Squalene Synthase: YM-53601 is an inhibitor of squalene synthase (farnesyl-diphosphate: farnesyl-diphosphate farnesyl-transferase, EC 2.5.1.21). (IC50 values: rat liver microsomes: 90 nM; hamster liver microsomes: 170 nM; guinea-pig liver microsomes: 46 nM; rhesus monkey liver microsomes: 45 nM; human HepG2 cell microsomes: 79 nM) [1]

Close-range IC50 values of 90, 170, 46, 45, and 79 nM were observed for YM-53601 squalene synthase activity in liver microsomes from hamsters, guinea pigs, rhesus monkeys, and human HepG2 cells[1]. YM-53601 also inhibits hamster delta squalene synthase, which converts [3H] farnesyl diphosphate into [3H] squalene, with an IC50 of 170 nM [2]. In H35 cells, YM-53601 (1 μM) increases thapsigargin cooking, while in HepG2 and H35 cells, it decreases cooking levels [4]. Assay for Cell Viability[4]

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Squalene Synthase Inhibition: YM-53601 inhibited squalene synthase activity in a dose-dependent manner in microsomes prepared from various species. The IC50 values were 90 nM in rat liver microsomes, 170 nM in hamster liver microsomes, 46 nM in guinea-pig liver microsomes, 45 nM in rhesus monkey liver microsomes, and 79 nM in human HepG2 cell microsomes. The inhibitory activity was comparable to or greater than that of the reference squalene synthase inhibitor FBQ. [1]

Suspension biosynthesis using YM-53601 suspension solution (ED50, 32 mg/kg)[1]. In addition, YM-53601 decreased curtain non-HDL cholesterol levels in hamsters by almost 70% over the course of five days at a dose of 50 mg/kg/day [2]. In vivo doxorubicin-induced hepatocellular carcinoma (HCC) development and cell death are enhanced by YM-53601 [4].

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Inhibition of Cholesterol Biosynthesis in Rats: In rats, a single oral dose of YM-53601 (6.25, 12.5, 25, or 50 mg/kg) dose-dependently inhibited de novo cholesterol biosynthesis from [14C]-acetate. The ED50 for this inhibition was 32 mg/kg. [1]
- Plasma Lipid Lowering in High-Fat Diet Fed Rats: In rats fed a high-fat diet, YM-53601 (50 mg/kg/day for 7 days) reduced plasma nonHDL cholesterol by 44% and triglycerides by 33% compared to control. In contrast, pravastatin (50 mg/kg/day) had little effect on plasma lipid levels in this model. [1]
- Plasma Lipid Lowering in Guinea Pigs: In guinea pigs fed a normal diet, YM-53601 (10, 30, or 100 mg/kg/day for 14 days) produced dose-dependent reductions in total cholesterol (up to 44% at 100 mg/kg) and nonHDL cholesterol (up to 47% at 100 mg/kg). Pravastatin (100 mg/kg/day) reduced total and nonHDL cholesterol by 32% and 33%, respectively. YM-53601 did not affect plasma HDL cholesterol, whereas pravastatin significantly decreased HDL cholesterol. YM-53601 also reduced plasma triglycerides by 30% at the highest dose, while pravastatin increased triglycerides by 17%. [1]
- Plasma Lipid Lowering in Normal Diet Fed Hamsters: In hamsters fed a normal diet, YM-53601 (12.5, 25, or 50 mg/kg/day for 5 days) dose-dependently reduced plasma total cholesterol (39-57%), nonHDL cholesterol (57-74%), and triglycerides (75-81%). At 50 mg/kg, these reductions were significantly greater than those achieved with the reference squalene synthase inhibitor FBQ. [1]
- Plasma Lipid Lowering in High-Fat Diet Fed Hamsters: In hamsters fed a high-fat diet, YM-53601 (10, 30, or 100 mg/kg/day for 7 days) dose-dependently reduced plasma total cholesterol (24-59%), nonHDL cholesterol (31-76%), and triglycerides (35-73%). The triglyceride-lowering effect of YM-53601 (73% reduction at 100 mg/kg) was significantly greater than that of fenofibrate (53% reduction at 100 mg/kg). ED50 values for YM-53601 were 49 mg/kg for total cholesterol, 22 mg/kg for nonHDL cholesterol, and 23 mg/kg for triglycerides. For fenofibrate, ED50 values were 2221 mg/kg, 297 mg/kg, and 84 mg/kg, respectively. [1]
- Plasma Lipid Lowering in Rhesus Monkeys: In rhesus monkeys fed a normal diet, YM-53601 (12.5, 25, or 50 mg/kg twice daily for 3 weeks) reduced plasma nonHDL cholesterol in a dose-dependent manner (up to 37% reduction at 50 mg/kg). In a separate 4-week study, YM-53601 (50 mg/kg twice daily) reduced nonHDL cholesterol by 21%, which was significantly greater than the 13% reduction achieved with pravastatin (25 mg/kg twice daily). YM-53601 did not affect plasma HDL cholesterol levels in these studies. No indications of liver damage were observed with YM-53601 at the tested doses. [1]

