KRAS(G12C)

ARS-1620 produces average peak tumor concentrations of 1.5 μM (50 mg/kg) and 5.5 μM (200 mg/kg) after a single oral dose or five consecutive daily doses. This allows for significant KRAS^G12C target occupancy (>=70% G12C-TE at 200 mg/kg) for longer than 24 hours. ARS-1620, administered once daily at a dose of 200 mg/kg, significantly suppresses tumor growth (p<0.001) in MIAPaCa2 xenografts (p.G12C) in a dose-dependent manner. For the full three-week course of treatment, ARS-1620 is well tolerated in all tumor models used. Moreover, even at oral doses of up to 1,000 mg/kg given daily for seven days, no clinical signs or toxicity of ARS-1620 have been seen in CD-1 mice.

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ARS-1620 Displays Potent and Selective Anti-tumor Activity in PDX Tumor Models [1]
To further demonstrate therapeutic efficacy with ARS-1620, we profiled anti-tumor responses in a panel of patient-derived (PDX) tumor xenograft models harboring KRAS p.G12C (n = 4) compared to PDX models that lack the mutant allele (n = 3) (also see Table S4 for targeted sequencing results). The p.G12C PDX panel comprised three adenocarcinoma NSCLCs, which is a potential target indication with the greatest frequency of KRAS p.G12C mutations (11%–16%) in patients (Campbell et al., 2016, Jordan et al., 2017) and a pancreatic adenocarcinoma that represents a minor proportion (<2%) of KRAS mutant pancreatic cancers (Bailey et al., 2016). ARS-1620 induced significant TGI (p < 0.001) and marked regression in p.G12C PDX models following 3 weeks of treatment using a daily 200 mg/kg schedule, whereas non-G12C bearing xenografts lacked any response (Figure 7A). We confirmed on average ≥75% G12C target occupancy of ARS-1620 in tumors at the end of study (6 hr post last dose) using a daily 200 mg/kg treatment schedule (Figure 7B). Additionally, ARS-1620 significantly inhibited p-ERK and p-S6 across the p.G12C PDX models (p < 0.05) (Figures 7C, S6A, and S6B). This resulted in significant apoptosis induction (p = 0.0148) as monitored by immunohistochemistry (IHC) staining of cleaved caspase-3 (Figure 7D). We next assessed if ARS-1620 administered at higher doses or frequency could improve the target occupancy and efficacy in tumor-bearing animals. Both 200 mg/kg twice daily (b.i.d.) or 400 mg/kg once daily schedules of ARS-1620 provided improved response rates in p.G12C PDX models (Figure 7A) and were associated with stronger p-ERK inhibition (Figures S6A–S6C), consistent with greater G12C occupancy as observed in cell-line-derived xenograft models (Figures S5E–S5H). Across all tumor models employed, ARS-1620 was well tolerated over the entire 3-week treatment period (Figures S6D and S6E). Moreover, there were no observed clinical signs or toxicity of ARS-1620 in CD-1 mice even at oral doses up to 1,000 mg/kg administered daily over a 7-day period (see Animal Studies in the STAR Methods). Above 400 mg/kg, ARS-1620 exhibits PK saturation and hence no further enhancement of anti-tumor efficacy was achieved in cell line-derived and PDX-derived models (data not shown). In summary, ARS-1620 is generally well-tolerated and the maximum tolerated dose (MTD) was not reached in mice. Collectively, the in vivo efficacy and mutant selectivity observations of ARS-1620 across a variety of KRASG12C mouse cancer models support future therapeutic strategies for covalently targeting the S-IIP of KRAS.

