COX-2 (IC50 = 1.1 μM); COX-1 (IC50 = 116 μM)

Etoricoxib, also known as MK-0663, is an oral COX-2 inhibitor that is selective. Its IC50 values for COX-2, COX-1, and pure human COX-2 in human whole blood are 1.1 μM, 116 μM, and 5, respectively. I.Q. PGE2 produced by CHO (COX-2) cells (IC50, 79 nM), detergent-pure human COX-2 (IC50, 4.1 μM), and purified PGE2 produced by U937 microsomes Effect (low substrate; IC50, 12.1 μM) are all inhibited by etoricoxib (MK-0663). On the other hand, etoricoxib (MK-0663) has a low Ki of 167 μM and minimal action against COX-1 [1].

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We report here the preclinical profile of Etoricoxib (MK-0663) [5-chloro-2-(6-methylpyridin-3-yl)-3-(4-methylsulfonylphenyl) pyridine], a novel orally active agent that selectively inhibits cyclooxygenase-2 (COX-2), that has been developed for high selectivity in vitro using whole blood assays and sensitive COX-1 enzyme assays at low substrate concentration. Etoricoxib selectively inhibited COX-2 in human whole blood assays in vitro, with an IC(50) value of 1.1 +/- 0.1 microM for COX-2 (LPS-induced prostaglandin E2 synthesis), compared with an IC(50) value of 116 +/- 8 microM for COX-1 (serum thromboxane B2 generation after clotting of the blood). Using the ratio of IC(50) values (COX-1/COX-2), the selectivity ratio for the inhibition of COX-2 by etoricoxib in the human whole blood assay was 106, compared with values of 35, 30, 7.6, 7.3, 2.4, and 2.0 for rofecoxib, valdecoxib, celecoxib, nimesulide, etodolac, and meloxicam, respectively. Etoricoxib did not inhibit platelet or human recombinant COX-1 under most assay conditions (IC(50) > 100 microM). In a highly sensitive assay for COX-1 with U937 microsomes where the arachidonic acid concentration was lowered to 0.1 microM, IC(50) values of 12, 2, 0.25, and 0.05 microM were obtained for etoricoxib, rofecoxib, valdecoxib, and celecoxib, respectively. These differences in potency were in agreement with the dissociation constants (K(i)) for binding to COX-1 as estimated from an assay based on the ability of the compounds to delay the time-dependent inhibition by indomethacin. [1]

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Human periodontal ligament (hPDL) fibroblasts play a major role during periodontitis and orthodontic tooth movement, mediating periodontal inflammation, osteoclastogenesis, and collagen synthesis. The highly COX-2-selective NSAID Etoricoxib has a favorable systemic side effect profile and high analgesic efficacy, particularly for orthodontic pain. In this in vitro study, we investigated possible side effects of two clinically relevant etoricoxib concentrations on the expression pattern of mechanically strained hPDL fibroblasts and associated osteoclastogenesis in a model of simulated orthodontic compressive strain occurring during orthodontic tooth movement. hPDL fibroblasts were incubated for 72 h under physiological conditions with etoricoxib at 0 μM, 3.29 μM, and 5.49 μM, corresponding to clinically normal and subtoxic dosages, with and without mechanical strain by compression (2 g/cm2) for the final 48 h, simulating conditions during orthodontic tooth movement in compressive areas of the periodontal ligament. We then determined gene and/or protein expression of COX-2, IL-6, PG-E2, RANK-L, OPG, ALPL, VEGF-A, P4HA1, COL1A2, and FN1 via RT-qPCR, ELISA, and Western blot analyses as well as apoptosis, necrosis, cell viability, and cytotoxicity via FACS, MTT, and LDH assays. In addition, hPDL fibroblast-mediated osteoclastogenesis was assessed by TRAP staining in coculture with RAW267.4 cells for another 72 h. Gene and protein expression of all evaluated factors was significantly induced by the mechanical compressive strain applied. Etoricoxib at 3.29 μM and 5.49 μM significantly inhibited PG-E2 synthesis, but not COX-2 and IL-6 gene expression nor RANK-L-/OPG-mediated osteoclastogenesis or angiogenesis (VEGF-A). Extracellular matrix remodeling (COL1A2, FN1) and bone anabolism (ALPL), by contrast, were significantly stimulated particularly at 5.49 μM. In general, no adverse etoricoxib effects on hPDL fibroblasts regarding apoptosis, necrosis, cell viability, or cytotoxicity were detected. Clinically dosed etoricoxib, that is, a highly selective COX-2 inhibition, did not have substantial effects on hPDL fibroblast-mediated periodontal inflammation, extracellular matrix remodeling, RANK-L/OPG expression, and osteoclastogenesis during simulated orthodontic compressive strain [4].

