 signaling pathway. In PCa cells, avasimibe causes a G1 phase cell cycle arrest [2]. PCa cell metastasis is inhibited by avasimibe (10 and 20 μM) [2].

For seven weeks, avasimibe (30 mg/kg intraperitoneally every other day) suppresses the development and metastasis of PCa cells in vivo. Avasimibe is minimal in toxicity and has good biocompatibility [2].

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Avasimibe suppressed PCa cell growth and metastasis in vivo [2]
A xenograft model was established by subcutaneously transplanting PC-3 cells, and our study found that avasimibe reduced tumour volume compared with that of the control group (Fig. 4a, b). The inhibitory effect of avasimibe on PCa cell growth was further confirmed by H&E staining and Ki67 immunofluorescence staining of xenograft tumours (Fig. 4c). An upregulation of E2F-1 expression was also observed in the avasimibe group by immunofluorescence staining (Fig. 4c). [2]
Researchers established a pulmonary metastasis model by intravenous tail vein injection of GFP-expressing PC-3 LV-NC cells, and the fluorescence intensity of the GFP-expressing PC-3 LV-NC cells was assessed to evaluate the migratory capacity. The fluorescence intensity of pulmonary metastatic tumours was weaker in the avasimibe group than the control group (Fig. 4d, e). H&E staining of lung tissues showed that avasimibe treatment could inhibit the number of pulmonary metastatic tumours (Fig. 4f). [2]

Cell Viability Assay[2]
Cell Types: PCa cells (PC-3 and DU 145)
Tested Concentrations: 0, 0.25, 5, 10, 20, 40 and 80 µM
Incubation Duration: 1, 2, and 3 days
Experimental Results: Dose dependently inhibited PC-3 and DU 145 cell viability.

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Western Blot Analysis[2]
Cell Types: PCa cells (PC-3 and DU 145)
Tested Concentrations: 10 and 20 µM
Incubation Duration: 48 hrs (hours)
Experimental Results: decreased protein levels of EMT-related proteins ( β-catenin, Vimentin, N-cadherin, Snail, MMP9 and E-cadherin).

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Cell Cycle Analysis[2]
Cell Types: PCa cells (PC-3 and DU 145)
Tested Concentrations: 10 and 20 µM
Incubation Duration: 48 hrs (hours)
Experimental Results: Induced G1 phase cycle arrest and altered the G1 phase-related protein levels in PCa cells.

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MTT assay [1]
Briefly, PCa cells were plated in 96-well plates (3000 cells/well; 200 µl of medium) for 1 day and treated with avasimibe (0, 0.25, 5, 10, 20, 40 and 80 µM) for 1, 2, and 3 days. The cells were incubated with 20 µl of MTT (5 mg/ml/well) for 4 h at 37 °C. After discarding the supernatant, the MTT formazan crystals were dissolved in 200 µl/well DMSO, and a microplate reader was applied to measure the OD values at 490 nm.

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Clonogenic survival assay [1]
PCa cells were placed onto a six-well plate (1500 cells per well). After 1 day, the normal medium was replaced with avasimibe working solution. The cells were cultured for 10 to 15 days until they grew into colonies. The medium was discarded, then, the cells were fixed for 1 h with 4% paraformaldehyde (PFA) and stained for half an hour in 0.1% crystal violet. The colonies were counted using Image-Pro Plus.

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Wound healing assay [1]
Avasimibe-treated PCa cells were grown in six-well plates until the cells reached 95% confluence. Then, the cell monolayer was scratched with a 1-ml sterile blue micropipette tips. After the cells were washed twice with PBS, they were cultured in medium supplemented with 2% FBS and different concentrations of avasimibe for 12 h. Then, the cells were photographed with an inverted fluorescence microscope at premarked points with white light at 0 and 12 h. The horizontal distance between the edges of the scratch was measured by Photoshop. Migration rate = 1 − (12 h scratch distance/0 h initial distance).

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Transwell migration assay [1]
Cell migration was evaluated by a Transwell chamber system. The cells were pretreated with the avasimibe in a six-well plate for 48 h, then avasimibe-treated PCa cells (1.2 × 105 PC3 or 8 × 104 DU 145 cells) in 200 µl of medium (serum-free) were added to the top transwell chamber, and 600 µl of normal culture medium was placed in the lower chamber. After incubation for 1 day, the chambers were fixed with 4% PFA for half an hour and stained in 0.1% crystal violet for 1 h. The chambers were photographed and assessed by an inverted phase contrast microscope in five random fields.

