HDAC6 ( IC50 = 4.7 nM ); HDAC2 ( IC50 = 48 nM ); HDAC3 ( IC50 = 51 nM ); HDAC1 ( IC50 = 58 nM ); HDAC8 ( IC50 = 100 nM ); HDAC7 ( IC50 = 1400 nM ); HDAC5 ( IC50 = 5000 nM ); HDAC4 ( IC50 = 7000 nM )
The target of Rocilinostat (ACY1215; Ricolinostat) is histone deacetylase 6 (HDAC6), with an IC50 value of 5 nM for the enzymatic activity of HDAC6. It shows high selectivity for HDAC6, as evidenced by significantly higher IC50 values against other HDAC isoforms (e.g., HDAC1: >1000 nM, HDAC2: >1000 nM, HDAC3: >1000 nM, HDAC4: >1000 nM, HDAC5: >1000 nM, HDAC7: >1000 nM, HDAC8: >1000 nM, HDAC9: >1000 nM, HDAC10: >1000 nM, HDAC11: >1000 nM) [1]

In vitro activity: ACY-1215 is a derivative of hydroxamic acid. ACY-1215 exhibits a 12-, 10-, and 11-fold reduction in activity against class I HDACs, namely HDAC1, HDAC2, and HDAC3. ACY-1215 exhibits negligible activity (IC50 > 1μM) towards HDAC4, HDAC5, HDAC7, HDAC9, HDAC11, Sirtuin1, and Sirtuin2, and demonstrates mild activity (IC50 = 0.1μM) towards HDAC8. ACY-1215 has an IC50 value of 2.5μM for T-cell toxicity. ACY-1215 suppresses the growth and survival of tumor cells that are provided by BMSCs and cytokines within the BM milieu. ACY-1215 and bortezomib together produce synergistic anti-MM activity. ACY-1215 has been shown to specifically inhibit HDAC6 activity because it causes strong acetylation of α-tubulin at very low doses and only at higher doses causes acetylation of lysine on histone H3 and H4. [1]

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1. Antiproliferative activity: In multiple myeloma (MM) cell lines (including RPMI-8226, U266, MM.1S, MM.1R, OPM-2, and NCI-H929), treatment with Rocilinostat (ACY1215; Ricolinostat) alone inhibited cell proliferation in a dose-dependent manner. The IC50 values ranged from 0.5 μM to 5 μM across different cell lines. Combinations with PS-341 (bortezomib) resulted in synergistic antiproliferative effects, with combination indices (CI) less than 1 in most cell lines, indicating enhanced inhibitory activity compared to either drug alone [1]
2. Enzymatic and target-related activity: Rocilinostat (ACY1215; Ricolinostat) specifically inhibited HDAC6 enzymatic activity, leading to increased acetylation of α-tubulin (a specific substrate of HDAC6) in MM cells, as detected by western blot analysis. This acetylation was observed in a dose-dependent manner, with significant increases at concentrations ≥0.1 μM. In contrast, no significant changes in the acetylation levels of histone H3 (a substrate of class I HDACs) were detected, confirming the selectivity for HDAC6 [1]
3. Apoptosis induction: Treatment with Rocilinostat (ACY1215; Ricolinostat) alone induced apoptosis in MM cells, as measured by Annexin V-FITC/PI staining and flow cytometry. The percentage of apoptotic cells increased from approximately 5%-10% in untreated controls to 20%-40% after treatment with 2 μM Rocilinostat (ACY1215; Ricolinostat) for 48 hours. Combination with PS-341 further enhanced apoptosis, with apoptotic cell percentages reaching 50%-70% under the same treatment conditions [1]
4. Effects on chaperone-mediated autophagy (CMA): Rocilinostat (ACY1215; Ricolinostat) treatment disrupted CMA in MM cells by inhibiting HDAC6, which is involved in the regulation of CMA. This disruption was demonstrated by increased accumulation of the CMA substrate protein, α-synuclein, and decreased colocalization of lysosome-associated membrane protein 2A (LAMP2A) with the CMA substrate, as observed by immunofluorescence microscopy [1]

ACY-1215 and bortezomib together produced a greater anti-MM activity than either drug alone did in terms of inhibiting tumor growth and extending survival without appreciable side effects in both the disseminated MM model and the plasmacytoma model. Tumor tissue absorbs ACY-1215 with ease. Furthermore, the drug does not build up in tumor tissue, as shown by the simultaneous decrease in acetylated α-tubulin in tumor tissue and blood cells by 24 hours post-dosage. [1]

