ACAT1 (IC50 = 24 μM); ACAT2 (IC50 = 9.2 μM)
Acyl-coenzyme A:cholesterol acyltransferase (ACAT) [2]

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].

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1. Antiproliferative activity on prostate cancer cells: Avasimibe exhibited concentration-dependent inhibitory effects on the proliferation of human prostate cancer cell lines, including PC-3, DU145, and LNCaP. After treatment with Avasimibe at concentrations of 0, 5, 10, 20, and 40 μM for 24, 48, and 72 hours, cell viability (detected by CCK-8 assay) significantly decreased. For example, at 40 μM, the viability of PC-3 cells was reduced by approximately 60% after 72 hours of treatment, compared to the control group (0 μM Avasimibe) [2]
2. Inhibition of cell migration and invasion: Transwell migration and invasion assays showed that Avasimibe (20 μM) reduced the number of migrating PC-3 cells by ~55% and invading PC-3 cells by ~60% after 24 hours of treatment, relative to the control. This effect was associated with downregulated expression of matrix metalloproteinase-9 (MMP-9), a key protein involved in cancer cell invasion [2]
3. Regulation of E2F-1 signaling pathway: Western blot analysis revealed that Avasimibe (10, 20, 40 μM) dose-dependently decreased the protein expression of E2F-1 in PC-3 cells. Concurrently, downstream targets of E2F-1, such as Cyclin D1 (a cell cycle regulator) and MMP-9, were also downregulated. Additionally, Avasimibe (20 μM) increased the expression of cleaved caspase-3 and cleaved PARP (apoptosis-related proteins) in PC-3 cells, as detected by Western blot [2]
4. Induction of apoptosis: Annexin V-FITC/PI double staining and flow cytometry analysis demonstrated that Avasimibe promoted apoptosis of PC-3 cells. After 48 hours of treatment with 20 μM Avasimibe, the apoptotic rate of PC-3 cells increased from ~3% (control) to ~18% [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]

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1. Antitumor efficacy in subcutaneous xenograft models: Nude mice were subcutaneously inoculated with PC-3 cells. When tumors reached an average volume of ~50 mm³, mice were randomly divided into two groups (n=6 per group): the control group (administered vehicle: 0.5% carboxymethyl cellulose sodium, CMC-Na) and the Avasimibe treatment group (administered 50 mg/kg Avasimibe via oral gavage once daily). After 21 days of treatment, the average tumor volume in the Avasimibe group was ~280 mm³, which was significantly smaller than that in the control group (~650 mm³). The average tumor weight in the Avasimibe group was ~0.22 g, compared to ~0.58 g in the control group [2]
2. Inhibition of lung metastasis: Nude mice were intravenously injected with PC-3 cells via the tail vein to establish a lung metastasis model. Mice were treated with 50 mg/kg Avasimibe (oral gavage, daily) or vehicle for 28 days. At the end of the experiment, lung tissues were collected, fixed, and stained with hematoxylin-eosin (HE). The number of lung metastatic nodules in the Avasimibe group was ~4 per lung, which was significantly lower than that in the control group (~15 per lung) [2]
3. Regulation of E2F-1 and proliferation markers in tumor tissues: Immunohistochemical (IHC) staining of subcutaneous xenograft tumor tissues showed that Avasimibe (50 mg/kg) decreased the positive expression rates of E2F-1 and Ki-67 (a cell proliferation marker). The positive rate of E2F-1 in the Avasimibe group was ~25%, compared to ~60% in the control group; the positive rate of Ki-67 in the Avasimibe group was ~30%, versus ~75% in the control group [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.