In p.G12C mutant cancer cells, ARS-1620 covalently and with high potency selectively inhibits KRAS (G12C) activity. In line with its covalent mechanism of inhibition, ARS-1620 quickly engaged G12C in a concentration- and time-dependent manner. ARS-1620 showed near complete engagement at 3.0 μM and half maximal G12C target engagement (TE50) at ~0.3 μM after 2 hours of treatment, when compared to a panel of cell lines carrying the mutant variant. In H358 (p.G12C), but not in negative control lung cancer cell lines lacking p.G12C (A549, H460, and H441), RS-1620 selectively and dose-dependently inhibits RAS-GTP and the phosphorylation of MEK, ERK, RSK, S6, and AKT. The potency of ARS-1620 is sub-micromolar allele-specific (IC50 = 0.3 μM; IC90 = 1 μM). Cys-12 is covalently modified by ARS-1620 to mediate its activity, which is exclusive to the G12C allele.

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Cellular KRASG12C target engagement (G12C-TE) [1]
Cells (30-50 x103) were treated with indicated compounds for the times listed and subsequently washed twice with PBS and prepared for protein extraction as previously described (Patricelli et al., 2016). Following iodoacetamide alkylation and trypsin digestion, the samples were analyzed by targeted LC/MS-MS analysis on a Dionex RSLCnano LC using Skyline Targeted Mass Spec Environment v3.6 software (MacLean et al., 2010) coupled with a Q-Exactive quadrupole orbitrap mass spectrometer as previously described (Patricelli et al., 2016). Kinetic G12C target engagement was modeled with KinTek Global Kinetic Explorer (Johnson, 2009).

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Tumor KRASG12C target engagement (G12C-TE) [1]
Protein extracts from tumors were prepared in IP lysis buffer using a Precellys bead homogenizer. Approximately 400 μg protein was aliquoted and spiked with 1 picomole heavy isotopically labeled KRASG12C 1-169 his-tagged protein (Lys-13C6,15N2 and Arg-13C6,15N4) as an internal standard. Heavy isotopically labeled KRASG12C was produced in E. coli, with protein purity higher than 90% and an isotopic purity of more than 99%. Proteins were precipitated using acetone, and re-suspended in LDS sample/reducing buffer and separated by SDS-PAGE using a 10% NuPAGE Bis-Tris gel, and subsequently stained with Coomassie brilliant blue. A gel band covering 15 to 25 kDa was excised from each lane, followed by in-gel trypsin digestion of the gel-embedded proteins. Released peptides from the gel were analyzed by targeted LC/MS-MS analysis on a Dionex RSLCnano LC coupled with a Q-Exactive quadrupole-orbitrap mass spectrometer using Skyline Targeted Mass Spec Environment v3.6 software (MacLean et al., 2010) as previously described (Patricelli et al., 2016). Briefly, precursor reaction monitoring was used to quantify the endogenous and heavy isotopic labeled tryptic KRASG12C peptide LVVVGAC∗GVGK and KRAS-NRAS normalization peptide SYGIPFIETSAK. Peptide light / heavy (L/H) isotope ratios were calculated from the peak areas for the endogenous and heavy isotopic labeled protein. Percent engagement was determined according to the following formula:

After being seeded into 24 well ULA-plates, 5×10⁴ cells are left to rest for the entire night. After that, DMSO or ARS-1620 are applied to the cells. After two treatment days, the amount of apoptosis and cell death is assessed using flow cytometry to measure the percentage of sub-diploid events and DNA content (cell cycle) or by staining with annexin V-APC and prodidium iodide or 70% ethanol fixation followed by FxCycle Violet staining[1].

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Cell proliferation assays [1]
For comparison of anti-growth activity a CellTiter-Glo (CTG) luminescent based assay was used. Cells (800-1,200 per well) were seeded (using the same media) in standard tissue culture-treated 96-well format plates or ultra-low attachment surface 96-well format plates. The day after plating, cells were treated with a 9 point 3-fold dilution series of indicated compounds (100 μl final volume per well) and cell viability was monitored 5 days later according to the manufacturer’s recommended instructions, where 50 μl of CellTiter-Glo reagent was added, vigorously mixed, covered, and placed on a plate shaker for 20 min to ensure complete cell lysis prior to assessment of luminescent signal. For inducible KRASG12V rescue experiments, cells were seeded as 3D suspensions (ultra-low adherent plates) in the presence or absence of doxycycline (100 ng/ml), 90 μl final culture media volume. 24hr later indicated concentrations of compound or DMSO (in 10 μl) was added to the cultures. Remaining cell numbers was monitored 5 days later using the CTG assay as described above. For experiment using Ras-less MEFs, cells were seeded as 3D suspensions (ultra-low adherent plates). The day after plating, cells were treated with a 9 point 3-fold dilution series of indicated compounds (100 μl final volume per well) and cell viability was monitored 5 days later by the CTG assay.

