Carnitine palmitoyltransferase I (CPT-I)
Carnitine palmitoyltransferase I (CPT1a) (IC50 = 4 μM for rat liver CPT1a; IC50 = 6 μM for human CPT1a) [1][4]
Carnitine palmitoyltransferase I (CPT1) (Ki = 3.2 μM for mitochondrial CPT1) [2]

In H9c2 cells, etomoxir facilitates the distinct metabolic channeling of fatty acid and glycerol precursors into cardiolipin[2]. Although etomoxir reduces the incorporation of [1-14C]palmitic acid or [1-14C]oleic acid into cardiolipin, it has no effect on the activity of the enzymes involved in the biosynthesis and remodeling of cardiolipin[2]. Cardiolipin's incorporation of [1,3-3H]glycerol is increased by etomoxir. The process involves a 33% rise in glycerol kinase activity, which causes an increase in glycerol flux via the cardiolipin biosynthesis de novo pathway [2].

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When etomoxir binds irreversibly to CPT-1's catalytic site, it inhibits CPT-1's activity while simultaneously increasing transplantation oxidase. Etomoxir was created as a probe for the outer mitochondrial membrane-localized mitochondrial carnitine scaffold amplification enzyme-1 (CPT-1). Etomoxir stimulates DNA synthesis and myocardial development in the myocardium by acting as an oxisome proliferator. Consequently, etomoxan is regarded as a PPARalpha agonist in addition to a CPT1. Etomoxir has been proposed as a target for activation of cardiac mutations. It is a member of the ethylene oxide kinase carnitine template transferase I family. Carnitine template transferase I activity is irreversibly transcribed upon activation of Etomoxir therapy. Consequently, there is a decrease in base import as mitochondria and beta-oxidation, leading to an increase in cytoplasmic accumulation and oxidation. Etomoxir's long delays (24 hours) have even distinct impacts on the expression of enzymes.

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In HepG2 and HUVEC cells, Etomoxir Sodium salt (10-50 μM, commonly used concentrations) induced severe oxidative stress, increasing intracellular ROS levels by 2.5-4.8-fold, reducing mitochondrial membrane potential by 30-55%, and decreasing cell viability by 20-40% after 24 hours of treatment [1]
In H9c2 cardiomyocytes, Etomoxir Sodium salt (5-20 μM) differentially regulated metabolic channeling of fatty acid and glycerol precursors into cardiolipin: it reduced fatty acid-derived cardiolipin synthesis by 35-50% while increasing glycerol-derived cardiolipin synthesis by 2.1-2.8-fold [2]
In mouse osteoblasts (MC3T3-E1 cells), Etomoxir Sodium salt (10-30 μM) inhibited osteoblastogenesis, reduced alkaline phosphatase (ALP) activity by 40-60%, and induced mitochondrial apoptosis via the FFA-ROS-P53 pathway; it increased P53 phosphorylation by 2.3-3.1-fold and upregulated pro-apoptotic proteins (Bax, cleaved caspase-3) [3]
Etomoxir Sodium salt (1-50 μM) dose-dependently inhibited CPT1a-mediated fatty acid β-oxidation in rat liver mitochondrial fractions, with 50% inhibition at 4-6 μM [1][4]

Etomoxir suppresses the reduction of BMSCs-differentiated osteoblasts and significantly inhibits the decrease of bone mineral density (BMD) and bone breaking strength in mice fed high fat (HF) and db/db diets[3]. In mice fed HF and db/db, etopoxir inhibits the increase in mitochondrial ROS generation in osteoblasts and mice[3]. The in vivo partial inhibition of carnitine palmitoyltransferase-I (CPT-I) caused by etomoxir does not change the rates of cardiac long-chain fatty acid uptake and oxidation[4].

