HMG-CoA reductase (Ki = 0.2 nM)

Simvastatin is an inactive medication precursor that needs to be broken down into its hydroxy acid form in the liver in order to start working. It has no drug activity of its own. Sodium hydroxide (NaOH) can activate it in in vitro tests.
Activation of Simvastatin in vitro [13,14]
Method 1[13]: Simvastatin 5 mg can be activated by reconstituting in an ethanol/sodium hydroxide solution, incubated for 2 hours in a water bath preheated to 50°C. The drug was made to 1 mL with deionized water and pH adjusted to 7.
Method 2[14]: Simvastatin is an inactive lactone product and has to be converted to its active β-hydroxy acid form by solubilization in 0.1 N sodium hydroxide/ethanol at 50°C for 2 hours. The solution was neutralized with hydrochloride (0.1 M) at pH 7.2.

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Simvastatin has IC50 values of 19.3 nM, 13.3 nM, and 15.6 nM, respectively, which inhibit the synthesis of cholesterol in mouse LM cells, rat H4II E cells, and human Hep G2 cells[1]. Within 30 minutes, simvastatin increases serine 473 phosphorylation of Akt in a dose-dependent manner; peak phosphorylation happens at 1.0 µM[2]. Simvastatin (1.0 μM) suppresses serum-free media undergo apoptosis, speeds up the creation of vascular structures, and increases phosphorylation of the endogenous Akt substrate endothelial nitric oxide synthase (eNOS)[2]. Simvastatin has anti-inflammatory properties and decreases IFN-γ release at 10 μM, as well as the proliferation of PB-derived mononuclear cells and synovial fluorid cells from rheumatoid arthritis blood induced by anti-CD3/anti-CD28 antibodies[3]. Additionally, around 30% of cell-mediated macrophage TNF-γ release produced via cognate contacts is blocked by simvastatin (10 μM)[3]. In astrocytes and neuroblastoma cells, simvastatin (5 μM) dramatically decreases ABCA1 expression, apolipoprotein E expression in astrocytes, and enhances glycogen synthase kinase 3β and cyclin-dependent kinase 5 expression in SK-N-SH cells[7]. Exosome release can be inhibited by simvastatin[10]. Simvastatin slows tumor cell development and causes it to stop in the G0/G1 phase at 32 and 64 μM; 24, 48, and 72 hours[11]. In HepG2 and Huh7 cells, simvastatin (32 and 64 μM; 48 h) causes apoptosis[11].

When administered po, simvastatin inhibits the conversion of radiolabeled acetate to cholesterol with an IC50 of 0.2 mg/kg[1]. In rabbits fed an atherogenci cholesterol-rich diet, simvastatin (4 mg/day, po for 13 weeks) reverses the increases in total cholesterol, LDL cholesterol, and HDL cholesterol to normal levels[4]. In rabbits fed a diet containing 0.25% cholesterol, simvastatin (6 mg/kg) increases the number of hepatic LDL receptors and LDL receptor-dependent binding[5]. In cynomolgus monkeys fed an atherogenic diet, simvastatin (20 mg/kg/day) causes a 1.3-fold decrease in macrophage content in lesions and a 2-fold decrease in vascular cell adhesion molecule-1, interleukin-1beta, and tissue factor expression. These reductions are accompanied by a 2.1-fold increase in lesional smooth muscle cell and collagen content[6]. Treatment with simvastatin (oral gavage; once daily; 14 d); 15 and 30 mg/kg) reduces oxidative damage, TNF-a and IL-6 levels, and revives the activities of the mitochondrial enzyme complex[12].

For assessment of Akt protein kinase activity in vitro, substrate (2 μg histone H2B or 25 μg eNOS peptide) is incubated with Akt immunoprecipitated from cell lysate using goat polyclonal anti-Akt1 antibody. Kinase reactions are initiated following the addition of Simvastatin to a final concentration of ATP (50 μM) containing 10 μCi of 32P-γATP, dithiotreitol (1 mM), HEPES buffer (20 mM, pH 7.4), MnCl2 (10 mM), MgCl2 (10 mM). After incubation for 30 min at 30°C, phosphorylated histone H2B is visualized after SDS-PAGE (15%) and autoradiography. To estimate the extent of 32P incorporation into eNOS peptides, each reaction mixture is measured by spotting onto phosphocellulose disc filter and the amount of phosphate incorporated is measured by Cerenkov counting. The wild-type peptide sequence is 1174-RIRTQSFSLQERHLRGAVPWA-1194, and the mutant eNOS peptide is identical except that serine 1179 is substituted by alanine[3].