Squalene Synthase Assay: Microsomes were prepared from liver tissues of various species (rat, hamster, guinea-pig, dog, rhesus monkey) and from human HepG2 cells. The assay was conducted in HEPES buffer (pH 7.5) containing NaF, MgCl2, DTT, NADPH, 5 μM farnesyl pyrophosphate (FPP), [³H]-FPP, 10 μM NB-598 (a squalene epoxidase inhibitor to prevent further metabolism of squalene), and sodium pyrophosphate. After a 5-minute pre-incubation at 30°C, the reaction was started by adding microsomes (10 μg protein). The reaction proceeded for 20 minutes at 30°C and was terminated by adding a KOH:ethanol solution. Synthesized [³H]-squalene was extracted with petroleum ether and quantified by liquid scintillation counting. [1]

Cell Viability Assay[4]
Cell Types: H35 and HepG2 cells
Tested Concentrations: 1 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: diminished mitochondrial cholesterol levels in H35 and HepG2 cells.

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Cell Culture for Microsome Preparation: Human HepG2 cells, a hepatoma cell line, were cultured and harvested. Microsomes were prepared from these cells to assess squalene synthase inhibition. The specific culture conditions were not detailed in the text. [1]

Animal/Disease Models: SD (SD (Sprague-Dawley)) rat, body weight 150- 170 g[1]
Doses: 6.25, 12.5, 25 or 50 mg/kg
Route of Administration: Single oral administration
Experimental Results: In a dose-dependent manner in rats Acetate inhibits cholesterol biosynthesis. The ED50 value of YM-53601 for cholesterol biosynthesis inhibition is 32 mg/kg.

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Animal/Disease Models: Five to sixweeks old male BALB/c athymic (nu/nu) nude mice [4]
Doses: 15 mg/kg
Route of Administration: Daily po (oral gavage) treatment for 2 weeks
Experimental Results: Dramatically diminished intratumoral Cholesterol levels.