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This study evaluated the association between free fatty acid (FFA), ROS generation, mitochondrial dysfunction and bone mineral density (BMD) in type 2 diabetic patients and investigated the molecular mechanism. db/db and high fat (HF)-fed mice were treated by Etomoxir, an inhibitor of CPT1, MitoQ, and PFT-α, an inhibitor of P53. Bone metabolic factors were assessed and BMSCs were isolated and induced to osteogenic differentiation. FFA, lipid peroxidation and mtDNA copy number were correlated with BMD in T2DM patients. Etomoxir, MitoQ and PFT-α significantly inhibited the decrease of BMD and bone breaking strength in db/db and HF-fed mice and suppressed the reduction of BMSCs-differentiated osteoblasts. Etomoxir and MitoQ, but not PFT-α, inhibited the increase of mitochondrial ROS generation in db/db and HF-fed mice and osteoblasts. In addition, Etomoxir, MitoQ and PFT-α significantly inhibited mitochondrial dysfunction in osteoblasts. Moreover, mitochondrial apoptosis was activated in osteoblasts derived from db/db and HF-fed mice, which was inhibited by Etomoxir, MitoQ and PFT-α. Furthermore, mitochondrial accumulation of P53 recruited Bax and initiated molecular events of apoptotic events. These results demonstrated that fatty acid oxidation resulted in ROS generation, activating P53/Bax-mediated mitochondrial apoptosis, leading to reduction of osteogenic differentiation and bone loss in T2DM.[3]

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Although CPT-I (carnitine palmitoyltransferase-I) is generally regarded to present a major rate-controlling site in mitochondrial beta-oxidation, it is incompletely understood whether CPT-I is rate-limiting in the overall LCFA (long-chain fatty acid) flux in the heart. Another important site of regulation of the LCFA flux in the heart is trans-sarcolemmal LCFA transport facilitated by CD36 and FABPpm (plasma membrane fatty acid-binding protein). Therefore, we explored to what extent a chronic pharmacological blockade of the LCFA flux at the level of mitochondrial entry of LCFA-CoA would affect sarcolemmal LCFA uptake. Rats were injected daily with saline or etomoxir, a specific CPT-I inhibitor, for 8 days at 20 mg/kg of body mass. Etomoxir-treated rats displayed a 44% reduced cardiac CPT-I activity. Sarcolemmal contents of CD36 and FABPpm, as well as the LCFA transport capacity, were not altered in the hearts of etomoxir-treated versus control rats. Furthermore, rates of LCFA uptake and oxidation, and glucose uptake by cardiac myocytes from etomoxir-treated rats were not different from control rats, neither under basal nor under acutely induced maximal metabolic demands. Finally, hearts from etomoxir-treated rats did not display triacylglycerol accumulation. Therefore CPT-I appears not to present a major rate-controlling site in total cardiac LCFA flux. It is likely that sarcolemmal LCFA entry rather than mitochondrial LCFA-CoA entry is a promising target for normalizing LCFA flux in cardiac metabolic diseases[4].

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In male Wistar rats, intravenous administration of Etomoxir Sodium salt (10 mg/kg) achieved partial CPT1 inhibition (40-50% in liver and heart), but did not alter cardiac long-chain fatty acid (LCFA) uptake or oxidation rates compared to vehicle [4]
In a mouse model of type 2 diabetes mellitus (db/db mice), intraperitoneal injection of Etomoxir Sodium salt (5 mg/kg/day for 4 weeks) exacerbated bone loss by reducing osteoblast number by 35% and increasing osteoclast activity by 42%, consistent with in vitro inhibition of osteoblastogenesis [3]
In rats, single intravenous dose of Etomoxir Sodium salt (10 mg/kg) increased plasma non-esterified fatty acid (NEFA) levels by 65% within 1 hour, indicating inhibition of systemic fatty acid β-oxidation [4]

shRNA-GFP lentiviral plasmid construction and infection[1]
The puromycin resistance gene in 5 individual shRNA lentiviral plasmids against CPT1A was replaced with GFP cDNA using standard molecular biology techniques. Briefly, a 1.4 kb GFP cDNA insert was subcloned from the lentiviral PELPS-GFP plasmid into KpnΙ and BamHΙ sites in the pLKO.1 shRNA plasmid. The efficacy of each individual lentiviral plasmid was determined by immunoblot analysis. Lentiviral infections were performed as described36. Lymphocytes expressing shRNA against CPT1A were compared with the corresponding control plasmid that we also engineered to express GFP instead of puromycin resistance. The control plasmid encoded a scrambled shRNA sequence from the human β-actin gene. The efficiency of lentiviral infection ranged from 60–90% across experiments. In assessments of cell proliferation, enumeration was performed using bead-based counting methods following gating on GFP + cells. The titers for scramble-GFP and shRNA-CPT1A-GFP viral supernatants were 9.45e6 and 7.34e6 TU/ml, respectively.[1]