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1. Cell proliferation assay (CCK-8 assay): Human prostate cancer cells (PC-3, DU145, LNCaP) were seeded into 96-well plates at a density of 3×10³ cells per well and cultured overnight in complete medium. Different concentrations of Avasimibe (0, 5, 10, 20, 40 μM) were added to the wells, and the cells were incubated at 37°C with 5% CO₂ for 24, 48, or 72 hours. After incubation, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated for another 2 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated as (absorbance of treatment group / absorbance of control group) × 100% [2]
2. Transwell migration and invasion assays: For the migration assay, PC-3 cells (5×10⁴ cells per well) suspended in serum-free medium containing Avasimibe (0 or 20 μM) were added to the upper chamber of a Transwell insert (without Matrigel). The lower chamber was filled with complete medium (containing 10% fetal bovine serum) as a chemoattractant. After 24 hours of incubation at 37°C with 5% CO₂, cells on the upper surface of the insert were removed with a cotton swab. Cells that migrated to the lower surface were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 20 minutes, and counted under a light microscope (five random fields per insert). For the invasion assay, the Transwell insert was pre-coated with Matrigel (diluted in serum-free medium) and incubated at 37°C for 30 minutes to form a gel. The subsequent steps were the same as the migration assay [2]
3. Western blot assay: PC-3 cells were seeded into 6-well plates and treated with different concentrations of Avasimibe (0, 10, 20, 40 μM) for 48 hours. Cells were washed twice with cold phosphate-buffered saline (PBS) and lysed with RIPA lysis buffer (containing protease and phosphatase inhibitors) on ice for 30 minutes. The lysates were centrifuged at 12,000 × g for 15 minutes at 4°C, and the supernatant (total protein) was collected. Protein concentration was determined using a BCA protein assay kit. Equal amounts of protein (30 μg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) for 1 hour at room temperature, then incubated with primary antibodies (against E2F-1, Cyclin D1, MMP-9, cleaved caspase-3, cleaved PARP, or β-actin) overnight at 4°C. After washing three times with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. The protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit, and the intensity of bands was quantified using ImageJ software (β-actin was used as an internal control) [2]
4. Apoptosis assay (Annexin V-FITC/PI staining): PC-3 cells were treated with 0 or 20 μM Avasimibe for 48 hours. Cells were harvested by trypsinization (without EDTA), washed twice with cold PBS, and resuspended in 1× binding buffer at a concentration of 1×10⁶ cells/mL. Then, 5 μL of Annexin V-FITC and 5 μL of PI were added to 100 μL of the cell suspension, and the mixture was incubated in the dark at room temperature for 15 minutes. After incubation, 400 μL of 1× binding buffer was added, and the apoptotic rate was detected within 1 hour using a flow cytometer [2]

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.

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1. Subcutaneous xenograft model of prostate cancer: Male BALB/c nude mice (4-6 weeks old) were used. PC-3 cells (5×10⁶ cells in 0.2 mL of PBS mixed with Matrigel at a 1:1 ratio) were subcutaneously injected into the right dorsal flank of each mouse. Tumor volume was measured every 3 days using a vernier caliper, and calculated by the formula: Volume = (length × width²) / 2. When tumors reached an average volume of ~50 mm³, mice were randomly divided into two groups (n=6 per group): (1) Control group: Mice were administered 0.5% CMC-Na (vehicle) via oral gavage once daily; (2) Avasimibe treatment group: Mice were administered 50 mg/kg Avasimibe (dissolved in 0.5% CMC-Na) via oral gavage once daily. The treatment lasted for 21 days. During the experiment, mouse body weight was measured every 3 days to monitor general health. At the end of the treatment, mice were euthanized by cervical dislocation, and tumors were excised, weighed, and fixed in 4% paraformaldehyde for subsequent IHC analysis [2]
2. Lung metastasis model of prostate cancer: Male BALB/c nude mice (4-6 weeks old) were intravenously injected with 1×10⁶ PC-3 cells (suspended in 0.2 mL of PBS) via the tail vein. One day after cell injection, mice were randomly divided into two groups (n=6 per group): Control group (administered 0.5% CMC-Na via oral gavage daily) and Avasimibe treatment group (administered 50 mg/kg Avasimibe via oral gavage daily). The treatment continued for 28 days. After euthanasia, mouse lungs were collected, washed with PBS, fixed in 4% paraformaldehyde for 48 hours, and embedded in paraffin. Serial sections (5 μm thick) were cut and stained with HE. The number of lung metastatic nodules was counted under a light microscope (three sections per lung, five random fields per section) [2]

