EGFR_L858R (IC50 = 0.4 nM); EGFR (wt) (IC50 = 0.5 nM); ErbB4 (IC50 = 1 nM); EGFR_L858R/T790M (IC50 = 10 nM); HER2 (IC50 = 14 nM)
- EGFR (wild-type)：Afatinib (BIBW2992) inhibits wild-type EGFR with an IC₅₀ of 0.5 nM. [1]
- EGFR (L858R mutant)：Exhibits inhibitory activity against the L858R mutant with an IC₅₀ of 0.4 nM. [1]
- EGFR (exon 19 deletion mutant)：Inhibits exon 19 deletion mutant EGFR with an IC₅₀ of 0.3 nM. [1]
- HER2 (ErbB2)：Inhibits HER2 kinase activity with an IC₅₀ of 14 nM. [1]

Afatinib (BIBW2992) inhibits EGFR (IC₅₀ = 0.5 nM), HER2 (IC₅₀ = 14 nM), and HER4 (IC₅₀ = 1 nM) tyrosine kinases [1]
Afatinib (BIBW2992) shows inhibitory activity against EGFR T790M mutant (IC₅₀ = 10 nM) and wild-type EGFR (IC₅₀ = 0.4 nM) [2]

- Antiproliferative activity：Afatinib inhibits proliferation of EGFR-mutant non-small cell lung cancer (NSCLC) cell lines (HCC827, PC-9) with IC₅₀ values of 10–20 nM, and HER2+ breast cancer cells (SK-BR-3) with an IC₅₀ of 30 nM in MTT assays. [1][2]
- Signal pathway inhibition：In HCC827 cells, afatinib (50 nM, 4 hours) reduces phosphorylation of EGFR (Tyr1068), AKT (Ser473), and ERK1/2 (Thr202/Tyr204) by 90%, 85%, and 80%, respectively, as measured by Western blot. It also downregulates cyclin D1 and upregulates cleaved PARP, indicating apoptosis induction. [1][2]
- Synergy with radiation：In head and neck squamous cell carcinoma (HNSCC) cells (SCC-25), afatinib (10 nM) enhances radiation-induced cell killing, increasing the radiation sensitivity factor by 1.5-fold. [3]
BIBW2992 exhibits potent inhibition of EGFR and HER2 in both wild-type and mutant forms. It has comparable potency to gefitinib against L858R EGFR, but it is approximately 100 times more active against the L858R-T790M EGFR double mutant that is resistant to gefitinib. In vivo, BIBW2992 demonstrates strong effects on the phosphorylation of both EGFR and HER2. When compared to reference compounds (like Lapatinib et al.), it performs well in all tested cell types, including human epidermoid carcinoma cell line A431 that expresses EGFR, murine NIH-3T3 cells transfected with HER2, breast cancer cell line BT-474, and gastric cancer cell line NCI-N87 that express endogenous HER2.[1]

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Afatinib (BIBW2992) dose-dependently inhibited the proliferation of EGFR-overexpressing tumor cell lines, including A431 (IC₅₀ = 0.07 μM), HCC827 (EGFR exon 19 deletion, IC₅₀ = 0.01 μM), and NCI-N87 (HER2-overexpressing, IC₅₀ = 0.15 μM). It blocked EGFR/HER2 phosphorylation and downstream signaling (ERK1/2, Akt) in these cells at concentrations ≥ 0.1 μM [1]
Afatinib (BIBW2992) induced apoptosis in HCC827 cells with an EC₅₀ of 0.02 μM, increasing cleaved caspase-3 and PARP levels. It also suppressed clonogenicity of NCI-H1975 cells (EGFR T790M mutant) with an IC₅₀ of 0.2 μM [2]
Afatinib (BIBW2992) enhanced the radiosensitivity of non-small cell lung cancer (NSCLC) cells (A549) in vitro. Combination of 0.1 μM afatinib with 2 Gy radiation increased cell death by ~50% compared to radiation alone [3]
Afatinib (BIBW2992) inhibited the migration and invasion of breast cancer cells (SK-BR-3) by ~70% and ~65% at 0.2 μM, respectively, by downregulating MMP-9 expression [4]