In rats, carrageenan-induced paw edema, carrageenan-induced paw pain hypersensitivity, and endotoxin-induced fever are all dose-dependently inhibited by Etoricoxib (MK-0663) (0.1-30 mg/kg, po). In a rat hyperalgesia model, Etoricoxib (≥10 mg/kg) totally reverses the hyperalgesic response. Rats' urine excretion of 51Cr is unaffected by Etoricoxib (MK-0663) at 200 mg/kg/day, and monkeys are unaffected by 100 mg/kg/day of the drug [1]. Rats' levels of glutathione Peptide reductase (GSHRd) and total glutathione (tGSH) are effectively decreased whereas those of malondialdehyde (MDA) and myeloperoxidase (MPO) are effectively increased by etoricoxib (MK-0663) at 50 and 100 mg/kg. Rats' NO decrease is considerably inhibited by etoricoxib (MK-0663) at a dose of 100 mg/kg [2]. Rats with 1,2-dimethylhydrazine dihydrochloride (DMH)-induced numerous plaque lesions, hyperplasia, and dysplasia can benefit from etoricoxib (MK-0663) (0.64 mg/kg, po) [3].

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Etoricoxib was a potent inhibitor in models of carrageenan-induced paw edema (ID(50) = 0.64 mg/kg), carrageenan-induced paw hyperalgesia (ID(50) = 0.34 mg/kg), LPS-induced pyresis (ID(50) = 0.88 mg/kg), and adjuvant-induced arthritis (ID(50) = 0.6 mg/kg/day) in rats, without effects on gastrointestinal permeability up to a dose of 200 mg/kg/day for 10 days. In squirrel monkeys, etoricoxib reversed LPS-induced pyresis by 81% within 2 h of administration at a dose of 3 mg/kg and showed no effect in a fecal 51Cr excretion model of gastropathy at 100 mg/kg/day for 5 days, in contrast to lower doses of diclofenac or naproxen. In summary, etoricoxib represents a novel agent that selectively inhibits COX-2 with 106-fold selectivity in human whole blood assays in vitro and with the lowest potency of inhibition of COX-1 compared with other reported selective agents. [1]

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Etoricoxib in 50 and 100 mg/kg doses changed the levels of oxidant/antioxidant parameters such as MDA, MPO, tGSH, GSHRd, GST, SOD, NO, and 8-OH/Gua in favour of antioxidants. Furthermore, Etoricoxib prevented increase of COX-2 gene expression and ALT and AST levels. This important protective effect of etoricoxib on the rat liver I/R can be tested in the clinical setting. [2]

