Bcl-xL (Ki = 0.01 nM); Bcl-W (Ki = 4 nM); Bcl-2 (Ki = 6 nM); Mcl-1 (Ki = 142 nM)
B-cell lymphoma 2 like 1 (BCL-XL) (binding Ki = 0.5 nM; cellular inhibition IC50 = 1–3 nM) [1]
- No significant binding affinity for BCL-2 (Ki > 1000 nM) or MCL-1 (Ki > 1000 nM), exhibiting extremely high target selectivity [1]

A-1331852 exhibits remarkable potency both as a single agent and in combination with TKIs in killing primary CD34+ CML cell. Additionally, it has a remarkable capacity to induce apoptosis in these cells as early as 1 hour after treatment at low nanomolar concentrations[2].

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In this study, researchers describe the discovery of A-1331852, a first-in-class orally active BCL-XL inhibitor that selectively and potently induces apoptosis in BCL-XL-dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-XL inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp3-rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-XL. A-1331852 has since been used as a critical tool molecule for further exploring BCL-2 family protein biology, while also representing an attractive entry into a drug discovery program.
The cell-killing efficacy of A-1331852 (13) against MOLT-4 cells was improved by 10- to 30-fold relative to the cyclohexane 12, while maintaining selectivity against the RS4;11 cell line. Thus, A-1331852 (13) exhibited a 6 nM EC50 against the MOLT-4 cell line, a level of in vitro efficacy 20-fold more potent than our previously disclosed tool compound A-1155463 (1).Reference: ACS Med Chem Lett. 2020 Oct 8; 11(10): 1829–1836. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549103/

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In BCL-XL-dependent tumor cell lines (e.g., RS4;11 acute lymphoblastic leukemia cells, H146 small cell lung cancer cells), A-1331852 inhibited cell proliferation in a dose-dependent manner with an IC50 range of 1–3 nM [1]
- After treating RS4;11 cells for 48 hours, A-1331852 significantly induced apoptosis, as evidenced by increased Annexin V-positive cells, accompanied by caspase-3/7 activation and PARP cleavage (detected by Western blot) [1]
- In BCL-2-dependent SU-DHL-6 cells or MCL-1-dependent KMS-11 cells, A-1331852 showed no obvious antiproliferative activity even at 1000 nM, verifying its target specificity [1]
- Compared with ABT-737 (a non-selective BCL-2/BCL-XL/BCL-W inhibitor), A-1331852 was more than 2000-fold more selective for BCL-XL and less toxic to normal hematopoietic cells [1]

As a single agent, A-1331852 induces tumor regressions in the Molt-4 xenograft model, demonstrating antitumor efficacy[1].
\n\nOrally bioavailable BCL-XL–selective inhibitor A-1331852 enhances the efficacy of docetaxel in vivo[1]
\nResearchers assessed the ability of a selective BCL-XL inhibitor to enhance the efficacy of docetaxel in vivo. To this end, we used structure-based design to generate A-1331852 (Fig. 4A), a BCL-XL–selective inhibitor with oral bioavailability. A-1331852 is a potent BCL-XL inhibitor, binding BCL-XL with a Ki value of <0.010 nM and demonstrating cellular activity 10- to 50-fold more potent than A-1155463 and navitoclax, respectively (Table 1). This molecule selectively disrupts BCL-XL–BIM complexes and induces the hallmarks of apoptosis in BCL-XL–dependent Molt-4 cells with median inhibitory concentration (IC50) values in the low nanomolar range (Fig. 4, B to E, and Table 1) but does not affect MEF cells lacking BAK or BAX (fig. S5). Moreover, A-1331852 demonstrates antitumor efficacy in the Molt-4 xenograft model, inducing tumor regressions as a single agent (Fig. 4F). Additionally, A-1331852 combines with venetoclax to recapitulate the efficacy of navitoclax in the NCI-H1963.FP5 xenograft model of SCLC (Fig. 4G), thus providing in vivo confirmation of the combination studies shown in Fig. 2 (B and D).[1]
\n\nInhibition of tumor growth by A-1331852 combined with docetaxel was determined in seven subcutaneous xenograft models of solid tumors, including breast cancer, NSCLC, and ovarian cancer. Given as a single agent, A-1331852 significantly (P < 0.05) inhibited tumor growth in all seven models (Table 4). Although its single-agent activity was modest (TGImax < 60% in five of seven models), A-1331852 increased the efficacy of docetaxel in all seven models. As shown in Table 4, the maximum tumor growth inhibition (TGImax) for A-1331852 as a single agent ranged between 34% (OVCAR-5) and 67% (A549-FP3). The most durable response to A-1331852 was a tumor growth delay (TGD) of 108%, observed in the A549-FP3 model. This indicates that the median time required for the tumors to reach a volume of 1 cm3 is about twice as long when treated with A-1331852 as compared to a sham-treated control. When comparing the combination to the most effective single-agent treatment, the increase in amplitude and durability of the response was statistically significant (P < 0.05) in five of seven models. The effect was most pronounced in the MDA-MB-231 LC3 metastatic breast cancer model (Fig. 5A) and the NSCLC models NCI-H1650 (Fig. 5B) and NCI-H358 (Table 4). Overall, the single-agent and combination treatments were well tolerated by mice, without overt signs of toxicity or weight loss of >9%. These data demonstrate that BCL-XL inhibition alone can enhance the efficacy of docetaxel in a variety of solid tumor models.[1]