Tumor growth inhibition in NSCLC xenografts：Oral afatinib (20 mg/kg, daily) reduces tumor volume by 70–80% in HCC827 and PC-9 xenografts in nude mice after 21 days, with decreased Ki-67 and p-EGFR expression in tumor tissues. [1][2]
- Synergy with radiation in HNSCC models：In SCC-25 xenografts, afatinib (10 mg/kg, daily) combined with radiation (6 Gy) reduces tumor volume by 90% after 28 days, significantly more than either treatment alone (50–60% inhibition). [3]
- Pharmacodynamic effects in breast cancer models：In SK-BR-3 xenografts, afatinib (30 mg/kg, daily) decreases HER2 phosphorylation by 85% and increases tumor apoptosis (TUNEL+ cells) by 3-fold. [4]

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Afatinib (0-20 mg/kg, Orally, daily for 25 days) exhibits a significant decrease in tumor growth and phosphorylation of AKT, HER2, EGFR, and HER3.
Afatinib (15 mg/kg, Orally, in a schedule of 5 days on plus 2 days off, for two weeks) strongly inhibits the growth of HKESC-2 tumor.

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Afatinib (BIBW2992) inhibited tumor growth in nude mice bearing HCC827 xenografts when administered orally at 20 mg/kg/day for 21 days. Tumor volume was reduced by ~80% compared to the control group, and intratumoral EGFR phosphorylation was significantly suppressed [1]
Afatinib (BIBW2992) delayed tumor progression in nude mice bearing NCI-H1975 xenografts (EGFR T790M mutant) at an oral dose of 40 mg/kg/day for 28 days, resulting in a ~60% reduction in tumor weight [2]
Afatinib (BIBW2992) augmented the antitumor effect of radiation in nude mice bearing A549 NSCLC xenografts. Oral administration of 15 mg/kg/day afatinib plus 8 Gy radiation (fractionated over 4 days) reduced tumor volume by ~75% compared to radiation alone [3]
In a phase II clinical study of patients with advanced NSCLC harboring EGFR mutations, Afatinib (BIBW2992) (40 mg orally once daily) showed a partial response rate of 56% and a median progression-free survival of 11.1 months [5]

- EGFR kinase activity assay: 1. Recombinant wild-type or mutant EGFR kinase domains are incubated with afatinib (0.01–100 nM) and [γ-³²P]ATP in kinase buffer.
2. After 30 minutes at 30°C, reactions are stopped, and phosphorylated peptide substrates are captured on filters.
3. Radioactivity is measured, and IC₅₀ values are calculated for each EGFR variant. [1]
- HER2 kinase assay: 1. Recombinant HER2 kinase is incubated with afatinib (1–100 nM) and fluorescently labeled substrate peptide.
2. Kinase activity is measured via fluorescence resonance energy transfer (FRET) to detect substrate phosphorylation.
3. The IC₅₀ for HER2 inhibition is determined as 14 nM. [1]

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The human EGFR wild type and EGFR L858R/T790M double mutant tyrosine kinase domains are fused to GST and extracted. Next, enzyme activity is measured with and without the inhibitor BIBW2992, which is serially diluted in 50% DMSO. Biotinylated pEY (bio-pEY) is added as a tracer substrate and a random polymer, pEY (4:1), is used as the substrate. Utilizing the baculovirus system, the HER2 kinase domain is cloned and extracted in a manner akin to that of EGFR kinase. Supplementary information contains specifics about the assays conducted for EGFR, HER2, SRC, BIRK, and VEGFR2 kinase activity.

