VEGFR1 (IC50 = 33 nM); VEGFR2 (IC50 = 35 nM); VEGFR3 (IC50 = 0.5 nM)

Fruquintinib has a good pharmacokinetic profile across a variety of animal species. In mice, oral fruquintinib administration significantly reduced VEGF-induced VEGFR2 phosphorylation in the lung tissue. There was a strong correlation between drug exposure and the degree and duration of the inhibition of VEGFR2 phosphorylation. In several human tumor xenograft models, the potent anti-angiogenic effect led to strong anti-tumor efficacy with good dose response[1].

---

Fruquintinib potently inhibits VEGF induced KDR phosphorylation in lung tissue of mouse [1]
Fruquintinib inhibited KDR signaling in HUVEC and HEK293-KDR cells in vitro. To confirm such effect and establish PK/PD relationship in vivo, VEGF-A induced KDR phosphorylation in the lung tissue in mice was evaluated following oral administration of fruquintinib. As demonstrated in Figure 4A, after a single oral dose of fruquintinib at 2.5 mg/kg, VEGF stimulated KDR phosphorylation was completely suppressed and the effect sustained for at least 8 hours whereas the p-KDR level recovered at 16 hours after dose. Plasma samples were collected at 1, 4, 8, 16 and 24 hours post dosing to determine the concentrations of fruquintinib (Fig. 4B). Fruquintinib reached Cmax at 1 hour and accordingly achieved maximum level of inhibition of p-KDR, taking the p-KDR level below the basal level. At 8 hours the p-KDR inhibition still maintained at 86%, and the corresponding fruquintinib concentration in plasma was 176 ng/mL. Consistent with lack of inhibition of KDR phosphorylation in the lung tissue at 16 hours, the plasma concentration of fruquintinib was found to be below 10 ng/mL. At 24 hours, the plasma concentration of fruquintinib was below the low limit of quantification. These results demonstrated that the p-KDR inhibition in lung directly correlated with drug exposures in plasma and 176 ng/mL of fruquintinib in plasma could achieve greater than 80% target inhibition in lung tissue.

---

Fruquintinib inhibits tumor growth in multiple human xenograft models [1]
Anti-tumor activity of fruquintinib was evaluated in a variety of tumor xenografts along with measurements of plasma drug concentrations in an attempt to establish target inhibition-tumor growth inhibition relationship (Fig. 5A–5D and Table 2). The results from gastric cancer BGC-823 model seemed to indicate that the drug concentration needs to be at least maintained above EC85 (drug concentration required to inhibit KDR phosphorylation by 85%) for around 8 hours in order to achieve >80% tumor growth inhibition (clinically stable disease), and the longer target covering duration to achieve tumor regression (clinically partial response) (Fig. 5A–C). BGC-823 was found to be most sensitive to fruquintinib. In this model, fruquintinib inhibited tumor growth by 62.3% and 95.4∼98.6%, at 0.5 and 2 mg/kg once daily dosing, respectively (Fig. 5A and B, Table 2). When the dose was elevated to 5 mg/kg and 20 mg/kg, the tumors regressed by 24.1% and 48.6%, respectively (Fig. 5B, Table 2) presumably due to longer duration of target inhibition (Fig. 5C, plasma concentration of 5 and 20 mg/kg appeared to cover EC85 for about 12 and 18 hours, respectively). The level of anti-tumor growth activity of fruquintinib varied in different tumor xenograft models. In some cases, activation of tumor growth signaling pathways may play a role. For instance, in renal cell cancer Caki-1 xenograft model, fruquintinib at 2 mg/kg dose only produced a moderate TGI of 51.5%, comparing to almost complete tumor growth inhibition observed in BCG-823 model at this dose. (Fig. 5D, Table 2). Further analysis revealed that significant c-Met activation is present in Caki-1 cells. Rational combination in such tumors may provide optimal therapeutic effect. In fact, combination treatment of fruquintinib with a c-Met inhibitor in Caki-1 xenograft model produced significant synergy and led to complete tumor growth suppression.
To further confirm anti-angiogenic effect of fruquintinib in vivo, an endothelial cell surface marker CD31 (PECAM-1) in the Caki-1 tumor tissue was measured by immunohistochemistry (IHC) method after treatment with fruquintinib for 3 weeks. The results are shown in Figure 5D and E. A clear dose-dependent anti-angiogenic effect was observed in the Caki-1 tumor tissue, and the inhibition rate was 73.0%, 53.5% and 25.6% at 5, 2, and 0.8 mg/kg, respectively. Comparing to the vehicle-treated group, fruquintinib significantly decreased the micro-vessel density even at the lowest dose of 0.8 mg/kg (P < 0.05). These results suggested that fruquintinib inhibited tumor growth through inhibition of tumor angiogenesis.
Anti-tumor efficacy of fruquintinib combining with chemotherapeutic agents was also investigated in PDX models. As shown in Figure 6A, combination of fruquintinib with Taxotere (docetaxel) showed significantly enhanced anti-tumor effect in gastric cancer GAS1T0113P5, with a TGI of 73%, comparing to 44% and 46% when treated with fruquintinib and docetaxel alone, respectively. Similarly, the enhanced inhibitory effect of fruquintinib with oxaliplatin was seen in the colon cancer PDX model COL1T0117P4 (Fig. 6B). It was worth noting that fruquintinib did not increase animal body weight loss caused by docetaxel or oxalipatin in combinational use (Table 2).