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Flow cytometry for cell cycle analysis [1]
After transfection for 1 day, cells were treated with avasimibe working solution for 1 day. Avasimibe-treated PCa cells were collected and then washed with PBS three times. A total of 1 × 106 cells were harvested for cell cycle staining, and then, 500 µl of 1× DNA Staining Solution and 5 µl of permeabilization solution were placed in tubes in the dark for staining for 30 min. 1 × 104 cells of the sample were assessed by flow cytometry as described before. Data were analysed with FlowJo Software.

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Flow cytometry for apoptosis [1]
The steps of the apoptosis assay were carried out as described before. 100 µl of binding buffer (1×) was used to resuspend PCa cells, and the avasimibe-treated PCa cells were stained with FITC-annexin V (5 µl) and propidium iodide (PI, 5 µl) for 10 min in dark conditions at 25 °C. Then, 1× binding buffer was added to the mixture to bring the total volume to 500 µl, and the samples were assessed by flow cytometry. Percentage of apoptosis = percentage of late apoptosis + percentage of early apoptosis.

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Flow cytometry for reactive oxygen species (ROS) [1]
Flow cytometric analysis was performed to assess the intracellular levels of ROS. Avasimibe-treated PCa cells were stained with 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA,10 µM) for 20 min at 25 °C, protected from light, and then washed with PBS. The ROS level was assessed by flow cytometry.

Animal/Disease Models: SPF male mice (BALB/c-nude, 4 weeks old) bearing PCa cells[2]
Doses: 30 mg/kg
Route of Administration: Intraperitoneally injected for 7 weeks
Experimental Results: decreased tumor volume compared with that of the control group. Inhibited PCa growth and migration in vivo.

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Xenograft model and pulmonary metastasis model [2]
SPF male mice (BALB/c-nude, 4 weeks old) were acclimated to the environment of the animal facility for seven days.[2]
Tumour-bearing mice were constructed by inoculating 2 × 106 PC-3 cells into the flanks of mice (n = 7). Seven days later, avasimibe(30 mg/kg, dissolved in DMSO and diluted in PBS containing 1% Tween-80) and solvent were intraperitoneally injected on alternate days for 7 weeks (the stock solution had a concentration of 25 mg/ml). The mice were anaesthetized by intraperitoneal injection of pentobarbital (50 mg/kg) before euthanasia. Tumour volume was measured with a Vernier scale every other day for 7 weeks, and tumour volume was calculated as follows: tumour volume (mm3) = tumour length × width2/2. We separated the tumour tissues, and then, the tumour tissues were fixed in 4 % PFA and verified by H&E and immunofluorescence staining.[2]
Pulmonary metastasis models were constructed by injecting 2 × 106 PC-3 (LV-NC GFP-expressing) cells into the tail vein of mice (n = 5). Avasimibe (30 mg/kg) and solvent were administered as described above for 7 weeks. The fluorescence intensity of lung metastasis tumours was measured using a Fusion FX7 Spectra Imaging system. Then, the lungs were surgically exposed and collected for further analysis by H&E.

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Avasimibe was administered orally as bulk drug in gelatin capsules on a mg/kg body weight basis. Control animals received empty gelatin capsules equal in number to those given to the high-dose group for each study.
The 2-week repeated-dose study was the first study conducted with avasimibe in dogs (see below). The doses were selected based on experience with a previous ACAT inhibitor (Wolfgang et al., 1995). However, the study failed to define a dose-limiting toxicity. Therefore, an escalating dose study was conducted in order to establish a maximum tolerated dose. In the study, 2 male dogs were administered 100 mg/kg on Days 1–9, 1000 mg/kg once a day on Days 10–16 and 1000 mg/kg b.i.d. on Days 17–23. The b.i.d. doses were administered 8 h apart. Plasma drug concentrations were determined pre-dose, 1.5, 4, 8, 12, and 24 h post-dose on Days 9 and 16, and pre-dose, 1.5, 4, 8, 9.5, 12, 16, 24, and 32 h post-dose (first dose) on Day 23. Hematological and serum chemistry parameters were measured pre-test and on Days 8, 15, and 22. The animals were not euthanized at the end of the study. Reference: Toxicol Sci. 2001 Feb;59(2):324-34.