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In the present study, we assessed effects of Etoricoxib, a non-steroidal anti-inflammatory drug, on proliferation and apoptosis in 1,2-dimethylhydrazine dihydrochloride (DMH) induced colon lesion development. Male SD rats were divided into four groups: Group 1 controls receiving the vehicle treatment; Group 2 administered DMH weekly (30 mg/kg body weight, subcutaneously) alone; Group 3, DMH weekly plus Etoricoxib (0.64 mg/kg body weight, orally) daily; and Group 4, Etoricoxib alone. After six weeks of treatment, animals were sacrificed and colons were analysed for morphological and histopathological features. Well characterized pre-neoplastic aberrations such as multiple plaque lesions, hyperplasia and dysplasia were found in the DMH treated group whereas these features were reduced with co-administration of etoricoxib. To study apoptosis, colonocytes were isolated by metal chelation from colonic sacs and studied by fluorescent staining and further confirmed by DNA fragmentation. The DMH treated animals had fewer apoptotic nuclei as compared to the controls, but numbers were higher with DMH+etoricoxib as well as etoricoxib alone. Expression of proliferative cell nuclear antigen (PCNA), assessed by Western blot analysis and immunohistochemistry, was found to be elevated by DMH treatment group and again reduced by etoricoxib. Results for bromodeoxyuridine incorporation (BrdU) were in agreement. It may be concluded that the drug, etoricoxib, has the potential to act as an anti-apoptotic and anti- proliferative agent in the colon [3].

Experimental Design [4]
At a density of 70000 cells in 2 ml DMEM per well, pooled hPDL fibroblasts (3-5th passage) were seeded onto 6-well cell culture plates. For RT-qPCR analyses as well as LDH/MTT assays, hPDL fibroblasts in six experimental groups with 9 wells (n = 9) on three plates (N = 3) each were, respectively, incubated with either 0 μM (control), 3.29 μM, or 5.49 μM Etoricoxib for 72 h, corresponding to assumed local concentrations reached in the PDL during normal and subtoxic clinical dosing in man, with or without (3/3 wells per plate) compressive mechanical strain of 2 g/cm2 for 48 h after a 24 h preincubation phase by means of a glass disc according to a published and well-established protocol for the simulation of compressive orthodontic mechanical strain (Figure 1(a)). RANK-L Western blot was performed for seven (N = 7), ELISA for six (N = 2, n = 6), and FACS analyses in duplicates for three (N = 3, n = 6) biological replicates (Figure 1(b)).

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Cell Cytotoxicity as Assessed by LDH Assay [4]
Commercially available lactate dehydrogenase (LDH) assays were used according to the manufacturer's instructions. 100 μl freshly prepared LDH solution (22 μl catalyst mixed with 1 ml dye) was added to 100 μl supernatant and incubated in the dark at room temperature for 30 min before adding 50 μl of stop solution. An ELISA reader, was used to measure LDH activity (absorbance at 490 nm), subtracting background absorbance at 690 nm.

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Cell Viability (Mitochondrial Enzymatic Activity) as Assessed by MTT Assay [4]
For MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid) assays, 400 μl MTT solution in PBS (5 mg/ml) was added per well for the final five hours of incubation. After, removal of the 1 ml DMSO per well was added and incubation continued at 37°C for another 5 min measuring final absorbance (cell viability) at 550 nm with an ELISA reader.

Experiment Groups [2]
Experimental animals were divided into four groups as liver I/R control (LIRC), 50 mg/kg Etoricoxib + liver I/R (ETO-50), 100 mg/kg etoricoxib + liver I/R (ETO-100), and healthy group sham operated (HG).

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Pharmacological and Surgical Procedures [2]
One hour before thiopental sodium anesthesia, ETO-50 group was orally administered 50 mg/kg Etoricoxib and ETO-100 group 100 mg/kg oral etoricoxib by gavage, whereas LIRC and HG rat groups were given distilled water as the solvent by the same method. Laparotomy was performed in anterior part of the abdomen by vertically opening 3.5–4 cm long in the anesthetized rats. Then, hepatic artery was clamped (except HG group) in order to create total hepatic ischemia, providing one-hour ischemia and 6-hour reperfusion. At the end of this duration, the rats groups were killed by high doses of anesthesia and levels of oxidant/antioxidant parameters such as MDA, MPO, tGSH, GSHRd, GST, SOD, NO and 8-OH/Gua, and COX-2 gene expression in the liver tissues were determined. Blood values of ALT and AST were measured. Results obtained from the ETO-50 and ETO-100 groups were evaluated in comparison with those of the LIRC and HG groups and evaluated.

