FP ( Ki = 790 nM ); TP Receptor ( Ki = 2400 nM ); EP4 ( Ki = 1.3 nM ); EP3 ( Ki = 30 nM )

ONO-AE3-208 inhibits in vitro cell migration and invasion in a dose-dependent way while having no effect on cell proliferation[2]. When the EET synthesis inhibitor MS-PPOH is present, ONO-AE3-208 eliminates CTGF. Arachidonic acid (AA) causes the attached Af-Art to dilate in a dose-dependent manner; ONO-AE3-208 blocks this effect[3].

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An EP4 Antagonist ONO-AE3-208 Suppressed Invasion and Migration of Prostate Cancer Cells Without Affecting the Cell Proliferation[3]
By cell proliferation assays, we observed the effect of ONO-AE3-208 on the cell proliferation of PC3, LNCaP, LNCaP/mock, and LNCaP/EP4+. The proliferation rates of these cells were not changed by the administration of up to 10 µmol/L of ONO-AE3-208, despite the EP4 expression levels of the cells.

ONO-AE3-208 inhibits PC3 cell metastasis to the bone in vivo in mice[2]. When comparing the photon tumor burdens in the ONO-AE3-208-treated group and the control group, there is a significant time-dependent increase in the former. Compared to the latter, the former has a noticeably higher rate of metastasis formation. In the ONO-AE3-208-treated animals, the median time of metastasis formation is 29 days, whereas in the control group, it is 21 days[4].

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The EP4 inhibitor ONO-AE3-208 attenuates albuminuria in streptozotocin-diabetic eNOS knockout mice. ONO-AE3-208 reduces albuminuria and mesangial matrix accumulation in db/db mice. ONO-AE3-208 attenuates kidney injury in subtotally nephrectomized rats.[5]

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Experimental protocols for Connecting tubule glomerular feedback (CTGF) [2]
1)Time control for experiments #2 and #3: three consecutive concentration-response curves were generated by increasing luminal NaCl in the CNT from 10 to 80 mmol/L.
2)Effect of the EP4 antagonist ONO-AE3-208 on CTGF: three consecutive concentration-response curves were generated by increasing luminal NaCl in the CNT from 10 to 80 mmol/L. The EET synthesis inhibitor MS-PPOH (10−6 mol/L) was added to the second and third curves, and the EP4 antagonist ONO-AE3-208 (10−7 mol/L) to the third curve. This concentration of ONO-AE3-208 is 77 times its Ki for the EP4 receptor, and at least 100 times lower than its Ki for the EP2 receptor.
3)Effect of the EP4 antagonist L161982 on CTGF: this experiment was similar to #2 but the EP4 antagonist L161982 (10−5 mol/L) was used instead of ONO-AE3-208. This concentration of L161982 is 312 times its Ki for the EP4 receptor, and 6 times lower than its Ki for the EP2 receptor.
4)Effect of endothelium disruption on CTGF: CTGF was induced by increasing NaCl in the CNT from 10 to 80 mmol/L. Then a goat anti-human antibody against von Willebrand factor (14.29 mg/ml diluted 1:1000) plus 2% guinea pig complement were perfused into the lumen of the Af-Art for 10 minutes followed by a 20-minute wash-out period, and CTGF was induced again. To confirm complete functional removal of the endothelium, we added acetylcholine to the lumen of the Af-Art, 10−5 mol/L, a concentration we have repeatedly shown to be sufficient to dilate the Af-Art.
5)Effect of exogenous arachidonic acid (AA) in the CNT: After the Af-Art was preconstricted with NE, AA was added to the lumen of the CNT at increasing concentrations from 10−7 to 10−5 mol/L in the absence of NaCl. At the end of the experiment, we removed AA and switched the CNT luminal perfusate to 80 mmol/L NaCl.
6)Effect of an EP4 antagonist on CTGF induced by exogenous AA: this experiment was similar to #5, except that MS-PPOH was added to the lumen of the CNT and ONO-AE3-208 was added to the bath.
In all experiments, Af-Art diameter was measured in the region of maximal response to NE at three sites 3–5 μm apart and expressed as the average of these three measurements. Diameter was recorded at 5-second intervals with a video camera and measured with a computer equipped with Metavue image analysis software.