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Rat Cholesterol Biosynthesis Study:** Male SD rats (150-170 g) were housed under a reversed light cycle (lights off 07:30-20:30) for one week to increase daytime hepatic cholesterol biosynthesis. YM-53601 or FBQ was suspended in 0.5% methylcellulose and administered orally at 13:00 h at doses of 6.25, 12.5, 25, or 50 mg/kg. One hour later, [¹⁴C]-acetate (40.5 μCi/animal) was injected intraperitoneally. Rats were sacrificed 2 hours after drug treatment, and plasma [¹⁴C]-cholesterol was measured. [1]
- **Rat High-Fat Diet Study:** Male SD rats (5 weeks old) were fed a high-fat diet (CE-2 supplemented with 1.5% cholesterol, 0.5% cholate, and 10% coconut oil) for 7 days. YM-53601 (12.5, 25, or 50 mg/kg/day) or pravastatin (50 mg/kg/day) was administered orally once daily for the same period. Compounds were suspended in 0.5% methylcellulose. Blood samples were collected 2 hours after the final dose after an 18-hour fast. [1]
- **Guinea Pig Study:** Male Hartley guinea pigs (4 weeks old) were fed a normal diet (GC-4). YM-53601 (10, 30, or 100 mg/kg/day) or pravastatin (100 mg/kg/day) was administered orally once daily for 14 days. Compounds were suspended in 0.5% methylcellulose. Blood samples were collected 2 hours after the final dose after an 18-hour fast. [1]
- **Hamster Normal Diet Study:** Male Syrian golden hamsters (8 weeks old, ~140 g) were housed under a reversed light cycle for one week and fed a standard low-cholesterol diet (CE-2). YM-53601 (12.5, 25, or 50 mg/kg/day) or FBQ (50 mg/kg/day) was administered orally once daily for 5 days. Compounds were suspended in 0.5% methylcellulose. Blood samples were collected 2 hours after the final dose after an 18-hour fast. [1]
- **Hamster High-Fat Diet Study:** Male Syrian golden hamsters were fed a high-fat diet (CE-2 supplemented with 0.5% cholesterol and 5% coconut oil) for 11 days. Starting on day 5, YM-53601 or fenofibrate (10, 30, or 100 mg/kg/day) was administered orally once daily for 7 days. Blood samples were collected 2 hours after the final dose after an 18-hour fast. [1]
- **Rhesus Monkey Study:** Adult male rhesus monkeys (~4 kg) were fed a commercial diet (Primate Chow No. 5048) with banana supplementation. In the first experiment, YM-53601 (12.5, 25, or 50 mg/kg) was administered twice daily (09:00 and 17:00) by diet admixture for 3 weeks. In the second experiment, YM-53601 (25 or 50 mg/kg twice daily) or pravastatin (25 mg/kg twice daily) was administered by diet admixture for 4 weeks. Blood specimens were obtained at 09:00 after a 16-hour fast before, during, and after treatment. [1]

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Rat Cholesterol Biosynthesis Study: Male SD rats (150-170 g) were housed under a reversed light cycle (lights off 07:30-20:30) for one week to increase daytime hepatic cholesterol biosynthesis. YM-53601 or FBQ was suspended in 0.5% methylcellulose and administered orally at 13:00 h at doses of 6.25, 12.5, 25, or 50 mg/kg. One hour later, [¹⁴C]-acetate (40.5 μCi/animal) was injected intraperitoneally. Rats were sacrificed 2 hours after drug treatment, and plasma [¹⁴C]-cholesterol was measured. [1]
- Rat High-Fat Diet Study: Male SD rats (5 weeks old) were fed a high-fat diet (CE-2 supplemented with 1.5% cholesterol, 0.5% cholate, and 10% coconut oil) for 7 days. YM-53601 (12.5, 25, or 50 mg/kg/day) or pravastatin (50 mg/kg/day) was administered orally once daily for the same period. Compounds were suspended in 0.5% methylcellulose. Blood samples were collected 2 hours after the final dose after an 18-hour fast. [1]
- Guinea Pig Study: Male Hartley guinea pigs (4 weeks old) were fed a normal diet (GC-4). YM-53601 (10, 30, or 100 mg/kg/day) or pravastatin (100 mg/kg/day) was administered orally once daily for 14 days. Compounds were suspended in 0.5% methylcellulose. Blood samples were collected 2 hours after the final dose after an 18-hour fast. [1]
- Hamster Normal Diet Study: Male Syrian golden hamsters (8 weeks old, ~140 g) were housed under a reversed light cycle for one week and fed a standard low-cholesterol diet (CE-2). YM-53601 (12.5, 25, or 50 mg/kg/day) or FBQ (50 mg/kg/day) was administered orally once daily for 5 days. Compounds were suspended in 0.5% methylcellulose. Blood samples were collected 2 hours after the final dose after an 18-hour fast. [1]
- Hamster High-Fat Diet Study: Male Syrian golden hamsters were fed a high-fat diet (CE-2 supplemented with 0.5% cholesterol and 5% coconut oil) for 11 days. Starting on day 5, YM-53601 or fenofibrate (10, 30, or 100 mg/kg/day) was administered orally once daily for 7 days. Blood samples were collected 2 hours after the final dose after an 18-hour fast. [1]
- Rhesus Monkey Study: Adult male rhesus monkeys (~4 kg) were fed a commercial diet (Primate Chow No. 5048) with banana supplementation. In the first experiment, YM-53601 (12.5, 25, or 50 mg/kg) was administered twice daily (09:00 and 17:00) by diet admixture for 3 weeks. In the second experiment, YM-53601 (25 or 50 mg/kg twice daily) or pravastatin (25 mg/kg twice daily) was administered by diet admixture for 4 weeks. Blood specimens were obtained at 09:00 after a 16-hour fast before, during, and after treatment. [1]

