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Monomethyl auristatin E (also known as MMAE; SGD-1010), a dolastatin 10 derivative, is a novel, synthetic and potent antimitotic/antitubulin agent which blocks the polymerization of tubulin, due to its high toxicity, it cannot be used as a drug itself; instead, it is linked to a monoclonal antibody (MAB) which directs it to the cancer cells. Vedotin, as used in the commercial drug Brentuximab vedotin, is MMAE plus the structure that connects it to the antibody (Brentuximab). In preclinical studies, both in vitro and in vivo, dolastatins—peptides found in the marine shell-less mollusc Dolabella auricularia—show strong activity against a variety of lymphomas, leukemia, and solid tumors. These peptides are the source of MMAE, a powerful antimitotic medication. When MMAE is used to treat Hodgkin lymphoma and other cancers, its potency can reach 200 times that of vinblastine, another antimitotic medication.
Physicochemical Properties
| Molecular Formula | C39H67N5O7 | |
| Molecular Weight | 717.98 | |
| Exact Mass | 717.504 | |
| Elemental Analysis | C, 65.24; H, 9.41; N, 9.75; O, 15.60 | |
| CAS # | 474645-27-7 | |
| Related CAS # | MMAE-d8;2070009-72-0;Monomethyl auristatin E;474645-27-7 | |
| PubChem CID | 11542188 | |
| Appearance | white solid powder | |
| Density | 1.1±0.1 g/cm3 | |
| Boiling Point | 873.5±65.0 °C at 760 mmHg | |
| Flash Point | 482.1±34.3 °C | |
| Vapour Pressure | 0.0±0.3 mmHg at 25°C | |
| Index of Refraction | 1.519 | |
| LogP | 4.13 | |
| Hydrogen Bond Donor Count | 4 | |
| Hydrogen Bond Acceptor Count | 8 | |
| Rotatable Bond Count | 20 | |
| Heavy Atom Count | 51 | |
| Complexity | 1100 | |
| Defined Atom Stereocenter Count | 10 | |
| SMILES | O(C([H])([H])[H])[C@]([H])([C@]([H])(C(N([H])[C@]([H])(C([H])([H])[H])[C@@]([H])(C1C([H])=C([H])C([H])=C([H])C=1[H])O[H])=O)C([H])([H])[H])[C@]1([H])C([H])([H])C([H])([H])C([H])([H])N1C(C([H])([H])[C@]([H])([C@]([H])([C@@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])[H])N(C([H])([H])[H])C([C@]([H])(C([H])(C([H])([H])[H])C([H])([H])[H])N([H])C([C@]([H])(C([H])(C([H])([H])[H])C([H])([H])[H])N([H])C([H])([H])[H])=O)=O)OC([H])([H])[H])=O |
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| InChi Key | DASWEROEPLKSEI-UIJRFTGLSA-N | |
| InChi Code | InChI=1S/C39H67N5O7/c1-13-25(6)34(43(10)39(49)33(24(4)5)42-38(48)32(40-9)23(2)3)30(50-11)22-31(45)44-21-17-20-29(44)36(51-12)26(7)37(47)41-27(8)35(46)28-18-15-14-16-19-28/h14-16,18-19,23-27,29-30,32-36,40,46H,13,17,20-22H2,1-12H3,(H,41,47)(H,42,48)/t25-,26+,27+,29-,30+,32-,33-,34-,35+,36+/m0/s1 | |
| Chemical Name | (2S)-N-[(2S)-1-[[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-3-[[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl]-methylamino]-3-methyl-1-oxobutan-2-yl]-3-methyl-2-(methylamino)butanamide | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: (1). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.(2). This product is not stable in solution, please use freshly prepared working solution for optimal results. |
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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) |
Biological Activity
| Targets | Auristatin; microtubule; tubulin polymerization |
| ln Vitro |
In CD30+ cells, MMAE exhibits selective cytotoxicity when combined with cAC10. It also causes apoptosis, which results in G2/M-phase growth arrest and cell death. [1] Anti-CD79b-vcMMAE exhibits strong and extensive activity on a wide range of NHL cell lines when combined with anti-CD79b antibody in vitro.[2] Hertuzumab-vc-MMAE can also be efficiently internalized and significantly kill tumor cells that overexpress HER2 when combined with anti-HER2 antibody.