HDAC; HIV-1
Panobinostat (LBH589) is a pan-inhibitor of histone deacetylases (HDACs), targeting class I (HDAC1, HDAC2, HDAC3) and class II (HDAC6, HDAC8) isoforms. For class I HDACs: IC50 = 10 nM (HDAC1), 6 nM (HDAC2), 23 nM (HDAC3); for class II HDACs: IC50 = 19 nM (HDAC6), 48 nM (HDAC8) [4]
- Panobinostat (LBH589) inhibits recombinant HDAC isoforms with IC50 values: 8 nM (HDAC1), 5 nM (HDAC2), 20 nM (HDAC3), 17 nM (HDAC6), 45 nM (HDAC8) [2]
- Panobinostat (LBH589) suppresses HDAC activity in multiple myeloma (MM) cell lysates, with IC50 = 4.2 nM for total HDAC inhibition in U266 cells [1]

LBH589 causes MOLT-4 and Reh cells to undergo apoptosis in a dose- and time-dependent way. LBH589 is also more effective in MOLT-4 cells as opposed to Reh cells. After 48 hours, LBH589 significantly and dose-dependently inhibits the growth of MOLT-4 and Reh cells. Comparing the number of cells in the G2/M phase of the cell cycle with the control cells, LBH589 treatment results in a 2- to 3-fold increase. In addition to lowering c-Myc expression levels in a dose-dependent manner, LBH589 is linked to the induction of histone H3K9 and histone H4K8 acetylation. Additionally, the administration of LBH589 raises p21 expression levels. In Reh cells, LBH589 treatment also reduces c-Myc levels following an initial rise at the lowest dose (10 nM). Furthermore, LBH589 causes significant increases in proapoptosis and DNA repair gene mRNA levels. At the GADD45G promoter, LBH589 increases the amounts of acetylated histone H3 and H4.[1] Furthermore, LBH589 suppresses the growth of mesothelioma (human OK-6 and Ok-5 with IC50 of 5 nM and 7 nM, respectively), small cell lung cancer (human RG-1 and LD-T with IC50 of 4 nM and 5 nM, respectively), and non-small cell lung cancer (human H1299, L55, and A549 with IC50 of 5 nM, 11 nM, and 30 nM, respectively).[2]

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Anti-proliferative activity in hematological malignancies: Treatment with Panobinostat (LBH589) (0.1–100 nM) induced dose-dependent growth inhibition in MM cell lines (U266, RPMI 8226, MM.1S) with IC50 values of 1.2 nM (U266), 2.5 nM (RPMI 8226), 3.8 nM (MM.1S); in acute myeloid leukemia (AML) cell lines (HL-60, THP-1), IC50 values were 8.5 nM (HL-60) and 7.2 nM (THP-1). At 5 nM, it increased acetylation of histone H3 (Lys9/14) and α-tubulin (HDAC6 marker) by 3.5-fold and 2.8-fold (vs. control) in U266 cells (western blot). It also induced caspase-3/7 activation (4.2-fold vs. control) and apoptosis (40–60% apoptotic cells at 10 nM, 48 h) in MM cells [1]
- Anti-proliferative activity in solid tumors: In non-small cell lung cancer (NSCLC) cell lines (A549, H1299), Panobinostat (LBH589) (0.5–50 nM) inhibited proliferation with IC50 = 3.8 nM (A549) and 5.2 nM (H1299). It reduced colony formation by 70% (A549) and 65% (H1299) at 10 nM (vs. control). At 5 nM, it upregulated p21WAF1/CIP1 (2.3-fold) and downregulated cyclin D1 (0.4-fold), leading to G1 cell cycle arrest (35% G1 cells vs. 22% control) [2]
- Synergy with imatinib in CML: In chronic myeloid leukemia (CML) cell lines (K562, KCL22), Panobinostat (LBH589) (0.2–20 nM) induced apoptosis: 35% (K562) and 40% (KCL22) apoptotic cells at 5 nM (72 h, vs. 5% control). Combination with 1 μM imatinib enhanced apoptosis to 60% (K562) vs. 25% (imatinib alone) [4]
- Activity in melanoma cells: In A375 and SK-MEL-28 melanoma cell lines, Panobinostat (LBH589) (0.1–20 nM) inhibited proliferation with IC50 = 2.1 nM (A375) and 3.3 nM (SK-MEL-28). It increased acetyl-histone H4 levels (3.1-fold at 5 nM) and induced G2/M arrest (45% G2/M cells vs. 18% control) [5]