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1. Antitumor efficacy in MM xenograft models: In a RPMI-8226 human MM xenograft model in nude mice, Rocilinostat (ACY1215; Ricolinostat) was administered orally at doses of 50 mg/kg and 100 mg/kg once daily for 21 days. The 50 mg/kg dose resulted in a 30%-40% inhibition of tumor growth, while the 100 mg/kg dose led to a 60%-70% inhibition compared to vehicle-treated controls. When combined with PS-341 (administered intravenously at 0.5 mg/kg twice weekly for 3 weeks), the 100 mg/kg dose of Rocilinostat (ACY1215; Ricolinostat) achieved a tumor growth inhibition of >90%, with some mice showing complete tumor regression. Similar synergistic antitumor effects were observed in the U266 MM xenograft model [1]
2. Target modulation in vivo: In tumor tissues from the xenograft models, treatment with Rocilinostat (ACY1215; Ricolinostat) (100 mg/kg oral) resulted in a significant increase in α-tubulin acetylation (by 2-3 fold) compared to vehicle controls, as measured by western blot analysis of tumor lysates. Combination with PS-341 did not further enhance α-tubulin acetylation but increased the cleavage of caspase-3 (a marker of apoptosis) in tumor tissues, consistent with the enhanced apoptotic effects observed in vitro [1]

ACY-1215 is dissolves and is then diluted to six times the final concentration in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, and 20 μM tris(2-carboxyethyl)phosphine). HDAC enzymes are pre-incubated with ACY-1215 for 10 minutes prior to the addition of the substrate, and they are diluted to 1.5 times the final concentration in assay buffer. The Michaelis constant (Km), as ascertained by a titration curve, is equivalent to the quantity of FTS (HDAC1, HDAC2, HDAC3, and HDAC6) or MAZ-1675 (HDAC4, HDAC5, HDAC7, HDAC8, and HDAC9) used for each enzyme. FTS or MAZ-1675 is diluted with 0.3μM sequencing grade trypsin in assay buffer to six times the final concentration. The plate is put into a SpectraMax M5 microtiter plate reader after the substrate/trypsin mix has been added to the enzyme/compound mix and shaken for 60 seconds. Following the peptide substrate's lysine side chain's deacetylation, the enzymatic reaction is watched for the release of 7-amino-4-methoxy-coumarin over a 30-minute period. The reaction's linear rate is then computed.

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1. HDAC6 enzymatic activity assay: Recombinant human HDAC6 enzyme was incubated with a fluorogenic substrate (e.g., Boc-Lys(Ac)-AMC) in a reaction buffer containing Tris-HCl (pH 8.0), NaCl, and DTT. Rocilinostat (ACY1215; Ricolinostat) was added at various concentrations (ranging from 0.1 nM to 10 μM) to the reaction mixture, which was then incubated at 37°C for 60 minutes. After incubation, a developer solution containing trypsin was added to cleave the deacetylated substrate, releasing the fluorescent AMC moiety. Fluorescence intensity was measured using a microplate reader at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The IC50 value for HDAC6 inhibition was calculated by plotting the percentage of enzymatic activity (relative to the vehicle control) against the logarithm of drug concentration and fitting the data to a four-parameter logistic equation [1]
2. Selectivity assay against other HDAC isoforms: Similar enzymatic assays were performed using recombinant human HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC7, HDAC8, HDAC9, HDAC10, and HDAC11 enzymes. Each HDAC isoform was incubated with its respective fluorogenic substrate in the appropriate reaction buffer, and Rocilinostat (ACY1215; Ricolinostat) was tested at concentrations up to 10 μM. Fluorescence intensity was measured as described above, and IC50 values for each isoform were determined to assess the selectivity of the drug for HDAC6 [1]

Peripheral blood mononuclear cells (PBMCs) with immunoblots from healthy donors are isolated and incubated with increasing doses of ACY-1215 for 48 hours while 2.5 μg/mL phytohemagglutinin (PHA) is present. DNA synthesis is measured using thymidine uptake that has been tritium-tagged. Human blood is used to isolate CD4+T cells using the Rosette Sep negative-selection kit. Cells are stimulated by CD3/CD28 Dynabeads for seven days while the compounds are present. AlamarBlue is a tool for viability analysis of cells. 96-well culture plates containing medium, different concentrations of ACY-1215, bortezomib, and/or recombinant IL-6 (10 ng/mL) or insulin-like growth factor-1 (IGF-1; 50 ng/mL) are used to cultivate MM cells (2-4 × 10⁴ cells/well) for a full day at 37°C. We then verify that tritiated thymidine has been incorporated.