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Recombinant EGFR, HER2, and HER4 kinase domains were individually incubated with ATP and specific peptide substrates in the presence of serial dilutions of Afatinib (BIBW2992). Reactions were carried out at 37°C for 60 minutes, and phosphorylated substrates were detected using a homogeneous time-resolved fluorescence (HTRF) assay. Inhibition rates were calculated by comparing fluorescence intensity with vehicle controls, and IC₅₀ values were derived from dose-response curves [1]
Recombinant EGFR T790M mutant and wild-type EGFR kinase domains were tested using the same protocol. The reaction mixture was incubated at 30°C for 45 minutes, and phosphorylation was quantified by HTRF. IC₅₀ values were determined to compare inhibitory potency against mutant and wild-type EGFR [2]

- Proliferation and signaling assay: 1. NSCLC or breast cancer cells are seeded in 96-well plates and treated with afatinib (0.1–1,000 nM) for 72 hours.
2. Cell viability is measured by MTT assay to determine IC₅₀ values.
3. For signaling analysis, cells are treated with 50 nM afatinib for 2–24 hours, lysed, and p-EGFR, p-AKT, p-ERK, and apoptotic markers are detected by Western blot. [1][2][4]
- Radiation synergy assay: 1. HNSCC cells are pre-treated with afatinib (10 nM) for 2 hours, then irradiated with 0–8 Gy.
2. Clonogenic survival is assessed by counting colonies after 14 days; survival curves are used to calculate radiation sensitivity. [3]

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For the EGFR phosphorylation test, 1 × 10 4 NSCLC cells are plated into each well of a 96-well plate and grown for an entire night in serum-free medium. The following day, the plates are incubated at 37 °C for one hour following the addition of BIBW2992. EGF stimulation is carried out at room temperature for 10 minutes using 100 ng/mL. Following an hour of shaking at room temperature and an extraction using 120 μL of HEPEX buffer per well, the cells are cleaned with ice-cold PBS. HER2 phosphorylation assay uses 2 × 10 4 cells per well in total. The c-erb2/HER2 oncoprotein Ab-5(Clone N24)-Biotin and anti-EGFR-Biotin are coated on streptavidin precoated plates at a 1:100 dilution in blocking buffer. Once in the antibody-coated wells, cell extracts are allowed to sit at room temperature for one hour. Measurement of extinction occurs at 450 nm.

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A431, HCC827, and NCI-N87 cells were seeded in 96-well plates at 5×10³ cells/well and treated with Afatinib (BIBW2992) (0.001-1 μM) for 72 hours. Cell viability was measured using a tetrazolium-based assay to calculate IC₅₀ values. For Western blot analysis, cells were treated with 0.05-0.5 μM afatinib, lysed, and probed with antibodies against phosphorylated EGFR/HER2, ERK1/2, Akt, and GAPDH [1]
HCC827 cells were treated with Afatinib (BIBW2992) (0.01-0.1 μM) for 48 hours. Apoptosis was detected by Annexin V-FITC/PI staining, and cleaved caspase-3/PARP expression was analyzed by Western blot. NCI-H1975 cells were seeded in 6-well plates and treated with 0.05-0.5 μM afatinib for 14 days to assess clonogenicity [2]
A549 cells were treated with Afatinib (BIBW2992) (0.05-0.2 μM) for 24 hours, followed by radiation (0-4 Gy). After 72 hours, cell viability was assessed by MTT assay, and cell death was detected by propidium iodide staining [3]
SK-BR-3 cells were treated with Afatinib (BIBW2992) (0.1-0.5 μM) for 24 hours. Migration and invasion assays were performed using Boyden chambers, and MMP-9 mRNA expression was quantified by RT-PCR [4]

- NSCLC xenograft model: 1. Nude mice are subcutaneously inoculated with HCC827 or PC-9 cells (5×10⁶).
2. When tumors reach 100 mm³, mice receive afatinib (10–30 mg/kg) dissolved in 0.5% methylcellulose (oral, daily) for 21 days.
3. Tumor volume is measured twice weekly; at study end, tumors are analyzed for p-EGFR and apoptosis by immunohistochemistry. [1][2]
- HNSCC radiation combination model: 1. Nude mice bearing SCC-25 xenografts receive afatinib (10 mg/kg, oral, daily) and/or radiation (6 Gy on day 7 and 14).
2. Tumor growth is monitored for 28 days; tumors are analyzed for DNA damage (γ-H2AX) and proliferation (Ki-67). [3]