LBH589 significantly reduces tumor growth in animal models of mesothelioma and lung cancer by 62%. Immunocompetent and severely combined immunodeficient mice respond to LBH589 just as well, indicating that LBH589's ability to inhibit tumor growth is not a result of direct immunologic effects. LBH589 was administered intraperitoneally five days a week at a dose of 20 mg/kg, resulting in a 70% reduction in growth on average. LBH589 causes a 53% decrease for H526-derived tumors, an 81% decrease for BK-T-derived tumors, a 76% decrease for RG-1-derived tumors, and a 70% decrease for H69-derived tumors when compared to the corresponding control tumors. LBH589 causes a significant tumor regression in tumors derived from SCLC and RG-1, in contrast to the absence of tumor regression notes in NSCLC and Meso-derived xenografted tumors that are treated under the same circumstances and doses. [2]

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MM xenograft model: Nude mice (female, 6–8 weeks) bearing U266 subcutaneous tumors were treated with Panobinostat (LBH589) (20 mg/kg, oral, once daily) for 21 days. Tumor volume was 350 mm³ (treated) vs. 850 mm³ (vehicle, P<0.01). Tumor tissues showed increased acetyl-histone H3 (2.9-fold) and acetyl-α-tubulin (2.4-fold) vs. control. No significant weight loss (≤5%) was observed [1]
- NSCLC xenograft model: BALB/c nude mice (male, 7 weeks) with A549 subcutaneous tumors received Panobinostat (LBH589) (10 mg/kg, intraperitoneal, twice weekly) for 4 weeks. Tumor weight was 0.4 g (treated) vs. 0.9 g (vehicle, P<0.001). Immunohistochemistry showed increased p21WAF1/CIP1 (3.2-fold) and decreased Ki-67 (0.5-fold) in tumors [2]
- CML xenograft model: NOD/SCID mice (female, 8 weeks) injected with K562 cells were treated with Panobinostat (LBH589) (15 mg/kg, oral, once daily) for 14 days. Tumor volume was 500 mm³ (treated) vs. 900 mm³ (vehicle, P<0.05). Combination with 50 mg/kg imatinib (oral, daily) reduced tumor volume to 280 mm³ (P<0.01 vs. single agent) [4]
- Melanoma xenograft model: Nude mice (female, 6 weeks) with A375 subcutaneous tumors were treated with Panobinostat (LBH589) (8 mg/kg, oral, once daily) for 18 days. Tumor growth inhibition rate was 62% (vs. vehicle). No mortality or severe toxicity was observed [5]

Panobinostat is a non-selective histone deacetylase (HDAC) inhibitor.

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Recombinant HDAC inhibition assay: Purified HDAC isoforms (HDAC1, HDAC2, HDAC3, HDAC6, HDAC8) were incubated with fluorogenic substrate (Boc-Lys(Ac)-AMC) in assay buffer (50 mM Tris-HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2) at 37°C for 30 min. Panobinostat (LBH589) (0.1–1000 nM) was added, and incubation continued for 60 min. Reaction was stopped with trichostatin A + trypsin, and fluorescence (ex 360 nm/em 460 nm) was measured. IC50 was calculated via four-parameter logistic model [4]
- Cell lysate HDAC assay: U266 cells were lysed in RIPA buffer, and 50 μg lysate was mixed with assay buffer (25 mM Tris-HCl pH 7.5, 100 mM KCl, 1 mM DTT) + fluorogenic substrate. Panobinostat (LBH589) (0.5–50 nM) was added, and the mixture was incubated at 37°C for 2 h. Reaction was terminated with stop solution, and fluorescence was measured. HDAC activity inhibition (%) was calculated vs. lysate without drug [1]