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Cell cycle and apoptosis assays [1]
5x104 cells were seeded into 24 well ULA-plates and allowed to rest overnight. Cells were then treated with DMSO or indicated compounds. After 2 days of treatment, apoptosis and cell death was measured by staining with annexinV-APC and prodidium iodide or by 70% ethanol fixation followed by FxCycle Violet staining to measure DNA content (cell cycle) and percentage of sub-diploid events by flow cytometry. Data was acquired on a MACSQuant flow cytometer and analyzed with FlowJo software V.10.1.

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Immunoblotting and RAS-GTP pulldown [1]
1X Lysis buffer (25 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5 mM MgCl2, 5% glycerol, 1% NP40) from Active Ras Detection kit was supplemented with phosphatase inhibitors and EDTA-free protease inhibitors (Protease inhibitor cocktail tables) and used for cell lysis. For lysates where RAS-GTP was assessed, we followed the manufacturers’ recommended procedure. In brief, 0.5-1x106 pre-adhered cells (for 24 hr prior) were rinsed with ice-cold PBS, or if in 3D-spheroid suspensions (0.5-1x106) cells (seeded in ultra-low adherent plates 24 hr prior) were pelleted at 300 x g for 3 min and washed with ice-cold PBS. Following this cells were lysed with 1ml (or 0.5ml) of lysis buffer containing 80 μg/ml of GST-tagged RAF-RBD for 10 min on ice. Remaining adherent cells were scraped off and lysate was centrifuged at 14,000 rpm for 5 min at 4°C. 90% of the pre-cleared lysates were subsequently added to pre-washed glutathione agarose beads for 1 hour at 4°C under constant rocking. The beads were subsequently pelleted and washed 3 times and eluted for western blotting with 40-60 μl of 1X SDS-PAGE sample buffer. The other remaining 10% of lysate was used to determine protein concentration by a Bradford protein assay and western blotting for indicated signaling markers. For time course experiments extending beyond 24 hours, a minor modification to the RAS-GTP pulldown assay was made to account for significant differences in cell number and/or apoptosis induction caused by the treatment. To account for this, cell lysates were processed with half volume lysis buffer without RBD added. A small sample of lysate was saved for protein concentration determination and the rest of the lysate was snap frozen. To ensure equal amount of protein undergoes RBD pulldown; lysates were subsequently thawed (at RT) and adjusted to 1 mg/ml with lysis buffer (0.5 mL volume). Equal amounts of lysate were then added to 0.5 mL lysis buffer containing RAF-RBD (1 mL total volume). Lysates were vortexed, incubated for 10 min on ice and subsequently pre-cleared at 14,000 rpm for 5 min at 4°C. The remaining steps proceeded similar as stated above.

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Cysteine selectivity profiling [1]
H358 cells (5x106) were treated with the indicated compounds and concentrations for 4 hours. Cells were subsequently washed and harvested for proteomic analysis by LC/MS-MS as previously described (Patricelli et al., 2016). Briefly, solvent exposed cysteines in cell lysates were labeled using 100 μM iodoacetamide desthiobiotin. Following trypsin digestion, desthiobiotinylated peptides were enriched using high-capacity streptavidin agarose. Peptide samples were analyzed using a Dionex RSLCnano LC coupled to a Q-Exactive Plus mass spectrometer. Progenesis LC-MS for proteomics v3.0 software was used for automated run alignments, peak picking, normalization, and peak abundance calculations across the dataset. Identification of desthiobiotinylated peptides was obtained by database searching using Proteome Discoverer v1.4. Normalized peak abundances for each peptide were exported to Excel 2013. Mean values and %CV were calculated for each sample group each containing 3 biological replicates. Log2 fold changes were calculated between DMSO control and treated sample groups. A two-tailed t test for each peptide was performed to determine the statistical significance between treated and DMSO control sample groups assuming equal variance.