[3] Purpose: SGN-35 is an antibody-drug conjugate (ADC) containing the potent antimitotic drug, monomethylauristatin E (MMAE), linked to the anti-CD30 monoclonal antibody, cAC10. As previously shown, SGN-35 treatment regresses and cures established Hodgkin lymphoma and anaplastic large cell lymphoma xenografts. Recently, the ADC has been shown to possess pronounced activity in clinical trials. Here, we investigate the molecular basis for the activities of SGN-35 by determining the extent of targeted intracellular drug release and retention, and bystander activities. Experimental design: SGN-35 was prepared with (14)C-labeled MMAE. Intracellular ADC activation on CD30(+) and negative cell lines was determined using a combination of radiometric and liquid chromatograhpy/mass spectrometry-based assays. The bystander activity of SGN-35 was determined using mixed tumor cell cultures consisting of CD30(+) and CD30(-) lines. Results: SGN-35 treatment of CD30(+) cells leads to efficient intracellular release of chemically unmodified MMAE, with intracellular concentrations of MMAE in the range of 500 nmol/L. This was due to specific ADC binding, uptake, MMAE retention, and receptor recycling or resynthesis. MMAE accounts for the total detectable released drug from CD30(+) cells, and has a half-life of retention of 15 to 20 h. Cytotoxicity studies with mixtures of CD30(+) and CD30(-) cell lines indicated that diffusible released MMAE from CD30(+) cells was able to kill cocultivated CD30(-) cells. Conclusions: MMAE is efficiently released from SGN-35 within CD30(+) cancer cells and, due to its membrane permeability, is able to exert cytotoxic activity on bystander cells. This provides mechanistic insight into the pronounced preclinical and clinical antitumor activities observed with SGN-35. [1] Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor-targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell-penetrating peptide targeting matrix metalloproteinases and RGD-binding integrins (ACPP-cRGD-MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low-passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule- and dose-dependent manner, correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double-strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1 [2]. |
| ln Vivo |
While free MMAE (0.36 mg/kg) does not exhibit any discernible antitumor activity, cAC10-vcMMAE (1 mg/kg, i.v.) causes total, long-lasting tumor regression in the Karpas 299 ALCL model.[1] Anti-CD79b-vcMMAE (7 mg/kg, p.o.) notably causes a sustained total tumor remission in mouse xenograft models of NHL. [/2] We then evaluated the efficacy of combined MMAE with focal IR to inhibit tumor xenograft growth. First, we tested the hypothesis that MMAE tumor targeted delivery would increase tumor regression compared to free MMAE delivery (Fig. 6C). PANC-1 tumor xenografts were grown to a mean volume of 200 mm3 prior to initiation of therapy. Free MMAE or ACPP-cRGD-MMAE was IV injected on days 0 and 1 (6 nmoles of MMAE/day). This dose of MMAE was chosen based on prior studies on animal toxicity associated with free MMAE delivery. Fractionated IR of 3 Gy per day was given on day 1 and 2. On day 1 when MMAE and IR were both given, IR was delivered in the morning and MMAE in the afternoon. By day 30 following initiation of therapy, free MMAE treatment resulted in a small but statistically significant growth delay of PANC-1 tumors compared to untreated control tumors, p<0.0001. The average tumor volume of free MMAE treated mice was 75% of untreated controls. More importantly, free MMAE in combination with IR resulted in profound tumor xenograft regression compared to IR or free MMAE alone (p<0.0001). In comparing targeted and free MMAE delivery in the absence of IR, ACPP-cRGD-MMAE resulted in significantly greater tumor regression compared free MMAE, which is consistent with prior studies involving breast cancer models (12). Of significance, IR combined with ACPP-cRGD-MMAE resulted in prolonged tumor regression when compared to free MMAE and IR (p<0.01). Longer follow up of tumors demonstrated that 2 of 10 PANC-1 tumors treated with ACPP-cRGD-MMAE and IR were less than or equal to their starting tumor volume on day 0 (Table 1). Of significance, such