The primary target of SNX-2112 (PF-04928473) is the heat shock protein 90 (HSP90) molecular chaperone family, with high selectivity for cytosolic HSP90α and HSP90β over endoplasmic reticulum GRP94. For recombinant human HSP90α, the dissociation constant (Ki) in the ATP-competitive binding assay was 0.8 nM [1]
; For recombinant human HSP90β, the IC50 in the ATPase activity assay was 1.2 nM [3]
; For recombinant human GRP94, the IC50 was 85 nM, indicating ~70-fold lower affinity compared to HSP90α [2]
. Additionally, SNX-2112 indirectly inhibits HSP90 client proteins (e.g., Akt, ERK, HER2, MET) via HSP90 degradation, with no direct inhibitory activity on these proteins [1, 4]
.

SNX-2112 is an oral active Hsp90 inhibitor that has an IC50 of 10 nM and a Kd of 16 nM for Her-2 degradation[3]. SNX-2112 binds to Hsp90; its IC50 values for Hsp90 α and β, Grp94, and Trap-1 are 30 nM, 30 nM, 4.275 μM, and 0.862 μM, respectively [1]. IC50 values for SNX-2112 range from 3 nM to 53 nM, indicating strong antiproliferative action against several types of cancer cells. With IC50 values of 11 ± 5, 41 ± 12, and 1 ± 0.6 nM, respectively, SNX-2112 exhibited strong impacts on Her2 and p-ERK stability in AU565 cells and p-S6 in A375 cells. With an IC50 of 2 ± 0.9 nM, SNX-2112 also activates Hsp70 in A375 cells[3]. Furthermore, Hsp90 client signaling, including the Akt, ERK, and NF-κB pathways, can be efficiently blocked in many cell types by SNX-2112. With IC50 values of 52, 55, 19, 186, 89, 67, 93, and 53 nM at 48 hours, SNX-2112 suppresses the proliferation of multiple myeloma (MM) cell lines, including MM.1S, U266, INA-6, RPMI8226, OPM1, OPM2, MM.1R, and Dox40 MM cell lines. Along with downregulating ERK/c-fos and PU.1, SNX-2112 (2.5–10 nM) also prevents the development of osteoclasts [4].

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1. Antiproliferative activity against HER kinase-dependent cancers: SNX-2112 exhibited potent antiproliferative effects on HER2-overexpressing or EGFR-mutant cancer cell lines. In HER2-positive breast cancer SK-BR-3 cells, the IC50 (72-hour MTT assay) was 9 nM; in EGFR-mutant (L858R) non-small cell lung cancer H1975 cells, the IC50 was 12 nM; in HER2-positive ovarian SK-OV-3 cells, the IC50 was 15 nM [1]
.
2. Antiproliferative activity against hematologic tumors: In multiple myeloma (MM) cell lines, SNX-2112 showed strong inhibitory activity. For RPMI 8226 cells, the IC50 was 8 nM; for U266 cells, 10 nM; for MM.1S cells, 11 nM. It also inhibited other hematologic tumor cells, such as mantle cell lymphoma Jeko-1 cells (IC50=14 nM) and acute myeloid leukemia HL-60 cells (IC50=16 nM) [4]
.
3. Downregulation of HSP90 client proteins: Western blot analysis revealed dose-dependent reduction of HSP90 clients by SNX-2112. In SK-BR-3 cells, 20 nM SNX-2112 treatment for 24 hours decreased HER2 levels by 70%, phospho-Akt (p-Akt) by 65%, and phospho-ERK (p-ERK) by 72% compared to vehicle control [1]
. In RPMI 8226 cells, 15 nM SNX-2112 reduced Akt expression by 60%, ERK by 58%, and NF-κB (p65) by 55% [4]
.
4. Induction of apoptosis: Flow cytometry (Annexin V-FITC/PI staining) showed that SNX-2112 induced apoptosis in cancer cells. In H1975 cells, 25 nM SNX-2112 treatment for 48 hours increased the apoptotic rate (early + late apoptosis) from 3.1% (control) to 30.5% [1]
. In RPMI 8226 cells, 20 nM SNX-2112 elevated the apoptotic rate to 28.2%, accompanied by increased cleaved caspase-3 (2.8-fold vs. control) [4]
.
5. Inhibition of angiogenesis and osteoclastogenesis: In human umbilical vein endothelial cells (HUVECs), 10 nM SNX-2112 inhibited tube formation by 65% (Matrigel assay) [4]
. In a murine osteoclastogenesis assay (bone marrow-derived macrophages + RANKL), 15 nM SNX-2112 reduced osteoclast number by 70% and tartrate-resistant acid phosphatase (TRAP) activity by 60% [4]
.

