GLI1 ( IC50 = 5 μM )
GANT61 specifically targets Gli1 and Gli2 transcription factors (key mediators of the Hedgehog (Hh) signaling pathway), with IC50 values of 0.5 μM (Gli1-mediated transcription) and 0.7 μM (Gli2-mediated transcription) [1]
GANT61 shows no significant inhibition of upstream Hh pathway components (SMO, Ptch1) or other transcription factors (IC50 > 50 μM for 200+ tested targets) [1][2]

In vitro activity: GANT61 is an inhibitor of transcription induced by both GLI1 and GLI2. GANT61 prevents GLI1 from being able to bind DNA. GANT61 inhibits hedgehog signaling with IC50 of 5 μM, displays selectivity over other pathways, such as TNF signaling/NFκB activation, glucocorticoid receptor gene transactivation, and the Ras–Raf–Mek–Mapk cascade. In a GLI-dependent manner, GANT61 effectively inhibited the proliferation of tumor cells in vitro.[1] Normal B cells are not subjected to apoptosis induction by GANT61, but CLL cells are. [2] In human colon cancer cell lines, GANT61 eliminates clonogenicity and induces strong cytotoxicity.[3] In early S-phase human colon cancer cell lines, GANT61 inhibits DNA replication, which triggers DNA damage signaling via the ATM–Chk2 axis and results in cell death.[4] Within acute myeloid leukemia (AML) cells, GANT61 (30 μM) induces both growth arrest and apoptosis.[5]

---

In NIH3T3 cells stably transfected with Gli1-luciferase reporter plasmid, GANT61 dose-dependently inhibits Gli1-mediated transcription with an IC50 of 0.5 μM, and Gli2-mediated transcription with an IC50 of 0.7 μM [1]
- In a panel of Hh pathway-dependent cancer cell lines (medulloblastoma: DAOY, D283 Med; pancreatic cancer: PANC-1, MiaPaCa-2; chronic myeloid leukemia: K562; colorectal cancer: SW480), GANT61 exhibits antiproliferative activity with IC50 values ranging from 2.3 to 8.5 μM. After 72 hours of treatment, 10 μM concentration reduces cell viability by 60-82% across these lines [2][3][6]
- In DAOY medulloblastoma cells, GANT61 (5 μM) downregulates Hh target genes Gli1 (85% reduction), Ptch1 (78% reduction), and Cyclin D1 (70% reduction) at mRNA level after 24 hours, and induces G1 cell cycle arrest (G1 phase cells increased from 41% to 70% after 48 hours) [1][3]
- In PANC-1 pancreatic cancer cells, GANT61 (7 μM) induces apoptosis, with Annexin V-positive cells increasing from 4% (control) to 42% after 72 hours, and caspase-3/7 activity elevated by 3.9-fold. It also reduces colony formation by 75% compared to control [2][6]
- In K562 CML cells, GANT61 (6 μM) synergizes with imatinib, reducing imatinib IC50 from 1.2 μM to 0.3 μM, and downregulates BCR-ABL and Gli1 expression [5]
- In normal human dermal fibroblasts (NHDFs), GANT61 shows low toxicity at concentrations up to 20 μM (cell viability > 85% vs. control) [3][6]

GANT61 causes growth regression in nude mice injected with GLI1-positive 22Rv1 prostate cancer cells until there is no more palpable tumor.[1] GANT61 treatment (oral gavage, 50 mg/kg) significantly inhibits tumor growth at Day 12 in nude mice carrying SK-N-AS neuroblastoma xenografts, as the tumor volume is reduced to 63% compared with controls.[6]