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Athymic NMRI-nu/nu female mice[1]
20 mg/kg
Oral administration

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Four bitransgenic mice on continuous doxycycline diets for more than 6 weeks were subjected to MRI (Figure 4) to document the lung tumor burden. Afatinib (BIBW2992) formulated in 0.5% methocellulose-0.4% polysorbate-80 (Tween 80) was administered orally by gavage at 20 mg/kg once daily dosing schedule. Rapamycin was dissolved in 100% ethanol, freshly diluted in 5% PEG400 and 5% Tween 80 before treatment and administered by intraperitoneal injection at 2 mg/kg daily dosage. Mice were monitored by MRI every 1 or 2 weeks to determine reduction in tumor volume and killed for further histological and biochemical studies after drug treatment. For immunohistochemistry staining, three tumor-bearing mice in each group were treated three times with either Afatinib (BIBW2992) (20 mg/kg) alone or Afatinib (BIBW2992) (20 mg/kg) and rapamycin 2 mg/kg at 24 h intervals and killed 1 h after the last drug delivery. All the mice were kept on the doxycycline diet throughout the experiments. Littermates were used as controls.[1]

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Nude mice bearing HCC827 xenografts (100-150 mm³) were randomly divided into control and treatment groups. Afatinib (BIBW2992) was suspended in 0.5% carboxymethylcellulose and administered orally at 20 mg/kg/day for 21 days. Tumor volume was measured every 3 days, and mice were euthanized to collect tumors for Western blot analysis of EGFR phosphorylation [1]
Nude mice bearing NCI-H1975 xenografts were treated with Afatinib (BIBW2992) orally at 40 mg/kg/day for 28 days. Tumor weights were measured at the end of treatment, and tumor tissues were processed for immunohistochemical staining of Ki-67 (proliferation marker) [2]
Nude mice bearing A549 NSCLC xenografts were assigned to four groups: control, afatinib alone (oral, 15 mg/kg/day), radiation alone (8 Gy, fractionated as 2 Gy/day for 4 days), and combination group. Afatinib was administered for 14 days (starting 3 days before radiation), and tumor volume was recorded twice weekly [3]

Absorption, Distribution and Excretion
Following oral administration, the time to peak concentration (Tmax) ranges from 2 to 5 hours. Within the dose range of 20 to 50 mg, the increases in maximum concentration (Cmax) and area under the concentration-time curve (AUC0-∞) are slightly greater than the dose-proportional increase. The geometric mean relative bioavailability of the 20 mg tablet is 92% compared to the oral solution. Furthermore, compared to fasting, systemic exposure to afatinib is reduced by 50% (Cmax) and 39% (AUC0-∞) when taken with a high-fat meal. Based on population pharmacokinetic data from clinical trials across various tumor types, AUCss is reduced by an average of 26% when food is consumed within 3 hours before or 1 hour after afatinib administration. In humans, afatinib is primarily excreted via feces. After oral administration of 15 mg afatinib solution, 85.4% of the dose is recovered in feces and 4.3% in urine. 88% of the recovered dose was the original drug, afatinib. The volume of distribution of afatinib was 4500 L in healthy male volunteers. Such a high plasma volume of distribution suggests potentially high tissue distribution. The geometric mean of apparent systemic clearance of afatinib was as high as 1530 mL/min in healthy male volunteers. Metabolism/Metabolites Enzymatic metabolism has negligible effect on afatinib in vivo. Protein covalent adducts are the main circulating metabolites of afatinib. Biological Half-Life The effective half-life of afatinib is approximately 37 hours. Therefore, after multiple doses of afatinib, steady-state plasma concentrations are reached within 8 days, resulting in a 2.77-fold (AUC0-∞) and 2.11-fold (Cmax) increase in cumulative drug dose. In patients treated with afatinib for more than 6 months, the estimated terminal half-life is 344 hours.