Male BALB/c mice that are 6 to 8 weeks old are used for pharmacokinetic (PK) studies. Mice are given ARS-1620 by oral gavage administration at a dose of 10 mg/kg or a single intravenous (IV) bolus to assess oral bioavailability. The amount of ARS-1620 in plasma is measured using LC-MS/MS techniques. Mean plasma concentration-time profiles are used to estimate pharmacokinetic parameters. Using the linear trapezoidal rule, the area under the curve (AUC) is computed from time versus concentration data. The ratio of the AUC for ARS-1620 from the oral and IV dosage is used to compute the oral bioavailability. Relative doses are used to normalize the computation[1].

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\n\nPharmacokinetic (PK) studies in mice [1]
\nFor pharmacokinetic (PK) studies 6- to 8-week-old male BALB/c mice were used. To determine oral bioavailability, mice were treated with ARS-1620 by a single intravenous (IV) bolus or oral gavage administration at the doses of 2 and 10 mg/kg, respectively. ARS-1620 was formulated in water solution with 1% N-methyl-2-pyrrolidone, 19% polyethylene glycol 400, and 10% cyclodextrin and then sterilized by filtration for IV dosing. Oral formulation was prepared in solution (100% Labrasol®, Gattefossé). Drug concentration in plasma was quantified by LC-MS/MS-based methods. Pharmacokinetic parameters were estimated using Phoenix WinNonlin from mean plasma concentration-time profiles. The area under the curve (AUC) was calculated from time versus concentration data using the linear trapezoidal rule. The oral bioavailability is calculated as the ratio of AUC for ARS-1620 from oral and IV dosage. The calculation is normalized by relative doses. In all experiments, ARS-1620 displayed greater than 50% oral bioavailability. For PK analysis from tumor samples, vials containing tumor samples were added with 5-fold water and homogenized with a bead mill homogenizer. For calibration standard or QC samples, 40 μl of blank tumor homogenate was transferred into each well of a 96-well removable tube plate and spiked with 10 μl of 5X standard stock spiking solution of ARS-1620 prepared in 100% DMSO. For PK samples, 40 μl of tumor homogenate was transferred into each well of a 96-well removable tube plate and spiked with 10 μl of 100% DMSO. All samples were added with 150 μl of 100% ice-cold acetonitrile containing internal standard and vortexed to ensure thorough mixing. Samples were centrifuged at 3,400 rpm for 10 min and the clean supernatants (30 μl) were transferred into 96-well plate containing 170 μl water with 0.1% formic acid. The plate was capped and briefly vortexed to ensure thorough mixing of the extracted samples. The samples were subjected to LC-MS/MS analysis using an Agilent Technologies 6430 Triple Quad LC/MS system.
\n\nA Phenomenex Gemini-NX column (C18, 3 μm, 110 Å, 20 mm x 2.0 mm) was used for the LC-MS/MS analysis with mobile phase A containing 10 mM NH4HCO3 in water (pH 10, adjusted by NH4OH) and mobile phase B containing 100% acetonitrile. The LC gradient started with 10% B at time zero till 0.3 minutes, and then was increased to 90% B at 2 minutes. The gradient was decreased from 90% B to 10% B from 2.4 minutes to 2.5 minutes, and then the column was equilibrated at 10% B till 3 minutes. The mass peak of ARS-1620 was monitored by multiple reaction monitoring (MRM) using transition of 431.1 > 124.1 amu. Chromatogram signals were integrated and calibrated using Agilent MassHunter Workstation Software B.06.00. Pharmacokinetic parameters were derived by non-compartmental analysis using Phoenix WinNonlin (version 6.3) from individual tumor concentration versus time profiles. Results are expressed as mean ± s.d. No further statistical analysis was performed.\n