Cell Proliferation Assay[3]
Cell counting kit-8 (CCK8) assay was performed to assess the effect of ONO-AE3-208 on cell proliferation. 5 × 103 cells (PC3 cells) and 1 × 104 cells (LNCaP, LNCaP/mock and LNCaP/EP4+ cells) were seeded in 96-well plates. Then, every 24 h for 72 h, a batch of cells was stained with 10 μl of CCK8 regent at 37° for 2 h. The coloring reaction was quantified with an automatic plate reader at 450 nm. Each experiment was triplicated and performed three times independently.

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Invasion Assay[3]
Cell invasion activity of prostate cancer cells was assessed by BD BioCoat Matrigel Invasion Chambers. The cells were washed with PBS and resuspended in media without FBS at a density of 3 × 104cells/ml. 500 μl of cell suspension was put onto the upper chamber coated matrigel, and 750 μl of culture media with 1 % FBS were added to the lower chamber of the transwell. After 24 h incubation at 37 °C in 5 % CO2 incubator, the cells on the upper surface of the filters were removed by wiping with a cotton swab. The filters were fixed in 70 % ethanol and stained with hematoxylin. The stained cells were counted under a microscope in six randomly selected fields. At least three chambers from three different experiments were analyzed.

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Wound-Healing Assay[3]
Wound-healing assay was performed as previously described. Subconfluent PC3, LNCaP, LNCaP/mock, and LNCaP/EP4+ cells in 6-well culture dishes were scratched with a plastic pipette tip and cultured for 24 h. The widths of the “wound” (scratched areas) were measured by image J (http://rsbweb.nih.gov/ij/), and proportion of the wound healing was calculated by the following formula: 100 %—(width after 24 h/width at the beginning) × 100 %. Each experiment was triplicated and performed three times independently.

Animal model 1: Induction of colitis.[1]
DSS of the average molecular weight of 5,000 was administered to 8-week-old mice for 7 days at either 3% (low dose) or 7% (high dose) concentration in the drinking water. The addition of DSS or any drugs mentioned below to the drinking water did not affect water consumption of mice. Indomethacin also was added to the drinking water at a dose of 4 mg/kg/day and administered to the animals during the entire experimental period. This dose of indomethacin was reported to inhibit PGE2 production in rats and mice in vivo. An EP4 antagonist, ONO-AE3-208, 4-{4-Cyano-2-[2-(4-fluoronaphthalen-1-yl) propionylamino] phenyl} butyric acid (AE3-208), and an EP4 agonist, ONO-AE1-734, methyl-7-[(1R, 2R, 3R)-3-hydroxy-2-[(E)-(3S)-3-hydroxy-4-(m-methoxymethylphenyl)-1-butenyl]-5-oxocyclopenthl]-5-thiaheptanoate (AE1-734), were used. The Ki values of ONO-AE3-208 obtained by competition-binding isotherms to displace the radioligand binding to the respective prostanoid receptor are 1.3, 30, 790, 2,400 nM for EP4, EP3, FP, and TP, respectively, and more than 10,000 nM for the other prostanoid receptors. The Ki values of AE1-734 are 0.7, 56, and 620 nM for EP4, EP3, and EP2, respectively, and more than 10,000 nM for the rest of the prostanoid receptors. ONO-AE3-208 was administered (10 mg/kg/day) orally in the drinking water. When this compound was administered orally at 10 mg/kg as a bolus, a peak plasma concentration of 677 ng/ml was attained in 0.25 hours after the administration with 18% of bioavailability. The plasma half-life of this compound measured in an experiment of intravenous injection was 0.2 hours. AE1-734 was administered subcutaneously twice a day (0.1 mg/kg/each) from 1 day before the DSS treatment until the end of experiment. When AE1-734 was injected subcutaneously at this dose, the peak plasma concentration of 100 ng/ml was attained at 10 minutes after the injection with more than 70% for bioavailability. The plasma concentration declined with a half-life of 30 minutes.
To evaluate mucosal integrity, the FITC-dextran assay was used. Wild-type C57BL/6 mice were administered with either ONO-AE3-208 or vehicle in the drinking water. After 1 day, both groups of mice were fed with 3% DSS in the drinking water in the continued presence or absence of ONO-AE3-208, and 24 hours later, 200 μl of FITC-dextran (average molecular weight, 4,400) (2 mg/ml in saline) was administered orally. Serum concentration was determined 4 hours after the administration of FITC-dextran. EP4–/– mice and their wild-type control mice were similarly treated with 3% DSS and administered with FITC-dextran. The colon was snap-frozen and cryostat sections of 10-μm thickness were used for fluorescent microscopic analysis.

