HMG-CoA reductase (Ki = 0.2 nM)

In hCM cells treated with benzyl alcohol sulfate, simvastatin acid (0.1–20 μM; 24 h) significantly decreased ROS generation by 8.9% to 43% [2]. hCM- was altered by simvastatin acid (0.1–20 μM; 24 h).

Alzheimer's disease (AD) is a neurodegenerative disease characterised by the presence of β-amyloid plaques and acetylcholine depletion leading to neurobehavioral defects. AD was contributed also with downregulation of TGF-β1/SMAD2 and GSK3β/β-catenin pathways. Simvastatin (SMV) improved memory function experimentally and clinically. Hence, this study aimed to investigate the mechanistic role of SMV against aluminium chloride (AlCl3) induced neurobehavioral impairments. AD was induced by AlCl3 (50 mg/kg) for 6 weeks. Mice received Simvastatin (10 or 20 mg/kg) or Donepezil (3 mg/kg) for 6 weeks after that the histopathological, immunohistochemical and biochemical test were examined. Treatment with SMV improved the memory deterioration induced by AlCl3 with significant recovery of the histopathological changes. This was concomitant with the decrease of AChE and Aβ (1-42). SMV provides its neuroprotective effect through upregulating the protein expression of β-catenin, TGF-β1 and downregulating the expression of GSK3β, TLR4 and p-SMAD2.https://pubmed.ncbi.nlm.nih.gov/37454825/

OATP3A1 functional assays[2]
The fluorescent substrate sodium fluorescein was used for characterization of OATP3A1 function in hCMs, HEK293-NEO (empty vector), and HEK293-OATP3A1-transfected cells. Cells (500,000 cells/dish) were grown on 100 mm dishes. Sodium fluorescein is a general substrate of OATP3A1 (Patik et al., 2015). Cells were pre-incubated with Dulbecco's Phosphate Buffered Saline (DPBS) pH 7.4 and pH 5.5 for 10 min at 37 °C in a 5% CO2 atmosphere as several studies have reported increased OATP transport activity at acidic pH (Kobayashi et al., 2003, Nozawa et al., 2004, Varma et al., 2011). Cells were then treated with sodium fluorescein (2 μM) for 30 min (uptake period) (Wen et al., 2014). Only hCMs were incubated in the presence or absence of cyclosporine (suspected transport inhibitor) (10 μM) or simvastatin acid (10 μM) at 37 °C in a 5% CO2 atmosphere for 30 min. After washing with DPBS pH 7.4 or 5.5, all cells were collected and lysed with 0.1 N NaOH and the intensity of fluorescence was quantified on a BioTek Synergy™ 4 Hybrid Microplate Reader using Gen5 1.10 software at excitation/emission 460/515 nm.

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[3H]Simvastatin acid uptake experiments in OATP3A1 expressing cells[2]
Uptake experiments were conducted at pH 7.4 and pH 5.5 at 37 °C in a 5% CO2 atmosphere. Cellular uptake of radiolabeled simvastatin acid was measured in monolayer cultures of HEK293-NEO (empty vector) and HEK293-OATP3A1 transfected cells (100,000 cells/well) grown on 24-well plates. After two days of culture (Chiba et al., 2013), cells were washed once and pre-incubated in DPBS at pH 7.4 and pH 5.5 for 10 min at 37 °C in a 5% CO2 atmosphere. Uptake was then evaluated by adding radiolabeled simvastatin acid (0.05 μM) in DPBS. At indicated times (1, 2, 5, 15, 30, 45 min), uptake was terminated by replacement of the transport buffer with ice-cold DPBS. After washing two times in ice-cold DPBS, the cells were lysed in 0.1 N NaOH. The radioactivity in the cells was determined by liquid scintillation counting on a Beckman LS 6000IC scintillation counter. For kinetic analyses, cells were pre-incubated with unlabeled simvastatin acid (0.01, 0.05, 0.1, 0.5, 1 μM) for 30 min (uptake period) in DPBS prior to evaluating the uptake of radiolabeled simvastatin acid (0.05 μM). The data were plotted to determine Vmax and Km using nonlinear regression.