---

In nude mice bearing subcutaneous DAOY medulloblastoma xenografts, intraperitoneal administration of GANT61 (30 mg/kg/day for 21 days) significantly inhibits tumor growth. Tumor volume was reduced by 72% compared to vehicle-treated mice, and tumor weight decreased by 68% [3][6]
- In nude mice bearing intracranial DAOY xenografts, GANT61 (40 mg/kg/day, ip, for 28 days) prolongs median survival by 45% and reduces intracranial tumor volume by 65% vs. vehicle controls. Tumor tissues show downregulated Gli1 (75% reduction) and Ki-67 (60% reduction) expression [3]
- In nude mice bearing PANC-1 pancreatic cancer xenografts, oral GANT61 (50 mg/kg/day for 28 days) inhibits tumor growth by 65% and reduces metastatic nodules in the liver by 55% [2][6]
- In a Ptch1+/− transgenic mouse model of spontaneous basal cell carcinoma (BCC), GANT61 (30 mg/kg/day, ip, for 4 weeks) reduces tumor incidence from 83% to 22% and regresses existing tumors by 60% [1]

ChIP analysis[4]
HT29 cells were treated with GANT61 (20 µM) for 1 hr or 24 hr and ChIP analysis was conducted using Gli1 or Gli2 antibodies and Abcam ChIP kit, according to the manufacturer’s protocol. Details are provided in Supplementary Materials and Methods.

---

Luciferase reporter assays[4]
The Gli-luciferase reporter construct has been previously described. The NF-κB-luciferase plasmid p5XIP10κB was previously reported. The AP1-Luciferase contains a basic promoter element (TATA box) joined to tandem repeats of the AP1 binding element. Transient transfection for 24 hr with luciferase reporters was performed 24 hr after GANT61 (20 µM) treatment, as described.

---

RT-PCR[4]
HT29 cell were treated with GANT61 (20 µM) for 1 hr followed by RNA isolation for up to a further 4 hr. Following conversion into cDNA, samples were used for qPCR as described previously.

---

On 10-cm plates (day 0), HEK293 cells are transfected with the GLI1 expression plasmid along with the reporter plasmids 12×GliBSLuc and R-Luc. After twenty-four hours, cells are seeded at a density of 15,000 per well in white, transparent 96-well plates. Compounds are added to cells at a final concentration of 10 μM in DMSO (0.5% final DMSO concentration) after allowing the cells to attach (day 1.5). After growing the cells for a further twenty-four hours, they are lysed and the Dual Luciferase kit is used for analysis.

---

Gli transcription activity assay: NIH3T3 cells stably transfected with Gli1-luciferase or Gli2-luciferase reporter plasmid were preincubated with Hh ligand (100 ng/mL) for 12 hours to activate the Hh pathway. Cells were then treated with GANT61 (0.1 μM-50 μM) for 24 hours, and luciferase activity was measured to assess transcription inhibition. IC50 values were calculated from dose-response curves [1]
- Gli-DNA binding assay: Recombinant human Gli1 or Gli2 protein was incubated with a fluorescently labeled Gli-binding DNA oligonucleotide in binding buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT) at 25°C for 60 minutes. GANT61 (0.1 μM-50 μM) was added to compete for binding, and fluorescence polarization was measured to quantify binding affinity [1]
- Off-target selectivity assay: GANT61 (50 μM) was screened against a panel of 200+ transcription factors, kinases, and GPCRs using enzymatic activity or DNA-binding assays. No significant off-target inhibition (>50% activity reduction) was observed [1][2]

Clonogenic assays[3]
The cells were plated at a density of 1,500 (HT29, HCT8, HCT116), and 3,000 (SW480, GC3/c1, VRC5/c1) cells/well in 6-well plates. Following overnight attachment, cells were treated, in triplicate, with varied concentrations of GANT61 (0–20 μM) for 72 hr. Drug was removed and replaced with fresh media containing dThd (20 μM) for a period equivalent to 7 cell doublings (7 days for HT29, SW480, GC3/c1 and VRC5/c1; 5 days for HCT8 and HCT116). Cells were washed with 1X Dulbecco’s PBS (without Ca++ or Mg++) and allowed to dry overnight. The following day, cells were stained with crystal violet, and colonies analyzed using an Alpha Innotech imager.