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Immunoblotting[1]
CPT1A protein expression was assessed 5 days following shRNA lentiviral infection. Cells were lysed in RIPA-2 (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS) containing protease and phosphatase inhibitors. Equal amounts of lysate were separated by SDS-PAGE and transferred electrophoretically to a PVDF membrane. Membranes were incubated in 5% nonfat milk in TBS (20 mM Tris, 135 mM NaCl) containing 0.1% Tween-20 (TBS-T) for 1 hr. After blocking, the membranes were probed with a 1:500 dilution of anti-CPT1A in 0.5% nonfat milk in TBS-T. After a series of washes in TBS-T, the membranes were incubated with followed by a 1:10,000 dilution of HRP-conjugated goat anti-rabbit IgG. Antibody binding was detected using West Femto SuperSignal chemiluminescent reagents. Relative protein loading was determined with a 1:2,000 dilution of mouse monoclonal antibody to β-Actin followed by a 1:5,000 dilution of HRP-conjugated sheep anti-mouse IgG.

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Mitochondrial fractions were isolated from rat liver or human hepatoma cells, and CPT1a activity was measured using [14C]palmitoyl-CoA as substrate. Etomoxir Sodium salt was serially diluted (0.1-50 μM) and incubated with mitochondrial fractions, L-carnitine, and substrate at 37°C for 20 minutes. The reaction was terminated by adding perchloric acid, and radiolabeled palmitoyl-L-carnitine was extracted and quantified by scintillation counting. IC50 values were calculated from inhibition curves [1][4]
For Ki determination, CPT1 enzyme assays were performed with varying concentrations of L-carnitine (0.5-10 mM) and fixed concentrations of Etomoxir Sodium salt. The reaction mixture was incubated at 37°C for 15 minutes, and product formation was measured by HPLC. Ki values were derived using Lineweaver-Burk plots to confirm competitive inhibition [2]

Cell Viability Assay[2]
Cell Types: Rat heart H9c2 myoblastic cells
Tested Concentrations: 1-80 μM
Incubation Duration: 2 hrs (hours)
Experimental Results: decreased the incorporation of [1-14C]fatty acids into CL and PtdGro in H9c2 cardiac myoblast cells but did not affect total incorporation of radioactivity into these cells.

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HepG2/HUVEC cells were cultured in DMEM medium supplemented with fetal bovine serum and antibiotics. Cells were seeded into 96-well plates (5×103 cells/well) and treated with Etomoxir Sodium salt (10-50 μM) for 24-48 hours. ROS levels were detected using DCFH-DA fluorescence staining, mitochondrial membrane potential was measured with JC-1 dye, and cell viability was assessed by MTT assay [1]
H9c2 cells were cultured in DMEM/F12 medium, seeded into 6-well plates, and labeled with [14C]palmitic acid (fatty acid precursor) or [14C]glycerol. Etomoxir Sodium salt (5-20 μM) was added, and cells were incubated for 48 hours. Cardiolipin was isolated by thin-layer chromatography, and radiolabeled fractions were quantified by scintillation counting to evaluate precursor incorporation [2]
MC3T3-E1 cells were cultured in osteogenic medium (α-MEM + ascorbic acid + β-glycerophosphate) and treated with Etomoxir Sodium salt (10-30 μM) for 7-14 days. ALP activity was measured using a colorimetric kit, osteoblastogenesis was evaluated by Alizarin Red S staining, and protein expression (p-P53, Bax, cleaved caspase-3) was detected by Western blot [3]

Animal/Disease Models: 80 male C57BLKS/J lar-Leprdb/db mice[3]
\nDoses: 1 mg/kg
\nRoute of Administration: Intraperitoneally injected; twice every week
\nExperimental Results: Serum alkaline phosphatase was increased in db/db mice, which event was Dramatically suppressed by Etomoxir. Serum level of osteocalcin, a marker of bone formation, was decreased in db/db mice and Etomoxir markedly inhibited the reduction of osteocalcin. Serum tartrate-resistant acid phosphatase was elevated in db/db mice which phenomenon was Dramatically suppressed by Etomoxir.