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Drug-drug interaction screening[2]
Cellular uptake of radiolabeled simvastatin acid was measured in monolayer cultures of HEK293-NEO (empty vector) and HEK293-OATP3A1 transfected cells (100,000 cells/well) grown on 24-well plates. After two days of culture (Chiba et al., 2013), cells were washed once and pre-incubated in DPBS pH 7.4 or pH 5.5 for 10 min at 37 °C in a 5% CO2 atmosphere. In order to evaluate the potential for competitive inhibition, cells were treated in the presence or absence of prototypical substrates [benzylpenicillin (10 and 100 μM), cyclosporine (10 and 100 μM), estrone-3-sulfate (10 and 100 μM), and indoxyl-sulfate (10 and 100 μM)] for 30 min (uptake period) in DPBS. Uptake was then evaluated by adding radiolabeled simvastatin acid (0.05 μM) in DPBS. After 1 min, uptake was terminated by replacement of the uptake solution with ice-cold DPBS. After washing two times in ice-cold DPBS, the cells were lysed in 0.1 N NaOH. The radioactivity in the cells was determined by liquid scintillation counting on a Beckman LS 6000IC scintillation counter

Western Blot Analysis[2]
Cell Types: hCM and HEK293 (transfected with OATP3A1)
Tested Concentrations: 0.1, 1, 10 and 20 μM
Incubation Duration: 24 h
Experimental Results: OATP3A1 expression was diminished in a dose-dependent manner by 1.5% to 90% in both hCM and OATP3A1-expressing cells.

Simvastatin/SMV was dissolved in 0.5% carboxymethylcellulose (CMC), while AlCl3, and DPZ were dissolved in water.
Mice were assigned randomly to five groups (6 mice per group): Group (1); normal untreated group received water daily for 6 weeks, Group (2–5); positive control group received AlCl3 (50 mg/kg/day, p.o.) for 6 weeks (Al-Amin et al., 2019, Li et al., 2018, Singh and Goel, 2015), Group (3−4); mice received SMV (10 or 20 mg/kg/day, p.o.), respectively (Jin et al., 2016), 45 min before AlCl3 (Nampoothiri et al., 2015), Group (5); mice received donepezil (3 mg/kg/day, p.o.) (Shin et al., 2018), 45 min before AlCl3.
After six weeks, mice were tested for the assessment of memory using Barnes Maze test (a test for spatial learning & memory), T Maze Spontaneous Alternation test (a test for spatial memory), and Novel Object Recognition Test (a test for different phases of learning and memory). After the behavior tests, the rodents were anesthetized using I.P. anaesthetic dose of 100 mg/kg ketamine and 10 mg/kg xylazine, then sacrificed by cervical displacement. The brains were removed immediately and cut into halves. Each mouse's right cerebral hemisphere is dissected, fixed with a 10% neutral buffer formalin, and used for histological and immunohistochemical dyeing. Hippocampus extracted from the left cerebral hemisphere was refrigerated and saved at − 80 °C, then used later for biochemical analysis.
https://pubmed.ncbi.nlm.nih.gov/37454825/

[1]. HMG-CoA Reductase inhibitors: an updated review of patents of novel compounds and formulations (2011-2015). Expert Opin Ther Pat. 2016 Nov;26(11):1257-1272.

[2]. Characterization of simvastatin acid uptake by organic anion transporting polypeptide 3A1 (OATP3A1) and influence of drug-drug interaction. Toxicol In Vitro. 2017 Dec;45(Pt 1):158-165.