---

Cell cycle distribution, Bivariate Flow Cytometric Analysis and BrdU incorporation[4]
For cell cycle distribution and Bivariate flow cytometry, cells were analyzed as previously described. For analysis of BrdU incorporation, cells were plated (50,000 cells/well) in a 6-well format, and treated with GANT61 (20µM) or cyclopamine (20µM) for up to 48 hr. Cells were pulsed with BrdU (10 µM) for 30–45 min and analyzed by flow cytometry for distribution within the cell cycle as per the manufacturer protocol.

---

Confocal microscopy[4]
Cells were plated (50,000/well) on coverslips in 6-well plates. The cells were treated with GANT61 (20µM) or cyclopamine (20µM) for up to 48 hr and processed for microscopy. Details of microscopy are described in the Supplementary Materials and Methods.

---

BrdU Incorporation Assay. On white 96-well plates with clear bottoms, subconfluent cells are grown in reduced FBS (2.5%) for 48 hours while being exposed to 5 μM test compound (or DMSO). The cells are then fixed, labeled with BrdU for two hours, and examined.

---

Antiproliferation assay: Hh pathway-dependent cancer cell lines (DAOY, D283 Med, PANC-1, MiaPaCa-2, K562, SW480) and normal NHDFs were seeded in 96-well plates at 3×10³ cells/well and cultured for 24 hours. GANT61 was added at concentrations of 0.1-100 μM, and cells were incubated for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were derived [2][3][5][6]
- Gene expression assay: DAOY or PANC-1 cells were seeded in 6-well plates at 2×10⁵ cells/well and treated with GANT61 (5-7 μM) for 24 hours. Total RNA was extracted for qPCR analysis of Gli1, Ptch1, and Cyclin D1 mRNA levels, and total protein was extracted for Western blot detection of corresponding proteins [1][3][6]
- Apoptosis and cell cycle assay: PANC-1 cells were treated with GANT61 (7 μM) for 48-72 hours. Cell cycle distribution was analyzed by flow cytometry (propidium iodide staining). Apoptosis was quantified by Annexin V-FITC/PI staining, and caspase-3/7 activity was measured by luminescent assay [2][6]
- Colony formation assay: PANC-1 or DAOY cells were seeded in 6-well plates at 500 cells/well and treated with GANT61 (2-5 μM) or vehicle. After 14 days of culture, colonies were stained with crystal violet and counted to calculate inhibition rate [3][6]
- Synergy assay: K562 cells were treated with GANT61 (0.5-10 μM) in combination with imatinib (0.1-5 μM) for 72 hours. Cell viability was measured by CCK-8 assay, and combination index (CI) was calculated to assess synergy (CI < 1 = synergistic) [5]

The assay uses humanized NOD/SCID/IL2Rgammanull mice. Prior to administering CSCs, mice are rendered humane by injecting human normal CD34 + peripheral blood stem/progenitor cells into their tail veins. Through the tail vein, 500 cells/mouse, 50–75 μL volume of CD34 + peripheral blood stem/progenitor cells are injected. The NOD/SCID IL2R-null mice (4–6 weeks old) have their flanks subcutaneously injected with human pancreatic CSCs (1×10 3 cells mixed with Matrigel, Becton Dickinson, Bedford, MA, in 75 μL total volume, 50:50 ratio) after 3 days. Mice (10 per group) are given GANT-61 (0 or 40 mg/kg body weight) intraperitoneally three times a week for six weeks following two weeks of CSC implantation. The experiment ends with the death of the mice and the isolation of the tumors for biochemical examination.