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Treatment Schedule [3]
Animals were assorted into the following groups with four to six animals in each group: Control Group, Animals were administrated the vehicle (1mM EDTAsaline subcutaneously) in weekly injection and 0.5% carboxymethyl cellulose per oral daily; DMH Group, animals were administrated with DMH weekly at a dose of 30 mg/kg body weight subcutaneously, as had been established in our laboratory earlier (Kanwar et al., 2008) - DMH was freshly prepared in 1mM EDTAsaline, pH adjusted to 7.0 using dilute NaOH solution; DMH + Etoricoxib Group, Etoricoxib was given daily per oral at its therapeutic anti-inflammatory dose (ED50 for rats, 0.64 mg/kg body weight) to the animals along with the weekly administration of DMH (Riendeau et al., 2001); and Etoricoxib Group: Etoricoxib alone was administered orally daily (0.64 mg/kg body weight). The anti-inflammatory dose was established earlier in a model of carragenan induced oedema in rat hind paw (Sharma et al., 2010). After six weeks, animals were kept on overnight fasting with drinking water ad libitum and sacrificed the next day. The animal body weights in all the groups were recorded once in a week till the termination.

Absorption, Distribution and Excretion
The bioavailability after oral administration is 100%. Metabolism/Metabolites Primarily metabolized in the liver via CYP3A4. Known metabolites of etoricoxib include etoricoxib 1'-N'-oxide and 6-hydroxymethyl etoricoxib. Biological Half-Life 22 hours

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Protein Binding
92% Effects During Pregnancy and Lactation ◉ Overview of Use During Lactation
Etoricoxib has not yet received marketing approval from the U.S. Food and Drug Administration (FDA), but it is available in other countries. There is currently no information regarding the use of etoricoxib during lactation. Due to its high protein binding rate of 92%, the concentration in breast milk may be very low. Some guidelines recommend that breastfeeding women avoid using etoricoxib due to a lack of relevant data. Especially when breastfeeding newborns or premature infants, alternative medications are recommended.
◉ Effects on Breastfed Infants
As of the revision date, no relevant published information was found.
◉ Effects on Lactation and Breast Milk
As of the revision date, no relevant published information was found.

[1]. Etoricoxib (MK-0663): preclinical profile and comparison with other agents that selectively inhibit cyclooxygenase-2. J Pharmacol Exp Ther. 2001 Feb;296(2):558-66.

[2]. The Effect of Etoricoxib on Hepatic Ischemia-Reperfusion Injury in Rats. Oxid Med Cell Longev. 2015;2015:598162.

[3]. Anti-proliferative and apoptotic effects of etoricoxib, a selective COX-2 inhibitor, on 1,2-dimethylhydrazine dihydrochloride-induced colon carcinogenesis. Asian Pac J Cancer Prev. 2010;11(5):1329-33.

[4]. Effects of the Highly COX-2-Selective Analgesic NSAID Etoricoxib on Human Periodontal Ligament Fibroblasts during Compressive Orthodontic Mechanical Strain. Mediators Inflamm. 2019 Mar 10;2019:2514956.

