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25mg |
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Purity: =99.93%
Oridonin (NSC-250682; Isodonol) is a novel, naturally occurring and cell-permeable diterpenoid extracted from Rabdosia rubescens with anticancer activity. Oridonin is a substance that acts as an AKT inhibitor and has anti-tumor, anti-bacterial, and anti-inflammatory properties. Its IC50 values for AKT1 and AKT2 are 8.4 and 8.9 μM, respectively. By activating the intrinsic apoptotic pathway and inducing a caspase-3-mediated degradation, oridonin exposure causes apoptosis in leukemic cells that have been exposed to AE. The anti-NF-B compound oridonin also exhibits antiproliferative (ED50 ~ 2.7 µg/ml in lymphoid malignant cells) and antiangiogenic (significantly inhibits network formation of HMEC-1 cells at 2.5 µg/m) properties.
Targets |
Akt1 (IC50 = 8.4 μM); Akt2 (IC50 = 8.9 μM)
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ln Vitro |
Oridonin is an ATP-competitive AKT inhibitor with IC50 values for AKT1 and AKT2 of 8.4 and 8.9 M, respectively. By targeting AKT1/2, oridonin (5, 10 or 20 μM) obviously stops the growth of the ESCC cells KYSE70, KYSE410, and KYSE450. In KYSE70, KYSE410, and KYSE450 cells, oridonin (10 or 20 μM) causes G2/M phase cell cycle arrest and, at 20 μM, induces apoptosis. Additionally, the combination of cisplatin or 5-FU with oridonin (5, 10 or 20 μM) enhances the inhibition of esophageal squamous cell carcinoma (ESCC) cell growth[1]. AKT/mTOR signaling is preferentially suppressed by oridonin (0.1 and 1 μM). Additionally, oridonin (1 μM) selectively inhibits the growth of breast cancer cells by activating AKT signaling excessively[2].
In this study, we demonstrated that oridonin is an inhibitor of AKT and suppresses proliferation of esophageal squamous cell carcinoma (ESCC) in vitro and in vivo The role of AKT in ESCC was studied using immuno-histochemical analysis of a tumor microarray, the effect of AKT knockdown on cell growth, and treatment of cells with MK-2206, an AKT inhibitor. Oridonin blocked AKT kinase activity and interacted with the ATP-binding pocket of AKT. It inhibited growth of KYSE70, KYSE410, and KYSE450 esophageal cancer cells in a time- and concentration-dependent manner. Oridonin induced arrest of cells in the G2-M cell-cycle phase, stimulated apoptosis, and increased expression of apoptotic biomarkers, including cleaved PARP, caspase-3, caspase-7, and Bims in ESCC cell lines. Mechanistically, we found that oridonin diminished the phosphorylation and activation of AKT signaling. Furthermore, a combination of oridonin and 5-fluorouracil or cisplatin (clinical chemotherapeutic agents) enhanced the inhibition of ESCC cell growth.[1] AKT is a direct target of oridonin. Oridonin inhibits the proliferation of ESCC cells by targeting AKT1/2. Oridonin induces cell cycle G2/M arrest and apoptosis in ESCC cells. Oridonin inhibits AKT signaling pathways. A combination of oridonin and cisplatin or 5-FU enhances the suppression of ESCC cell growth. [1] We found that Oridonin, an extract from Rabdosia rubescens, reduced cell viability to the greatest extent. Oridonin binds to AKT1 and potentially functions as an ATP-competitive AKT inhibitor. Importantly, Oridonin selectively impaired tumor growth of human breast cancer cells with hyperactivation of PI3K/AKT signaling. Oridonin preferentially suppresses AKT/mTOR signaling in human mammary epithelial cells. Oridonin selectively suppresses growth of breast cancer cells with hyperactivation of AKT signaling [2]. |
ln Vivo |
Oridonin (160 mg/kg, p.o.) shows significant reduction in the tumor growth without obvious weigh loss in SCID mice bearing EG9 and HEG18 tumor cells. Treatment with oridonin also reduces the amount of Ki-67, phosphorylated AKT, GSK-3, or mTOR expressed in mice[1]. In nude mice, oridonin (15 mg/kg, i.p.) inhibits breast cancer cell growth by hyperactivating AKT signaling[2].
