MIP-1α-CCR5 ( IC50 = 1 nM ); MIP-1β-CCR5 ( IC50 = 1 nM ); RANTES-CCR5 ( IC50 = 1.4 nM ); MCP-1-CCR2b ( IC50 = 27 nM ); R5 HIV-1 (Ba-L) ( EC50 = 1.2 nM ); R5 HIV-1 (Ba-L) ( EC90 = 5.7 nM ); R5 HIV-1 (Ba-L) ( EC50 = 3.7 nM ); R5 HIV-1 (Ba-L) ( EC90 = 12.8 nM ); R5 HIV-1 (KK) ( EC50 = 1.6 nM ); R5 HIV-1 (KK) ( EC90 = 20.8 nM ); R5 HIV-1 (HHA) ( EC50 = 3.2 nM ); R5 HIV-1 (HHA) ( EC90 = 7.5 nM ); R5 HIV-1 (CTV) ( EC50 = 3.5 nM ); R5 HIV-1 (CTV) ( EC90 = 27 nM ); mCXCR3( IC50 = 369 nM )
TAK-779 deters R5 HIV-1 in MAGI-CCR5 cells. It is a strong and selective nonpeptide antagonist of CCR5, with a Ki of 1.1 nM, with an effective and selective EC50 and EC90 of 1.2 nM and 5.7 nM, respectively. In CHO/CCR2b cells, TAK-779 less effectively inhibits the binding of [¹²⁵I]-monocyte chemotactic protein 1 to CCR2b, with an IC50 of 27 nM for CCR2b. With an IC50 of 1.4 nM, TAK-779 also totally prevents [¹²⁵I]-RANTES from binding to CHO/CCR5 cells. CCR5-mediated Ca²⁺-signaling is specifically inhibited by TAK-779 (20 nM). Furthermore, on X4 HIV-1 strains, TAK-779 exhibits no inhibition[1]. As an antagonist of CXCR3, TAK-779 prevents T cell migration but not proliferation[2].

TAK-779 deters R5 HIV-1 in MAGI-CCR5 cells. It is a strong and selective nonpeptide antagonist of CCR5, with a Ki of 1.1 nM, with an effective and selective EC50 and EC90 of 1.2 nM and 5.7 nM, respectively. In CHO/CCR2b cells, TAK-779 less effectively inhibits the binding of [¹²⁵I]-monocyte chemotactic protein 1 to CCR2b, with an IC50 of 27 nM for CCR2b. With an IC50 of 1.4 nM, TAK-779 also totally prevents [¹²⁵I]-RANTES from binding to CHO/CCR5 cells. CCR5-mediated Ca²⁺-signaling is specifically inhibited by TAK-779 (20 nM). Furthermore, on X4 HIV-1 strains, TAK-779 exhibits no inhibition[1]. As an antagonist of CXCR3, TAK-779 prevents T cell migration but not proliferation[2].

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Treatment with TAK-779 did not reduce the proliferation of anti-MOG T cells in response to MOG 35-55 peptide stimulation. Interestingly, an increased proliferative response was observed in T cells from TAK-779-treated mice. [3]
TAK-779 did not affect the production of IFN-γ by MOG-specific T cells when co-cultured with antigen-presenting cells (APCs) in the presence of MOG. [3]
TAK-779 treatment did not influence the capacity of APCs from immunized mice to produce IL-12 when co-cultured with MOG-specific T cells. [3]
TAK-779 treatment did not significantly affect the production of IL-17 by splenocytes or lymph node cells from immunized mice upon MOG stimulation. [3]
The expression of mRNA for CXCR3 and CCR5 in purified T cells from immunized mice was not affected by TAK-779 treatment. [3]

TAK-779 (10 mg/kg daily, s.c.) considerably increases the rat intestinal transplantation model's allograft survival. Additionally, TAK-779 reduces the quantity of CD4⁺ and CD8⁺ T cells in the recipient's mesenteric lymph nodes (MLN), blood, and spleen[2]. TAK-779 (150 µg/mouse, s.c.) inhibits the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice that have been immunized against myelin oligodendrocyte glycoprotein (MOG). TAK-779 reduces the amount of leukocytes that carry CXCR3 and CCR5 infiltrating the spinal cord. TAK-779 has no effect on immune responses specific to myelin oligodendrocyte glycoprotein (MOG) or on the ability of T cells specific to MOG to transmit experimental autoimmune encephalomyelitis (EAE)[3].