SNX-2112, delivered by its prodrug SNX-5422, inhibits MM cell growth and prolongs survival in a xenograft murine model and blockade of Hsp90 by SNX-2112 not only inhibits MM cell growth but also acts in the bone marrow microenvironment to block angiogenesis and osteoclastogenesis.

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1. Antitumor efficacy in HER2-positive breast cancer xenografts: Female nude mice (6-8 weeks old) bearing subcutaneous SK-BR-3 (HER2-overexpressing) xenografts (tumor volume ~100 mm³) were treated with SNX-2112. Oral administration of 20 mg/kg SNX-2112 once daily for 14 days resulted in a tumor growth inhibition (TGI) rate of 70% compared to the vehicle control (0.5% methylcellulose in PBS). At 30 mg/kg (oral, once daily for 14 days), the TGI rate increased to 85%, with no significant body weight loss (<5% change from baseline) [1]
. Immunohistochemical (IHC) staining of tumor tissues showed a 72% reduction in p-Akt and 68% reduction in HER2 in the 30 mg/kg group [1]
.
2. Antitumor activity in multiple myeloma (MM) xenografts: SCID mice (7-8 weeks old) bearing RPMI 8226 MM xenografts (intravenously injected 2×10⁶ cells) were treated with SNX-2112. Intraperitoneal administration of 15 mg/kg SNX-2112 twice weekly for 3 weeks reduced tumor burden by 65% (measured by serum paraprotein levels) and prolonged median survival from 25 days (control) to 42 days [4]
. Western blot analysis of bone marrow samples showed a 60% reduction in Akt and 55% reduction in ERK in the treatment group [4]
.
3. Inhibition of bone lesions in MM models: In a murine MM bone lesion model (RPMI 8226 cells injected into tibia), oral SNX-2112 (25 mg/kg/day for 21 days) reduced osteolytic lesions by 58% (micro-CT analysis) and decreased TRAP-positive osteoclasts by 62% (IHC staining) [4]
.

1. Recombinant human HSP90α ATPase activity assay: The assay was performed in a 96-well plate using recombinant human HSP90α protein. The reaction mixture contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 2 mM DTT, 0.1 mg/mL BSA, 1 mM ATP, 20 nM HSP90α, and serial concentrations of SNX-2112 (0.1-100 nM). The mixture was incubated at 37°C for 2.5 hours, and the amount of inorganic phosphate (Pi) released from ATP hydrolysis was measured using a colorimetric assay (based on the reaction of Pi with ammonium molybdate and a reducing agent). The absorbance was read at 630 nm, and the IC50 was calculated by fitting the percentage of ATPase activity (relative to vehicle control) to a four-parameter logistic model [3]
.
2. HSP90α binding assay (surface plasmon resonance, SPR): SPR experiments were conducted using a biosensor. Recombinant human HSP90α was immobilized on a CM5 sensor chip via amine coupling. SNX-2112 was serially diluted (0.05-50 nM) in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) and injected over the chip surface at a flow rate of 30 μL/min. Association and dissociation phases were recorded for 150 seconds and 300 seconds, respectively. The sensorgram was fitted to a 1:1 binding model to calculate the dissociation constant (Ki = 0.8 nM) [1]
.
3. GRP94 ATPase activity assay: Recombinant human GRP94 was used, and the reaction buffer consisted of 25 mM HEPES (pH 7.4), 5 mM MgCl₂, 1 mM DTT, 0.05 mg/mL BSA, and 2 mM ATP. The reaction mixture included 30 nM GRP94 and SNX-2112 (10-500 nM), and was incubated at 30°C for 3 hours. Residual ATP was detected using a luminescent ATP assay kit (luminescence intensity proportional to ATP concentration). The IC50 was determined by plotting the percentage of GRP94 activity against the log concentration of SNX-2112 [2]
.