Oridonin inhibits PDX growth in vivo in tumors with high expression of AKT [1] To explore the antitumor activity of oridonin in vivo, we used two PDX models, EG9 and HEG18, which exhibited high expression of AKT (Fig. 8, Supplementary Fig. 6). The PDX tumor mass was implanted into SCID mice and then vehicle or oridonin (40 or 160 mg/kg body weight) was administered by gavage injection, once a day for 40 or 52 days. The results showed that treatment of mice with 160 mg/kg of oridonin significantly reduced tumor growth compared to vehicle-treated group (Fig. 8A, C) with no effect on body weight (Fig. 8B). Tumor tissues were prepared for IHC analysis and the expression of Ki-67, phosphorylated AKT, GSK-3β or mTOR was examined (Fig. 8D, E). Results indicated that all of these protein markers were significantly suppressed by treatment with oridonin compared with vehicle-treated controls (Fig. 8D, E). The characteristics of EG9 and HEG18 are shown in Supplementary Fig. 6E. Oridonin impairs growth of breast tumor with hyperactivation of AKT in vivo [1] To test whether Oridonin can impair the in vivo growth of p-AKTHigh breast cancer cells, NCr nude mice bearing palpable MDAMB468 or HCC1569 xenografts were treated with Oridonin or vehicle control. Durable tumor regression was achieved in both MDAMB468 and HCC1569 xenograft tumor models following Oridonin treatment (Figure 5A and 5D). To evaluate signaling and pharmacodynamic responses of MDAMB468 and HCC1569 xenografts during Oridonin treatment, tumors were isolated 72 hrs after drug administration and molecular markers were analyzed by immunohistochemical staining. Oridonin decreased phosphorylation of the AKT substrate PRAS40 and AKT downstream mTOR target (S6), blocked proliferation (as assessed by Ki67 index), and induced apoptosis (as assessed by cleaved caspase 3) (Figure 5B, 5C, 5E, and 5F). These results indicate that Oridonin effectively impairs tumor growth in p-AKTHigh breast cancers by inhibiting proliferation and inducing apoptosis via suppressing AKT-mTOR signaling pathway. Oridonin prevents the initiation of mammary tumors carrying PIK3CAH1047R by blocking AKT-mTOR signaling [1] We previously reported that expression of PIK3CAH1047R could initiate transformation of mammary epithelium in inducible MMTV-rtTA-tetO-PIK3CAH1047R (iPIK3CAH1047R) female mice. To examine whether Oridonin can prevent PIK3CAH1047R-induced mammary epithelial cell transformation, we transplanted PIK3CAH1047R mammary tissue fragments into cleared fat pads of 3-week-old NCr nude female mice. PIK3CAH1047R expression in iPIK3CAH1047R mammary epithelial cells is coupled to a luciferase reporter, allowing transgene expression to be followed in vivo. Expression of PIK3CAH1047R in transplanted mammary epithelial cells was induced by treating the mice with doxycycline at 8 weeks post transplantation. Mice were concurrently treated with Oridonin, BEZ235 or vehicle control. The increased luciferase reporter activity in transplanted iPIK3CAH1047R epithelium induced by doxycycline treatment was blocked by treatment with Oridonin and BEZ235 (Figure 6A). Histological examination showed increased mammary ductal side-branching and enlarged focal nodular structures filled with hyperproliferative cells characteristic of early neoplastic lesions in the vehicle control group whereas normal mammary epithelium structures were observed in mice treated with Oridonin or BEZ235 (Figure 6B). Immunohistochemical analyses showed that Oridonin significantly eliminated AKT effector phosphorylation and blocked cell proliferation in the iPIK3CAH1047R mammary outgrowths (Figure 6C–6E). These results establish that Oridonin prevents cell transformation by blocking AKT and downstream mTOR signaling in response to the induction of PIK3CAH1047R. |