Biochemical assays [1]
For human kinase assay in HMP, all recombinant kinases were purchased commercially. The kinase activity was determined by Z’-lyte or Transcreener fluorescence polarization according to manufacturers’ instruction. Fruquintinib selectivity was further assessed at 1 μmol/L against a panel of 253 kinases using [32p-ATP] incorporation method. The UBI protocols are available at www.millipore.com/drugdiscovery/ KinaseProfiler.

---

Fruquintinib suppresses VEGF/VEGFR cell signaling in human umbilical vein endothelial cells (HUVEC) and human lymphatic endothial cells (HLEC) with an IC50 at low nanomolar level in in vitro enzymatic and cellular assays. Out of the 253 kinases tested, only a small number of them, besides VEGFRs, are inhibited. Fruquintinib is a highly potent inhibitor of angiogenesis induced by VEGF.

In flat-bottomed 96-well plates, 100 mL of media containing 0.5% foetal bovine serum (FBS) was used to seed primary HUVEC cells at a density of 2 × 10⁴ cells/well. The following day, Fruquintinib was applied to the cells for eighteen hours at 37 degrees Celsius. The AlamarBlue assay was used to assess cell survival. After three hours of incubation at 37 C, the fluorescence value of the plates was measured on Tecan at Ex 530 nm and Em 590 nm.

---

Cell proliferation assay [1]
Primary HUVECs or HLECs in exponential phase were suspended in 100 μL of RPMI-1640 media containing 0.5% FBS, and seeded at 5 × 103 cell/well in 96-well plates pre-coated with 0.2% gelatin or fibronectin, and incubated overnight in a 5% CO2, 37°C incubator. Fruquintinib and VEGF-A165 or VEGF-C (50 ng/mL) were added and incubated for 48 hours. Viability of the cells was determined using CCK-8 assay format.

---

HUVEC cytotoxicity assay [1]
Primary HUVEC cells at 2 × 104 cells/well were seeded in flat bottomed 96-well plates with 100 μL media containing 0.5% FBS. The next day, cells were treated with Fruquintinib for 18 hours at 37°C. The cell survival was determined by AlamarBlue assay. The plates were incubated for 3 hours at 37°C and fluorescence value was read at Ex 530 nm and Em 590 nm on Tecan.