Statins are remarkably safe and efficient medications that are the mainstay of hypercholesterolemia treatment and have proven to be an invaluable tool to lower the risk of acute cardiovascular events. These compounds are inhibitors of 3-hydroxy-methylglutaryl CoA reductase (HMG-R), the rate-limiting enzyme in cholesterol biosynthesis. In spite of their success, they present undesirable side effects and are now loosing patent protection, which provides a great opportunity for the development of new and improved statins. Areas covered: This review summarizes the new patents for HMG-R inhibitors for the 2011-2015 period. Combinations of existing statins with other drugs are also addressed, as well as novel applications of existing statins. Expert opinion: Recent efforts for the discovery of HMG-CoA-R inhibitors has resulted in several new molecules. Most of these are based on commercially available statins, including sterol and terpenoid derivatives. A few peptides have also been patented. However, the origin of the side effects caused by previous statins continues to be, to a large extent, unknown. Although the patents published in the past 5 years are promising, and might result in new drugs, there is still no way to know if they will present reduced toxicity. Only future clinical trials will answer this question.[1]

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Human organic anion transporting polypeptide 3A1 (OATP3A1) is predominately expressed in the heart. The ability of OATP3A1 to transport statins into cardiomyocytes is unknown, although other OATPs are known to mediate the uptake of statin drugs in liver. The pleiotropic effects and uptake of simvastatin acid were analyzed in primary human cardiomyocytes and HEK293 cells transfected with the OATP3A1 gene. Treatment with simvastatin acid reduced indoxyl sulfate-mediated reactive oxygen species and modulated OATP3A1 expression in cardiomyocytes and HEK293 cells transfected with the OATP3A1 gene. We observed a pH-dependent effect on OATP3A1 uptake, with more efficient simvastatin acid uptake at pH5.5 in HEK293 cells transfected with the OATP3A1 gene. The Michaelis-Menten constant (Km) for simvastatin acid uptake by OATP3A1 was 0.017±0.002μM and the Vmax was 0.995±0.027fmol/min/105 cells. Uptake of simvastatin acid was significantly increased by known (benzylpenicillin and estrone-3-sulfate) and potential (indoxyl sulfate and cyclosporine) substrates of OATP3A1. In conclusion, the presence of OATP3A1 in cardiomyocytes suggests that this transporter may modulate the exposure of cardiac tissue to simvastatin acid due to its enrichment in cardiomyocytes. Increases in uptake of simvastatin acid by OATP3A1 when combined with OATP substrates suggest the potential for drug-drug interactions that could influence clinical outcomes.[2]

C25H39O6-.H4N+

453.61202

453.309

C, 66.20; H, 9.56; N, 3.09; O, 21.16

139893-43-9

Simvastatin hydroxy acid sodium;101314-97-0;Simvastatin acid;121009-77-6;Simvastatin acid-d6 ammonium

10961424

White to off-white solid powder

4.429

3

6

10

32

689

7

CCC(C)(C)C(=O)O[C@H]1C[C@H](C=C2[C@H]1[C@H]([C@H](C=C2)C)CC[C@H](C[C@H](CC(=O)[O-])O)O)C.[NH4+]

FFPDWNBTEIXJJF-OKDJMAGBSA-N

InChI=1S/C25H40O6.H3N/c1-6-25(4,5)24(30)31-21-12-15(2)11-17-8-7-16(3)20(23(17)21)10-9-18(26)13-19(27)14-22(28)29;/h7-8,11,15-16,18-21,23,26-27H,6,9-10,12-14H2,1-5H3,(H,28,29);1H3/t15-,16-,18+,19+,20-,21-,23-;/m0./s1

Ammonium (3R,5R)-7-[(1S,2S,6R,8S,8aR)-8-(2,2-dimethylbutanoyloxy)-2,6-dimethyl-1,2,6,7,8,8a-hexahydronaphthalen-1-yl]-3,5-dihydroxyheptanoate

MK-733; Synvinolin; MK-733; Simvastatin ammonium salt; 139893-43-9; Tenivastatin ammonium; UNII-76RD797JAX; 76RD797JAX; Ammonium simvastatin; EC 604-165-5; Simvastatin Carboxylic Acid Ammonium Salt; Sinvacor; MK 733; MK733;

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 : ~11.11 mg/mL (~24.49 mM)

Solubility in Formulation 1: ≥ 1.11 mg/mL (2.45 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 11.1 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: ≥ 1.11 mg/mL (2.45 mM) (saturation unknown) in 10% DMSO + 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 11.1 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: ≥ 1.11 mg/mL (2.45 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 11.1 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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