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Animal model 2: Bone Metastasis Animal Model and Bioluminescent Imaging[3]
To establish bone metastasis, 1 × 105 PC3/Luc cells suspended in 100 µl of PBS were inoculated into the left heart ventricle (day 0) of 5-week-old male nude mice (NU/NU) as previously described. Mice were separated to two groups (9 mice/group) one day before inoculating with cancer cells (day-1), then given a daily dose of 10 mg/kg of ONO-AE3-208 intraperitoneally to the treatment group and distilled water to the control group. Assessment of subsequent metastasis was monitored by measuring photon flux using the IVIS 100 in vivo imaging system 7 min after injecting luciferin intraperitoneally every 5–10 days for up to 60 days on mice anesthetized by exposure to 1–3 % isoflurane.

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Thirty-four 6-week old nude mice were divided into an experimental and a control group of equal number to be treated by intraperitoneal injection of ONO-AE3-208 and double distilled water, respectively. Then PC3/LUC cells were constructed by stably transfecting luciferin to prostate cancer PC3 cells and inoculated into the left ventricle of the mice to establish an animal model of systemic bone metastasis. The time of metastasis formation, photon tumor burdens, and changes of the survival curves after modeling were compared between the two groups of mice.[4]

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Animal model 3: Streptozotocin (STZ)-diabetic eNOS−/− mice[5]
Male C57BL/6 and eNOS−/− (C57BL/6 genetic background) mice were studied at eight weeks of age. Mice received a daily i.p. injection of STZ (55 mg/kg in 0.1 M citrate buffer, pH 4.5) or citrate buffer alone after a 4 hour fast for five consecutive days. Animals received ONO-AE3-208 at a dose of 10 mg/kg/day in drinking water or drinking water alone, for three weeks beginning on the day of the first injection of STZ. In a previous report, ONO-AE3-208 administered orally to mice as a 10 mg/kg bolus achieved a peak plasma concentration of 677 ng/ml after 0.25 hours with 18% bioavailability. Urine nephrin content (Exocell, Philadelphia, PA) and urine albumin excretion were determined by ELISA after housing mice in individual metabolic cages for 24 hours. Blood glucose was determined by OneTouch UltraMini. To determine the effect of broadspectrum COX inhibition, male control and STZ-diabetic/6 and eNOS−/− mice were treated with either indomethacin (4 mg/kg/day in drinking water44, Cayman Chemical, Ann Arbor, MI) or drinking water alone beginning with the first i.p. injection of STZ and continued for two weeks (n = 10/group).

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Animal model 4: db/db mice[5]
Male db/m and db/db mice on a BKS background aged eight weeks were randomly allocated to receive either ONO-AE3-208 (10 mg/kg/day in drinking water) or drinking water alone for eight weeks. An additional group of db/db mice were treated contemporaneously with captopril at a dose of 20 mg/kg/day in drinking water18. Blood glucose and urine albumin excretion were determined as already described. SBP was determined using a CODA non-invasive blood pressure system. Serum creatinine was determined by HPLC. For silver staining, urine volumes containing 0.5 µg creatinine were solubilized in sample buffer and separated by SDS-PAGE before staining with a ProteoSilver Stain kit.