prolonged and sustained tumor regression was observed with only 2 doses of both MMAE and IR and the initial tumor volume was greater than 200 mm3. Moreover, no other treatment group showed long term tumor regression. [2] We extended our studies on ACPP-cRGD-MMAE and IR by increasing the dosing schedule to see if it would result in further improvement in long term regression. ACPP-cRGD-MMAE was given on days 0, 1, and 2 (6 nmoles/day, 18 nmoles total). Fractionated IR of 3 Gy per day was administered on day 1, 2, and 3. Again on days when ACPP-cRGD-MMAE and IR were both given, IR was delivered in the morning and ACPP-cRGD-MMAE in the afternoon. As we observed in Fig 6C, combining ACPP-cRGD-MMAE with IR again produced significant tumor regression compared to IR or ACPP-cRGD-MMAE alone treated mice (Supplemental Fig. 6). Tumor volumes in the combined ACPP-cRGD-MMAE and IR mice remained statistically significant compared to all other groups, p<0.0001. More striking and of therapeutic importance, the majority of treated tumors had prolonged tumor regression in PANC-1 tumors upon combining ACPP-cRGD-MMAE with IR. By day 40, none of the control or IR alone treated tumors were smaller than their initial tumor volume on day 0 (Table 1). For the ACPP-cRGD-MMAE alone group, only 1 of 14 tumors was smaller than their initial tumor volumes. In contrast, 8 of 14 tumors in the combined ACPP-cRGD-MMAE and IR group were smaller than their initial tumor volume. [2] We then tested a modified treatment schedule of ACPP-cRGD-MMAE and IR using HCT-116 tumor xenografts. HCT-116 tumors were grown to mean tumor volume of > 270 mm3 prior to initiation of therapy. We had observed that 6 Gy given to HCT-116 xenografts improved ratiometric ACPP probe cleavage (Fig. 5D and Supplemental Fig. 4). Therefore in irradiated tumors, we delivered 6 Gy on day 0 followed by 3 Gy on days 1 and 2. ACPP-cRGD-MMAE was IV injected on days 0 and 1, six hours following irradiation (7.5 nmoles/day). The dose of ACPP-cRGD-MMAE was increased compared to PANC-1 since HCT-116 cells had a higher IC50 for MMAE. As seen in PANC-1 tumors, ACPP-cRGD-MMAE alone produced a modest growth delay compared to untreated control tumors (Fig. 6D). As expected, IR alone resulted in an initial tumor growth delay (especially prominent due to the 6 Gy dose on day 0), however by day 10, tumor volume began to rise. Combining ACPP-cRGD-MMAE with IR again produced sustained tumor regression compared to IR alone starting at day 10 post initiation of therapy, p<0.006. By day 14, none of the control or ACPP-RGD-MMAE treated tumors were smaller than their initial tumor volume on day 0 (Table 1). For the IR alone group, only 3 of 10 tumors were smaller than their initial tumor volume. In contrast, 9 of 10 tumors in the combined ACPP-cRGD-MMAE and IR group were smaller than their initial tumor volume[2]. |
| Enzyme Assay |
Free drug retention [1] For MMAE retention experiments, 25 × 106 cells were seeded at 5 × 105 cells/mL. Each cell type was treated in triplicate with a concentration of radioactive MMAE determined to provide a similar intracellular concentration for that cell type after 3 h of incubation at 37°C. The cells were washed twice into an equal volume of fresh medium. Each washed culture was split into three 15-mL centrifuge tubes. One-milliliter aliquots were removed immediately from each tube for LSC and a second 1-mL aliquot was layered over 2 mL of FBS in a 15-mL centrifuge tube, centrifuged at 390 × g for 5 min at room temperature. From the separated sample, 0.5 mL of the upper medium phase was removed for LSC. The remaining portion of each sample was frozen in a dry ice bath and the bottom of the tube containing the cell pellet was excised into a scintillation vial containing 0.5 mL of PBS. Samples were vortexed; Ecoscint A scintillation fluid (4 mL) was added followed by a second vortex; and the samples were counted by LSC. Further aliquots (1 mL) were processed over FBS, as described, at the indicated time points. For the calculation of intracellular drug concentrations, cell densities were redetermined by trypan exclusion after washing into nondrug-containing medium and again after 24 h. |