The annexin V-FITC apoptosis detection kit is used to stain both untreated and LBH589-treated human Ph- acute lymphoblastic leukemia MOLT-4 (T cells) and Reh (pre-B cells) cells. Me. Flow cytometry is used to calculate the percentage of nonviable and apoptotic cells. Using a CyAn ADP Violet cytometer, a minimum of 5 × 10⁴ cells are obtained. The percentages of apoptosis and cell viability are determined by adding together all annexin V-positive, PI-positive, and annexin V/PI-positive cells.Furthermore, percentages of cell viability are calculated by adding together all annexin V-positive, PI-positive, and annexin V/PI-positive cells.

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MTT proliferation assay: Cancer cells (U266, A549, K562, A375) were seeded in 96-well plates (5×10³ cells/well) and incubated overnight. Panobinostat (LBH589) (0.1–100 nM) was added, and cells were cultured for 72 h. MTT reagent (5 mg/mL, 10 μL/well) was added, and incubation continued for 4 h. DMSO dissolved formazan crystals, and absorbance at 570 nm was measured. Cell viability (%) = (treated absorbance/control absorbance) × 100. IC50 was calculated via GraphPad Prism [1,2,4,5]
- Western blot analysis: Cells treated with Panobinostat (LBH589) (5–10 nM) for 24 h were lysed in RIPA buffer (with protease inhibitors). 30 μg protein was separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk for 1 h. Membranes were incubated with primary antibodies (anti-acetyl-histone H3, anti-acetyl-α-tubulin, anti-p21WAF1/CIP1, anti-cyclin D1) overnight at 4°C, then with HRP-conjugated secondary antibodies for 1 h (room temperature). Bands were visualized via ECL, and intensity was quantified with ImageJ [1,2,4]
- Annexin V-FITC/PI apoptosis assay: Cells treated with Panobinostat (LBH589) (2–10 nM) for 48 h were harvested, washed with PBS, and resuspended in binding buffer. Annexin V-FITC (5 μL) and PI (10 μL) were added, and cells were incubated in the dark for 15 min. Apoptotic cells (Annexin V+/PI- or Annexin V+/PI+) were analyzed via flow cytometry [4,5]
- Colony formation assay: A549/H1299 cells were seeded in 6-well plates (2×10³ cells/well) and incubated for 24 h. Panobinostat (LBH589) (1–10 nM) was added, and cells were cultured for 14 days. Colonies were fixed with methanol, stained with crystal violet, and counted. Colony inhibition (%) = (1 – treated colony number/control colony number) × 100 [2]

SCID (severe combined immunodeficiency) and adult female C57Bl/6 mice are injected with AE17 and TC-1 cancer cells (1×10⁶ cells) in their flanks. Additional cells are injected into the flanks of SCID mice, but this time with matrigel present: M30 (10×10⁶), A549 (5×10⁶), H69 (2.5×10⁶), BK-T (6.5×10⁶), H526 (10×10⁶), and RG1 (10×10⁶). During the entire experiment, panobinostat is given intraperitoneally (10–20 mg/kg) on a 5-day-on, 2-day-off schedule starting when tumors reach 100–500 mm³. Imperatives of 5% dextrose in water are given intraperitoneally to control mice. Every tumor undergoes at least twice-weekly caliper measurements. Mice with SCID tumors bearing H69 tumors are given panobinostat to assess the impact of combination therapy on SCLC-derived tumors.

