HMG-CoA reductase (Ki = 0.2 nM)

Simvastatin acid (0.1–20 μM; 24 hours) significantly decreased ROS generation in hCM cells treated with indoxyl sulfate by 8.9% to 43% [2]. The protein expression of OATP3A1 in hCM and OATP3A1-expressing HEK293 cells is altered by simvastatin acid (0.1–20 μM; 24 hours) [2].

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.

---

[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.

---

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 hrs (hours)
Experimental Results: OATP3A1 expression diminished from 1.5% to 90% in a dose-dependent manner for 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]

---

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]

C50H84CAO15

965.270977020264

964.544

530112-57-3

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

71587759

Typically exists as solid at room temperature

5.349

7

15

20

66

689

14

[Ca+2].O(C(C(C)(C)CC)=O)[C@H]1C[C@@H](C)C=C2C=C[C@H](C)[C@H](CC[C@H](C[C@H](CC(=O)[O-])O)O)[C@@H]12.O(C(C(C)(C)CC)=O)[C@H]1C[C@@H](C)C=C2C=C[C@H](C)[C@H](CC[C@H](C[C@H](CC(=O)[O-])O)O)[C@@H]12.O.O.O

ZZURQDTUMWWURH-XVIDMKIDSA-L

InChI=1S/2C25H40O6.Ca.3H2O/c2*1-6-25(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;;;;/h2*7-8,11,15-16,18-21,23,26-27H,6,9-10,12-14H2,1-5H3,(H,28,29);;3*1H2/q;;+2;;;/p-2/t2*15-,16-,18+,19+,20-,21-,23-;;;;/m00..../s1

calcium;(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;trihydrate

Tenivastatin calcium hydrate; UNII-QSL5L73L0O; 530112-57-3; QSL5L73L0O; calcium;(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;trihydrate; DTXSID10201061; Q27287478; calcium;(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;trihydrate; 1-NAPHTHALENEHEPTANOIC ACID, 8-(2,2-DIMETHYL-1-OXOBUTOXY)-1,2,6,7,8,8A-HEXAHYDRO-.BETA.,.DELTA.-DIHYDROXY-2,6-DIMETHYL-, CALCIUM SALT, HYDRATE (2:1),(.BETA.R,.DELTA.R,1S,2S,6R,8S,8AR)-; Simvastatin acid calcium hydrate;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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