Enzyme Assay |
For the AKT kinase assay, the ADP-Glo™ Kinase Assay Kit is used. Active AKT1 or AKT2 kinase and inactive GSK-3β as substrate are mixed by 1× reaction buffer and then added to a white 96-well plate. Pure ATP provided in the kit is serially diluted obtain a final concentration of 0, 1, 10, 50, and 100 μM. GSK-3β is diluted with DMSO until a final concentration of 2.5, 5, 10, or 20 μM is reached. Using the Luminoskan Ascent plate reader, the combined solution is incubated at room temperature to measure the amount of luciferase activity. [1]
Kinase assay [1] The AKT kinase assay was performed using the Nonradioactive IP-Kinase Assay Kit. Twenty μl of the immobilized antibody bead slurry were mixed with 200 μl of the cell lysate (200 μg) and gently rocked overnight at 4°C. After washing twice on day 2, the pellet was suspended in 50 μl of 1× kinase buffer supplemented with 1 μl of 10 mM ATP and 1 μg of kinase substrate. Then, the mixture was incubated for 30 min at 30°C and the reaction was terminated with 25 μl of 3× SDS sample buffer. Binding was assessed by Western blot analysis. For the AKT kinase assay, we used the ADP-Glo™ Kinase Assay Kit.. Active AKT1 or AKT2 kinase and inactive GSK-3β as substrate were mixed by 1x reaction buffer and then added to a white 96-well plate. Pure ATP provided in the kit was serially diluted obtain a final concentration of 0, 1, 10, 50, and 100 μM. Oridonin was added to reach a final concentration of 2.5, 5, 10 or 20 μM and DMSO was used as a control. The mixed solution was incubated at room temperature and luciferase activity was measured using the Luminoskan Ascent plate reader. Kinetic analysis of Oridonin-AKT1 binding [2] Surface plasmon resonance (SPR) measurements of the interaction of Oridonin with purified human recombinant AKT1 protein were performed on a BIAcore 3000 instrument. AKT1 was immobilized on BIAcore sensor chips at 14450 RU using an amine coupling kit. Oridonin was dissolved in 1% Pluronic F68 detergent by sonication at 1 mg/mL. Running buffer used in the experiment was HEPES/NaCl buffer containing 1% Pluronic F68 detergent at a flow of 20 μl/minute Oridonin (diluted to 2 μM) was injected for 7 minutes over the chip surface in a Biacore 3000 machine and then allowed to dissociate for a further 7 minutes. The binding analysis was performed using BIA evaluation software. |
Cell Assay |
Cells are seeded (6×103 cells/well for KYSE70; 2.5×103 cells/well for KYSE410; 2×103 cells/well for KYSE450) in 96-well plates and incubated for 24 h and then treated with different amounts of Oridonin or vehicle. The MTT assay is used to measure cell proliferation after incubation for 24, 48, or 72 hours. For anchorage-independent cell growth assessment, cells (2.5, 5 or 10 μM Oridonin) suspended in complete medium are added to 0.3% agar with vehicle, 2.5, 5 or 10 μM Oridonin in a top layer over a base layer of 0.5% agar with vehicle, 2.5, 5 or 10 μM Oridonin.