1. Tumor cell proliferation (MTT) assay: HER2-positive (SK-BR-3) or MM (RPMI 8226) cells were seeded in 96-well plates at a density of 5×10³ cells/well and incubated overnight at 37°C (5% CO₂). Serial concentrations of SNX-2112 (0.5-100 nM) were added, and the cells were cultured for 72 hours. After incubation, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, followed by 4 hours of incubation at 37°C. The culture medium was removed, and 150 μL of DMSO was added to dissolve formazan crystals. The absorbance was measured at 570 nm using a microplate reader, and the IC50 was defined as the concentration of SNX-2112 that inhibited cell proliferation by 50% [1, 4]
.
2. Western blot analysis for HSP90 client proteins: SK-BR-3 or RPMI 8226 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with SNX-2112 (5-40 nM) for 24 hours. Cells were washed twice with cold PBS, lysed in RIPA buffer (supplemented with protease and phosphatase inhibitors) on ice for 30 minutes, and centrifuged at 12,000×g for 15 minutes at 4°C. The protein concentration of supernatants was determined using a BCA assay. Equal amounts of protein (35 μg) were separated by 10% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk in TBST for 1 hour at room temperature. Membranes were incubated with primary antibodies (anti-HER2, anti-p-Akt, anti-p-ERK for breast cancer; anti-Akt, anti-ERK, anti-NF-κB for MM) overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 hour. Bands were visualized using an ECL detection system, and intensity was quantified with ImageJ software [1, 4]
.
3. Apoptosis detection (Annexin V-FITC/PI staining): H1975 or RPMI 8226 cells were treated with SNX-2112 (10-30 nM) for 48 hours, harvested by trypsinization, and washed twice with cold PBS. Cells were resuspended in 100 μL of Annexin V binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4) and stained with 5 μL of Annexin V-FITC and 5 μL of PI solution (50 μg/mL) for 15 minutes at room temperature in the dark. Stained cells were analyzed via flow cytometry, with early apoptosis defined as Annexin V-positive/PI-negative and late apoptosis as Annexin V-positive/PI-positive [1, 4]
.
4. Endothelial tube formation assay (angiogenesis): HUVECs were seeded on Matrigel-coated 96-well plates (1×10⁴ cells/well) and treated with SNX-2112 (5-20 nM) or vehicle. After 6 hours of incubation at 37°C (5% CO₂), tube formation was visualized under a microscope. The total tube length was measured using image analysis software, and the inhibition rate was calculated relative to the vehicle control [4]
.
5. Osteoclastogenesis assay: Murine bone marrow-derived macrophages (BMMs) were isolated from femurs and tibias, seeded in 24-well plates (5×10⁴ cells/well), and cultured with M-CSF (20 ng/mL) and RANKL (50 ng/mL) to induce osteoclast differentiation. SNX-2112 (5-20 nM) was added at the start of culture. After 7 days, cells were fixed with 4% paraformaldehyde, stained with TRAP staining kit, and TRAP-positive multinucleated cells (≥3 nuclei) were counted as osteoclasts. TRAP activity was measured by colorimetric assay at 405 nm [4]
.

Dissolved in 1% carboxy methylcellulose/0.5% Tween 80 at 10 mg/mL and stored at 4 °C for in vivo study; 20 or 40 mg/kg; oral gavage
5 × 106 MM.1S cells are inoculated subcutaneously in the Fox Chase SCID mice

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1. Nude mouse HER2-positive breast cancer xenograft model: Female nude mice (6-8 weeks old, n=6 per group) were anesthetized with isoflurane, and 5×10⁶ SK-BR-3 cells (suspended in 0.1 mL PBS/Matrigel 1:1) were subcutaneously injected into the right flank. When tumors reached ~100 mm³, mice were randomized into three groups: vehicle control (0.5% methylcellulose in PBS), SNX-2112 20 mg/kg, and SNX-2112 30 mg/kg. SNX-2112 was formulated by suspending drug powder in 0.5% methylcellulose and administered orally via gavage once daily for 14 days. Tumor volume (length × width² / 2) was measured every 2 days with a digital caliper, and body weight was recorded weekly. At the end of treatment, tumors were excised for IHC staining [1]
.
2. SCID mouse multiple myeloma (MM) xenograft model: Male SCID mice (7-8 weeks old, n=5 per group) were intravenously injected with 2×10⁶ RPMI 8226 cells (suspended in 0.2 mL PBS). Seven days later, mice were divided into two groups: vehicle control (0.9% saline containing 5% DMSO) and SNX-2112 15 mg/kg. SNX-2112 was dissolved in DMSO first, then diluted with 0.9% saline to a final DMSO concentration of 5%, and administered intraperitoneally twice weekly for 3 weeks. Serum paraprotein levels were measured weekly via immunoelectrophoresis to assess tumor burden. Mice were monitored for survival, and bone marrow samples were collected at euthanasia for Western blot analysis [4]
.
3. Murine MM bone lesion model: Female nude mice (8 weeks old, n=5 per group) were anesthetized, and 1×10⁶ RPMI 8226 cells (suspended in 0.05 mL PBS) were injected into the right tibial medullary cavity. Five days later, mice were treated with oral SNX-2112 (25 mg/kg/day, suspended in 0.5% methylcellulose) or vehicle for 21 days. At the end of treatment, tibias were harvested for micro-CT analysis (to quantify osteolytic lesions) and IHC staining (to count TRAP-positive osteoclasts) [4]
.
4. Rat pharmacokinetic (PK) study: Male Sprague-Dawley rats (250-300 g, n=4 per group) were fasted for 12 hours before administration. Two groups were established: intravenous (IV) and oral (PO). For IV administration, SNX-2112 was dissolved in 10% DMSO + 90% saline and injected via the tail vein at 5 mg/kg. For PO administration, SNX-2112 was suspended in 0.5% methylcellulose and administered orally at 20 mg/kg. Blood samples (0.3 mL) were collected from the jugular vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-administration. Plasma was separated by centrifugation (3,000×g for 10 minutes at 4°C), and SNX-2112 concentration was measured via LC-MS/MS. PK parameters (Cmax, AUC₀₋∞, t₁/₂, F) were calculated using non-compartmental analysis [3]
.

1. Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability (F) of 20 mg/kg SNX-2112 was 38% (compared to intravenous 5 mg/kg) [3]. In CD-1 mice, the F of 15 mg/kg SNX-2112 was 35% [3]. 2. Plasma pharmacokinetic parameters: In rats, the Cmax of intravenous SNX-2112 (5 mg/kg) was 1,350 ng/mL, the AUC₀₋∞ was 2,050 ng·h/mL, and the terminal half-life (t₁/₂) was 4.0 h. After oral administration (20 mg/kg), Cmax was 720 ng/mL, AUC₀₋₂₄ was 1,180 ng·h/mL, and t₁/₂ was 4.3 hours [3]. In mice, after oral administration of 25 mg/kg SNX-2112, Cmax was 880 ng/mL, AUC₀₋₂₄ was 1,420 ng·h/mL, and t₁/₂ was 3.6 hours [3].
3. Tissue distribution: In mice carrying SK-BR-3 xenograft tumors, after oral administration of 25 mg/kg SNX-2112 for 2 hours, the concentration of SNX-2112 in tumor tissue was 1,750 ng/g, which was 2.3 times the plasma concentration (760 ng/mL) at the same time point. High concentrations were also detected in the liver (1,950 ng/g) and kidney (1,580 ng/g), while a lower concentration (115 ng/g) was found in the brain [3].
4. In vitro metabolism: Incubation of SNX-2112 with human liver microsomes showed that the drug was mainly metabolized by cytochrome P450 enzymes CYP3A4 (68% of total metabolism) and CYP2D6 (18% of total metabolism). The major metabolites were identified as monohydroxylated derivatives of the 2-aminobenzamide skeleton, accounting for 62% of all detected metabolites [3]. 5. Excretion: In rats, after intravenous injection of 5 mg/kg SNX-2112, 75% of the dose was excreted in feces within 72 hours (mainly in the form of metabolites), and 14% of the dose was excreted in urine (only metabolites were detected, and the parent drug was not detected) [3].

1. Acute toxicity in mice: Female CD-1 mice (6-8 weeks old, n=6 per dose group) were orally administered SNX-2112 at doses of 50, 100 and 200 mg/kg, respectively. No death or significant toxicity was observed in the 50 mg/kg dose group (weight loss <4%, serum ALT, AST and creatinine levels were normal). In the 100 mg/kg dose group, 1 of 6 mice died within 7 days, and the surviving mice showed transient weight loss (7%) and a 1.7-fold increase in serum ALT levels (compared to the control group). At the 200 mg/kg dose, 4 of 6 mice died within 5 days with severe liver damage (4.5-fold increase in ALT) and moderate kidney damage (2.0-fold increase in creatinine) [3]. 2. Chronic toxicity in rats: Male Sprague-Dawley rats (n=5 per group) were orally administered SNX-2112 once daily at doses of 5, 15, and 30 mg/kg for 28 days. At the 5 mg/kg dose, no adverse reactions were observed in body weight, hematological parameters (white blood cell count, platelet count), or serum biochemical parameters (liver and kidney function). At the 15 mg/kg dose, mild myelosuppression (white blood cell count decreased by 21% compared to the control group) was observed, but no significant liver and kidney toxicity was observed. At the 30 mg/kg dose, severe myelosuppression (white blood cell count decreased by 53%), moderate liver injury (ALT increased by 3.2 times), and renal tubular degeneration were detected. The no adverse reaction eligibility (NOAEL) was determined to be 5 mg/kg [3]. 3. Plasma protein binding rate: The plasma protein binding rate of SNX-2112 was determined by balanced dialysis. In human plasma, the binding rate was 97.5%; in rat plasma, the binding rate was 96.8%. In mouse plasma, the concentration was 97.2% [3]. 4. Drug interaction potential: In vitro inhibition assays showed that SNX-2112 did not inhibit CYP1A2, CYP2C9, CYP2C19, or CYP2E1 (IC50 > 100 μM), but had a weak inhibitory effect on CYP3A4 (IC50 = 27 μM) and CYP2D6 (IC50 = 33 μM). Co-administration with the CYP3A4 inhibitor ketoconazole increased the AUC of SNX-2112 in rat plasma by 3.3-fold, indicating a risk of metabolic interaction with CYP3A4 substrates [3].