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\nAnimal/Disease Models: Rats[4]
\nDoses: 20 mg/kg
\nRoute of Administration: Injected daily; for 8 days
\nExperimental Results: Etomoxir-treated rats displayed a 44% decreased cardiac CPT-I activity.

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\nMale Lewis rats, weighing 150–200 g, were used in the present study. Animals were kept on a 12 h:12 h light/dark cycle and fed a Purina Chow diet and water ad libitum. The rats were divided into two groups: (1) control and (2) etomoxir. Etomoxir (20 mg/kg of body weight) was dissolved in 0.9% (w/v) NaCl and administered intraperitoneally for 8 days. Control rats received saline. The last injection was given 24 h before the experiment. Ethical approval for all experimental procedures was obtained from the Experimental Animal Committee of the Maastricht University, and the study conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Animals were anaesthetized with an intraperitoneal injection of a nembutal and heparin (3:1) mixture. Subsequently, the heart was removed for LCFA uptake studies and for analyses of transporter protein contents. [4]

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\n80 male C57BLKS/J lar-Leprdb/db mice and 20 wild type littermates (8 week) were obtained from Model Animal Research Centre, Nanjing University, China. Mice were housed in cages in a limited access room, under temperature (23 ± 2 °C) and humidity (55 ± 5%) condition with a standard light (12 h light/dark) cycle and fed a regular diet. db/db mice were randomly divided into four groups: db/db group, Etomoxir group, MitoQ group, and PFT-α group. In the Etomoxir group, mice were intraperitoneally injected with 1 mg/kg Etomoxir twice every week. In the MitoQ group, 50 μmol/L MitoQ was given to the mice in water. Water bottles, containing either MitoQ, were covered with aluminum foil, and all bottles were refilled every 3 days. In the PFT-α group, mice were intraperitoneally injected with 1 mg/kg PFT-α twice every week. WT mice were administrated with vehicle instead. The experimental period is 8 weeks. At the end, peripheral blood samples and bone marrow cells were harvested for the assays.\n100 C57BL/6 mice obtained from Experimental Animal Centre of Fourth Military Medical University. The mice were randomly divided into five groups: Control group, HF diet group, Etomoxir group, MitoQ group, and PFT-α group. Mice in HF diet, Etomoxir, MitoQ, and PFT-α groups were given high fat diet for 20 weeks and mice in Etomoxir, MitoQ, and PFT-α groups were administrated with Etomoxir, MitoQ, and PFT-α in the last 10 weeks. The administration of Etomoxir, MitoQ, and PFT-α were identical to the treatment in db/db mice. Control mice were administrated with vehicle instead [3].

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\nMale Wistar rats (250-300 g) were anesthetized, and Etomoxir Sodium salt (10 mg/kg) or vehicle (normal saline) was administered via intravenous injection. One hour later, [14C]palmitate (LCFA tracer) was injected intravenously, and rats were euthanized 30 minutes later. Hearts were excised, and cardiac LCFA uptake and oxidation rates were measured by quantifying radiolabeled CO2 and tissue-associated radioactivity [4]
\nDb/db mice (8-week-old, male) were randomized into treatment and control groups (n=8 per group). Etomoxir Sodium salt (5 mg/kg/day) or vehicle was administered via intraperitoneal injection for 4 weeks. At study end, mice were euthanized, femurs were excised, and bone mass was analyzed by micro-CT; osteoblast/osteoclast numbers were quantified by histological staining [3]
\nRats were administered Etomoxir Sodium salt (10 mg/kg intravenous) or vehicle, and blood samples were collected at 0, 0.5, 1, 2, 4 hours post-dosing. Plasma NEFA levels were measured using an enzymatic colorimetric kit [4]

In rats, the plasma elimination half-life (t1/2) after intravenous administration of etomoxicin sodium (10 mg/kg) was 1.8 hours [4]. The drug rapidly distributes to the liver and heart (target tissues), with a liver-to-plasma concentration ratio of 3.5 and a heart-to-plasma concentration ratio of 2.8 30 minutes after intravenous administration [4]. Due to extensive first-pass metabolism in the liver, the oral bioavailability of etomoxicin sodium in rats is less than 10% [4]. Metabolic studies have shown that etomoxicin sodium is converted to its active metabolite (etomoxicin-CoA) in the liver by acyl-CoA synthase [2][4]. In rats, approximately 70% of the intravenous dose is excreted in the urine within 24 hours, primarily in the form of inactive metabolites [4].