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Subcutaneous administration of TAK-779 (150 μg per mouse daily) significantly reduced the incidence and severity of MOG-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. It delayed disease onset and decreased the maximum mean clinical score. [3]
TAK-779 treatment prevented body weight loss in EAE mice. [3]
TAK-779 treatment did not affect the potential of MOG-specific T cells to adoptively transfer EAE to recipient mice. The onset, severity, and duration of disease in recipients were similar whether they received T cells from vehicle-treated or TAK-779-treated donors. [3]
TAK-779 treatment markedly inhibited the migration of CXCR3/CCR5-bearing CD4⁺, CD8⁺ T cells, and CD11b⁺ leukocytes into the spinal cord, as shown by histopathology, flow cytometry, and real-time PCR analysis. [3]
TAK-779 treatment significantly decreased the mRNA expression of several chemokines (CCL2, CCL3, CCL4, CXCL9, CXCL10) and cytokines (IFN-γ, IL-17) in the spinal cord of EAE mice, but did not change CCL5 expression. It also reduced the expression of CCR2 mRNA. [3]

The beta-chemokine receptor CCR5 is considered to be an attractive target for inhibition of macrophage-tropic (CCR5-using or R5) HIV-1 replication because individuals having a nonfunctional receptor (a homozygous 32-bp deletion in the CCR5 coding region) are apparently normal but resistant to infection with R5 HIV-1. In this study, we found that TAK-779, a nonpeptide compound with a small molecular weight (Mr 531.13), antagonized the binding of RANTES (regulated on activation, normal T cell expressed and secreted) to CCR5-expressing Chinese hamster ovary cells and blocked CCR5-mediated Ca2+ signaling at nanomolar concentrations. The inhibition of beta-chemokine receptors by TAK-779 appeared to be specific to CCR5 because the compound antagonized CCR2b to a lesser extent but did not affect CCR1, CCR3, or CCR4. Consequently, TAK-779 displayed highly potent and selective inhibition of R5 HIV-1 replication without showing any cytotoxicity to the host cells. The compound inhibited the replication of R5 HIV-1 clinical isolates as well as a laboratory strain at a concentration of 1.6-3.7 nM in peripheral blood mononuclear cells, though it was totally inactive against T-cell line-tropic (CXCR4-using or X4) HIV-1[1].

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Binding Assays[1]
CHO-K1 and CCR5-expressing CHO (CHO/CCR5) cells (5 × 104 cells per 100 μl) were cultured in a microtiter tray. After a 24-h incubation at 37°C, culture medium was replaced with the binding buffer [Ham’s F-12 medium containing 20 mM Hepes and 0.5% BSA (pH 7.2)]. Binding reactions were performed at room temperature for 40 min in the presence of [125I]-RANTES (specific activity: 2,000 Ci/mmol; Amersham Pharmacia) and various concentrations of the test compound. The binding reaction was terminated by washing out the free ligand with cold PBS, and the cell-associated radioactivity was counted by Top-count scintillation counter. Binding assays for other receptors, CCR1, CCR2b, CCR3, CCR4, and CXCR4, were carried out in a similar way.