---

HUVEC Tube formation [1]
Flat bottomed 96-well plates were pre-coated with 70 μL of basement membrane matrix for 30 minutes at 37°C to form gelling. Primary HUVECs in exponential phase were seeded at 2 × 104 cells/well in 100 μL RPMI-1640 media containing 0.5% FBS. The cells, with or without Fruquintinib treatment, were incubated in a 5% CO2, 37°C incubator for 18 hours. The result was recorded by photographing under a microscope with 40× magnification. The total length of tubes in the presence of the compound was compared to that in the absence of the compound by using Image-Pro Plus software, and the inhibition rate was calculated based on the total tube length (TTL) under the microscope using the formula below: Inhibition rate (%) = (1 − TTL of compound treatment/TTL of control) ×100%.

---

Chick embryo chorioallantoic membrane (CAM) assay [1]
Fertilized chicken eggs with 6 day incubation were used. Forty eggs were divided into 4 groups and incubated at 37°C with 50% humidity for 24 hours. On the following day, a small window (1 × 1 cm2) was made in the shell under aseptic conditions. The slides loaded with 10 μL of physiological saline containing various concentrations of Fruquintinib were placed on the top of the growing CAMs. Pseudolarix acid B (PAB) was applied as a positive control. The window was resealed with an adhesive tape and the eggs were returned to the incubator. Upon 48 hours of incubation, the CAMs were photographed using an Olympus Live View Digital camera.

Mice: The xenograft models derived from patients are created subsequent to the primary tumor undergoing multiple in vivo passages. When tumors reach a size of 100–300 mm³, the animals are divided into groups of 6–8 at random. For three weeks, the mice are given either the vehicle (treated group) or a single daily dose of fruquintinib (0.5–20 mg/kg) suspended in the vehicle (control group). In combination studies, intravenous injections of either oxaliplatin (10 mg/kg) or docetaxel (Taxotere, 5 mg/kg) are given once a week to nude mice. Three times a week, body weight and tumor size are measured. TVs, or tumor volumes, are computed.

---

In vivo target inhibition assay (p-KDR inhibition) [1]
Female Balb/c nude mice at the age of 10∼11 weeks were treated with a single oral dose of fruquintinib at 2.5 mg/kg suspended in 0.5% aqueous CMC-Na. At 1, 4, 8, 16 and 24 hours post dose, the plasma and lung samples were harvested for fruquintinib exposure and p-KDR analyses, respectively. Each group of designated time points was composed of 3 animals. Recombinant mouse VEGF was intravenously injected to the animals at the dose of 0.5 μg/mouse in the study groups, while animals in the control group received the same volume of PBS instead. All animals were anaesthetized with CO2 and sacrificed 5 minutes after VEGF injection. The left lobes of the harvested lungs were lysed to determine p-KDR and β-actin by Western blots. The p-KDR and β-actin bands were visualized with Odyssey Infrared Imaging system and quantified with QuantityOne4.31 software. The inhibitory effect of fruquintinib was evaluated by quantification of normalized p-KDR signal over β-actin of the fruquintinib-treated groups relative to that derived from VEGF stimulated vehicle-treated group.

---

In vivo anti-tumor efficacy assessment in human tumor xenograft models [1]
Human gastric cancer BGC-823 cell line was obtained from Shanghai Institutes for Biological Sciences in China. Large cell lung cancer NCI-H460, colon cancer HT-29, and clear cell renal cancer Caki-1 cell lines were purchased from ATCC. The tumor cells, at a density of 1∼5 × 106 cells/mouse, were subcutaneously inoculated to the right flanks of nude mice. The patient derived xenograft models were established after the primary tumor adopted serial passages in vivo. Once tumors have grown to 100∼300 mm3, the animals were randomly assigned with 6∼8 animals per group. The mice were treated orally with the vehicle (control group) or fruquintinib at a dose range of 0.5∼20 mg/kg suspended in the vehicle (treated group) once daily for 3 weeks. In combination studies, docetaxel (Taxotere, 5 mg/kg) or oxaliplatin (10 mg/kg) was administered to nude mouse via intravenous injection, once a week. Tumor size and body weights were measured 3 times a week. Tumor volumes (TV) were calculated using the formula: TV = (length × [width]2)/2. The percentage of tumor growth inhibition (%TGI = 100 × [1− (TV final - TVinitial for drug treated group)/(TVfinal – TVinitial for the control group)]) was used for evaluation of anti-tumor efficacy.