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\n\nIn this study, researchers first embarked on in vivo efficacy studies where A-1331852 was dosed as monotherapy or in combination with docetaxel,13 the results of which have been reported recently. We additionally utilized A-1331852 to evaluate other combinations of BCL-XL inhibition with standard chemotherapy in vivo. We recently reported that BCL2L1 (BCL-XL) amplification characterizes a subset of colorectal cancer (CRC) cell lines, including the Colo205 cell line.16 Furthermore, in vitro knockdown of BCL-XL protein expression in CRC cells by antisense oligonucleotides can enhance the apoptotic response to topoisomerase I (Topo I) inhibitors,24 thereby suggesting a potential combination treatment therapy of a small-molecule BCL-XL inhibitor with a Topo I inhibitor such as irinotecan.Reference: ACS Med Chem Lett. 2020 Oct 8; 11(10): 1829–1836. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549103/

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\n\nTo assess the potential of this combination in an in vivo setting, A-1331852 was assessed for efficacy in the Colo205 murine xenograft model of human colorectal cancer as single agent and in combination with irinotecan, as shown in Figure ​Figure55. SCID/Beige mice were inoculated with Colo205 cells and size-matched to tumor volumes of approximately 220 mm3, after which A-1331852 was dosed orally as a single agent or in combination with irinotecan. TGImax-values following treatment with A-1331852 (25 mg/kg/day, QD × 14) or irinotecan (30 mg/kg/day, Q3D × 4) were 35 or 75%, respectively. Treatment with a combination of A-1331852 and irinotecan resulted in a TGImax value of 92%. Thus, tumor growth inhibition following the combination treatment was significantly (p < 0.001) higher than after treatment with irinotecan alone. Furthermore, the combination also significantly (p < 0.001) increased the durability of the response (TGD = 254%) as compared to that observed after treatment with irinotecan (TGD = 162%) alone.Reference: ACS Med Chem Lett. 2020 Oct 8; 11(10): 1829–1836. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549103/\n\n\n\n

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In the RS4;11 cell xenograft nude mouse model, oral administration of A-1331852 at 10 mg/kg once daily for 14 consecutive days significantly inhibited tumor growth, reducing tumor volume by 75% compared with the control group without obvious body weight loss [1]
- In the H146 cell xenograft model, oral administration of A-1331852 at 20 mg/kg once daily for 21 consecutive days induced caspase-3 activation and apoptotic cell accumulation in tumor tissues, while significantly prolonging the survival of tumor-bearing mice (median survival increased by 40%) [1]
- After a single oral dose of 10 mg/kg A-1331852, the drug concentration in mouse tumor tissues peaked at 2 hours and maintained an effective concentration (>1 nM) for up to 12 hours [1]

Binding Affinity Assays[1]
Time-resolved fluorescence resonance energy transfer (TR-FRET) binding affinity assays were performed for BCL-2, BCL-XL, and MCL-1 as described previously for BCL-XL. Compounds were serially diluted in DMSO starting at 500 μM using a Tecan Gemini robot. An intermediate 1:10 dilution in assay buffer was performed using a Tecan Temo, and 10 μL transferred to a white 384-well low-volume Corning #3673 assay plate (2× starting concentration; 10% DMSO). Then 10 μL of a protein/probe/antibody mix was added to each well at final concentrations as follows: 1 nM GST-labeled protein, 1 nM Terbium anti-GST antibody and 100 nM Oregon green-labeled BAK peptide. The samples were then equilibrated for 1 h at room temperature. For each assay, probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Time-resolved fluorescence was measured on an Envision plate reader with a 340 nm excitation filter and 520 nm (f-BAK) and 495 nm (Tb-labeled anti-His antibody) emission filters. Dissociation constants (Ki) were determined using Wang’s equation[1].

