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
Bioavailability is 100% following oral administration.

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Metabolism / Metabolites
Hepatic, primarily via CYP3A4.
Etoricoxib has known human metabolites that include Etoricoxib 1'-N'-oxide and 6-Hydroxymethyletoricoxib.

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Biological Half-Life
22 hours

Protein Binding
92%

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Etoricoxib is not approved for marketing in the United States by the U.S. Food and Drug Administration but is available in other countries. No information is available on the use of etoricoxib during breastfeeding. Because it is 92% protein bound, the amounts in milk are likely to be very low. Some guidelines recommend avoiding etoricoxib because of a lack of data during breastfeeding. An alternate drug may be preferred, especially while nursing a newborn or preterm infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

[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 is a member of the class of bipyridines that is 2,3'-bipyridine which is substituted at the 3, 5, and 6' positions by 4-(methylsulfonyl)phenyl, chlorine, and methyl groups, respectively. It has a role as a cyclooxygenase 2 inhibitor and a non-steroidal anti-inflammatory drug. It is a sulfone, a member of bipyridines and an organochlorine compound.
Etoricoxib is a new COX-2 selective inhibitor. Current therapeutic indications are: treatment of rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, chronic low back pain, acute pain and gout. Like any other COX-2 selective inhibitor, Etoricoxib selectively inhibits isoform 2 of cyclo-oxigenase enzyme (COX-2) to reduce the generation of prostaglandins (PGs) from arachidonic acid. It is approved in more than 60 countries worldwide but not in the US.
Etoricoxib is a synthetic, nonsteroidal anti-inflammatory drug (NSAID) with antipyretic, analgesic, and potential antineoplastic properties. Etoricoxib specifically binds to and inhibits the enzyme cyclooxygenase-2 (COX-2), resulting in inhibition of the conversion of arachidonic acid into prostaglandins. Inhibition of COX-2 may induce apoptosis and inhibit tumor cell proliferation and angiogenesis.
A sulfone and pyridine derivative that acts as a cyclooxygenase-2 inhibitor. It is used as a NSAID for the treatment of pain associated with RHEUMATOID ARTHRITIS and ANKYLOSING SPONDYLITIS. It is also used for the short-term treatment of moderate postoperative dental pain.

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Drug Indication
For the treatment of rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, chronic low back pain, acute pain and gout.

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Mechanism of Action
Like any other COX-2 selective inhibitor Etoricoxib selectively inhibits isoform 2 of cyclo-oxigenase enzyme (COX-2), preventing production of prostaglandins (PGs) from arachidonic acid.

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Pharmacodynamics
Etoricoxib is a COX-2 selective inhibitor (approximately 106 times more selective for COX-2 inhibition over COX-1).

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Blood ALT and AST activities which are measured to evaluate protective effect of Etoricoxib in the liver I/R damage are the most commonly used parameters in evaluation of the hepatic function. Tuncer et al. reported that levels of ALT and AST are elevated during the hepatic I/R damage. Also ALT and AST have been experimentally shown to significantly increase in the hepatic oxidative tissue damage caused by I/R. Free oxygen radicals whose production is stimulated in the liver I/R damage are accused of the increase in ALT and AST activities. ALT and AST activities were found to be significantly lower and very close to the baseline values in the etoricoxib group than the LIRC group. This can be considered to indicate that liver functions in rats were much more different in the etoricoxib group than in the LIRC group. In conclusion, etoricoxib prevented the liver oxidative damage due to I/R. Oxidant/antioxidant balance changed in favour of oxidants in the LIRC and in favour of antioxidants in the etoricoxib group. In addition, etoricoxib improved hepatic dysfunctions caused by I/R. This information suggests that etoricoxib can be beneficial in prevention of the damage which may emerge in the clinical I/R process. [2]