---

In vivo anti-angiogenesis analysis [1]
At the end of Caki-1 anti-tumor efficacy study, the subcutaneous tumors from control- and fruquintinib-treated groups were collected and fixed in Zinc-formalin, and paraffin embedded sections were prepared. Immunohistochemistry staining of CD31 was analyzed. In each section, 5 non-necrosis areas were randomly chosen and CD31 positive area was determined by NIKON BR-3.0 software. The anti-angiogenic effect of fruquintinib was evaluated by CD31 inhibition calculated with the formula: Inhibition% = 100 × [1− (CD31 positive area/tumor area for compound treated group)/(CD31 positive area/tumor area for the control group)].

Absorption, Distribution and Excretion
The fruquintinib steady-state geometric mean (% coefficient of variation [CV]) maximum concentration (Cmax) is 300 ng/mL (28%) and the area under the concentration-time curve for the dosing interval (AUC0-24h) is 5880 ng∙h/mL (29%) at the recommended dosage. The fruquintinib Cmax and AUC0-24h are dose-proportional across the dosage range of 1 to 6 mg (0.2 to 1.2 times the recommended dosage). Fruquintinib steady state is achieved after 14 days with a mean AUC0-24h accumulation of 4-fold. The fruquintinib median (min, max) time to Cmax is approximately 2 hours (0, 26 hours). No clinically significant differences in fruquintinib pharmacokinetics were observed following administration of a high-fat meal (800 to 1000 calories, 50% fat).
Following oral administration of a 5 mg radiolabeled fruquintinib dose, approximately 60% of the dose was recovered in urine (0.5% unchanged) and 30% of the dose was recovered in feces (5% unchanged).
The mean (SD) apparent volume of distribution of fruquintinib is approximately 46 (13) L.
The apparent clearance (SD) of fruquintinib is 14.8 (4.4) mL/min.

---

Metabolism / Metabolites
Fruquintinib is primarily eliminated by CYP450 and non-CYP450 (i.e., sulfation and glucuronidation) metabolism. CYP3A and to a lesser extent CYP2C8, CYP2C9, and CYP2C19 are the CYP450 enzymes involved in fruquintinib's metabolism.

---

Biological Half-Life
The fruquintinib's mean (SD) elimination half-life is approximately 42 (11) hours.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of fruquintinib during breastfeeding. Because fruquintinib is 95% bound to plasma proteins, the amount in milk is likely to be low. However, the manufacturer recommends that breastfeeding be discontinued during fruquintinib therapy and for 2 weeks after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

---

Protein Binding
The plasma protein binding of fruquintinib is approximately 95%.

[1]. Discovery of fruquintinib, a potent and highly selective small molecule inhibitor of VEGFR 1, 2, 3 tyrosine kinases for cancer therapy. Cancer Biol Ther. 2014;15(12):1635-45.