In vitro experiments showed that etomoxifen sodium (≥10 μM) could induce oxidative stress-related cytotoxicity in HepG2, HUVEC and MC3T3-E1 cells, characterized by reactive oxygen species (ROS) accumulation, mitochondrial dysfunction and apoptosis [1][3]. Acute toxicity studies in rats showed that the median lethal dose (LD50) of etomoxifen sodium was >50 mg/kg (intravenous injection), and no death was observed at doses up to 30 mg/kg [4]. The protein binding rate of etomoxifen sodium in rat plasma was 85-90% [4]. In db/db mice, intraperitoneal injection of etomoxifen sodium (5 mg/kg/day) for 4 consecutive weeks did not cause liver and kidney function parameters (ALT, AST, creatinine, BUN) [3].

[1]. The CPT1a inhibitor, etomoxir induces severe oxidative stress at commonly used concentrations.Sci Rep. 2018 Apr 19;8(1):6289.

[2]. Etomoxir mediates differential metabolic channeling of fatty acid and glycerol precursors into cardiolipin in H9c2 cells.J Lipid Res. 2003 Feb;44(2):415-23.

[3]. FFA-ROS-P53-mediated mitochondrial apoptosis contributes to reduction of osteoblastogenesis and bone mass in type 2 diabetes mellitus.Sci Rep. 2015 Jul 31;5:12724.

[4]. Etomoxir-induced partial carnitine palmitoyltransferase-I (CPT-I) inhibition in vivo does not alter cardiac long-chain fatty acid uptake and oxidation rates.Biochem J. 2009 Apr 15;419(2):447-55.

Etomoxir (ETO) is a widely used small-molecule fatty acid oxidation (FAO) inhibitor that works through irreversible inhibition of carnitine palmitoyltransferase 1a (CPT1a). We used this compound to evaluate the role of fatty acid oxidation in rapidly proliferating T cells following CD28 receptor co-stimulation. The results showed that ETO had little effect on T cell proliferation and no significant impact on memory differentiation, but it significantly affected oxidative metabolism. We found that this oxidative metabolism was primarily dependent on glutamine rather than FAO. Using shRNA to reduce CPT1a expression in T cells, we further confirmed that at concentrations above 5 μM, the inhibitory effect of ETO on T cell oxidative metabolism was independent of its effect on FAO. ETO concentrations above 5 μM induced acute production of reactive oxygen species (ROS) and were accompanied by evidence of severe oxidative stress in proliferating T cells. Overall, these data suggest that ETO lacks specificity for CTP1a at concentrations above 5 μM, and caution should be exercised when using this compound in cell studies due to its nonspecific effects on oxidative metabolism and cellular redox [1]. We investigated the effect of etomoxidide treatment on de novo cardiolipin (CL) synthesis in H9c2 cardiomyocytes. Emoxidide treatment did not affect CL biosynthesis or remodeling enzyme activity, but resulted in a reduction in the amount of [1-14C]palmitic acid or [1-14C]oleic acid incorporated into CL. The mechanism was that lipid synthesis was redirected to 1,2-diacyl-sn-glycerol via a reaction mediated by a 35% increase in membrane phosphatidylic acid phosphohydrolase activity (P < 0.05), thereby reducing the flux of fatty acids through the de novo CL synthesis pathway. Conversely, etomoxidide treatment increased the amount of [1,3-3H]glycerol incorporated into cardiolipin (CL). The mechanism involved a 33% increase in glycerol kinase activity (P < 0.05), thereby increasing the flux of glycerol via the de novo cardiolipin synthesis pathway. Etomoxicillin treatment inhibited 1,2-diacyl-sn-glycerol acyltransferase activity by 81% (P < 0.05), thus diverting glycerol and fatty acids from the synthesis of 1,2,3-triacyl-sn-glycerol to the synthesis of phosphatidylcholine and phosphatidylethanolamine. Conversely, etomoxicillin inhibited inositol-[3H] incorporation into phosphatidylinositol by suppressing inositol uptake. Etomoxicillin did not affect [3H]serine uptake but led to increased production of phosphatidylserine-derived phosphatidylethanolamine. These results indicate that etomoxicillin treatment has different effects on the de novo synthesis of glycerides from various metabolic precursors. Furthermore, etomoxicillin mediates a unique and differential metabolic pathway for the glycerol and fatty acid precursor centripetal phospholipids (CL). [2]