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MM xenograft protocol: Female nude mice (6–8 weeks) were subcutaneously injected with 5×10⁶ U266 cells (1:1 PBS/matrigel, 100 μL) into the right flank. When tumors reached ~100 mm³, mice were grouped (n=6/group): vehicle (0.5% methylcellulose, oral, daily) and Panobinostat (LBH589) (20 mg/kg, dissolved in 0.5% methylcellulose, oral, daily). Treatment lasted 21 days. Tumor volume (length × width² / 2) was measured every 3 days; body weight was recorded weekly. Mice were euthanized, and tumors were excised for western blot [1]
- NSCLC xenograft protocol: Male BALB/c nude mice (7 weeks) were subcutaneously injected with 1×10⁷ A549 cells (100 μL PBS/matrigel). When tumors reached ~150 mm³, mice were grouped (n=5/group): vehicle (PBS + 0.1% DMSO, intraperitoneal, twice weekly) and Panobinostat (LBH589) (10 mg/kg, dissolved in PBS + 0.1% DMSO, intraperitoneal, twice weekly). Treatment lasted 4 weeks. Mice were euthanized, and tumors were weighed/processed for immunohistochemistry [2]
- CML xenograft protocol: Female NOD/SCID mice (8 weeks) were intravenously injected with 2×10⁶ K562 cells. After 7 days, mice were grouped (n=5/group): vehicle (0.2% Tween 80 in PBS, oral, daily), Panobinostat (LBH589) (15 mg/kg, dissolved in 0.2% Tween 80 in PBS, oral, daily), and combination (15 mg/kg Panobinostat (LBH589) + 50 mg/kg imatinib, oral, daily). Treatment lasted 14 days. Tumor volume was monitored, and tumors were excised for analysis [4]
- Melanoma xenograft protocol: Female nude mice (6 weeks) were subcutaneously injected with 3×10⁶ A375 cells (100 μL PBS/matrigel). When tumors reached ~80 mm³, mice were grouped (n=6/group): vehicle (0.5% methylcellulose, oral, daily) and Panobinostat (LBH589) (8 mg/kg, dissolved in 0.5% methylcellulose, oral, daily). Treatment lasted 18 days. Tumor volume was measured every 2 days; body weight was recorded weekly [5]

Absorption, Distribution and Excretion
Following an administration of 20 mg pabisostat, absorption is rapid, reaching maximum absorption within 2 hours. Metabolism/Metabolites Pabisostat is extensively metabolized into 77 metabolites. 2% and 3% of unmetabolized pabisostat are recovered in urine and feces, respectively. The main metabolic pathways of pabisostat include reduction, hydrolysis, oxidation, and glucuronidation. CYP and non-CYP enzymes play important roles in metabolism, while CYP2D6 and CYP2C19 have relatively minor roles.

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Biological half-life>
30 hours

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Rat pharmacokinetic characteristics: Oral administration of pabisostat (LBH589) (10 mg/kg) showed an oral bioavailability of 25%, Cmax = 80 ng/mL (Tmax = 1.5 hours), terminal t1/2 = 4.2 hours, and AUC0-24h = 350 ng·h/mL. Intravenous injection (5 mg/kg) showed Cmax = 220 ng/mL, t1/2 = 3.8 h, AUC0-∞ = 480 ng·h/mL [3]
- Mouse pharmacokinetic characteristics: Oral administration of pabilistat (LBH589) (15 mg/kg) showed oral bioavailability = 18%, Cmax = 65 ng/mL (Tmax = 2 h), t1/2 = 3.5 h, AUC0-24h = 290 ng·h/mL [4]
- Plasma protein binding: Pabilistat (LBH589) was 98% bound to human plasma proteins (mainly albumin) [3]
- Metabolism: In human liver microsomes, pabilistat (LBH589) is metabolized by CYP3A4 and CYP2D6; CYP1A2, CYP2C9 or CYP2C19 It has no significant effect on its metabolism [3]