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Animal model 5: Subtotally nephrectomized rats[5]
Male Sprague Dawley rats aged eight weeks underwent sham or subtotal nephrectomy surgery as previously described45. Briefly, for subtotal nephrectomy surgeries, under isoflurane anesthesia, the right kidney was removed via subcapsular nephrectomy and infarction of two thirds of the left kidney was achieved by selective ligation of two out of three of the branches of the left renal artery. Sham surgery involved laparotomy and manipulation of both kidneys prior to wound closure. One week later, rats were randomized to receive ONO-AE3-208 (1 mg/kg/day or 10 mg/kg/day) in drinking water or drinking water alone and they were followed for a further seven weeks. SBP was determined by tail cuff plethysmography as previously described46. GFR was determined by single shot FITC inulin clearance with repeated sampling via the tail vein as previously described. Urine protein excretion was determined using the benzethonium chloride method after 24 hour metabolic caging and urine creatinine was determined by autoanalyzer

[1]. The prostaglandin receptor EP4 suppresses colitis, mucosal damage and CD4 cell activation in the gut. J Clin Invest. 2002 Apr;109(7):883-93.

[2]. Prostaglandin E2 mediates connecting tubule glomerular feedback. Hypertension. 2013 Dec;62(6):1123-8.

[3]. An EP4 Antagonist ONO-AE3-208 Suppresses Cell Invasion, Migration, and Metastasis of Prostate Cancer. Cell Biochem Biophys. 2014 Apr 18.

[4]. Inhibitory effect of ONO-AE3-208 on the formation of bone metastasis of prostate cancer in mice. Zhonghua Nan Ke Xue. 2014 Aug;20(8):684-9.

[5]. EP4 inhibition attenuates the development of diabetic and non-diabetic experimental kidney disease. Sci Rep. 2017 Jun 13;7(1):3442.

Researchers used mice deficient in each of the eight types and subtypes of prostanoid receptors and examined the roles of prostanoids in dextran sodium sulfate-induced (DSS-induced) colitis. Among the prostanoid receptor-deficient mice, only EP4-deficient mice and not mice deficient in either DP, EP1, EP2, EP3, FP, IP, or TP developed severe colitis with 3% DSS treatment, which induced only marginal colitis in wild-type mice. This phenotype was mimicked in wild-type mice by administration of an EP4-selective antagonist (AE3-208). The EP4 deficiency impaired mucosal barrier function and induced epithelial loss, crypt damage, and aggregation of neutrophils and lymphocytes in the colon. Conversely, administration of an EP4-selective agonist (AE1-734) to wild-type mice ameliorated severe colitis normally induced with 7% DSS, while that of AE3-208 suppressed recovery from colitis and induced significant proliferation of CD4+ T cells. In vitro AE3-208 enhanced and AE1-734 suppressed the proliferation and Th1 cytokine production of lamina propria mononuclear cells from the colon. DNA microarray analysis revealed elevated expression of genes associated with immune response and reduced expression of genes with mucosal repair and remodeling in the colon of EP4-deficient mice. We conclude that EP4 maintains intestinal homeostasis by keeping mucosal integrity and downregulating immune response.[1]

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Connecting tubule glomerular feedback (CTGF) is a mechanism in which Na reabsorption in the connecting tubule (CNT) causes afferent arteriole (Af-Art) dilation. CTGF is mediated by eicosanoids, including prostaglandins and epoxyeicosatrienoic acids; however, their exact nature and source remain unknown. We hypothesized that during CTGF, the CNT releases prostaglandin E2, which binds its type 4 receptor (EP4) and dilates the Af-Art. Rabbit Af-Arts with the adherent CNT intact were microdissected, perfused, and preconstricted with norepinephrine. CTGF was elicited by increasing luminal NaCl in the CNT from 10 to 80 mmol/L. We induced CTGF with or without the EP4 receptor blocker ONO-AE3-208 added to the bath in the presence of the epoxyeicosatrienoic acid synthesis inhibitor MS-PPOH. ONO-AE3-208 abolished CTGF (control, 9.4 ± 0.5; MS-PPOH+ONO-AE3-208, -0.6 ± 0.2 μm; P<0.001; n=6). To confirm these results, we used a different, specific EP4 blocker, L161982 (10(-5) mol/L), that also abolished CTGF (control, 8.5 ± 0.9; MS-PPOH+L161982, 0.8 ± 0.4 μm; P<0.001; n=6). To confirm that the eicosanoids that mediate CTGF are released from the CNT rather than the Af-Art, we first disrupted the Af-Art endothelium with an antibody and complement. Endothelial disruption did not affect CTGF (7.9 ± 0.9 versus 8.6 ± 0.6 μm; P=NS; n=7). We then added arachidonic acid to the lumen of the CNT while maintaining zero NaCl in the perfusate. Arachidonic acid caused dose-dependent dilation of the attached Af-Art (from 8.6 ± 1.2 to 15.3 ± 0.7 μm; P<0.001; n=6), and this effect was blocked by ONO-AE3-208 (10(-7) mol/L). We conclude that during CTGF, the CNT releases prostaglandin E2, which acts on EP4 on the Af-Art inducing endothelium-independent dilation. [2]