Fruquintinib is a member of the class of quinazolines that is quinazoline substituted by [2-methyl-3-(methylcarbamoyl)-1-benzofuran-6-yl]oxy, methoxy, and methoxy groups at positions 4, 6, and 7, respectively. It is a highly potent and selective inhibitor of VEGFR 1, 2 and 3 (IC50 = 33, 0.35, and 35 nM) used for the treatment of metastatic colorectal cancer. It has a role as an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor, an antineoplastic agent and a vascular endothelial growth factor receptor antagonist. It is a member of quinazolines, an aromatic ether, a member of 1-benzofurans and a secondary carboxamide.
Fruquintinib is a novel small-molecule anti-VEGFR that targets VEGFR-1,-2, and -3 to inhibit angiogenesis. Tumor angiogenesis is one of the most critical biological processes for increasing oxygen and nutrient supply to cancer cells, and the VEGF/VEGFR pathway is one of the most critical pathways for this phenomenon. Indeed, oncogenic activation, loss of tumor suppressor function, and hypoxia, usually facilitated by cancer cells, are known to upregulate VEGF. There are 2 major approaches to combatting tumor angiogenesis: neutralization of VEGF/VEGFR activity through monoclonal antibodies or blockage of VEGFR kinase activity through small-molecule inhibitors. The first approach can be exemplified by [bevacizumab], a VEGF-A trap antibody. Although [bevacizumab] is successful in sustaining target inhibition, mandatory intravenous dosing, immunogenicity, and the potential to induce autoimmune diseases hinder its clinical application. For the small-molecule approach, most earlier generations of VEGFR inhibitors such as [sunitinib], [sorafenib], [regorafenib], and [pazopanib] have poor selectivity, thus increasing the risk of off-target toxicity. Therefore, the advent of fruquintinib, a new generation of VEGFR inhibitors with a high kinome selectivity, demonstrated the feasibility of the small-molecule inhibitor approach. On November 8th, 2023, fruquintinib was approved by the FDA under the brand name Fruzaqla for the treatment of adult patients with metastatic colorectal cancer (mCRC) who received prior fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy, an anti-VEGF therapy, and, if RAS wild-type and medically appropriate, an anti-EGFR therapy. This approval is based on favorable results obtained from the FRESCO and FRESCO-2 trials, where an increase in overall survival rate was observed in both trials.
Fruquintinib is an orally available, small molecule inhibitor of vascular endothelial growth factor receptors (VEGFRs), with potential anti-angiogenic and antineoplastic activities. Upon oral administration, fruquintinib inhibits VEGF-induced phosphorylation of VEGFRs 1, 2, and 3 which may result in the inhibition of migration, proliferation and survival of endothelial cells, microvessel formation, the inhibition of tumor cell proliferation, and tumor cell death. Expression of VEGFRs may be upregulated in a variety of tumor cell types.

---

Drug Indication
Fruquintinib is indicated for the treatment of adult patients with metastatic colorectal cancer (mCRC) who have been previously treated with fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy, an anti-VEGF therapy, and, if RAS wild-type and medically appropriate, an anti-EGFR therapy.
Treatment of colorectal carcinoma

---

Mechanism of Action
Fruquintinib is a small-molecule kinase inhibitor of vascular endothelial growth factor receptors (VEGFR)-1, -2, and -3 with IC₅₀ values of 33, 35, and 0.5 nM, respectively

---

Pharmacodynamics
In vitro studies showed fruquintinib inhibited VEGF-mediated endothelial cell proliferation and tubular formation, while in vivo studies demonstrated fruquintinib-mediated tumor growth inhibition in a tumor xenograft mouse model of colon cancer. Inhibition of VEGF-induced VEGFR-2 phosphorylation was illustrated in both in vitro and in vivo studies. Fruquintinib exposure-response relationships and the time course of pharmacodynamic response are unknown. A mean increase in QTc interval >20 milliseconds (ms) was not observed at the approved recommended dosage.

---

VEGF/VEGFR signal axis has been proven to be an important target for development of novel cancer therapies. One challenging aspect in small molecular VEGFR inhibitors is to achieve sustained target inhibition at tolerable doses previously seen only with the long-acting biologics. It would require high potency (low effective drug concentrations) and sufficient drug exposures at tolerated doses so that the drug concentration can be maintained above effective drug concentration for target inhibition within the dosing period. Fruquintinib (HMPL-013) is a small molecule inhibitor with strong potency and high selectivity against VEGFR family currently in Phase II clinical studies. Analysis of Phase I pharmacokinetic data revealed that at the maximum tolerated dose of once daily oral administration fruquintinib achieved complete VEGFR2 suppression (drug concentrations were maintained above that required to produce >85% inhibition of VEGFR2 phosphorylation in mouse) for 24 hours/day. In this article, the preclinical data for fruquintinib will be described, including kinase enzyme activity and selectivity, cellular VEGFR inhibition and VEGFR-driven functional activity, in vivo VEGFR phosphorylation inhibition in the lung tissue in mouse and tumor growth inhibition in a panel of tumor xenograft and patient derive xenograft models in mouse. Pharmacokinetic and target inhibition data are also presented to provide a correlation between target inhibition and tumor growth inhibition. [1]