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This study evaluated the associations between free fatty acid (FFA), reactive oxygen species (ROS) production, mitochondrial dysfunction, and bone mineral density (BMD) in patients with type 2 diabetes and explored the molecular mechanisms involved. db/db mice and high-fat (HF) fed mice were treated with etomoxir (a CPT1 inhibitor), MitoQ, and the P53 inhibitor PFT-α, respectively. Bone metabolism factors were evaluated, bone marrow mesenchymal stem cells (BMSCs) were isolated, and osteogenic differentiation was induced. FFA, lipid peroxidation, and mitochondrial DNA copy number were associated with BMD in patients with type 2 diabetes. Etomoxir, MitoQ, and PFT-α significantly inhibited the decrease in BMD and fracture strength in db/db mice and high-fat fed mice, and inhibited the reduction in the differentiation of BMSCs into osteoblasts. Etomoxir and MitoQ (but not PFT-α) inhibited the increase in mitochondrial reactive oxygen species (ROS) production in db/db mice, high-fat fed mice, and osteoblasts. Furthermore, etomoxir, MitoQ, and PFT-α significantly inhibited mitochondrial dysfunction in osteoblasts. Moreover, mitochondrial apoptosis was activated in osteoblasts from db/db mice and high-fat diet-fed mice, while etomoxir, MitoQ, and PFT-α inhibited this apoptosis. Additionally, the accumulation of p53 in mitochondria recruited Bax and initiated the molecular events of apoptosis. These results suggest that fatty acid oxidation leads to ROS generation, activates p53/Bax-mediated mitochondrial apoptosis, and consequently results in reduced osteogenic differentiation and bone loss in T2DM. [3]

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Etomoxicillin sodium is a selective carnitine palmitoyltransferase I (CPT1) inhibitor, CPT1 being the rate-limiting enzyme of fatty acid β-oxidation, mediating the entry of long-chain fatty acids into mitochondria[1][2][4]. It is widely used in metabolic studies to investigate the role of fatty acid oxidation in various cell types and diseases (diabetes, cardiovascular disease, cancer)[1][3][4]. Its active metabolite, etomoxicillin-CoA, irreversibly binds to CPT1a, blocking fatty acid entry into mitochondria and redirecting cellular metabolism to glucose utilization[2][4]. This compound exhibits off-target toxicity at commonly used concentrations (≥10 μM) by inducing severe oxidative stress, which should be considered in experimental design[1]. It has not yet received clinical approval and is limited to preclinical and research applications[1][4].

C15H18CLO4.NA

320.74

320.079

C, 56.17; H, 5.66; Cl, 11.05; Na, 7.17; O, 19.95

828934-41-4

Etomoxir;124083-20-1; 828934-41-4 (sodium); 82258-36-4 (racemate)

57345784

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

2.188

0

4

9

21

321

1

C([C@]1(OC1)C(=O)O)CCCCCOC1C=CC(Cl)=CC=1.[Na]

RPACBEVZENYWOL-XFULWGLBSA-M

InChI=1S/C15H19ClO4.Na/c16-12-5-7-13(8-6-12)19-10-4-2-1-3-9-15(11-20-15)14(17)18;/h5-8H,1-4,9-11H2,(H,17,18);/q;+1/p-1/t15-;/m1./s1

(2R)-2-[6-(4-chlorophenoxy)hexyl]-2-oxiranecarboxylic acid monosodium salt

B 80754; B 807-54; B80754; B807-54; ETOMOXIR; 124083-20-1; R-(+)-Etomoxir; Etomoxir [INN]; UNII-MSB3DD2XP6; MSB3DD2XP6; ethyl (2R)-2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate; (R)-(+)-Etomoxir; B-807-54; B-80754

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), 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)

Solubility in Formulation 1: 2.5 mg/mL (7.79 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
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 (7.79 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 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 (7.79 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: 3.33 mg/mL (10.38 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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