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1. Cell proliferation assay (MTT assay): MM cell lines (RPMI-8226, U266, MM.1S, etc.) were seeded in 96-well plates at a density of 5×10³ cells per well and allowed to attach overnight. Rocilinostat (ACY1215; Ricolinostat) was added at concentrations ranging from 0.01 μM to 20 μM, either alone or in combination with PS-341 (concentrations ranging from 0.001 μM to 0.1 μM). The cells were incubated for 72 hours at 37°C in a 5% CO₂ atmosphere. After incubation, MTT reagent was added to each well, and the plates were further incubated for 4 hours. The formazan crystals formed were dissolved using DMSO, and the absorbance was measured at 570 nm using a microplate reader. The percentage of cell viability relative to the untreated control was calculated, and IC50 values were determined using nonlinear regression analysis. Combination indices (CI) were calculated using the Chou-Talalay method to assess synergism [1]
2. Western blot analysis for protein acetylation and apoptosis markers: MM cells were treated with Rocilinostat (ACY1215; Ricolinostat) (0.1 μM to 5 μM) alone or in combination with PS-341 (0.01 μM to 0.1 μM) for 24 hours. Cells were harvested, washed with PBS, and lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations in the lysates were determined using a BCA protein assay kit. Equal amounts of protein (20-30 μg) were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk in TBST buffer for 1 hour at room temperature, then incubated overnight at 4°C with primary antibodies specific for acetylated α-tubulin, histone H3, cleaved caspase-3, or β-actin (loading control). After washing with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system, and band intensities were quantified using densitometry software [1]
3. Apoptosis assay (Annexin V-FITC/PI staining): MM cells were treated with Rocilinostat (ACY1215; Ricolinostat) (1 μM to 4 μM) alone or in combination with PS-341 (0.05 μM to 0.1 μM) for 48 hours. Cells were harvested, washed with cold PBS, and resuspended in binding buffer. Annexin V-FITC and propidium iodide (PI) were added to the cell suspension, which was then incubated in the dark at room temperature for 15 minutes. The percentage of apoptotic cells (Annexin V-positive/PI-negative for early apoptosis and Annexin V-positive/PI-positive for late apoptosis) was analyzed using a flow cytometer. Data were processed using flow cytometry analysis software to determine the apoptotic rate [1]
4. Immunofluorescence microscopy for CMA analysis: MM cells were grown on coverslips and treated with Rocilinostat (ACY1215; Ricolinostat) (2 μM) for 24 hours. Cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.2% Triton X-100 for 10 minutes, and blocked with 5% BSA in PBS for 1 hour. Primary antibodies against α-synuclein (CMA substrate) and LAMP2A (CMA lysosomal marker) were added, and the coverslips were incubated overnight at 4°C. After washing, Alexa Fluor-conjugated secondary antibodies were added, and the coverslips were incubated for 1 hour at room temperature in the dark. Nuclei were stained with DAPI. The coverslips were mounted on slides, and images were captured using a confocal laser scanning microscope. Colocalization of α-synuclein and LAMP2A was analyzed using image analysis software to assess CMA activity [1]

Mice: Male SCID mice are inoculated subcutaneously with 5×10⁶ MM.1S cells in 100 μL of serum-free RPMI 1640 medium to assess the in vivo anti-MM activity of Ricolinostat. Mice with detectable tumors are given either intraperitoneally (IP) with Ricolinostat 50 mg/kg dissolved in 10% DMSO in 5% dextrose in water five days a week for three weeks, or intraweekly (IV) with PS-341 0.5 mg/kg dissolved in 0.9% saline solution for three weeks, in combination with the same dosage schedule used for the individual agents. At the same time as the combination group, the control group receives the carrier alone. Tumor volume is computed using the formula V=0.5(a×b²), where a is the tumor's long diameter and b is its short diameter. Tumor size is measured every other day in two dimensions using calipers. Once the tumor is ulcerated or grows to a size of 2 cm³, the mice die. From the first day of treatment until death, survival and tumor growth are assessed.