Safety in Rhesus Monkeys: In the rhesus monkey study, no indications of liver damage were observed following administration of YM-53601 at doses up to 50 mg/kg twice daily. In contrast, increased plasma alanine aminotransferase (ALT), an indicator of liver damage, was observed with pravastatin at 25 mg/kg twice daily, indicating that this dose was at the upper safety limit for pravastatin. [1]

[1]. YM-53601, a novel squalene synthase inhibitor, reduces plasma cholesterol and triglyceride levels in several animal species. Br J Pharmacol. 2000 Sep;131(1):63-70.

[2]. Syntheses of 3-ethylidenequinuclidine derivatives as squalene synthase inhibitors. Part 2: enzyme inhibition and effects on plasma lipid levels. Bioorg Med Chem. 2003 Aug 15;11(17):3735-45.

[3]. Farnesyl-diphosphate farnesyltransferase 1 regulates hepatitis C virus propagation. FEBS Lett. 2014 May 2;588(9):1813-20.

[4]. Mitochondrial cholesterol contributes to chemotherapy resistance in hepatocellular carcinoma. Cancer Res. 2008 Jul 1;68(13):5246-56.

Background: YM-53601 is a novel squalene synthase inhibitor. Unlike HMG-CoA reductase inhibitors (statins), squalene synthase inhibitors do not lower the levels of ubiquinone and dolichol, which are essential for cell growth and viability. [1]
- Mechanism of Action: YM-53601 inhibits squalene synthase, the enzyme that catalyzes the dimerization of two farnesyl pyrophosphate molecules to form squalene, a key step in cholesterol biosynthesis. By inhibiting this step, YM-53601 reduces cholesterol biosynthesis and subsequently lowers plasma cholesterol levels. It also demonstrates a triglyceride-lowering effect, the mechanism of which is not fully understood but appears distinct from that of fibrates based on correlation analyses. [1]
- Therapeutic Potential: YM-53601 reduces both plasma cholesterol and triglyceride levels in animal models, showing superior efficacy to pravastatin (an HMG-CoA reductase inhibitor) in lowering nonHDL cholesterol in rhesus monkeys and superior efficacy to fenofibrate (a fibrate) in lowering triglycerides in hamsters. These findings suggest YM-53601 may be a promising lipid-lowering therapy for the treatment of both hypercholesterolemia and hypertriglyceridemia in humans, potentially offering a combination therapy in a single agent. [1]

C21H22CLFN2O

372.86

372.14

182959-33-7

YM-53601 free base;182959-28-0

60196293

Off-white to light brown solid powder

5.389

2

3

3

26

528

0

C1=CC=C2C(=C1)C3=C(C=C(C=C3)OC/C(=C/4\CN5CCC4CC5)/F)N2.Cl

JWXYVHMBPISIJQ-TVWXOORISA-N

InChI=1S/C21H21FN2O.ClH/c22-19(18-12-24-9-7-14(18)8-10-24)13-25-15-5-6-17-16-3-1-2-4-20(16)23-21(17)11-15;/h1-6,11,14,23H,7-10,12-13H2;1H/b19-18-;

2-[(2E)-2-(1-azabicyclo[2.2.2]octan-3-ylidene)-2-fluoroethoxy]-9H-carbazole;hydrochloride

YM-53601 YM53601 YM 53601

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~268.20 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.)