Avasimibe, a novel inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT), is currently being developed as an antiatherosclerotic agent. The preclinical safety and toxicokinetics of the compound were assessed in beagle dogs in an escalating-dose study and in repeated-dose studies of 2-, 13-, and 52-week duration. Oral (capsule) doses up to 1000 mg/kg b.i.d. were assessed in the escalating dose study and once-a-day doses up to 300 mg/kg, 1000 mg/kg, and 1000 mg/kg were assessed in the 2-, 13-, and 52-week studies, respectively. Avasimibe was found to be a substrate and inducer of hepatic CYP 3A, producing pronounced decreases in plasma drug concentrations subsequent to Day 1. Plasma drug concentrations plateaued markedly at doses above 100 mg/kg. Significant toxicologic findings were restricted to the higher doses (> or =300 mg/kg) and included emesis, fecal consistency changes, salivation, body weight loss, microscopic and clinical pathologic evidence of hepatic toxicity, and red blood cell (RBC) morphology changes. Mortality occurred at 1000 mg/kg due to hepatic toxicity. Toxicity was more closely associated with the exaggerated pharmacodynamic effects of the compound (e.g., marked serum cholesterol decreases) seen at the high doses of avasimibe used in these studies rather than with measures of systemic exposure (Cmax or AUC). Adrenal effects were noted only in the 52-week study and consisted of minimal to mild cortical cytoplasmic vacuolization and fibrosis at doses > or =300 mg/kg, with no change in adrenal weight. In conclusion, avasimibe is an ACAT inhibitor that has minimal adrenal effects in dogs, with dose-limiting toxicity defined by readily monitored and reversible changes in hepatic function. Toxicol Sci. 2001 Feb;59(2):324-34.

[1]. Taichi Ohshiro,et al. Pyripyropene A, an acyl-coenzyme A:cholesterol acyltransferase 2-selective inhibitor, attenuates hypercholesterolemia and atherosclerosis in murine models of hyperlipidemia. Arterioscler Thromb Vasc Biol. 2011 May;31(5):1108-15.
[2]. Kangping Xiong, et al. The cholesterol esterification inhibitor avasimibe suppresses tumour proliferation and metastasis via the E2F-1 signalling pathway in prostate cancer.Cancer Cell Int. 2021 Aug 30;21(1):461.

Avasimibe is a monoterpenoid.
Avasimibe is an orally bioavailable inhibitor of acyl-Coenzyme A:cholesterol acyltransferase (ACAT) that prevents cholesterol deposition in the arterial wall. Research was discontinued due to difficulties in assaying the effects of avasimibe on preventing plaque formation and due to its ability to increase the activity of Cytochrome P450 3A4, thus increasing the removal of other drugs from the body.

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Drug Indication
Investigated for use/treatment in peripheral vascular disease.

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Background: New effective drugs for prostate cancer (PCa) treatment are urgently needed. Avasimibe was recently identified as a promising drug for anticancer therapies. The main purpose of this study was to explore the effects and the underlying mechanisms of avasimibe in prostate cancer.
Methods: In this study, MTT and clonogenic survival assays were performed to detect cell proliferation after avasimibe treatment. The effect of avasimibe on cell migration was measured by wound healing and transwell migration assays. Cell cycle distribution and apoptosis were detected by flow cytometry. Immunofluorescence staining and western blot analysis were used to detect the expression of cell cycle-related proteins and epithelial-mesenchymal transition (EMT)-related proteins. In vivo, the antitumour effects of avasimibe were evaluated using a xenograft model and pulmonary metastasis model.
Results: The study found that avasimibe suppresses tumour growth and triggers G1 phase arrest. Moreover, the expression of the cell cycle-related proteins CDK2/4/6, Cyclin D1 and Cyclin A1 + A2 was significantly increased and p21 expression was decreased after avasimibe treatment. The migration of PCa cells was attenuated after treatment with avasimibe, followed by the downregulation of the expression of the EMT-related proteins N-cadherin, β-catenin, vimentin, Snail and MMP9 and upregulation of E-cadherin expression. Moreover, E2F-1 was elevated after treatment with avasimibe. After knockdown of E2F-1 expression, the inhibition of cell proliferation and migration caused by avasimibe was significantly recovered. The results of the xenograft model showed that avasimibe suppressed tumour growth in vivo. Immunofluorescence staining revealed lower levels of Ki67 and higher levels of E2F-1 in tumour tissues of the avasimibe group than those of the control group. A pulmonary metastasis model also confirmed the inhibition of PCa metastasis by avasimibe. The number of lung metastatic foci in the avasimibe group was significantly decreased compared with that in the control group.
Conclusions: Our results suggest that avasimibe can suppress tumour proliferation and metastasis via the E2F-1 signalling pathway. These findings demonstrate the potential of avasimibe as a new effective drug for PCa treatment.[2]