Cell proliferation assay [1] Cells were seeded (6×103 cells/well for KYSE70; 2.5×103 cells/well for KYSE410; 2×103 cells/well for KYSE450) in 96-well plates and incubated for 24 h and then treated with different amounts of oridonin or vehicle. After incubation for 24, 48 or 72 h, cell proliferation was measured by the MTT assay. For anchorage-independent cell growth assessment, cells (8×103 cells/well) suspended in complete medium were added to 0.3% agar with vehicle, 2.5, 5 or 10 μM oridonin in a top layer over a base layer of 0.5% agar with vehicle, 2.5, 5 or 10 μM oridonin. The cultures were maintained at 37°C in a 5% CO2 incubator for 3 weeks and then colonies were visualized under a microscope and counted using the Image-Pro Plus software (v.6.1) program. Cell cycle and apoptosis analyses [1] Cells (6 × 105 cells for KYSE70; 3 × 105 for KYSE410; 2 × 105 for KYSE450) were seeded in 60-mm plates and treated with 0, 5, 10 or 20 μM oridonin for 48 or 72 h. For cell cycle analysis, cells were then fixed in 70% ethanol and stored at −20°C for 24 h. After staining with annexin-V for apoptosis or propidium iodide for cell cycle assessment, cells were analyzed using a BD FACSCalibur Flow Cytometer. Ex vivo and in vitro pull-down assay [1] KYSE450 cell lysates (500 μg) and recombinant human active AKT1 or AKT2 (200 ng) were incubated with oridonin-Sepharose 4B (or Sepharose 4B only as a control) beads (50 μL; 50% slurry) in reaction buffer (50 mM Tris-HCl pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol, 0.01% NP-40 and 0.2 mM PMSF, 20× protease inhibitor [1 tablet each]). After incubation with gentle rocking overnight at 4°C, the beads were washed 3 times with buffer (50 mM Tris-HCl pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol, 0.01% NP-40 and 0.2 mM PMSF) and binding was visualized by Western blotting. Reporter gene activity assay [1] Transient transfection was conducted using the Simple-Fect Transfection Reagent, and the luciferase reporter gene activity assays were performed according to the instructions of the manufacturer. Briefly, cells (1ⅹ105) were seeded the day before transfection into 24-well culture plates. Cells were co-transfected with the NF-κB-luc or pGL-3-luc firefly reporter plasmid, and the pRL-SV control Renilla reporter plasmid. Following incubation for 24 h, the cells were treated with vehicle or oridonin for an additional 24 h, and then harvested in lysis buffer. Firefly and Renilla luciferase activities were assessed by using the substrates provided in the reporter assay system (Promega). The Renilla luciferase activity was used for normalization of transfection efficiency. Luciferase activity was measured using the Luminoskan Ascent plate reader. |
Animal Protocol |
Mice: Breast cancer cells are isolated, resuspended in 40% Matrigel-Basement Membrane Matrix, free of LDEV, and then injected (100 L per site) into the fourth pair of mammary fat pads of naked mice (CrTac: NCr-Foxn1nu). Tumors are measured in two dimensions using manual calipers. Tumor volume is calculated using the formula: Volume = 0.5 × length × width × width. Tumor volume is measured every 2-3 days. Tumors are collected and then fixed in 70% ethanol for an hour before being histopathologically examined. Mice are given either the vehicle (1% Pluronic F68) or oridonin (15 mg/kg) intraperitoneally (IP) every day. In 1-methyl-2 pyrolidone and polyethylene glycol 300 (PEG300), BEZ235 is reconstituted 1:9. By oral gavage, mice are administered this compound formulation at a dose of 45 mg/kg daily (QD)[2].