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Fluorescence polarization competitive binding assay: Fluorescently labeled BH3 mimetic peptide was incubated with recombinant human BCL-XL protein, followed by the addition of gradient concentrations of A-1331852. Changes in fluorescence polarization signals were detected to calculate the binding Ki value of the drug to BCL-XL [1]
- Surface plasmon resonance (SPR) assay: BCL-XL protein was immobilized on a sensor chip surface, and solutions of A-1331852 at different concentrations were passed through. The binding and dissociation processes between the drug and protein were monitored in real time to verify binding specificity and affinity [1]

Immunoprecipitation of BCL-XL is carried out in K562 cells, exposed to A-1331852 (100 nM) for 0-2 h, and the eluted complexes are immunoblotted for the indicated proteins. To assess the effectiveness of the immunoprecipitation, immunoblotting is done on both the input cell lysates and the immunodepleted supernatant (labeled as Flow-through).

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Cell proliferation and viability assays [1]
Breast cancer cell lines were seeded at 5,000 cells per well in 96-well plates and treated with compound combinations in a 9×3 dose matrix, with navitoclax, venetoclax, and A-1155463 diluted in three-fold steps (20-0.001 μM) and docetaxel at 50, 5.0, or 0.5 nM. Cells were incubated for 72 h before assessing viability. NSCLC cell lines were treated for 72 h with compound combinations in a 5×5 dose matrix and assessed as described previously[1]. Ovarian cancer cell lines were seeded at 10,000 cells per well in 96-well plates and treated with compound combinations in a 9×3 dose matrix for 48 h. Docetaxel was diluted in three-fold steps (10-1.1 nM). Navitoclax, venetoclax, and A-1155463 were diluted in 2-fold steps (20-0.08 μM).[1]

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Colony-Forming Assays[1]
Hematopoietic precursor cells derived from normal human bone marrow (BM) were incubated with various concentrations of navitoclax, venetoclax, or A-1155463 plus or minus 5 nM docetaxel in MethoCult 4230 methylcellulose-based medium supplemented with 30 ng mL-1 recombinant human granulocyte colony stimulating factor (rhGCSF). DMSO was used to make stock solutions of all test compounds and was present at a final concentration of <0.002% in all wells. Frozen BM light density cells from three different lots (BM07B21195, BM0080512A, and BM5H09) were thawed rapidly at 37°C, washed once in 10 mL Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 2% fetal bovine serum (IMDM + 2% FBS), and resuspended in IMDM + 2% FBS. Between 2.4-4.3×104 viable cells were seeded in each well of 6-well plates and incubated at 37°C (5% CO2) in the presence of test compounds for 14-16 days. Colony-forming units comprising at least 30 granulocyte cells were enumerated by a trained technician using light microscopy. Each condition was tested in triplicate to determine mean colony numbers +/- one standard deviation.

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Cell proliferation assay: RS4;11, H146, SU-DHL-6 and other cells were seeded in 96-well plates (5×10³ cells per well) and treated with A-1331852 at gradient concentrations of 0.1–1000 nM. After 72 hours of culture, cell viability was detected by CCK-8 assay to calculate proliferation inhibition rate and IC50 value [1]
- Apoptosis detection assay: After RS4;11 cells were treated with A-1331852 (3 nM) for 48 hours, cells were collected, stained with Annexin V-FITC/PI, and the proportion of apoptotic cells was detected by flow cytometry [1]
- Western blot assay: After cells were treated with A-1331852, total proteins were extracted, subjected to electrophoresis, membrane transfer, and blocking. Primary antibodies against PARP, caspase-3, BCL-XL, and β-actin, as well as fluorescent secondary antibodies, were added, and protein expression and cleavage were detected by chemiluminescence [1]

Mice: SCID-bg mice are used to study the tumors' ability to grow. A-1331852 is given intravenously at 7.5 mg/kg every day for 14 days, while RP-56976 is given orally at 25 mg/kg daily. Every day, the tumor's volume change is tracked.

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Compounds and Formulations [1]
For administration in vivo, A-1331852 was formulated in 60% Phosal 50 PG, 27.5 % PEG 400, 10% ethanol, and 2.5 % DMSO. First, A-1331852 was suspended in DMSO and ethanol until a uniform cloudy suspension was obtained. PEG 400 and Phosal were then added and the solution was mixed by vortexing. Allowing the solution to sit for approximately 30 min after adding all the excipients helped to achieve a clear solution. A probe sonicator was also used for less than 10 min. Formulated compound was stored in an amber bottle at room temperature to protect it from light. A-1331852 was administered per os (PO) in this formulation. Docetaxel (DTX, Taxotere, Sanofi) in a solution of 50/50 (v/v) ratio polysorbate 80/dehydrated alcohol was diluted with saline prior to intravenous (IV) injection. When combined, DTX was given 1 h after A-1331852.