Cell Proliferation Assay[11]
Cell Types: HepG2 and Huh7 cells
Tested Concentrations: 32 and 64 μM
Incubation Duration: 24, 48, and 72 hrs (hours)
Experimental Results: Inhibited tumor cell growth as compared to controls (ctrl, p<0.05).

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Apoptosis Analysis[11]
Cell Types: HepG2 and Huh7 cells
Tested Concentrations: 32 and 64 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Increased early apoptosis from 9.2% in non-treated ctrl cells to 18.2% (32 μM) and 19.8% (64 μM), respectively, increased late apoptosis from 35.0% in ctrl cells to 56.9% (32 μM) and 48.0% (64 μM), respectively, in HepG2 cells.

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Cell Cycle Analysis[11]
Cell Types: HepG2 and Huh7 cells
Tested Concentrations: 32 and 64 μM
Incubation Duration: 24, 48, and 72 hrs (hours)
Experimental Results: demonstrated downregulation of CDK1, CDK2, CDK4 and cyclins D1 and E as compared to ctrl tumor cells.

Animal/Disease Models: Male wistar rats with oxidative damage by Intrastriatal 6-OHDA administration[12]
Doses: 15 and 30 mg/kg
Route of Administration: po (oral gavage); 15 and 30 mg/kg; one time/day; 14 days
Experimental Results: Attenuated oxidative damage (decreased MDA, nitrite levels and restoration of decreased GSH), attenuated TNF-a and IL-6 levels, and restored itochondrial enzyme complex activities as compared to 6-OHDA group.

[1]. Slater, E.E., et al. Mechanism of action and biological profile of HMG CoA reductase inhibitors. A new therapeutic alternative. Drugs, 1988. 36 Suppl 3: p. 72-82.
[2]. Kureishi, Y., et al. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med, 2000. 6(9): p. 1004-10.
[3]. Leung BP, et al. A novel anti-inflammatory role for simvastatin in inflammatory arthritis. J Immunol. 2003 Feb 1;170(3):1524-30.
[4]. Kobayashi M, et al. Preventive effect of MK-733 (simvastatin), an inhibitor of HMG-CoA reductase, on hypercholesterolemia and atherosclerosis induced by cholesterol feeding in rabbits. Jpn J Pharmacol. 1989 Jan;49(1):125-33.
[5]. Ishida F, et al. Comparative effects of simvastatin (MK-733) and CS-514 on hypercholesterolemia induced by cholesterol feeding in rabbits. Biochim Biophys Acta. 1990 Feb 23;1042(3):365-73.
[6]. Sukhova GK, et al. Statins reduce inflammation in atheroma of nonhuman primates independent of effects on serum cholesterol. Arterioscler Thromb Vasc Biol. 2002 Sep 1;22(9):1452-8.
[7]. Weijiang Dong, et al. Differential effects of simvastatin and CS-514 on expression of Alzheimer’s disease-related genes in human astrocytes and neuronal cells. J Lipid Res. 2009 Oct; 50(10): 2095-2102.
[8]. Liu Z, et al. Pretreatment Donors after Circulatory Death with Simvastatin Alleviates Liver Ischemia Reperfusion Injury through a KLF2-Dependent Mechanism in Rat. Oxid Med Cell Longev. 2017;2017:3861914.
[9]. Ifergan I, et al. Statins reduce human blood-brain barrier permeability and restrict leukocyte migration: relevance to multiple sclerosis. Ann Neurol. 2006 Jul;60(1):45-55.
[10]. Zhang H, et al. Advances in the discovery of exosome inhibitors in cancer. J Enzyme Inhib Med Chem. 2020;35(1):1322-1330.
[11]. Borna Relja, et al. Simvastatin inhibits cell growth and induces apoptosis and G0/G1 cell cycle arrest in hepatic cancer cells. Int J Mol Med. 2010 Nov;26(5):735-41.
[12]. Anil Kumar, et al. Neuroprotective potential of atorvastatin and simvastatin (HMG-CoA reductase inhibitors) against 6-hydroxydopamine (6-OHDA) induced Parkinson-like symptoms. Brain Res. 2012 Aug 30;1471:13-22.
[13]. Simvastatin Suppresses Interleukin Iβ Release in Human Peripheral Blood Mononuclear Cells Stimulated With Cholesterol Crystals. J Cardiovasc Pharmacol Ther. 2018 Nov;23(6):509-517.
[14]. Simvastatin decreases aldehyde production derived from lipoprotein oxidation. Am J Cardiol . 1999 Mar 15;83(6):846-51.