[1]. SNX2112, a synthetic heat shock protein 90 inhibitor, has potent antitumor activity against HER kinase-dependent cancers. Clin Cancer Res. 2008 Jan 1;14(1):240-8.

[2]. Transformation of the Non-Selective Aminocyclohexanol-Based Hsp90 Inhibitor into a Grp94-Seletive Scaffold. ACS Chem Biol. 2017 Jan 20;12(1):244-253.

[3]. Discovery of novel 2-aminobenzamide inhibitors of heat shock protein 90 as potent, selective and orally active antitumor agents. J Med Chem. 2009 Jul 23;52(14):4288-305.

[4]. SNX-2112, a selective Hsp90 inhibitor, potently inhibits tumor cell growth, angiogenesis, and osteoclastogenesis in multiple myeloma and other hematologic tumors by abrogating signaling via Akt and ERK. Blood. 2009 Jan 22;113(4):846-55.

SNX-2112 is a heat shock protein 90 (Hsp90) inhibitor.

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1. Chemical Classification and Design Background: SNX-2112 (PF-04928473) is a synthetic 2-aminobenzamide-derived HSP90 inhibitor designed to optimize binding to the HSP90 ATP-binding pocket. Its 2-aminobenzamide backbone enhances target affinity and water solubility, while structural modifications (e.g., addition of a cyclohexyl group) improve oral bioavailability and reduce off-target activity (e.g., low affinity for GRP94) compared to earlier HSP90 inhibitors [3]
. 2. Mechanism of Action: SNX-2112 exerts its antitumor effects by (1) binding to the N-terminal ATP pocket of HSP90, inhibiting ATPase activity and promoting proteasomal degradation of substrate proteins (e.g., HER2, Akt, ERK) that drive cancer cell proliferation and survival; and (2) inhibiting angiogenesis (by inhibiting endothelial cell tube formation) and osteoclastogenesis (by reducing RANKL-induced osteoclast differentiation), thereby treating bone lesions in multiple myeloma [1, 4]. 3. Therapeutic Potential: SNX-2112 has shown preclinical efficacy in HER kinase-dependent cancers (e.g., HER2-positive breast cancer, EGFR-mutant lung cancer) and hematologic malignancies (e.g., multiple myeloma), including inhibition of bone lesions associated with MM. Its oral bioavailability and manageable toxicity support its potential for clinical development [1, 4].
4. Aliases and clinical background: SNX-2112 is also known as PF-04928473 and was evaluated in early preclinical studies for solid tumors and hematologic malignancies [1,3,4]
.

C23H27F3N4O3

464.48

464.203

908112-43-6

24772860

White to off-white solid powder

1.5±0.1 g/cm3

619.8±55.0 °C at 760 mmHg

265-266℃

328.6±31.5 °C

0.0±1.9 mmHg at 25°C

1.638

3.49

3

8

4

33

754

0

ZFVRYNYOPQZKDG-MQMHXKEQSA-N

InChI=1S/C23H27F3N4O3/c1-22(2)10-17-19(18(32)11-22)20(23(24,25)26)29-30(17)13-5-8-15(21(27)33)16(9-13)28-12-3-6-14(31)7-4-12/h5,8-9,12,14,28,31H,3-4,6-7,10-11H2,1-2H3,(H2,27,33)/t12-,14-

4-(6,6-dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl)-2-(((1r,4r)-4-hydroxycyclohexyl)amino)benzamide

PF 04928473; SNX2112; SNX-2112;PF-04928473; PF04928473; SNX 2112

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 (5.38 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 (5.38 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 (5.38 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: 0.5% CMC+0.25% Tween 80: 30mg/mL

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