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EP4 is one of the prostaglandin E2 receptors, which is the most common prostanoid and is associated with inflammatory disease and cancer. We previously reported that over-expression of EP4 was one of the mechanisms responsible for progression to castration-resistant prostate cancer, and an EP4 antagonist ONO-AE3-208 in vivo suppressed the castration-resistant progression regulating the activation of androgen receptor. The aim of this study was to analyze the association of EP4 with prostate cancer metastasis and the efficacy of ONO-AE3-208 for suppressing the metastasis. The expression levels of EP4 mRNA were evaluated in prostate cancer cell lines, LNCaP, and PC3. EP4 over-expressing LNCaP was established, and their cell invasiveness was compared with the control LNCaP (LNCaP/mock). The in vitro cell proliferation, invasion, and migration of these cells were examined under different concentrations of ONO-AE3-208. An in vivo bone metastatic mouse model was constructed by inoculating luciferase expressing PC3 cells into left ventricle of nude mice. Their bone metastasis was observed by bioluminescent imaging with or without ONO-AE3-208 administration. The EP4 mRNA expression levels were higher in PC3 than in LNCaP, and EP4 over-expression of LNCaP cells enhanced their cell invasiveness. The in vitro cell invasion and migration were suppressed by ONO-AE3-208 in a dose-dependent manner without affecting cell proliferation. The in vivo bone metastasis of PC3 was also suppressed by ONO-AE3-208 treatment. EP4 expression levels were correlated with prostate cancer cell invasiveness and EP4 specific antagonist ONO-AE3-208 suppressed cell invasion, migration, and bone metastasis, indicating that it is a potential novel therapeutic modality for the treatment of metastatic prostate cancer. [3]

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Objective: To examine the effect of ONO-AE3-208, an EP4 antagonist, on the formation of bone metastasis from prostate cancer in mice. Methods: Thirty-four 6-week old nude mice were divided into an experimental and a control group of equal number to be treated by intraperitoneal injection of ONO-AE3-208 and double distilled water, respectively. Then PC3/LUC cells were constructed by stably transfecting luciferin to prostate cancer PC3 cells and inoculated into the left ventricle of the mice to establish an animal model of systemic bone metastasis. The time of metastasis formation, photon tumor burdens, and changes of the survival curves after modeling were compared between the two groups of mice. Results: At 30 days after modeling, bioluminescence imaging analysis showed that the photon tumor burdens were significantly increased in a time-dependent manner in the control group in comparison with those in the experimental group (P < 0.01). The rate of metastasis formation was significantly higher in the former than in the latter (93.3% vs 33.3%, P < 0.001). The median time of metastasis formation was 29 d (95% CI 26.547 - 35.262) in the experimental animals as compared with 21 d (95% CI 17.213 -24.787) in the controls (P < 0.001). Conclusion: EP4 antagonist ONO-AE3-208 can inhibit the formation of bone metastasis from prostate cancer in mice.[4]