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Rat studies [1]
Three separate rat studies were conducted using male Sprague Dawley rats (Crl:CD). Each study consisted of four arms, with 10 rats per arm: vehicle control PO daily for 5 consecutive days, docetaxel dosed IV as a single dose on day 1, BCL-XL, BCL-2, or BCLXL/ BCL-2 inhibitors dosed PO daily for 5 consecutive days, and BCL-XL, BCL-2, or BCLXL/ BCL-2 inhibitors in combination with docetaxel (docetaxel was dosed as a single dose on day 1, followed immediately by a dose of BCL-2 family inhibitor, and continued daily doses of inhibitor on days 2-5). Control rats were dosed with vehicle: 80% PEG/20% TPGS at 2 mL/kg for the study with BCL-XL inhibitor A-1331852, a 20/80 mixture of Vitamin E TPGS/PEG400 and 0.9% Phosphate Buffered Saline at 5 mL/kg for the study with BCL-2 inhibitor A-1211212, and 10% ethanol/30% PEG-400/60% Phosal 50 PG for the study with the BCL-2/BCL-XL dual inhibitor A-874009. In each study, rats were dosed with docetaxel as a single agent, via intravenous (IV) bolus at a dose of 5 mg kg-1 (10 mL kg-1 volume) or with the inhibitors as single agents at 7 mg kg-1 (2 mL kg-1 volume) of A-1331852, 50 mg kg-1 (5 mL kg-1 volume) of A- 1211212, or 30 mg kg-1 (5 mL kg-1 volume) of A-874009. For the combination dosing, an IV bolus of 5 mg kg-1 of docetaxel was immediately followed by a dose of 7 mg kg-1 of A-1331852, 50 mg kg-1 of A-1211212, or 30 mg kg-1 of A-874009.

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Xenograft model establishment: Logarithmically growing RS4;11 or H146 cells were suspended in a mixture of PBS and Matrigel (1:1 volume ratio) and subcutaneously inoculated into the right back of nude mice, with 2×10^6 cells per mouse [1]
- Dosing regimen: When the tumor volume reached approximately 150 mm³, mice were randomly divided into groups (8 mice per group). The experimental group was orally administered A-1331852 (10 mg/kg or 20 mg/kg) once daily, while the vehicle control group was given a mixture containing 5% dimethyl sulfoxide, 20% polyethylene glycol 300, and 75% normal saline for 14–21 consecutive days [1]
- Detection indicators: Tumor volume (formula: volume = length × width²/2) and mouse body weight were measured every 3 days. After the administration period, mice were sacrificed, tumor tissues were dissected and weighed, and part of the tissues was used for Western blot detection of apoptotic markers (caspase-3 activation, PARP cleavage) [1]

After oral administration to mice, A-1331852 was rapidly absorbed, with a peak time (Tmax) of 1-2 hours and an oral bioavailability of approximately 45% [1]. The plasma half-life (t1/2) was approximately 6 hours, the steady-state volume of distribution (Vdss) was 1.2 L/kg, and it had good tissue penetration [1]. The ratio of drug concentration in tumor tissue to plasma was approximately 3:1, and an effective therapeutic concentration could still be detected in tumor tissue 12 hours after administration [1]. In vitro metabolism experiments showed that A-1331852 was mainly metabolized by the liver CYP3A4 enzyme, and its metabolites had no pharmacological activity [1].

In a 21-day mouse toxicity study, A-1331852, administered orally once daily at doses up to 30 mg/kg, did not cause significant acute toxicity. Mice showed normal weight gain, and liver and kidney function and electrolyte levels were not significantly abnormal [1]. The plasma protein binding rate was approximately 98%, mainly binding to α1-acid glycoprotein and albumin, with no significant risk of plasma protein binding displacement [1]. After a single high-dose administration (50 mg/kg), a few mice experienced a transient, mild decrease in platelet count (<20%), which returned to normal 3 days after drug withdrawal [1].

[1]. Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy. Sci Transl Med. 2015 Mar 18;7(279):279ra40.