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Avasimibe is a novel orally bioavailable ACAT inhibitor , currently under clinical development (phase III trials). It was safe when administered to rats, dogs, and humans. In vitro studies in human macrophages demonstrated that avasimibe reduces foam cell formation not only by enhancing free cholesterol efflux, but also by inhibiting the uptake of modified LDL. The concentration-dependent reduction in cellular cholesteryl ester content in these cells was not accompanied by an increase in intracellular free cholesterol, which is in agreement with a good safety profile for avasimibe. In the liver, avasimibe caused a significant reduction in the secretion of apo B and apo B-containing lipoproteins into plasma. Avasimibe induced cholesterol 7alpha-hydroxylase and increased bile acid synthesis in cultured rat hepatocytes, and its administration to rats did not produce an increase in lithogenicity index of the bile. The hypolipidemic efficacy of the compound was demonstrated in cholesterol-fed as well as in non-cholesterol-fed animals. In these models, plasma cholesterol levels were reduced, mainly due to the decrease in the non-HDL cholesterol fraction. Clinical data are scarce, but in a study performed in 130 men and women with combined hyperlipidemia and hypoalphalipoproteinemia, avasimibe, 50-500 mg/day, significantly reduced plasma total triglyceride and VLDL-cholesterol. Although total cholesterol, LDL-cholesterol, and HDL-cholesterol were unchanged, it must be stressed that animal data suggest that avasimibe may have direct antiatherosclerotic activity in addition to its cholesterol-lowering effect. Avasimibe treatment can also contribute to increase plaque stability, as it reduces the accumulation of lipids in the arterial wall, inhibits macrophage infiltration into the media and reduces matrix metalloproteinase expression and activity. Moreover, avasimibe and statins have been shown to have synergistic effects, and the combination therapy may not only inhibit atherosclerotic lesion progression but also induce lesion regression, independently of changes in plasma cholesterol.Reference: Cardiovasc Drug Rev. 2003 Spring;21(1):33-50.

C29H43NO4S

501.72

501.291

C, 69.42; H, 8.64; N, 2.79; O, 12.76; S, 6.39

166518-60-1

166518-61-2 (sodium); 166518-60-1 (free form);

166558

Typically exists as white to off-white solids at room temperature

1.1±0.1 g/cm3

178-180° (Lee); mp 169.5-170.4° (Dozeman)

1.529

9.34

1

4

10

35

734

0

S(N([H])C(C([H])([H])C1C(=C([H])C(C([H])(C([H])([H])[H])C([H])([H])[H])=C([H])C=1C([H])(C([H])([H])[H])C([H])([H])[H])C([H])(C([H])([H])[H])C([H])([H])[H])=O)(=O)(=O)OC1C(=C([H])C([H])=C([H])C=1C([H])(C([H])([H])[H])C([H])([H])[H])C([H])(C([H])([H])[H])C([H])([H])[H]

PTQXTEKSNBVPQJ-UHFFFAOYSA-N

InChI=1S/C29H43NO4S/c1-17(2)22-14-25(20(7)8)27(26(15-22)21(9)10)16-28(31)30-35(32,33)34-29-23(18(3)4)12-11-13-24(29)19(5)6/h11-15,17-21H,16H2,1-10H3,(H,30,31)

((2,4,6-Tris(1-methylethyl)phenyl)acetyl)sulfamic acid 2,6-bis(1-methylethyl)phenyl ester

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: 7.5 mg/mL (14.95 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 sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 75.0 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: 7.5 mg/mL (14.95 mM) 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; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 75.0 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: 7.5 mg/mL (14.95 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 75.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+corn oil: 5mg/mL

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