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In recent years an intimate linkage was established among oncogenes, anti-oncogenes, and malignancy in the context of cell growth, differentiation, proliferation and apoptosis (Evan and Vousdan, 2001). In the present study we attempted to assess the effect of Etoricoxib which is a specific COX-2 inhibitor on the status of genomic DNA, proliferation markers (PCNA and BrdU) and apoptosis in DMH induced colon cancer in rat model. Histopathologically, there was seen a marked dysplasia as well as hyperplasia in DMH treated rats as also the morphologically identified neoplastic growth on MPLs. Oral administration of Etoricoxib was able to weaken these features prominently indicating its efficiency as a chemopreventive agent at the present dose for a period of six weeks, which can be considered as the early stage of carcinogenesis (Tanwar et al., 2009; Sharma et al., 2010). As proliferation is a key event in the development and normal functioning of intestine, several reports from animal studies showed that experimental colonic tumors induced by DMH are of epithelial origin and results in increased colonic crypt cellularity and colonic crypt cell proliferation (Richards, 1977; Heitman et al., 1983). Expression of PCNA by cells during the S and G2 phases of the cell cycle makes the protein a good cell proliferation marker. It also actively participates in a number of the molecular pathways responsible for the life and death of the mammalian cell (Paunesku et al., 2001). In addition to decreasing the incidence of MPLs and histopathological changes, the results from PCNA immunohistochemistry and fluorescent staining indicate that Etoricoxib reduced the proliferation and apoptosis in cancer cells, respectively. The data suggest that the cells don’t undergo apoptosis in DMH treated animals as compared to control whereas simultaneous administration of Etoricoxib increased the number of apoptotic cells. It had been reported that there ought to be a balance between the antiproliferative and apoptotic effect of the NSAID such as sulindac sulphide in cultured cells and indicate that apoptotic cells may strongly express the proliferation biomarkers Ki-67 and PCNA (Qias et al., 1997). NS-398, which is a specific COX-2 inhibitor, was also described to reduce cell proliferation of MC-26 cell line (Yao et al., 2004). This effect was associated with a reduction of PCNA, thus increasing the chaperoning of DNA polymerase. Interestingly, meloxicam was also able to downregulate PCNA and cyclin A in HepG2 cell line (hepatocellular carcinoma cells) leading to an inhibition of the cell proliferation (Li et al., 2006). BrdU is a thymidine analogue which, after incorporation into normal and malignant cells during S-phase of the cell cycle, can be detected using a monoclonal antibody and has several advantages over thymidine autoradography (Ma et al., 2002). Immunohistochemistry of BrdU demonstrates that cell proliferation to be maximum in DMH treated animals alone. As BrdU is thought to be incorporated in the cells during S-phase of cell division, therefore by allowing the knowledge of the percentage of BrdU positive cells the proliferation index can be easily deduced. Moreover, the number of BrdU positive cells was found to be maximum in DMH treated cells followed by Etoricoxib treated cells. Increased number of apoptotic cells and fragmentation of DNA afterEtoricoxib co-treatment reveal proapoptotic efficacy of NSAID in colon cancer.
In conclusion, our data suggest that the proliferation marker PCNA and BrdU as found in DMH treated animals strongly indicating the proliferating events in an early stage of carcinogenesis in 6 weeks. However, the Etoricoxib showed the anti-proliferative effect on DMH and Etoricoxib co-treated animals by dominantly downregulating these antigens. Similarly, apoptosis was found to be reduced in DMH-treated animals and Etoricoxib showed the revival of such effect [3].

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Based on our results, it seems likely that Etoricoxib medication, that is a highly selective inhibition of COX-2, at cell medium concentrations corresponding to clinically administered dosages and associated local tissue concentrations within the periodontal ligament does not have substantial effects on hPDL fibroblast-mediated periodontal inflammation, extracellular matrix remodeling, RANK-L/OPG expression, and osteoclastogenesis during simulated orthodontic compressive force application. Due to its also favorable side effect profile and high analgesic efficacy, particularly for orthodontic pain, it could therefore be a valid analgesic during orthodontic treatment.[4]

C18H15N2O2SCL

358.8419

358.054

C, 60.25; H, 4.21; Cl, 9.88; N, 7.81; O, 8.92; S, 8.94

202409-33-4

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

123619

Light yellow to yellow solid powder

1.3±0.1 g/cm3

510.0±50.0 °C at 760 mmHg

134-135°C

262.2±30.1 °C

0.0±1.3 mmHg at 25°C

1.601

2.21

0

4

3

24

514

0

O=S(C1=CC=C(C2=CC(Cl)=CN=C2C3=CC=C(C)N=C3)C=C1)(C)=O

MNJVRJDLRVPLFE-UHFFFAOYSA-N

InChI=1S/C18H15ClN2O2S/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

5-Chloro-6'-methyl-3-[4-(methylsulfonyl)phenyl]-2,3'-bipyridine

L-791456, L791456, Etoricoxib; 202409-33-4; Arcoxia; Tauxib; 5-Chloro-6'-methyl-3-(4-(methylsulfonyl)phenyl)-2,3'-bipyridine; Etoricoxibe; Etropain; Torcoxia; L 791456, MK-0663, MK 0663, MK0663; Arcoxia

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)

DMSO : ~100 mg/mL (~278.68 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.97 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 (6.97 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 (6.97 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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 (Please use freshly prepared in vivo formulations for optimal results.)