---

The potent in vitro activity against VEGFR of fruquintinib was confirmed in vivo following oral administration. Fruquintinib was found to inhibit VEGF stimulated VEGFR2 phosphorylation (p-KDR) in lung tissue of mice in an exposure dependent manner. A single oral dose of fruquintinib at 2.5 mg/kg in mice resulted in near complete (>85%) inhibition of p-KDR and the inhibition was maintained for at least 8 hours in the lung tissue. At that time point the corresponding fruquintinib plasma concentration was determined to be 176 ng/mL (Effective Concentration for 85% target inhibition, or EC85). In anti-tumor efficacy studies, at the dose of around 2 mg/kg given orally once daily, statistically significant tumor growth inhibition was achieved in a variety of human tumor xenograft models in mice, indicating that maintaining complete suppression of VEGFR2 pathway approximately 8 hours or longer could produce significant anti-tumor efficacy, although it would be preferable to sustain the complete target inhibition for 24 hours/day similar to antibodies to achieve the maximum anti-tumor activity. The EC85 (176 ng/mL for fruquintinib) is useful for estimating the duration of target inhibition at the recommended doses in clinical trials. According to pharmacokinetic data in phase I clinical trial, the plasma exposures of fruquintinib at the maximum tolerated dose achieved the EC85 concentration for 24 hours per day with once daily dosing schedule, indicating that fruquintinib potentially could introduce continuous target inhibition in cancer patients. In summary, fruquintinib is a potent, highly selective and orally active inhibitor of VEGFR1, 2, 3 tyrosine kinases. Fruquintinib inhibited VEGF-induced VEGFR2 phosphorylation, endothelial cell proliferation, and tubule formation in vitro and VEGFR2 phosphorylation in lung tissue. Fruquintinib demonstrated potent tumor growth inhibition activity in a panel of human tumor xenografts in mice. Enhanced tumor growth inhibition was observed in several PDX models when fruquintinib was combined with chemotherapeutic agents. The equally potent inhibitory activity against VEGFR2 and VEGFR3 of fruquintinib could potentially offer strong anti-tumor growth and metastasis benefits. These attractive attributes coupled with favorable pharmacokinetic properties and toxicity profile make fruquintinib a promising candidate for clinical investigation. [1]

C21H19N3O5

393.39

393.132

C, 64.12; H, 4.87; N, 10.68; O, 20.34

1194506-26-7

44480399

White to off-white solid powder

1.3±0.1 g/cm3

600.5±55.0 °C at 760 mmHg

317.0±31.5 °C

0.0±1.7 mmHg at 25°C

1.639

3.08

1

7

5

29

579

0

O1C(C([H])([H])[H])=C(C(N([H])C([H])([H])[H])=O)C2C([H])=C([H])C(=C([H])C1=2)OC1C2=C([H])C(=C(C([H])=C2N=C([H])N=1)OC([H])([H])[H])OC([H])([H])[H]

BALLNEJQLSTPIO-UHFFFAOYSA-N

InChI=1S/C21H19N3O5/c1-11-19(20(25)22-2)13-6-5-12(7-16(13)28-11)29-21-14-8-17(26-3)18(27-4)9-15(14)23-10-24-21/h5-10H,1-4H3,(H,22,25)

6-(6,7-dimethoxyquinazolin-4-yl)oxy-N,2-dimethyl-1-benzofuran-3-carboxamide

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: ≥ 0.59 mg/mL (1.50 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 5.9 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 0.59 mg/mL (1.50 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 5.9 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.

---

View More

Solubility in Formulation 3: ≥ 0.59 mg/mL (1.50 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 5.9 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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