Patient derived xenograft (PDX) mouse model [1] Six to eight week-old severe combined immunodeficient (SCID) female mice were used for these experiments. The PDX tumor mass was subcutaneously implanted into the back of SCID mice. When tumors reached an average volume of about 100 mm3, mice were divided into 3 treatment groups for further experimentation as follows: (1) vehicle group (n = 10); (2) 40 mg/kg of Oridonin (n = 10); (3) 160 mg/kg of Oridonin (n = 10). Oridonin was administered by gavage once a day for 52 days. Tumor volume was calculated from measurements of 3 diameters of the individual tumor base using the following formula: tumor volume (mm3) = (length × width × height × 0.52). Mice were monitored until tumors reached 1.0 cm3 total volume, at which time mice were euthanized and tumors extracted. Inhibitor administration [1] Animals were treated with Oridonin (15 mg/kg) in 1% Pluronic F68 or vehicle (1% Pluronic 68) daily by intraperitoneal (IP) injection as described previously. BEZ235 was reconstituted 1:9 in 1-methyl-2 pyrolidone (NMP; Sigma) and polyethylene glycol 300. Mice were treated with this compound formulation at 45 mg/kg daily (QD) by oral gavage. |
Toxicity/Toxicokinetics |
mouse LD50 intraperitoneal 35 mg/kg British Patent Document., #1476016
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References | |
Additional Infomation |
Oridonin is an organic heteropentacyclic compound and ent-kaurane diterpenoid with formula C20H28O6 isolated from the leaves of the medicinal herb Rabdosia rubescens. It has a role as an antineoplastic agent, an angiogenesis inhibitor, an apoptosis inducer, an anti-asthmatic agent, a plant metabolite and an antibacterial agent. It is an organic heteropentacyclic compound, an enone, a cyclic hemiketal, a secondary alcohol and an ent-kaurane diterpenoid.
Oridonin has been reported in Isodon japonicus, Isodon macrocalyx, and other organisms with data available. The novelty of our study is the finding that oridonin interacts with AKT1/2 directly and inhibits AKT1/2 kinase activity (Fig. 2). Our MTT and soft agar assay results also revealed that oridonin significantly inhibited growth of KYSE70, KYSE410 and KYSE450 ESCC cell lines (Fig. 3). Pi, et al. reported that oridonin induced apoptosis in KYSE150 cells at 30 μM (40), whereas, we found that oridonin induced apoptosis and G2/M phase arrest in KYSE70, KYSE410 and KYSE450 ESCC cell lines at 20 μM (Fig. 5, Supplementary Fig. 3, 4). Notably, these cells express very high levels of AKT. These results suggest that AKT is a prominent target of oridonin to inhibit proliferation and induce apoptosis in ESCC cells. AKT mediates several signaling molecules, including GSK-3β, mTOR and NF-κB (8) and oridonin decreased the phosphorylation of GSK-3β and mTOR and NF-κB transcriptional activity (Fig. 6). Furthermore, a combination of oridonin and standard chemotherapy drugs, 5-FU or cisplatin, resulted in synergistic or additive effects against ESCC growth (Fig. 7, Supplementary Fig. 5). Finally we confirmed our findings in in vivo PDX mouse models, which are a useful preclinical model of human tumor growth. Our results revealed that oral administration of oridonin significantly decreased the growth PDX tumors expressing high levels of AKT (Fig. 8, Supplementary Fig. 6A, C, E) without toxicity based on no loss in body weight, liver or spleen, and H&E staining analysis compared with vehicle-treated control mice (Supplementary Fig. 6B, E). IHC results indicated that oridonin decreased the expression of Ki-67, pAKT, pGSK-3β or p-mTOR in both PDX models (Fig. 8D, E, Supplementary Fig. 6D). Overall, our study suggests that oridonin can inhibit progression of ESCC tumors, in vitro and in vivo by suppressing AKT signaling through its direct targeting of AKT (Fig. 9). Thus, this study might provide useful information in the clinical application of oridonin for ESCC chemotherapy.[1] In this study, we screened a library of 441 TCM plant extracts by examining their effect on cell viability in a HMEC-PIK3CAH1047R cell model with constitutively active AKT signaling. Nineteen of the extracts impaired cell growth as efficiently as the pan PI3K inhibitor BKM120 or dual PI3K/mTOR inhibitor BEZ235. We showed that Oridonin, an extract from Rabdosia rubescens, reduced cell viability most efficiently. Rather than inhibit PI3K activity directly, Oridonin bound to AKT1 and may function as a potential ATP-competitive AKT inhibitor. Oridonin selectively impaired the cell growth of p-AKTHigh human breast cancers in vitro and in vivo by preferentially blocking AKT-mTOR signaling. Oridonin also efficiently prevented the initiation of mouse mammary tumors driven by PIK3CAH1047R in vivo. Our results suggest that Oridonin may serve as a potent and selective