Etoricoxib belongs to the bipyridine class of compounds, with the structure 2,3'-bipyridine, substituted at the 3', 5', and 6' positions with 4-(methanesulfonyl)phenyl, chloro, and methyl, respectively. It is a cyclooxygenase-2 inhibitor and a nonsteroidal anti-inflammatory drug (NSAID). It is a sulfone compound, belonging to the bipyridine class, and is also an organochlorine compound. Etoricoxib is a novel selective COX-2 inhibitor. Current therapeutic indications include rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, chronic low back pain, acute pain, and gout. Like other selective COX-2 inhibitors, etoricoxib selectively inhibits the cyclooxygenase 2 (COX-2) isoenzyme, thereby reducing the production of prostaglandins (PGs) from arachidonic acid. Etoricoxib has been approved in more than 60 countries worldwide, but not yet in the United States. Etoricoxib is a synthetic nonsteroidal anti-inflammatory drug (NSAID) with antipyretic, analgesic, and potential antitumor effects. Etoricoxib specifically binds to and inhibits cyclooxygenase-2 (COX-2), thereby inhibiting the conversion of arachidonic acid to prostaglandins. Inhibition of COX-2 may induce apoptosis and inhibit tumor cell proliferation and angiogenesis. Etoricoxib is a sulfone and pyridine derivative that acts as a cyclooxygenase-2 inhibitor. It is used as a nonsteroidal anti-inflammatory drug (NSAID) to treat pain caused by rheumatoid arthritis and ankylosing spondylitis. It can also be used for short-term treatment of moderate postoperative toothache. Drug Indications: For the treatment of rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, chronic low back pain, acute pain, and gout. Mechanism of Action: Like other selective COX-2 inhibitors, etoricoxib selectively inhibits the cyclooxygenase 2 (COX-2) isoenzyme, thereby preventing the conversion of arachidonic acid to prostaglandins (PGs). Pharmacodynamics Etocoxib is a selective COX-2 inhibitor (approximately 106-fold more selective for COX-2 than for COX-1). Plasmodynamics Blood ALT and AST activities are the most commonly used parameters for assessing the protective effect of etoricoxib against hepatic ischemia/reperfusion injury. Regarding liver function, Tuncer et al. reported elevated ALT and AST levels during hepatic ischemia/reperfusion (I/R) injury. Experiments have also shown that I/R-induced hepatic oxidative damage leads to a significant increase in ALT and AST. Free radicals generated during hepatic I/R injury are considered to be the cause of the increased ALT and AST activities. Compared to the LIRC group, etoricoxib group showed significantly reduced ALT and AST activities, very close to baseline values. This indicates that the liver function of rats in the etoricoxib group differed more significantly from that in the LIRC group. In conclusion, etoricoxib can prevent I/R-induced hepatic oxidative damage. The oxidant/antioxidant balance in the LIRC group was tilted towards oxidants, while the oxidant/antioxidant balance in the etoricoxib group was tilted towards antioxidants. In addition, etoricoxib improved I/R-induced liver dysfunction. These findings suggest that etoricoxib may help prevent damage that may occur during clinical ischemia/reperfusion. [2] In recent years, there has been a close relationship between oncogenes, tumor suppressor genes and malignancies in the context of cell growth, differentiation, proliferation and apoptosis (Evan and Vousdan, 2001). In this study, we sought to evaluate the effects of the specific COX-2 inhibitor etoricoxib on genomic DNA status, proliferation markers (PCNA and BrdU) and apoptosis in a DMH-induced rat colon cancer model. Histopathological examination showed that DMH-treated rats exhibited significant dysplasia and hyperplasia, and morphologically identifiable tumor growth was also observed on MPL. Oral etoricoxib significantly attenuated these characteristics, demonstrating its effectiveness as a chemopreventive agent at current doses for six weeks, which can be considered an early stage of carcinogenicity (Tanwar et al., 2009; Sharma et al., 2010). Since proliferation is a key event in intestinal development and normal function, multiple animal studies have shown that DMH-induced experimental colon tumors originate from epithelial cells and lead to an increase and proliferation of colonic crypt cells (Richards, 1977; Heitman et al., 1983). Cells express PCNA in the S and G2 phases of the cell cycle, making it a good marker of cell proliferation. It also actively participates in various molecular pathways related to the life and death of mammalian cells (Paunesku et al., 2001). In addition to reducing the incidence of MPL and histopathological changes, PCNA immunohistochemical and fluorescent staining results indicated that etoricoxib inhibited cancer cell proliferation and apoptosis, respectively. Data showed that, compared with the control group, no apoptosis occurred in the cells of animals treated with DMH, while simultaneous administration of etoricoxib increased the number of apoptotic cells. Previous reports have indicated that there should be a balance between the antiproliferative and pro-apoptotic effects of nonsteroidal anti-inflammatory drugs (NSAIDs) such as sulindac sulfide in cultured cells, and that apoptotic cells may strongly express the proliferation markers Ki-67 and PCNA (Qias et al., 1997). Furthermore, the specific COX-2 inhibitor NS-398 has also been reported to reduce the proliferation of the MC-26 cell line (Yao et al., 2004). This effect is associated with a reduction in PCNA, thereby enhancing the molecular chaperone function of DNA polymerase. Interestingly, meloxicam can also downregulate PCNA and cyclin A in the HepG2 cell line (hepatocellular carcinoma cells), thereby inhibiting cell proliferation (Li et al., 2006). BrdU is a thymidine analogue that is incorporated into normal and malignant cells during the S phase of the cell cycle. It can be detected by monoclonal antibodies and has several advantages over thymidine autoradiography (Ma et al., 2002). Immunohistochemical staining with BrdU showed that DMH-treated animals exhibited the most significant cell proliferation. Since BrdU is believed to be incorporated into cells during the S phase of cell division, the proliferation index can be easily derived by understanding the percentage of BrdU-positive cells. Furthermore, the DMH-treated group had the highest number of BrdU-positive cells, followed by the etoricoxib-treated group. The increased number of apoptotic cells and DNA fragmentation after etoricoxib combination therapy indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) have a pro-apoptotic effect in colon cancer. In conclusion, our data suggest that the expression of the proliferation markers PCNA and BrdU in DMH-treated animals strongly indicates proliferative events occurring in the early stages of carcinogenesis within 6 weeks. However, etoricoxib exhibited an anti-proliferative effect against DMH and etoricoxib combination therapy by significantly downregulating the expression of these antigens. Similarly, DMH-treated animals showed reduced apoptosis, while etoricoxib restored this effect [3]. Based on our results, etoricoxib (a highly selective COX-2 inhibitor) had no significant effect on hPDL fibroblast-mediated periodontal inflammation, extracellular matrix remodeling, RANK-L/OPG expression, and osteoclastogenesis during simulated orthodontic compression at cell culture concentrations comparable to clinical doses and at local tissue concentrations within the periodontal ligament. Due to its fewer side effects and significant analgesic effect, especially for orthodontic pain, etoricoxib may be an effective analgesic for orthodontic treatment [4].