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Ca2+ Mobilization Assays. [1]
CHO/CCR5 cells (5 × 106 cells/ml) were suspended in the assay buffer (5 mM KCl/147 mM NaCl/0.22 mM KH2PO4/1.1 mM Na2HPO4/5.5 mM glucose/0.3 mM MgSO4/1 mM MgCl2/10 mM Hepes, pH 7.4). The cells were loaded with 5 μM Fura-PE3AM at 37°C for 30 min, were washed twice with the assay buffer containing 1 mM CaCl2, and were resuspended at 1 × 107 cells/ml in the same buffer. At 150 sec after exposure to TAK-779, 20 nM RANTES was added, and relative increase of cytoplasmic Ca2+ levels was monitored by F-2000 fluorescence spectrometer. Calibration was carried out with 50 μM ionomycin for total Ca2+ release and with 5 mM EGTA for Ca2+ chelating.

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To evaluate receptor binding, Chinese hamster ovary (CHO) cells stably expressing human CCR5 (CHO/CCR5) were used. Cells were cultured in microtiter trays. After incubation, the medium was replaced with a binding buffer. Binding reactions were performed at room temperature for 40 minutes in the presence of a radiolabeled chemokine ligand (e.g., [¹²⁵I]-RANTES) and various concentrations of TAK-779. The reaction was terminated by washing with cold PBS, and cell-associated radioactivity was measured using a scintillation counter. Specific binding was calculated by subtracting nonspecific binding (to parental CHO-K1 cells). Inhibition constants (Ki) were calculated using the Cheng-Prusoff derivation from competitive binding experiments.[1]

The test compounds' (TAK-779, etc.) anti-HIV-1 activities are predicated on the suppression of p24 antigen production in PBMCs and the inhibition of virus-induced infectious focus formation in MAGI-CCR5 cells. To put it briefly, MAGI-CCR5 cells are grown in a microtiter tray with 1 × 10⁴ cells per well. The culture supernatants are replaced with new culture media containing the virus (approximately 300 focus forming units per well) and different concentrations of the test compounds (TAK-779, etc.) after a 24-hour incubation at 37°C. Following a two-day incubation period, 5-bromo-4-chloro-3-indolyl-β-d-galactosidase is used to fix and stain the cells. Microscopically, the number of infected (blue) cells is counted. In the PBMC assays, in different concentrations of the test compounds (TAK-779, etc.), HIV-1 is infected into phytohemagglutinin-stimulated PBMCs (2.5 × 10⁵ cells per 500 μl). The virus used for infection typically contains 1–10 ng of p24 per 2.5 × 10⁵ cells, depending on how replicable each strain is. The cells are first cultured with culture media containing the same concentrations of the compounds used during viral adsorption, and then they are subjected to a thorough washing process to remove any remaining viral particles, all while maintaining a 37°C incubation period. The culture supernatants are obtained on day 6 following viral infection, and using a sandwich ELISA kit, the p24 antigen levels are ascertained. The compounds' cytotoxicities and antiviral properties are assessed simultaneously. Their foundation lies in the survival and growth of simulated infected cells [1].

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Lymphocyte Proliferation Assay: Draining lymph node cells (2 × 10⁵ per well) from immunized mice were cultured with various concentrations of MOG 35-55 peptide or medium alone in 96-well plates. After 60 hours, 0.5 μCi per well of [³H]-thymidine was added, and cells were incubated for an additional 12 hours. Cells were harvested onto filters, and incorporated radioactivity was measured using a liquid scintillation counter. [3]
Cytokine Detection by ELISA (IFN-γ, IL-12p70, IL-17): For IFN-γ, purified T cells (4 × 10⁵ per well) from immunized mice were co-cultured with APC-enriched cells (1 × 10⁵ per well) from normal mice in the presence or absence of MOG (10 μg·mL⁻¹). Supernatants were harvested at 48 hours. For IL-12p70, APC-enriched cells (1 × 10⁵ per well) from immunized mice were co-cultured with purified T cells (4 × 10⁵ per well) from immunized mice with or without MOG (10 μg·mL⁻¹). Supernatants were harvested at 24 hours. For IL-17, splenocytes or lymph node cells (4 × 10⁵ per well) from immunized mice were cultured with or without MOG (10 μg·mL⁻¹). Supernatants were harvested at 72 hours. Cytokine levels in all supernatants were measured using specific ELISA kits. [3]
RNA Extraction and PCR Analysis: Cells or tissues were lysed, and total RNA was extracted. RNA was reverse-transcribed into cDNA. For standard RT-PCR, cDNA was amplified with specific primers, and products were separated on agarose gels and visualized. For quantitative real-time PCR, reactions were performed using a SYBR Green-based system, and mRNA levels were normalized to β-actin. Specific primers were used for genes including CD4, CD8, CD11b, CCR2, CCR5, CXCR3, CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, IFN-γ, and IL-17. [3]
Flow Cytometry Analysis of CNS-infiltrating Cells: Mononuclear cells were isolated from the spinal cord by enzymatic digestion and Percoll gradient centrifugation. Cells were blocked, stained with fluorochrome-conjugated antibodies against surface markers (e.g., CD4, CD8, CD11b, CXCR3, CCR5), washed, and analyzed by flow cytometry. The absolute number of a specific leukocyte subtype was calculated by multiplying its percentage by the total number of infiltrated leukocytes. [3]