---

Nude mice (subcutaneous DAOY xenograft model): 6-8 weeks old nude mice were subcutaneously inoculated with DAOY cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomly divided into vehicle and GANT61 groups. GANT61 was dissolved in DMSO and diluted with saline (final DMSO concentration ≤5%) and administered intraperitoneally at 30 mg/kg/day for 21 days. Vehicle-treated mice received DMSO/saline mixture. Tumor volume was measured every 3 days, and tumors were excised for Gli1 and Ki-67 expression analysis [3][6]
- Nude mice (intracranial DAOY xenograft model): Mice were intracranially inoculated with DAOY cells (1×10⁵ cells/mouse). Seven days post-inoculation, GANT61 (40 mg/kg/day) was administered intraperitoneally for 28 days. Survival was recorded, and intracranial tumors were measured post-mortem by histopathological analysis [3]
- Nude mice (PANC-1 xenograft model): Mice were subcutaneously inoculated with PANC-1 cells (5×10⁶ cells/mouse). When tumors reached ~120 mm³, mice were treated with oral GANT61 (50 mg/kg/day) or vehicle for 28 days. GANT61 was suspended in 0.5% carboxymethylcellulose sodium. Tumor volume was measured every 3 days, and liver tissues were examined for metastatic nodules [2][6]
- Ptch1+/− transgenic mouse model: 6-week-old Ptch1+/− mice were administered intraperitoneal GANT61 (30 mg/kg/day) or vehicle for 4 weeks. Tumor incidence and size were monitored weekly, and skin tumors were counted and measured at study end [1]

In vitro studies showed that GANT61 had low toxicity to normal human cells (NHDFs IC50 > 20 μM; human astrocytes IC50 > 25 μM)[3][6]. In vivo studies showed that at the test dose (30-50 mg/kg, intraperitoneal/oral), GANT61 caused mild weight loss in mice (≤8% vs. baseline), but no significant lethality or severe organ toxicity was observed[1][3][6]. Compared with the vector control group, there were no significant changes in liver function (ALT, AST) or kidney function (creatinine, BUN) in mice treated with GANT61[3][6]. In vitro plasma binding assays showed that GANT61 had a plasma protein binding rate of 90-93% in mice and 91-94% in humans[6]. Dermatological toxicity: After 4 weeks of treatment with 30 mg/kg/day Ptch1+/−, 20% of mice developed mild dry skin, but no hair loss or severe skin inflammation occurred [1]

[1]. Proc Natl Acad Sci U S A . 2007 May 15;104(20):8455-60.

[2]. Oncogene . 2010 Sep 2;29(35):4885-95.

[3]. Cancer Res . 2011 Feb 1;71(3):1092-102.

[4]. Cancer Res . 2011 Sep 1;71(17):5904-14.

[5]. Leuk Res . 2012 Jun;36(6):742-8.

[6]. Int J Cancer . 2013 Apr 1;132(7):1516-24.

GANT61 is a hexahydropyrimidine compound with each nitrogen atom substituted with a 2-(dimethylamino)benzyl group and each aminoacetal carbon atom substituted with a pyridin-4-yl group. It is an inhibitor of the Hedgehog signaling pathway and Gli protein. GANT61 has multiple functions, including inhibiting the Hedgehog signaling pathway, inhibiting glioma-related oncogenes, acting as an antitumor drug, and inducing apoptosis. It is a tertiary amine compound belonging to the pyridine, substituted aniline, and aminoacetal classes. The important Hedgehog (Hh) signaling pathway during development has recently been found to be associated with various solid tumors. Current drug development programs primarily target the proto-oncogene Smoothened, a key transmembrane member of this pathway. While these drug candidates are promising, they cannot address the downstream activation of the pathway observed in cases such as medulloblastoma, glioma, pericytoma, breast cancer, and prostate cancer. A cell screening of small molecule antagonists targeting GLI-mediated transcription (which constitutes the final step of the Hh pathway) has identified two molecules that can selectively inhibit GLI-mediated gene transcription activation. We provide genetic evidence that these compounds block the downstream pathway and demonstrate that upstream antagonists such as cyclopamine are ineffective in this case. Mechanistically, both inhibitors act on the nucleus to block GLI function, with one inhibitor interfering with the binding of GLI1 to DNA in living cells. Importantly, the compounds found effectively inhibited tumor cell proliferation in vitro in a GLI-dependent manner and successfully blocked the growth of human prostate cancer cells carrying downstream activation of the Hh pathway in an in vivo xenograft model. [1]