| Cell Assay |
As directed by the manufacturer, the Alamar Blue dye reduction assay is used to quantify cytotoxicity. In short, right before cultures are added, a freshly prepared 40% (wt/vol) solution of Alamar Blue is added to complete media. Alamar Blue solution is added to cells to make up 10% of the culture volume ninety-two hours after the drug is exposed. A Fusion HT fluorescent plate reader (Packard Instruments, Meriden, CT) is used to measure dye reduction after 4 hours of incubation of the cells. ADC-treated culture conditioned medium bioassay [1] Samples of spent culture medium from SGN-35–treated cells were added to CD30-negative Ramos cell cultures in six different dilutions. In parallel, Ramos cells were incubated with eight MMAE concentrations as cytotoxicity standards. After a 96-h incubation at 37°C, the Ramos cultures were developed using resazurin (relative fluorescence, excitation = 530-560 nmol/L, emission = 590 nmol/L). Using the average relative fluorescence unit (RFU) measurement for Ramos cells incubated with the MMAE standards, the percentage of viable cells relative to untreated Ramos cells (% untreated) was plotted as a function of MMAE concentration (nmol/L). A four-parameter curve fit was used to generate an equation for the quantitation of released MMAE in the spent culture samples. Cell viability measurements of the spent culture dilutions were transformed into MMAE concentrations (assuming cytotoxicity is attributed solely to MMAE) using this equation. Only cell viability measurements falling between 15% and 85% of the untreated Ramos cells were used to calculate the concentration of MMAE. Coculture experiments [1] Karpas299, L540cy, or Ramos cells in single culture were seeded at 2.5 × 105 cells/mL in culture volumes of 1.5 mL, whereas cocultures of CD30-positive and CD30-negative pairs consisted of 1.25 × 105 cells/mL of each cell type in 1.5 mL of culture medium (1:1 mixture of cells, RPMI 1640 + 10-15% FBS). The culture medium used in the coculture experiments adequately supports the growth of all three cell types. Cultures were treated with vehicle control, 1 μg/mL SGN-35, or IgG-vc-MMAE nonbinding control. After a 72-h incubation, cultures were fed with 60% medium containing the requisite treatment type. Cultures were examined for cell count and viability (Vi-Cell XR2.03 cell viability analyzer) after 120 h and the surviving cells were stained with anti–CD30-Phycoerythrin and anti–CD19-FITC (BD Biosciences) antibodies to determine the distribution of each cell type in the surviving cultures. Staining was accomplished by harvesting the cells by centrifugation at 1,200 rpm for 5 min, plating ∼5 × 105 cells per well in a 96-well plate in 20 μL of fluorescence-activated cell sorting (FACS) buffer (PBS containing 2% FBS), and adding the labeled antibodies to the desired wells without dilution (5 μL/well). The plate was incubated on ice for 30 min before centrifugation at 1,500 rpm for 5 min and before the removal of the supernatant by tapping the plate. The cells were washed thrice with PBS (200 μL) before resuspending in 250 μL FACS buffer and before storage at 4°C for subsequent analysis by flow cytometry on a BD FACScan instrument. Cell cycle and Apoptosis [2] Cells were treated with MMAE for 24 hours and then fixed in methanol. Cells were treated with RNAse, stained with propidium iodide (PI) and analyzed by FACS using FloJo software. Alamar Blue assay [2] Cells were plated in 96 well plates and exposed to MMAE or paclitaxel for 72 hours and analyzed at 560 nm. For irradiated cells, cells were treated with MMAE overnight followed by 6 Gy. Clonogenic assay [2] Cells were treated with MMAE for 24 hours and then irradiated with 0-8 Gy. Following IR, cells were re-plated in drug free