Mice: The mice receive a TAK-779 or vehicle injection in addition to their MOG immunization. After receiving the MOG immunization, the mice (N = 10) are given one daily subcutaneous injection of 150 µg TAK-779 (dissolved in 5% mannitol solution) in a volume of 100 µL. Starting on the first day following immunization, TAK-779 injections are administered once a day for 22 days. Based on the findings of previous studies, which showed that a dose of more than 100 µg per mouse is needed to produce significant inhibition, the dose of 150 µg was chosen. A dose of 50 µg per mouse was found to be ineffective. In addition to being used in asthma and allograft rejection models, the dose of 150 µg per mouse has also been utilized in other mouse experimental models. The control mice (N = 10) receive a daily injection of an equivalent volume of PBS containing 5% mannitol as a control[3].

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EAE Induction and TAK-779 Treatment in C57BL/6 Mice: Female C57BL/6 mice (6-8 weeks old) were immunized subcutaneously at three sites with an emulsion containing 200 μg of MOG 35-55 peptide and 400 μg of Mycobacterium tuberculosis H37Ra in complete Freund's adjuvant. Additionally, each mouse received 400 ng of pertussis toxin intraperitoneally on the day of immunization and 72 hours later. Starting from day 0 post-immunization, mice were treated daily with a subcutaneous injection of 150 μg of TAK-779 (dissolved in 5% mannitol solution) or an equal volume of vehicle control (5% mannitol in PBS) for 22 days. Clinical signs of EAE were scored daily. [3]
Adoptive Transfer EAE Experiment: Donor C57BL/6 mice were immunized with MOG 35-55 and treated with TAK-779 or vehicle. Ten days later, draining lymph node cells were harvested, and lymphocytes were cultured with MOG 35-55 (50 μg·mL⁻¹) and murine recombinant IL-12 (20 ng·mL⁻¹) for 4 days. Recipient C57BL/6 mice each received 1 × 10⁷ of these activated cells intravenously, along with two intraperitoneal injections of pertussis toxin (200 ng each) immediately after transfer and 48 hours later. Recipients were monitored for clinical signs of EAE for up to 24 days. [3]
Histopathological Assessment: At the peak of disease (day 17 post-immunization), mice were perfused with fixative. Spinal cords were collected, sectioned, and stained with hematoxylin and eosin (H&E). Inflammation in the spinal cord quadrants was scored microscopically based on mononuclear cell infiltration. [3]
Isolation of CNS-infiltrating Cells for Analysis: Mice were perfused with cold PBS. Spinal cords were extruded, digested with collagenase, and passed through a cell strainer. Mononuclear cells were isolated by centrifugation over a Percoll gradient for subsequent flow cytometry or other analyses. [3]

[1]. A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity. Proc Natl Acad Sci U S A. 1999 May 11;96(10):5698-703.