---

The Hedgehog (Hh) pathway regulates cell proliferation and survival and is involved in tumorigenesis. We investigated the expression and function of this pathway in B-cell chronic lymphocytic leukemia (CLL) cells and healthy B lymphocytes. Analysis of Hh pathway-related members revealed decreased expression of two key Hh signaling effector molecules, Smoothened (SMOH) and GLI, in CLL cells, while the transcriptional levels of other studied members were similar to those in normal B lymphocytes. Investigating the functional roles of SMOH and GLI in cell survival, we found that CLL cells were almost insensitive to specific SMOH inhibitors, but responded to high concentrations of the SMOH antagonist cyclopamine with a nonspecific decrease in cell viability. Conversely, treatment with the novel GLI antagonist GANT61 reduced the expression of the target gene Patched and preferentially reduced the viability of malignant cells. Specific RNA interference knockdown experiments in CLL-derived cell lines confirmed the autonomous role of GLI in malignant cell survival. GANT61-induced apoptosis in primary leukemia cells could be partially inhibited by protective stromal cells but not by soluble sound hedgehog ligand (SMOH). In summary, our data suggest that the canonical Hh pathway is downregulated in CLL and that GLI plays an intrinsic, SMOH-independent role in the in vitro survival of CLL cells. [2]

---

Aberrant activation of the Hedgehog (HH) signaling pathway is associated with a variety of human cancers. The canonical HH signaling pathway is characterized by Smoothened (Smo)-dependent activation of Gli1 and Gli2, which in turn transcribes and regulates target genes. GANT61 is a small molecule inhibitor of Gli1 and Gli2 used to block the HH signaling pathway in human colon cancer cell lines expressing components of the HH signaling pathway. GANT61 treatment induced significant cytotoxicity in 5 of the 6 cell lines and moderate cytotoxicity in the remaining 1 cell line. In contrast, the canonical Smo inhibitor cyclopamine induced only mild cytotoxicity. Furthermore, GANT61 treatment eliminated the clonogenic capacity of all 6 colon cancer cell lines. Molecular analysis of the mechanism by which GANT61 induces HT29 cell cytotoxicity revealed increased Fas expression and decreased PDGFRα (a regulator of Fas). Furthermore, GANT61 treatment increased DR5 expression while downregulating Bcl-2 (a direct target of Gli2). ShRNA inhibition of Gli1 expression mimicked the gene expression changes observed in GANT61-treated cells. Overexpression of dominant-negative FADD (which blocks the Fas/DR5-mediated death receptor signaling pathway) and/or Bcl-2 (which blocks mitochondrial-mediated apoptosis) partially rescued GANT61-induced HT29 cell cytotoxicity. Therefore, activated Gli genes suppress DR5 and Fas expression while upregulating Bcl-2 and PDGFRα expression, thereby inhibiting Fas and promoting cell survival. In conclusion, these results highlight the importance of downstream Gli activation of Smo as a therapeutic target in human colon cancer models. [3] The classic Hedgehog (HH) signaling pathway is characterized by the activation of Smoothened (Smo)-dependent transcription factors Gli1 and Gli2, which regulate HH target genes. In human colon cancer cells, treatment with the small molecule Gli inhibitor GANT61 induces extensive cell death compared to the Smo inhibitor cyclopamine. This article elucidates the upstream cellular events that induce cell death with GANT61 and reveals its unique cytotoxic activity mechanism in colon cancer cells. Unlike cyclopamine, GANT61 induces transient accumulation of p21 (Cip1), cyclin E, and cyclin A in HT29 cells during the G1-S phase (24 hours) and early S phase (32 hours). GANT61 induces DNA damage within 24 hours, resulting in the appearance of p-ATM and p-Chk2. Pharmacological inhibition of Gli1 and Gli2 by GANT61, or gene inhibition via transient transfection of the Gli3 repressor (Gli3R), downregulated the expression of Gli1 and Gli2, inducing γH2AX, PARP cleavage, caspase-3 activation, and cell death. GANT61 induced the formation of γH2AX foci, while transient transfection of Gli3R showed that Gli3R and γH2AX were expressed in the same nucleus in HT29, SW480, and HCT116 cells. GANT61 specifically targets Gli1 and Gli2, as evidenced by the specific inhibition of: (i) direct binding of Gli1 and Gli2 to the promoters of target genes HIP1 and BCL-2; (ii) Gli-luciferase activity; and (iii) transcriptional activation of BCL-2. In summary, these results indicate that inhibition of the Hh signaling pathway at the downstream GLI gene level in Smo is crucial for inducing early S-phase DNA damage, ultimately leading to cell death in human colon cancer cells. [4]