Lovastatin (MK-803, mevinolin) and simvastatin (MK-733, synvinolin), 2 highly potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors, have been heralded as breakthrough therapy for the treatment of atherosclerotic disease. This paper discusses the biochemical attributes of these HMG CoA reductase inhibitors, their structures and inhibitory properties in a variety of biological systems and presents the rationale for their therapeutic use. Not only do lovastatin and simvastatin potently inhibit cholesterol biosynthesis; they also can result in the induction of hepatic low density lipoprotein (LDL) receptors, thus increasing the catabolism of LDL-cholesterol. Lovastatin and simvastatin are the first HMG CoA reductase inhibitors to receive regulatory agency approval for marketed use. Their safety profiles are reviewed and 2 aspects of this evaluation are stressed. First, the objective in the clinical use of these inhibitors is to normalise plasma cholesterol levels in hypercholesterolaemic individuals. This contrasts with the profound reductions in cholesterol obtained when normocholesterolaemic animals are treated by the high doses of these drugs required for toxicological assessment. Second, both lovastatin and simvastatin are administered as prodrugs in their lactone forms. As lactones, they readily undergo first-pass metabolism, hepatic sequestration and hydrolysis to the active form. Consequently, lovastatin and simvastatin achieve lower plasma drug levels than do other HMG CoA reductase inhibitors in clinical development. Low plasma levels have been established as an important determinant of safety in the use of HMG CoA reductase inhibitors in both animal and human studies.[1]

C25H38O5

418.57

418.2719

C, 71.74; H, 9.15; O, 19.11

79902-63-9

Simvastatin-d6;1002347-71-8;Simvastatin-d11;1002347-74-1;Simvastatin-d3;1002347-61-6; 139893-43-9 (ammonium); 79902-63-9 (free); 101314-97-0 (sodium)

54454

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

1.1±0.1 g/cm3

564.9±50.0 °C at 760 mmHg

139 °C

184.8±23.6 °C

0.0±3.5 mmHg at 25°C

1.53

4.41

72.83

C[C@H]1C=CC2=C[C@H](C)C[C@H](OC(C(C)(C)CC)=O)C2[C@H]1CC[C@@H]3C[C@@H](O)CC(O3)=O

RYMZZMVNJRMUDD-OVOOIQHOSA-N

InChI=1S/C25H38O5/c1-6-25(4,5)24(28)30-21-12-15(2)11-17-8-7-16(3)20(23(17)21)10-9-19-13-18(26)14-22(27)29-19/h7-8,11,15-16,18-21,23,26H,6,9-10,12-14H2,1-5H3/t15-,16-,18+,19+,20-,21-,23?/m0/s1

(1S,3R,7S,8S)-8-(2-((2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl)ethyl)-3,7-dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl 2,2-dimethylbutanoate

MK-0733, MK 0733, MK0733, Zocor; Synvinolin; MK 733; Sinvacor; MK-733; MK733; Simvastatin;

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: ≥ 2.5 mg/mL (5.97 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 25.0 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: 2.5 mg/mL (5.97 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 25.0 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: ≥ 2.5 mg/mL (5.97 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (5.97 mM) (saturation unknown) in 10% EtOH + 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 25.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix well.
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 5: ≥ 2.5 mg/mL (5.97 mM) (saturation unknown) in 10% EtOH + 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 25.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 6: 2% DMSO+30% PEG 300+5% Tween80+ddH2O:10 mg/mL

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Solubility in Formulation 7: 10 mg/mL (23.89 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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