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\n\nToxicity assessment in mice [1]
\nFor toxicity assessment 6- to 8- week-old male and female CD-1 mice were administered ARS-1620 via oral gavage at doses of 0, 200, 600, and 1,000 mg/kg per day in 100% Labrasol® at 10ml/kg (n = 12 mice per group). Mortality, clinical observations, body weight, hematology, clinical chemistry, organ weights, and toxicokinetics were evaluated. No test article-related clinical observations occurred during the study. No significant drop in body weight was observed. No adverse hematological findings were found except mild increases in neutrophils at 1,000 mg/kg. No test article-related changes in clinical chemistry were noted. No test article-related changes in gross organ weights were found. Mild macroscopic inflammation in the stomach occurred at 1,000 mg/kg/day. No other indications of systemic toxicity were observed. No test article-related changes in microscopic evaluations of organs other than stomach irritation were found. At 1,000 mg/kg/day, Cmax and AUC0-24hr in males and females at the end of study were 5,000-9,000 ng/ml and 35,000-131,000 ng⋅h/mL, respectively.

Following a single oral dose or 5 consecutive days of daily administration, the mean peak tumor concentrations of ARS-1620 were 1.5 μM (50 mg/kg) and 5.5 μM (200 mg/kg), respectively, which significantly increased the occupancy of the KRASG12C target (G12C-TE ≥70% at 200 mg/kg) for more than 24 hours (Figures 5B and S5A). At these exposure doses, ARS-1620 induced dose- and time-dependent inhibition of RAS-GTP, and covalent modification of G12C in MIA-PaCa2 and H358 xenografts was also associated with this after a single dose (Figures 5C, S5B, and S5C). The target coverage was also extended to a regimen of taking ARS-1620 (200 mg/kg) daily for 3 consecutive days, providing significant G12C target occupancy (75% to 90% G12C-TE), as well as RAS-GTP and downstream signaling inhibition (Figure 5D). [1]

[1]. Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor. Cell. 2018 Jan 25;172(3):578-589.e17.

ARS-1620 is a quinazoline derivative with chlorine and fluorine substituents at positions 6 and 8, respectively, a 2-fluoro-6-hydroxyphenyl group at position 7, and a 4-(propanoyl)piperazin-1-yl group at position 4. It is a highly potent, selective, and orally bioavailable covalent KRAS-G12C inhibitor that effectively inhibits the gene encoding the protein KRAS (Kirsten rat sarcoma virus) in cells and animals. It can be used as an inhibitor, antiviral agent, and antitumor agent. Recently, it was discovered that KRASG12C can be targeted by allele-specific covalent targeting of the Cys-12 site located near the inducible allosteric switch II pocket (S-IIP). The success of this method relies on the dynamic cycling of KRASG12C between the active GTP-bound state and the inactive GDP-bound state, as S-IIP accessibility is limited to the GDP-bound state. This strategy has been shown to effectively inhibit mutant KRAS in vitro; however, its applicability in vivo remains uncertain. This article describes the design and identification of structure-based ARS-1620, a covalent compound with high efficiency and selectivity for KRASG12C. ARS-1620 can rapidly and persistently occupy the target in vivo, thereby inducing tumor regression. We used ARS-1620 to resolve the dependence of oncogenic KRAS and demonstrated that the monolayer cell culture model significantly underestimates the dependence of KRAS in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals that ARS-1620 represents a new generation of KRASG12C-specific inhibitors with good therapeutic potential. [1]