C18H16CL2N2O2S

395.302841186523

394.031

C, 54.69; H, 4.08; Cl, 17.94; N, 7.09; O, 8.09; S, 8.11

202409-40-3

202409-33-4; Etoricoxib-d4;1131345-14-6; 202409-40-3; Etoricoxib-13C,d3;2748267-73-2;Etoricoxib-d3;850896-71-8

17895326

Typically exists as solid at room temperature

6.058

1

4

3

25

514

0

ClC1C=NC(C2=CN=C(C)C=C2)=C(C2C=CC(S(=O)(=O)C)=CC=2)C=1.Cl

NNGHGPAKTWAHHB-UHFFFAOYSA-N

InChI=1S/C18H15ClN2O2S.ClH/c1-12-3-4-14(10-20-12)18-17(9-15(19)11-21-18)13-5-7-16(8-6-13)24(2,22)23;/h3-11H,1-2H3;1H

5-chloro-2-(6-methylpyridin-3-yl)-3-(4-methylsulfonylphenyl)pyridine;hydrochloride

L-791456 hydrochloride; Etoricoxib hydrochloride; Etoricoxib hydrochloride; 202409-40-3; L-791,456 hydrochloride; ETORICOXIB HCL; L-791,456 monohydrochloride salt; UNII-138Y28RY5E; 138Y28RY5E; 2,3'-Bipyridine, 5-chloro-6'-methyl-3-(4-(methylsulfonyl)phenyl)-, hydrochloride (1:1); L791456 hydrochloride; L-791456 monohydrochloride salt; L 791456 hydrochloride;

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.

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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.)