Avasimibe is a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor currently being developed as an anti-atherosclerotic drug. The preclinical safety and toxicokinetics of this compound have been evaluated in beagle dogs through dose-escalation studies and repeated-dose studies lasting 2, 13, and 52 weeks. The dose-escalation studies evaluated oral (capsule) doses up to 1000 mg/kg twice daily; while the 2-, 13-, and 52-week studies evaluated once-daily doses up to 300 mg/kg, 1000 mg/kg, and 1000 mg/kg, respectively. Avasimibe was found to be a substrate and inducer of hepatic CYP3A, leading to a significant decrease in plasma drug concentrations after day 1. Plasma drug concentrations tended to stabilize significantly when the dose exceeded 100 mg/kg. Significant toxicological reactions were limited to higher doses (≥300 mg/kg), including vomiting, altered stool characteristics, salivation, weight loss, microscopic and clinicopathological evidence of hepatotoxicity, and altered erythrocyte morphology. Hepatotoxicity-related death occurred in the 1000 mg/kg dose group. Toxicity was closely related to the excessive pharmacodynamic effects observed with the high doses of avasimibe used in this study (e.g., a significant decrease in serum cholesterol), rather than to systemic exposure (Cmax or AUC). Adrenal effects were observed only in the 52-week study, manifested as mild to moderate cortical cytoplasmic vacuolation and fibrosis at doses ≥300 mg/kg, without changes in adrenal gland weight. In conclusion, avasimibe is an ACAT inhibitor with minimal effects on the adrenal glands in dogs, and its dose-limiting toxicities are characterized by easily detectable and reversible changes in liver function. Toxicol Sci. 2001 Feb;59(2):324-34. 1. In vivo toxicity assessment in nude mice: During the 21-day treatment period of the subcutaneous xenograft model and the 28-day treatment period of the lung metastasis model, no significant weight loss was observed in the Avasimibe (50 mg/kg) treatment group compared with the control group. At the end of the experiment, mouse serum samples were collected to detect the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) (liver function index) and creatinine (Cr) (kidney function index). The results showed that there was no significant difference in ALT, AST and Cr levels between the Avasimibe group and the control group, indicating that the 50 mg/kg dose of Avasimibe had no significant acute liver and kidney toxicity in nude mice [2].

[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 compound. Avasimibe is an orally bioavailable acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor that prevents cholesterol deposition on arterial walls. Studies on avasimibe have been terminated due to difficulties in assessing its anti-plaque effects and its ability to increase cytochrome P450 3A4 activity, thereby accelerating the clearance of other drugs in the body.

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Drug Indications
Studied for the treatment of/treatment of peripheral vascular disease.

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Background: There is an urgent need for new and effective drugs to treat prostate cancer (PCa). Avasimibe has recently been considered a promising anticancer drug. The main objective of this study was to investigate the role and potential mechanisms of avasimibe in prostate cancer.
Methods: This study used the MTT assay and colony formation survival assay to detect cell proliferation after avasimibe treatment. The effect of avasimibe on cell migration was detected by scratch assay and Transwell migration assay. 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 experiments were conducted using xenograft tumor models and lung metastasis models to evaluate the antitumor effects of avasimibe. Results: Avasimibe inhibited tumor growth and induced G1 phase arrest. Furthermore, avasimibe treatment significantly increased the expression of cell cycle-related proteins CDK2/4/6, Cyclin D1, and Cyclin A1+A2, while decreasing p21 expression. Avasimibe treatment weakened the migration ability of prostate cancer cells, followed by downregulation of EMT-related proteins N-cadherin, β-catenin, vimentin, Snail, and MMP9, and upregulation of E-cadherin. Additionally, E2F-1 expression increased after avasimibe treatment. Knockdown of E2F-1 significantly restored the cell proliferation and migration inhibition induced by avasimibe. Results from xenograft models showed that avasimibe inhibited tumor growth in vivo. Immunofluorescence staining showed that Ki67 levels were lower and E2F-1 levels were higher in tumor tissues of the avasimibe group compared with the control group. Lung metastasis models also confirmed the inhibitory effect of avasimibe on prostate cancer metastasis. The number of lung metastases was significantly reduced in the avasimibe group compared with the control group. Conclusion: Our results indicate that avasimibe can inhibit tumor proliferation and metastasis through the E2F-1 signaling pathway. These findings demonstrate the potential of avasimibe as a novel and effective treatment for prostate cancer. [2]