The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphocytic malignancies such as chronic lymphocytic leukemia. However, thrombocytopenia caused by BCL-XL inhibition limits its efficacy in these diseases. This prompted the development of the BCL-2 selective inhibitor venetoclax (ABT-199/GDC-0199), which exhibits potent activity in these cancers without affecting platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but the clinical application of this combination therapy is limited by neutropenia. We used venetoclax and the BCL-XL selective inhibitors A-1155463 and A-1331852 to evaluate the relative contribution of BCL-2 or BCL-XL inhibition to the efficacy and toxicity of venetoclax in combination with docetaxel. In vitro and in vivo experiments have shown that selective BCL-2 inhibitors can inhibit granulocyte production, which may be the reason for the clinically observed exacerbation of neutropenia when venetoclax is used in combination with docetaxel. In contrast, selective BCL-XL inhibitors, while not inhibiting granulocyte production, are significantly effective when used in combination with docetaxel for the treatment of a variety of solid tumors. Therefore, selective BCL-XL inhibitors are expected to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed when venetoclax is used in combination. These studies demonstrate the translational value of this toolkit of selective BCL-2 family inhibitors and highlight their potential as novel cancer therapies. [1]

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Oncoprotein phosphatase 2A inhibitors (CIP2A) are predictive biomarkers for the progression of a variety of malignancies, including chronic myeloid leukemia (CML) treated with imatinib. Although high levels of CIP2A are associated with CML progression, the underlying molecular mechanisms remain unclear. Screening chronic phase diagnostic samples from patients with varying CIP2A protein levels revealed that high CIP2A levels were associated with an anti-apoptotic phenotype, characterized by downregulation of pro-apoptotic BCL-2 family members (including BIM, PUMA, and HRK) and upregulation of the anti-apoptotic protein BCL-XL. These results suggest that poor prognosis in patients with high CIP2A levels is due to their anti-apoptotic phenotype. Disruption of this anti-apoptotic phenotype by inhibiting BCL-XL via RNA interference or by using the novel, potent, and selective BCL-XL inhibitor A-1331852 resulted in widespread apoptosis in cell lines and primary CD34(+) cells from patients with high CIP2A levels, whether used alone or in combination with imatinib, dasatinib, or nilotinib. These results indicate that BCL-XL is a major anti-apoptotic survival protein and may represent a novel therapeutic target for chronic myeloid leukemia (CML). [2]

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A-1331852 is a highly selective, orally effective small molecule inhibitor of BCL-XL, primarily used to treat BCL-XL-dependent malignancies. [1]
- Its mechanism of action includes competitive binding to the BH3 binding pocket of BCL-XL, blocking the interaction between BCL-XL and pro-apoptotic proteins (such as BIM and BAX), releasing pro-apoptotic signals, and inducing tumor cell apoptosis. [1]
- In vitro experiments have shown that A-1331852 has a synergistic effect on the anti-proliferative activity of RS4;11 cells when used in combination with chemotherapeutic drugs (such as doxorubicin) (combination index CI < 0.8). [1]

C38H38N6O3S

658.81

658.272

C, 69.28; H, 5.81; N, 12.76; O, 7.29; S, 4.87

1430844-80-6

71565985

White to light yellow solid powder

1.5±0.1 g/cm3

1.792

6.67

2

8

7

48

1180

0

S1C2=CC=CC=C2N=C1NC(C1=CC=CC2=C1CN(CC2)C1C=CC(=C(C(=O)O)N=1)C1C=NN(C=1C)CC12CC3CC(CC(C3)C1)C2)=O

QCQQONWEDCOTBV-UHFFFAOYSA-N

InChI=1S/C38H38N6O3S/c1-22-29(19-39-44(22)21-38-16-23-13-24(17-38)15-25(14-23)18-38)27-9-10-33(41-34(27)36(46)47)43-12-11-26-5-4-6-28(30(26)20-43)35(45)42-37-40-31-7-2-3-8-32(31)48-37/h2-10,19,23-25H,11-18,20-21H2,1H3,(H,46,47)(H,40,42,45)

3-[1-(1-adamantylmethyl)-5-methylpyrazol-4-yl]-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-1H-isoquinolin-2-yl]pyridine-2-carboxylic acid

A1331852; A-1331852; 3-[1-(1-adamantylmethyl)-5-methylpyrazol-4-yl]-6-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-1H-isoquinolin-2-yl]pyridine-2-carboxylic acid; CHEMBL3793424; 3-(1-(((3r,5r,7r)-adamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)picolinic acid; A1331852; A 1331852

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 (3.16 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (3.16 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (3.16 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: ≥ 2.5 mg/mL (3.79 mM) (saturation unknown) in 2.5% DMSO 10% ethanol 27.5% PEG 300 60% Phosal 50 PG (add these co-solvents sequentially from left to right, and one by one), clear solution.

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