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Connecting tubule glomerular feedback (CTGF) is a mechanism in which Na reabsorption in the connecting tubule (CNT) causes afferent arteriole (Af-Art) dilation. CTGF is mediated by eicosanoids, including prostaglandins and epoxyeicosatrienoic acids; however, their exact nature and source remain unknown. We hypothesized that during CTGF, the CNT releases prostaglandin E2, which binds its type 4 receptor (EP4) and dilates the Af-Art. Rabbit Af-Arts with the adherent CNT intact were microdissected, perfused, and preconstricted with norepinephrine. CTGF was elicited by increasing luminal NaCl in the CNT from 10 to 80 mmol/L. We induced CTGF with or without the EP4 receptor blocker ONO-AE3-208 added to the bath in the presence of the epoxyeicosatrienoic acid synthesis inhibitor MS-PPOH. ONO-AE3-208 abolished CTGF (control, 9.4 ± 0.5; MS-PPOH+ONO-AE3-208, -0.6 ± 0.2 μm; P<0.001; n=6). To confirm these results, we used a different, specific EP4 blocker, L161982 (10(-5) mol/L), that also abolished CTGF (control, 8.5 ± 0.9; MS-PPOH+L161982, 0.8 ± 0.4 μm; P<0.001; n=6). To confirm that the eicosanoids that mediate CTGF are released from the CNT rather than the Af-Art, we first disrupted the Af-Art endothelium with an antibody and complement. Endothelial disruption did not affect CTGF (7.9 ± 0.9 versus 8.6 ± 0.6 μm; P=NS; n=7). We then added arachidonic acid to the lumen of the CNT while maintaining zero NaCl in the perfusate. Arachidonic acid caused dose-dependent dilation of the attached Af-Art (from 8.6 ± 1.2 to 15.3 ± 0.7 μm; P<0.001; n=6), and this effect was blocked by ONO-AE3-208 (10(-7) mol/L). We conclude that during CTGF, the CNT releases prostaglandin E2, which acts on EP4 on the Af-Art inducing endothelium-independent dilation.[5]

C24H21FN2O3

404.4335

404.153

C, 71.27; H, 5.23; F, 4.70; N, 6.93; O, 11.87

402473-54-5

10111831

White to yellow solid powder

1.3±0.1 g/cm3

662.4±55.0 °C at 760 mmHg

354.4±31.5 °C

0.0±2.1 mmHg at 25°C

1.637

4.56

2

5

7

30

660

0

FC1=C([H])C([H])=C(C2=C([H])C([H])=C([H])C([H])=C21)C([H])(C([H])([H])[H])C(N([H])C1C([H])=C(C#N)C([H])=C([H])C=1C([H])([H])C([H])([H])C([H])([H])C(=O)O[H])=O

MTDIMKNAJUQTIO-UHFFFAOYSA-N

InChI=1S/C24H21FN2O3/c1-15(18-11-12-21(25)20-7-3-2-6-19(18)20)24(30)27-22-13-16(14-26)9-10-17(22)5-4-8-23(28)29/h2-3,6-7,9-13,15H,4-5,8H2,1H3,(H,27,30)(H,28,29)

4-[4-cyano-2-[2-(4-fluoronaphthalen-1-yl)propanoylamino]phenyl]butanoic acid

AE 3-208; AE-3-208; AE3-208; ONO AE3 208; ONO-AE3-208; 4-[4-cyano-2-[2-(4-fluoronaphthalen-1-yl)propanoylamino]phenyl]butanoic Acid; 4-Cyano-2-[[2-(4-fluoro-1-naphthalenyl)-1-oxopropyl]amino]benzenebutanoic acid; DTXSID20435810; 4-(4-Cyano-2-(2-(4-fluoronaphthalen-1-yl)propanamido)phenyl)butanoic acid; 4-Cyano-2-[[2-(4-fluoro-1-naphthalenyl)-1-oxopropyl]amino]Benzenebutanoic acid; ONO-AE-3-208; ONO-AE 3-208

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: ~33.33 mg/mL (~82.4 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.14 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 20.8 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: ≥ 2.08 mg/mL (5.14 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 5%DMSO + 40%PEG300 + 5%Tween 80 + 50%ddH2O: 4.05mg/ml (10.01mM)

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