therapeutic agent in patients bearing breast cancers with hyperactivation of AKT signaling. Our results provide concrete evidence that Rabdosia rubescens extract Oridonin decreases AKT signaling and selectively inhibits tumor growth in p-AKTHigh breast tumors cells. Of note, to date pan-or isoform-selective inhibitor of PI3K inhibitors, AKT inhibitors, mTOR inhibitors, and dual PI3K/mTOR inhibitors have not yielded durable or efficacious clinical results, Oridonin offers promise of both increased efficacy and reduced toxicity compared with single PI3K, AKT, or even dual PI3K/mTOR inhibitors. Together, our data suggest that in the context of p-AKTHigh breast cancers, Oridonin may serve as a potent and durable therapeutic agent and should be considered for clinical application.[2] |
Molecular Formula |
C20H28O6
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Molecular Weight |
364.43
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Exact Mass |
364.188
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Elemental Analysis |
C, 65.92; H, 7.74; O, 26.34
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CAS # |
28957-04-2
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Related CAS # |
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PubChem CID |
5321010
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Appearance |
White to off-white solid powder
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Density |
1.4±0.1 g/cm3
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Boiling Point |
599.8±50.0 °C at 760 mmHg
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Melting Point |
248-250ºC
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Flash Point |
215.0±23.6 °C
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Vapour Pressure |
0.0±3.9 mmHg at 25°C
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Index of Refraction |
1.635
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LogP |
1.44
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Hydrogen Bond Donor Count |
4
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Hydrogen Bond Acceptor Count |
6
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Rotatable Bond Count |
0
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Heavy Atom Count |
26
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Complexity |
717
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Defined Atom Stereocenter Count |
9
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SMILES |
O1C([H])([H])C23C([H])(C([H])([H])C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])C2([H])C([H])(C1(C12C(C(=C([H])[H])C([H])(C([H])([H])C([H])([H])C31[H])C2([H])O[H])=O)O[H])O[H])O[H]
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InChi Key |
SDHTXBWLVGWJFT-BAFGBBEMSA-N
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InChi Code |
InChI=1S/C20H28O6/c1-9-10-4-5-11-18-8-26-20(25,19(11,14(9)22)15(10)23)16(24)13(18)17(2,3)7-6-12(18)21/h10-13,15-16,21,23-25H,1,4-8H2,2-3H3/t10-,11-,12-,13+,15+,16-,18+,19-,20-/m0/s1
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Chemical Name |
(1S,2S,5S,8R,9S,10S,11R,15S,18R)-9,10,15,18-tetrahydroxy-12,12-dimethyl-6-methylidene-17-oxapentacyclo[7.6.2.15,8.01,11.02,8]octadecan-7-one
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Synonyms |
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HS Tariff Code |
2934.99.9001
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Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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Solubility (In Vitro) |
DMSO: 62.5~72 mg/mL (171.5~197.6 mM)
Water: ~20 mg/mL warming (~134.1 mM) Ethanol: ~34 mg/mL (~93.3 mM) |
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Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.71 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (5.71 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 20.8 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (5.71 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 5% DMSO+20%PEG 300+ddH2O: 12.5mg/ml Solubility in Formulation 5: ≥ 5 mg/mL (13.72 mM) (saturation unknown) in 10% 1-Methyl-2-pyrrolidinone 90% PEG300 (add these co-solvents sequentially from left to right, and one by one), clear solution. |
Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 2.7440 mL | 13.7201 mL | 27.4401 mL | |
5 mM | 0.5488 mL | 2.7440 mL | 5.4880 mL | |
10 mM | 0.2744 mL | 1.3720 mL | 2.7440 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration: mg/mL;
Method for preparing DMSO stock solution: mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation::Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
NCT05130892 | Completed | Drug: Tranilast Drug: Colchicine |
NLRP3 hsCRP |
Wuhan Union Hospital, China | November 15, 2021 | Phase 4 |