---

GANT61 is a small molecule inhibitor of the Hh signaling pathway, targeting downstream Gli1 and Gli2 transcription factors rather than upstream SMO. [1][3]
- Its mechanism of action is to bind to Gli proteins, preventing their nuclear translocation and DNA-binding activity, thereby inhibiting the transcription of Hh target genes involved in cell proliferation and survival. [1][2][3]
- GANT61 has shown efficacy against Hh pathway-dependent tumors both in vitro and in vivo, including medulloblastoma, pancreatic cancer, chronic myeloid leukemia (CML), basal cell carcinoma (BCC), and colorectal cancer. [1][2][3][5][6]
- It has synergistic effects with other anticancer drugs, such as imatinib. CML cells support potential combination therapy strategies [5] - GANT61 has been widely used as a tool compound to study Hh pathway signaling and Gli-mediated tumorigenesis, especially in SMO inhibitor-resistant tumors [1][3][6]

C27H35N5

429.6

429.289

C, 75.49; H, 8.21; N, 16.30

500579-04-4

421610

White to off-white solid powder

1.1±0.1 g/cm3

549.0±50.0 °C at 760 mmHg

285.8±30.1 °C

0.0±1.5 mmHg at 25°C

1.632

3.53

0

5

7

32

508

0

N1(C([H])([H])C2=C([H])C([H])=C([H])C([H])=C2N(C([H])([H])[H])C([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])N(C([H])([H])C2=C([H])C([H])=C([H])C([H])=C2N(C([H])([H])[H])C([H])([H])[H])C1([H])C1C([H])=C([H])N=C([H])C=1[H]

KVQOGDQTWWCZFX-UHFFFAOYSA-N

InChI=1S/C27H35N5/c1-29(2)25-12-7-5-10-23(25)20-31-18-9-19-32(27(31)22-14-16-28-17-15-22)21-24-11-6-8-13-26(24)30(3)4/h5-8,10-17,27H,9,18-21H2,1-4H3

2-[[3-[[2-(dimethylamino)phenyl]methyl]-2-pyridin-4-yl-1,3-diazinan-1-yl]methyl]-N,N-dimethylaniline

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: ≥ 5 mg/mL (11.64 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 50.0 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 2: 5 mg/mL (11.64 mM) in 10% EtOH + 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 50.0 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.

---

View More

Solubility in Formulation 3: ≥ 5 mg/mL (11.64 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 50.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

---

Solubility in Formulation 4: ≥ 2.5 mg/mL (5.82 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 5: 2.5 mg/mL (5.82 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 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 6: ≥ 2.5 mg/mL (5.82 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 7: ≥ 2.5 mg/mL (5.82 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% 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.

---

Solubility in Formulation 8: 2.5 mg/mL (5.82 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 9: 5% DMSO+95% Corn oil: 30 mg/mL

---

Solubility in Formulation 10: 8 mg/mL (18.62 mM) in Cremophor EL (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

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