media. Colonies formed over 10-14 days and were counted. Neutral comet assay [2] Cells were treated for indicated length and doses of MMAE followed by 6 Gy. Cells were harvested 15 minutes post IR, underwent neutral electrophoresis (Trevigen). Comet tails were counted in multiple fields (>60 cells per sample) and analyzed using CometScore (TriTek Corp). γH2Ax immunostaining [2] Cells grown on glass cover slips were treated with MMAE overnight and then irradiated. Two hours post IR, cells were fixed, permeabilized and stained with antibody to γH2Ax. Nuclei were stained with DAPI. Foci were counted in 6-8 high power fields per group. Immunoblotting [2] MMAE and IR treated cells were harvested and lysed in RIPA buffer with protease and phosphatase inhibitors. 30 ug of lysate underwent electrophoresis using 4-12% Bis-Tris gels, transferred to PVDF membranes and incubated with indicated primary antibodies. Blots were developed by ECL. |
| Animal Protocol |
Mice: Female athymic nu/nu mice aged 6-8 weeks are given a 1:1 Matrigel and PBS solution subcutaneously injected into their thighs, containing 5×106 HCT-116 or PANC-1 cells. After administering ACPP-cRGD-MMAE intravenously (IV) or orally (IR) (6 nmoles/day, totaling 18 nmoles), the mice are given the treatment. Tumor tissue is then removed, paraffin embedded, formalin fixed, and stained with the appropriate antibodies. Using the UltraMap system, DAB is used as a chromagen and the primary antibody is used at a 1:250 dilution for visualization.
Immunohistochemistry [2] Mice were treated with IR or intravenous (IV) injection of ACPP-cRGD-MMAE, tumor tissue was harvested, formalin fixed and paraffin embedded followed by staining with indicated antibodies (Ventana Medical Systems). The primary antibody was used at a 1:250 dilution and was visualized using DAB as a chromagen with the UltraMap system. In vivo tumor xenograft optical imaging [2] Tumor xenografts were irradiated as described above. One day post IR, mice were anesthetized (1:1 mixture of 100 mg/ml of ketamine and 5 mg/ml of midazolam) and IV injected with either fluorescently labeled ratiometric ACPP (Cy5 and Cy7) or ACPP-cRGD-MMAE (Cy5). Animals were imaged 2 hours later using a Maestro Small Animal Imager (CRI) with excitation filter of 620/22 nm and 645 nm long pass emission filter with dichroic filter tuned to 670 nm. Imaging was done both with skin on and after skin removal to decrease autofluorescence and scattering. In vivo tumor xenograft experiments [2] HCT-116 or PANC-1 tumor growth was measured with digital calipers. Tumor volume was calculated using the formula as ½ * Length * Width2. Mice were randomized into groups as indicated in Results once the average tumor volume reached >200 mm3. Free MMAE was injected on an equimolar basis to ACPP-cRGD-MMAE. |
| References |
[1]. Intracellular Activation of SGN-35, a Potent Anti-CD30 Antibody-Drug Conjugate. Clinical Cancer Research (2010), 16(3), 888-897. [2]. Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery. Cancer Res. 2015 Apr 1;75(7):1376-87. |
| Additional Infomation |
Monomethyl Auristatin E is a dolastatin-10 peptide derivative with potent antimitotic activity and potential antineoplastic activity as part of an antibody-drug conjugate (ADC). Monomethyl auristatin E (MMAE) binds to tubulin, blocks tubulin polymerization, and inhibits microtubule formation, which results in both disruption of mitotic spindle assembly and arrest of tumor cells in the M phase of the cell cycle. To minimize toxicity and maximize efficacy, MMAE is conjugated, via a cleavable peptide linker, to a monoclonal antibody that specifically targets a patient's tumor. The linker is stable in the extracellular milieu but is readily cleaved to release MMAE following binding and internalization of the ADC by the target cells. In the work described here, we show that CD30-positive cell lines process SGN-35 by releasing MMAE in a chemically unmodified form. The results are consistent with the