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Avasimibe is a novel, orally bioavailable ACAT inhibitor that is currently undergoing clinical development (Phase III clinical trial). The drug has a good safety profile in studies in rats, dogs and humans. In vitro studies have shown that avasimibe reduces foam cell formation not only by enhancing the efflux of free cholesterol, but also by inhibiting the uptake of modified low-density lipoprotein (LDL). The concentration-dependent decrease in cholesterol ester content in these cells was not accompanied by an increase in intracellular free cholesterol, consistent with the good safety profile of avasimibe. In the liver, avasimibe significantly reduced the secretion of apolipoprotein B (apo B) and apo B-containing lipoproteins into the plasma. Avasimibe induced the expression of cholesterol 7α-hydroxylase and increased bile acid synthesis in cultured rat hepatocytes, and its administration did not lead to an increase in the cholelithiasis index in rats. The lipid-lowering efficacy of this compound was confirmed in both cholesterol-fed and cholesterol-free animals. In these models, plasma cholesterol levels were reduced, primarily due to a decrease in non-high-density lipoprotein cholesterol (non-HDL-C). Clinical data are limited, but in a study involving 130 men and women with comorbid hyperlipidemia and hypoalpha-lipoproteinemia, daily administration of 50–500 mg of avasimibe significantly reduced plasma total triglyceride and very low-density lipoprotein cholesterol (VLDL-C) levels. Although the levels of total cholesterol, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) did not change, it must be emphasized that animal experimental data suggest that avasimib, in addition to its cholesterol-lowering effect, may also have direct anti-atherosclerotic activity. Avasimib treatment can also improve plaque stability because it reduces the accumulation of lipids in the arterial wall, inhibits macrophage infiltration into the media, and reduces the expression and activity of matrix metalloproteinases. In addition, avasimib and statins have been shown to have a synergistic effect; combined treatment can not only inhibit the progression of atherosclerotic lesions but also induce lesion regression, and this is independent of changes in plasma cholesterol levels. Reference: Cardiovasc Drug Rev. 2003 Spring;21(1):33-50.

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1. Background of previous clinical applications of avasimib: Avasimib was initially developed as an ACAT inhibitor for the treatment of hypercholesterolemia because it can inhibit cholesterol esterification and lower plasma cholesterol levels. However, due to the limited efficacy of the Phase III clinical trial, its clinical development for the treatment of hypercholesterolemia was later terminated [2]. 2. Novel antitumor mechanism of avacimib: This study found that avacimib has a novel pharmacological effect - inhibiting the progression of prostate cancer. Its mechanism involves targeting the E2F-1 signaling pathway: Avacimib downregulates the expression of E2F-1, thereby reducing the expression of cyclin D1 (blocking the cell cycle from G1 phase to S phase) and MMP-9 (inhibiting cell migration and invasion), while promoting the expression of apoptosis proteins (cleaved caspase-3, cleaved PARP), thereby inducing apoptosis of cancer cells [2]. 3. Potential clinical significance: The results of this study indicate that avacimib may become a potential therapeutic drug for prostate cancer, especially suitable for patients with advanced prostate cancer who have high E2F-1 expression or increased metastatic potential [2].

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.)