[2]. Effects of a calcineurin inhibitor, FK506, and a CCR5/CXCR3 antagonist, TAK-779, in a rat small intestinal transplantation model. Transpl Immunol. 2011 Jul;25(1):49-55.

[3]. The chemokine receptor antagonist, TAK-779, decreased experimental autoimmune encephalomyelitis by reducing inflammatory cell migration into the central nervous system, without affecting T cell function. Br J Pharmacol. 2009 Dec;158(8):2046-56.

[4]. The unique target specificity of a nonpeptide chemokine receptor antagonist: selective blockade of two Th1 chemokine receptors CCR5 and CXCR3. J Leukoc Biol. 2003 Feb;73(2):273-80.

The β-chemokine receptor CCR5 is considered an ideal target for inhibiting macrophage-tropic (CCR5 or R5) HIV-1 replication because individuals carrying a non-functional receptor (homozygous deletion of the 32 bp coding region of CCR5) are phenotypically normal but resistant to R5 HIV-1 infection. This study found that the low molecular weight (Mr 531.13) non-peptide compound TAK-779 antagonized the binding of RANTES (activation regulators, normal T cell expression and secretion) to CCR5-expressing Chinese hamster ovary cells and blocked CCR5-mediated Ca2+ signaling at nanomolar concentrations. The inhibitory effect of TAK-779 on the β-chemokine receptor appears to be CCR5 specific, as the compound showed weak antagonism towards CCR2b but no effect on CCR1, CCR3, or CCR4. Therefore, TAK-779 exhibits highly efficient and selective inhibition of R5 HIV-1 replication without cytotoxicity to host cells. The compound inhibited the replication of R5 HIV-1 clinical isolates and laboratory strains in peripheral blood mononuclear cells at concentrations of 1.6–3.7 nM, but was completely ineffective against T-cell tropism (CXCR4 receptor or X4 receptor) HIV-1. [1]

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This study used a rat intestinal transplantation model to investigate the effects of the CCR5 and CXCR3 antagonists FK506 and TAK-779. The small intestine of DA rats was ectopically transplanted into LEW rats. Recipient rats were treated with FK506 (1 mg/kg/day, day 0–5) and TAK-779 (10 mg/kg/day, day 0–10), respectively. Transplantation survival and immune response to these materials were assessed by mixed lymphocyte response and IFN-γ production. The expression of chemokine receptors on lymphocytes was also detected. The mean survival times for the allogeneic group, FK506 group, TAK-779 group, and dual-drug combination group were 7.0±0.3 days, 12.0±1.0 days, 9.8±0.5 days, and 18.0±1.5 days, respectively. Compared with the TAK-779 group, cell proliferation and IFN-γ production were significantly inhibited in the FK506 group. Furthermore, the inhibitory effect of the dual-drug combination group was stronger than that of the FK506 monotherapy group, consistent with in vivo experimental and histopathological data. FACS analysis showed that the number of CD4(+) and CD8(+) cells in the recipient blood of the FK506 and TAK-779 groups was significantly reduced, and the number of CD4(+) and CD8(+) cells in the transplanted mesenteric lymph nodes of the TAK-779 group was also significantly reduced, while this phenomenon was not observed in the FK506 group. In addition, double staining of transplanted mesenteric infiltrating lymphocytes showed that the number of lymphocytes expressing CCR5 and CXCR3 was significantly reduced in the TAK-779 group compared with the allogeneic group, while no such phenomenon was observed in the FK506 group. Although FK506 inhibits cell proliferation and effector function, it has little effect on the expression of CCR5 and CXCR3 in lymphocytes. Further exploration of the effect of FK506 combined with TAK-779 treatment is expected to provide a new treatment method for intestinal transplantation. [2]