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Due to the lack of pharmacological tools, the strength and extent of KRAS dependence in KRAS-mutant cancers in vivo have not been fully studied. RNAi-based methods have been used to screen KRAS mutant cancer cell lines and large-scale shRNAs, revealing differences in sensitivity to KRAS silencing (Hayes et al., 2016; Lamba et al., 2014; McDonald et al., 2017; Singh et al., 2009; Sunaga et al., 2011; Vartanian et al., 2013). Differences in sensitivity to persistent mutant KRAS expression have also been confirmed under adherent monolayer and three-dimensional culture conditions (Fujita-Sato et al., 2015; Patricelli et al., 2016; Vartanian et al., 2013). In this report, we investigated KRAS dependence in the KRAS p.G12C cancer cell line. Using ARS-1620 as a pharmacological tool, this study systematically demonstrated the correlation between KRAS dependence in in vitro systems and in vivo non-small cell lung cancer (NSCLC) tumor models. We included G12C mutant cancer cells, which exhibited varying sensitivities to KRAS depletion, as confirmed by a meta-analysis of the Project DRIVE sensitivity network (McDonald et al., 2017). Using this cell line, we demonstrated differences in the sensitivity to KRAS inhibition using ARS-1620 in vitro, with varying KRAS-dependent scores across these cell lines, and also confirmed the culture-dependent effects of KRAS dependence in monolayer and 3D spheroid cultures. We can now extend these KRAS-dependent relationships to the in vivo environment. The high efficacy of ARS-1620 as a single agent in multiple cell lines and patient-derived mouse xenograft models underscores the central role of mutant KRAS in driving tumor growth and survival in vivo. Furthermore, our results not only demonstrate that 3D spheroid culture better predicts the in vivo sensitivity of KRAS-mutant cancer cells to ARS-1620, but also confirm that in vitro studies using adherent monolayer cell cultures to assess KRAS dependence significantly underestimate in vivo KRAS dependence. This has important translational implications for explaining the in vitro synthetic lethality of KRAS as a driver oncogene. Although 3D culture is increasingly valued and utilized to better mimic the in vivo environment and response to chemotherapy (Selby et al., 2017) and other therapeutic targets (e.g., HER2 and EGFR) (Ekert et al., 2014, Howes et al., 2014, Pickl and Ries, 2009, Weigelt et al., 2010), we have not yet found any approved oncology drugs exhibiting such significant activity differences between 2D and 3D cultures as KRAS inhibition. Patient response rates in future clinical trials of KRASG12C-targeted drugs will be an excellent test of the value of using 3D culture to predict clinical treatment response. In summary, the in vivo evidence of the broad efficacy of ARS-1620 as a single agent in non-small cell lung cancer (NSCLC) models demonstrates that a significant proportion of patients carrying p.G12C KRAS mutations may benefit from KRAS p.G12C targeted therapy. Our study provides the first in vivo evidence that S-IIP targeted therapy may be a promising treatment strategy for patients with KRAS p.G12C-mutant cancers. [1]

C21H17CLF2N4O2

430.8351

430.1

C, 58.54; H, 3.98; Cl, 8.23; F, 8.82; N, 13.00; O, 7.43

1698055-85-4

ARS-1323; 1698024-73-5; ARS-1630; 1698055-86-5

137003167

Off-white to light yellow solid powder

4

1

7

3

30

636

0

ClC1=C(C2C(=CC=CC=2O)F)C(=C2C(=C1)C(=NC=N2)N1CCN(C(C=C)=O)CC1)F

ZRPZPNYZFSJUPA-UHFFFAOYSA-N

InChI=1S/C21H17ClF2N4O2/c1-2-16(30)27-6-8-28(9-7-27)21-12-10-13(22)17(19(24)20(12)25-11-26-21)18-14(23)4-3-5-15(18)29/h2-5,10-11,29H,1,6-9H2

1-[4-[6-chloro-8-fluoro-7-(2-fluoro-6-hydroxyphenyl)quinazolin-4-yl]piperazin-1-yl]prop-2-en-1-one

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.83 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 (4.83 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.83 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 4: 5%DMSO + 40%PEG300 + 5%Tween 80 + 50%ddH2O: 4.3mg/ml (9.98mM)

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