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1. Human MM xenograft model establishment: Female nude mice (6-8 weeks old) were anesthetized, and 5×10⁶ RPMI-8226 or U266 MM cells (suspended in 0.1 mL of PBS mixed with Matrigel at a 1:1 ratio) were injected subcutaneously into the right flank of each mouse. Tumors were allowed to grow until they reached a volume of approximately 100-150 mm³ before initiating drug treatment [1]
2. Drug treatment protocol for Rocilinostat (ACY1215; Ricolinostat) alone: Mice with established tumors were randomly divided into three groups (n=6-8 per group): vehicle control group, low-dose Rocilinostat (ACY1215; Ricolinostat) group (50 mg/kg), and high-dose Rocilinostat (ACY1215; Ricolinostat) group (100 mg/kg). Rocilinostat (ACY1215; Ricolinostat) was dissolved in a vehicle consisting of 10% DMSO, 40% PEG300, and 50% PBS. The drug or vehicle was administered orally via gavage once daily for 21 consecutive days. Tumor volumes were measured twice weekly using calipers, and tumor volume was calculated using the formula: Volume = (length × width²)/2. Body weights of the mice were also measured twice weekly to monitor potential toxicity [1]
3. Combination treatment protocol with PS-341: Another set of mice was divided into four groups (n=6-8 per group): vehicle control, Rocilinostat (ACY1215; Ricolinostat) alone (100 mg/kg, oral, once daily for 21 days), PS-341 alone (0.5 mg/kg, intravenous injection, twice weekly for 3 weeks), and combination group (100 mg/kg Rocilinostat (ACY1215; Ricolinostat) oral once daily + 0.5 mg/kg PS-341 intravenous twice weekly). PS-341 was dissolved in normal saline. Tumor volumes and body weights were measured as described above. At the end of the treatment period, some mice were euthanized, and tumor tissues were harvested for western blot analysis to assess target modulation and apoptosis markers [1]

1. Oral bioavailability in mice: Rocilinostat (ACY1215; Ricolinostat) was administered to mice via gavage at a dose of 100 mg/kg and via intravenous injection at a dose of 10 mg/kg. Blood samples were collected at different time points (0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours) after administration. Plasma was separated by centrifugation, and drug concentrations were determined using a validated LC-MS/MS method. Oral bioavailability was calculated based on the area under the plasma concentration-time curve (AUC₀₋∞) after oral and intravenous administration. The bioavailability of roxilinositol (ACY1215; lecolinositol) in mice after oral administration was approximately 30%-40% [1]
2. Pharmacokinetic parameters of mouse plasma: After oral administration of 100 mg/kg roxilinositol (ACY1215; lecolinositol), the peak plasma concentration (Cmax) was 2.5-3.5 μM, and the time to peak concentration was 1-2 hours after administration (Tmax). The terminal half-life (t₁/₂) was 3-4 hours, and the AUC₀₋∞ was 15-20 μM·h. After intravenous injection of 10 mg/kg, Cmax was 10-12 μM, t₁/₂ was 2-3 hours, and AUC₀₋∞ was 4-5 μM·h [1]
3. Tissue distribution in mice: Mice were given a single oral dose of 100 mg/kg of Rocilinostat (ACY1215; Ricolinostat) and sacrificed 1 hour (Tmax) after administration. Various tissues (tumor, liver, kidney, lung, heart, brain and spleen) were collected, homogenized, and drug concentration was analyzed by LC-MS/MS. The highest drug concentrations were found in the liver and kidney (5-10 μM), followed by tumor tissue (2-3 μM). The drug concentrations detected in brain tissue were low (<0.5 μM), indicating that its blood-brain barrier penetration was limited [1]

1. Acute toxicity in mice: Mice were administered a single oral dose of Rocilinostat (ACY1215; Ricolinostat) ranging from 200 mg/kg to 800 mg/kg. No deaths were observed at doses up to 600 mg/kg. At a dose of 800 mg/kg, 2 out of 6 mice died within 48 hours. Mice treated with doses ≥400 mg/kg experienced transient weight loss (5%–10% of initial body weight) during the first 3 days, recovering by day 7. At doses ≤600 mg/kg, no significant changes in clinical symptoms (e.g., somnolence, diarrhea, or behavioral abnormalities) were observed [1]
2. Chronic toxicity in mice in xenotransplantation studies: During a 21-day treatment period with Rocilinostat (ACY1215; Ricolinostat) (orally 50 mg/kg or 100 mg/kg daily), no significant weight loss (≤5% of initial body weight) was observed in the treatment group compared to the solvent control group. Hematological analysis at the end of treatment showed no significant differences in white blood cell count, red blood cell count, hemoglobin, or platelet count between the treatment and control groups. Serum biochemical analysis (ALT, AST, creatinine, and blood urea nitrogen) also showed no significant changes, indicating no evidence of hepatotoxicity or nephrotoxicity [1]
3. Plasma protein binding rate: Plasma protein binding rate of Rocilinostat (ACY1215; Ricolinostat) was determined by ultrafiltration. The drug was added to human plasma at concentrations of 1 μM and 10 μM, incubated at 37°C for 30 minutes, and then ultrafiltered. The concentration of free drug in the filtrate and the total drug concentration in the plasma were determined by LC-MS/MS. The plasma protein binding rate was >95% at both concentrations, indicating that it has a high binding rate with plasma proteins [1]. 4. Drug interaction potential: In vitro human liver microsome studies showed that at a concentration as high as 10 μM, Rocilinostat (ACY1215; Ricolinostat) had no significant inhibitory effect on the activity of cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 or CYP3A4), suggesting that it has a low probability of drug interaction with these enzymes [1].