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Background and Objectives: CC chemokine receptor CCR5 and CXC chemokine receptor CXCR3 are involved in the regulation of T cell-mediated immune responses, as well as the migration and activation of these cells. In order to determine whether blocking these chemokine receptors can regulate the inflammatory response of the central nervous system (CNS), we investigated the effect of the non-peptide chemokine receptor antagonist TAK-779 on experimental autoimmune encephalomyelitis (EAE) mice. Methods: Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG) 35-55. Following immunization, TAK-779 was administered subcutaneously once daily. Inflammatory cells infiltrating the spinal cord were assessed histopathologically and by flow cytometry (FACS), mRNA expression was quantitatively detected by polymerase chain reaction (PCR), T cell proliferation was detected by [3H]-thymidine incorporation, and cytokine production was detected by enzyme-linked immunosorbent assay (ELISA). Disease incidence and severity were monitored (for 3 weeks). Main Results: TAK-779 treatment reduced the incidence and severity of experimental autoimmune encephalomyelitis (EAE). It significantly inhibited the migration of CD4+, CD8+, and CD11b+ leukocytes expressing CXCR3/CCR5 to the central nervous system (CNS). TAK-779 does not reduce the proliferation of anti-myelin oligodendrocyte glycoprotein (MOG) T cells, the production of interferon-γ (IFN-γ) by T cells, or the expression of CXCR3 on T cells. In addition, TAK-779 does not affect the production of IL-12 by antigen-presenting cells, the induction of CCR5 on T cells, or the ability of MOG-specific T cells to transfer EAEs. Conclusion and significance: TAK-779 inhibits the development of MOG-induced EAEs. This effect involves reducing the migration of inflammatory cells to the central nervous system without affecting the response of anti-MOG T cells or the ability of MOG-specific T cells to transfer EAEs. [3]

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TAK-779 is a synthetic non-peptide chemokine receptor antagonist that was initially developed for HIV treatment due to its CCR5 antagonistic activity. [3] In the context of experimental autoimmune encephalomyelitis (EAE), TAK-779 alleviates the disease by inhibiting the migration of inflammatory cells (T cells and monocytes/macrophages) expressing CXCR3 and/or CCR5 to the central nervous system without impairing the activation or effector function of the encephaloinflammatory T cells themselves. [3] This study demonstrates that chemokine receptors (such as CCR5, CXCR3, and CCR2) play a key role in the migration of leukocytes to the central nervous system during autoimmune neuroinflammation, and blocking these receptors represents a potential therapeutic strategy for treating tissue-specific autoimmune diseases such as multiple sclerosis. [3]

C33H39CLN2O2

531.127968072891

530.27

C, 74.63; H, 7.40; Cl, 6.67; N, 5.27; O, 6.02

229005-80-5

229005-80-5 (Cl); 263765-56-6 (cation)

183789

White to beige solid powder

8.171

1

3

6

38

769

0

C[N+](C1CCOCC1)(CC1C=CC(NC(=O)C2CCCC3=CC=C(C4C=CC(C)=CC=4)C=C3C=2)=CC=1)C.[Cl-]

VDALIBWXVQVFGZ-UHFFFAOYSA-N

InChI=1S/C33H38N2O2.ClH/c1-24-7-11-27(12-8-24)28-14-13-26-5-4-6-29(22-30(26)21-28)33(36)34-31-15-9-25(10-16-31)23-35(2,3)32-17-19-37-20-18-32;/h7-16,21-22,32H,4-6,17-20,23H2,1-3H3;1H

dimethyl-[[4-[[3-(4-methylphenyl)-8,9-dihydro-7H-benzo[7]annulene-6-carbonyl]amino]phenyl]methyl]-(oxan-4-yl)azanium;chloride

TAK-779 Chloride; TAK779; TAK 779

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ≥ 25 mg/mL (~47.1 mM)
H2O: ~16.7 mg/mL (~31.4 mM)

Solubility in Formulation 1: ≥ 2.58 mg/mL (4.86 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 25.8 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: ≥ 2.58 mg/mL (4.86 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 25.8 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: ≥ 2.58 mg/mL (4.86 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 25.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 50 mg/mL (94.14 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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