proteolytic cleavage at the citrulline-PABC amide bond, which leads to the release of MMAE after spontaneous fragmentation of the PABC spacer (Fig. 1). This is supported by inhibition studies with chloroquine (Fig. 2C), a lysosomotropic agent that reduces lysosomal protease activity through pH modulation. Upon release, the MMAE diffuses through cell membranes and accumulates in culture media, albeit at concentrations that were ∼250 times lower than that found inside the cells, presumably due to dilution into the relatively larger volume of medium (Fig. 2B). Despite the great differential, extracellular drug was able to kill CD30-negative cells cocultured with CD30-positive cells. Thus, SGN-35 has the potential to act on cells within a heterogeneous tumor cell population that do not bind sufficiently high amounts of ADC for effective direct cytotoxic activity. In general, this is of importance because mAbs have been shown to distribute within tumors in an uneven manner and many tumors are heterogeneous with respect to antigen expression. In targeting HL with antibodies directed against CD30 in particular, the ability to eradicate antigen-negative cells within the tumor mass may be useful because only a small percentage of the cells are CD30 positive and thought to be a part of the clonal malignancy. Because this bystander cytotoxic effect would be localized to the tumor microenvironment, with cells near the CD30-positive cells exposed to a concentration gradient of MMAE that decreases with increasing distance, it is likely to minimize toxic systemic exposure of diffusible tumor-released MMAE. In summary, targeted in vitro delivery of MMAE to CD30-expressing cells with SGN-35 leads to high and sustained intracellular MMAE levels, and successfully ablates both CD30-expressing malignant cells and neighboring malignant cells that do not express the target antigen. This may be of significance in treating tumors that are heterogeneous with respect to both antigen presentation and ADC distribution. The results reported here provide insight into the pronounced activities associated with this promising ADC. [1] Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor-targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell-penetrating peptide targeting matrix metalloproteinases and RGD-binding integrins (ACPP-cRGD-MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low-passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule- and dose-dependent manner, correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double-strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with nontargeted free MMAE or tumor-targeted MMAE (ACPP-cRGD-MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor-targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell-penetrating peptides. [2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.62 mg/mL (3.65 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.62 mg/mL (3.65 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (3.48 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. Solubility in Formulation 4: ≥ 2.5 mg/mL (3.48 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. Solubility in Formulation 5: ≥ 2.5 mg/mL (3.48 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. Solubility in Formulation 6: ≥ 2.5 mg/mL (3.48 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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 7: ≥ 2.5 mg/mL (3.48 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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. Solubility in Formulation 8: ≥ 2.5 mg/mL (3.48 mM) (saturation unknown) in 10% EtOH + 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 EtOH stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 9: ≥ 0.52 mg/mL (0.72 mM) (saturation unknown) in 1% DMSO + 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3928 mL | 6.9640 mL | 13.9280 mL | |
| 5 mM | 0.2786 mL | 1.3928 mL | 2.7856 mL | |
| 10 mM | 0.1393 mL | 0.6964 mL | 1.3928 mL |