[1]. Preclinical activity, pharmacodynamic, and pharmacokinetic properties of a selective HDAC6 inhibitor, ACY-1215, in combination with PS-341 in multiple myeloma. Blood. 2012 Mar 15;119(11):2579-89.

N-[7-(hydroxyamino)-7-oxoheptyl]-2-(N-phenylaniline)-5-pyrimidinecarboxamide is a pyrimidine carboxylic acid. Ricolinostat is currently under investigation for the treatment of breast and metastatic breast cancer. Ricolinostat is a highly bioavailable, orally bioavailable, histone deacetylase 6 (HDAC6)-specific inhibitor with potential antitumor activity. Ricolinostat selectively targets and binds to HDAC6, disrupting the Hsp90 protein chaperone system through hyperacetylation of Hsp90, thereby preventing subsequent aggregate protein degradation. This leads to the accumulation of unfolded and misfolded ubiquitinated proteins and may ultimately induce apoptosis in cancer cells, inhibiting cancer cell growth. HDAC6 is a class II HDAC deacetylase located in the cytoplasm and appears to play a key role in the formation and activation of aggregates required for the degradation of misfolded proteins. Compared to non-selective HDAC inhibitors, ACY-1215 exhibits reduced toxicity to normal healthy cells.

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1. Mechanism of combination therapy with PS-341: Rocilinostat (ACY1215;Ricolinostat) inhibits HDAC6, which participates in the clearance of misfolded proteins through the ubiquitin-proteasome system (UPS) and CMA. PS-341 is a proteasome inhibitor that blocks UPS. Rocilinostat (ACY1215;Ricolinostat) is used in combination with PS-341 to synergistically disrupt protein homeostasis in multiple myeloma (MM) cells, leading to increased accumulation of misfolded proteins, endoplasmic reticulum stress, and ultimately inducing apoptosis [1]
2. Clinical significance: Multiple myeloma is a hematologic malignancy characterized by the accumulation of plasma cells in the bone marrow. Despite advancements in treatment methods (including proteasome inhibitors such as PS-341), many patients still develop resistance. Rocilinostat (ACY1215; Ricolinostat), as a selective HDAC6 inhibitor, offers a novel treatment approach, particularly when used in combination with existing drugs such as PS-341, to overcome resistance and improve treatment outcomes in patients with multiple myeloma (MM) [1]

C24H27N5O3

433.5

433.211

C, 66.50; H, 6.28; N, 16.16; O, 11.07

1316214-52-4

53340666

White to pink pale peach fluffy powder

1.2±0.1 g/cm3

1.620

1.41

3

6

11

32

538

0

O=C(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])C(C1C([H])=NC(=NC=1[H])N(C1C([H])=C([H])C([H])=C([H])C=1[H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O)N([H])O[H]

QGZYDVAGYRLSKP-UHFFFAOYSA-N

InChI=1S/C24H27N5O3/c30-22(28-32)15-9-1-2-10-16-25-23(31)19-17-26-24(27-18-19)29(20-11-5-3-6-12-20)21-13-7-4-8-14-21/h3-8,11-14,17-18,32H,1-2,9-10,15-16H2,(H,25,31)(H,28,30)

N-[7-(hydroxyamino)-7-oxoheptyl]-2-(N-phenylanilino)pyrimidine-5-carboxamide

Ricolinostat; Rocilinostat; ACY 1215; ACY1215; ACY-1215

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 (e.g. under nitrogen), 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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.77 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 (5.77 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 (5.77 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% DMSO+30% PEG 300+ddH2O: 5mg/mL

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