Hepatotoxicity
Most clinical trials of pabilistat did not report the incidence of elevated serum enzymes during treatment, and it is often used in combination with other antitumor drugs that can cause elevated serum ALT and AST. In a large controlled trial of pabilistat versus placebo in combination with bortezomib and dexamethasone, the incidence of elevated ALT was similar in the pabilistat group (31%) and the placebo group (38%), and ALT values exceeding 5 times the upper limit of normal were uncommon (1.8% and 1.3%, respectively). Furthermore, there have been no reports of clinically significant liver injury with jaundice related to pabilistat treatment. Therefore, pabilistat appears to have low hepatotoxicity, and liver injury caused by pabilistat, even if it occurs, should be very rare. Probability Score: E (Unlikely to be the cause of clinically significant liver injury). Repeated-dose toxicity in rats: Oral administration of pabilistat (LBH589) (10–30 mg/kg/day) for 28 consecutive days showed a No Observed Adverse Effect Level (NOAEL) of 20 mg/kg/day. At a dose of 30 mg/kg/day, mild weight loss (approximately 8%), elevated serum ALT (2.5 times higher than the control group), and hepatocyte vacuolation (histopathology) were observed; no death or organ failure was observed [3]
- Mouse toxicity: Oral administration of pabisostat (LBH589) (15–25 mg/kg/day) for 14 consecutive days showed no observed adverse effects (NOAEL) at a dose of 15 mg/kg/day. At a dose of 25 mg/kg/day, mild diarrhea (30% of mice) and decreased white blood cell count (1.2 × 10⁹/L, compared to 2.5 × 10⁹/L in the control group) were observed [4]
- Normal cell toxicity: In human peripheral blood mononuclear cells (PBMCs), pabisostat (LBH589) (≤50 nM) showed ≥85% cell viability (compared to the control group), indicating low toxicity to normal cells [1]

[1]. Blood . 2008 May 15;111(10):5093-100.

[2]. Mol Cancer Ther . 2009 Aug;8(8):2221-31.

[3]. Haematologica . 2010 May;95(5):794-803.

[4]. Cancer Res . 2006 Jun 1;66(11):5781-9.

[5]. Cancer Lett . 2009 Aug 8;280(2):233-41.

Panobinostat is a hydroxamic acid formed by the condensation of the carboxyl group of (2E)-3-[4-({[2-(2-methylindole-3-yl)ethyl]amino}methyl)phenyl]prop-2-enoic acid with the amino group of hydroxylamine. It is a histone deacetylase inhibitor (in lactate form) often used in combination with bortezomib and dexamethasone to treat multiple myeloma. It has multiple functions, including as an EC 3.5.1.98 (histone deacetylase) inhibitor, an antitumor drug, and an angiogenesis modulator. It is a hydroxamic acid compound belonging to the cinnamamide, secondary amino, and methylindole classes. It is the conjugate base of panobistat (1+). Panobistat is a drug previously approved by the U.S. Food and Drug Administration (FDA) under the brand name Farydak for the treatment of certain types of cancer. Panobistat is currently being investigated as an investigational drug as part of a strategy to cure HIV infection. As an investigational HIV therapy, panobinostat belongs to a class of drugs known as latency reversal agents. Panobinostat is an oral deacetylase (DAC) inhibitor that received FDA approval on February 23, 2015, for the treatment of multiple myeloma. This approval was based on accelerated progression-free survival, and therefore the sponsor is conducting confirmatory trials to demonstrate its clinical efficacy in the treatment of multiple myeloma. Panobinostat is marketed by Novartis under the brand name Farydak. Panobinostat is a non-selective histone deacetylase inhibitor (pan-histone deacetylase inhibitor) and is currently the most effective histone deacetylase inhibitor on the market. Histone deacetylase (HDAC) inhibitors are histone deacetylase inhibitors. Their mechanism of action is the inhibition of histone deacetylase and cytochrome P450 2D6. Panobistat is an oral histone deacetylase inhibitor and antitumor drug approved for use in combination with other drugs to treat refractory or relapsed multiple myeloma. Panobistat causes a slight increase in serum enzymes during treatment, but has not been found to be associated with clinically significant liver injury. Panobistat is a cinnamic acid hydroxamic acid analog with potential antitumor activity. Panobistat selectively inhibits histone deacetylase (HDAC), inducing excessive acetylation of core histones, which may lead to the regulation of cyclin expression, cell cycle arrest at the G2/M phase, and apoptosis. Furthermore, the drug appears to regulate the expression of angiogenesis-related genes, such as hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF), thereby inhibiting endothelial cell chemotaxis and invasiveness. HDAC is an enzyme that deacetylates chromatin histones. Panobistat is an indole and hydroxamic acid derivative that acts as a histone deacetylase inhibitor. It is used in combination with bortezomib and dexamethasone as an anti-tumor drug for the treatment of multiple myeloma.
See also: Pabistat lactate (active ingredient).

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Drug Indications
Pabistat, in combination with dexamethasone and bortezomib, is indicated for the treatment of patients with multiple myeloma who have previously received at least two prior therapy regimens containing bortezomib and an immunomodulatory agent. This indication was granted accelerated approval on February 23, 2015, based on progression-free survival.
FDA Label
Farydak, in combination with bortezomib and dexamethasone, is indicated for the treatment of adult patients with relapsed and/or refractory multiple myeloma who have previously received at least two prior therapy regimens containing bortezomib and an immunomodulatory agent. Farydak, in combination with bortezomib and dexamethasone, is indicated for the treatment of adult patients with relapsed and/or refractory multiple myeloma who have previously received at least two prior therapy regimens containing bortezomib and an immunomodulatory agent.

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Mechanism of Action
Panobinostat is a deacetylase (DAC) inhibitor. DACs, also known as histone deacetylases (HDACs), are responsible for regulating the acetylation of approximately 1750 proteins in the body; their functions are involved in a variety of biological processes, including DNA replication and repair, chromatin remodeling, gene transcription, cell cycle progression, protein degradation, and cytoskeleton reorganization. In multiple myeloma, DAC proteins are overexpressed. Panobinostat inhibits class I (HDAC 1, 2, 3, 8), class II (HDAC 4, 5, 6, 7, 9, 10), and class IV (HDAC 11) proteins. The antitumor activity of panobinostat is thought to be attributed to epigenetic regulation of gene expression and inhibition of protein metabolism. Panobinostat also exhibits synergistic cytotoxic effects with bortezomib (a proteasome inhibitor also used to treat multiple myeloma). Mechanism of action: Pabilistat (LBH589) inhibits HDAC, leading to increased acetylation levels of histones and non-histone proteins (e.g., α-tubulin). This alters chromatin structure and regulates gene expression involved in cell cycle arrest, apoptosis, and differentiation [1,4]
- Combination therapy potential: Pabilistat (LBH589) has shown synergistic antitumor activity with imatinib (chronic myeloid leukemia), bortezomib (multiple myeloma), and cisplatin (non-small cell lung cancer), supporting the development of combination therapy clinical trials [2,4]
- Clinical development: Based on preclinical efficacy, pabilistat (LBH589) has been evaluated in Phase I/II clinical trials for hematologic malignancies (multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia) and solid tumors (non-small cell lung cancer, melanoma) [3,5]

C21H23N3O2

349.43

349.179

C, 72.18; H, 6.63; N, 12.03; O, 9.16

404950-80-7

404950-80-7;960055-56-5 (lactate); 960055-60-1 (mesylate);960055-50-9 (acetate); 960055-54-3 (fumarate); 960055-57-6

6918837

White to off-white solid powder

1.2±0.1 g/cm3

114-117?C

1.683

3.62

4

3

7

26

474

0

O=C(/C(/[H])=C(\[H])/C1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])N([H])C([H])([H])C([H])([H])C1=C(C([H])([H])[H])N([H])C2=C([H])C([H])=C([H])C([H])=C12)N([H])O[H]

FPOHNWQLNRZRFC-ZHACJKMWSA-N

InChI=1S/C21H23N3O2/c1-15-18(19-4-2-3-5-20(19)23-15)12-13-22-14-17-8-6-16(7-9-17)10-11-21(25)24-26/h2-11,22-23,26H,12-14H2,1H3,(H,24,25)/b11-10+

(E)-N-hydroxy-3-[4-[[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl]phenyl]prop-2-enamide

NVP-LBH589; NVP-LBH 589; LBH589; LBH 589; LBH-589; Panobinostat; Brand name Farydak

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: ≥ 2.5 mg/mL (7.15 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.15 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.15 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.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (7.15 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.

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Solubility in Formulation 5: 2.5 mg/mL (7.15 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.

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Solubility in Formulation 6: 2% DMSO+48% PEG 300+2% Tween 80+ddH2O: 5mg/mL

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