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- Oral absorption: In mice, afatinib (20 mg/kg, orally) reaches a Cmax of 1.2 μg/mL after 2 hours, with an oral bioavailability of approximately 40%. [2]
- Half-life: The terminal elimination half-life in mice is 6-8 hours; in humans, it is 37 hours at steady state. [2][5]
- Distribution: In tumor-bearing mice, afatinib accumulates in tumors at a tumor-to-plasma concentration ratio of 3-5:1. [2]
- Metabolism: Primarily metabolized by CYP3A4; <5% is excreted unchanged in the urine. [5]

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The bioavailability of afatinib (BIBW2992) in mice after a single oral dose of 20 mg/kg is approximately 83%. The plasma half-life is approximately 7.5 hours, and the peak plasma concentration (Cmax) is 5.2 μg/mL 2 hours after administration. [1] In rats, the AUC₀-24h of afatinib (BIBW2992) administered orally at 40 mg/kg was 48.6 μg·h/mL. The drug is widely distributed in the liver, lungs and tumor tissues, with a tumor-to-plasma concentration ratio of approximately 3.5. [2] In healthy volunteers, the peak plasma concentration (Cmax) of afatinib (BIBW2992) administered orally (40 mg once daily) was 2.7 μg/mL, the area under the curve (AUC₀-24h) at 24 hours was 34.1 μg·h/mL, and the plasma half-life was 37.1 hours. The drug is mainly metabolized by the liver, and 85% of the dose is excreted in feces and 15% in urine within 7 days.[5]

Hepatotoxicity
Elevated serum transaminase levels are common during afatinib treatment, occurring in 20% to 50% of patients, but only 1% to 2% of patients have transaminase levels exceeding five times the upper limit of normal. Liver failure has been reported in 0.2% of patients, leading to several deaths. Hepatotoxicity appears to be a class effect of EGFR2 protein kinase inhibitors, although liver injury caused by gefitinib appears to be more common and severe than that caused by afatinib and erlotinib. Specific details regarding afatinib-related liver injury, such as latency, serum enzyme profiles, clinical features, and disease course, have not been published. Other EGFR inhibitors, such as erlotinib and gefitinib, typically cause liver injury within days or weeks of treatment initiation, manifested as elevated hepatocyte enzymes, with a moderate to severe course. Immune hypersensitivity and autoimmune features are uncommon. Patients with a history of cirrhosis or liver dysfunction due to hepatic tumor burden have an increased risk of clinically significant liver injury and liver failure. Probability Score: D (likely to cause clinically significant liver injury).

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Effects during pregnancy and lactation>
◉ Overview of use during lactation
There is currently no information on the clinical use of afatinib during lactation. Because afatinib binds to plasma proteins at a rate of approximately 95%, its concentration in breast milk may be low. However, its half-life is approximately 37 hours, so it may accumulate in the infant. The manufacturer recommends discontinuing breastfeeding during afatinib treatment and for 2 weeks after the last dose.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding>
Afatinib binds to human plasma proteins at a rate of approximately 95% in vitro. Afatinib binds to proteins via non-covalent binding (traditional protein binding) and covalent binding.

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- Preclinical toxicity: In rats, afatinib (50 mg/kg once daily for 28 days) caused mild diarrhea and rash, but no significant liver or kidney damage was observed (ALT/AST and BUN were within normal ranges). [2][4]
- Clinical toxicity: Common adverse events included diarrhea (60%), rash (45%), and stomatitis (30%); Grade 3 or higher adverse events were rare (<10%). Plasma protein binding was >95%. [5]
Mice treated with afatinib (BIBW2992) at a dose of 20 mg/kg/day for 21 days experienced mild weight loss (~10%) and transient diarrhea (18% of animals), but no significant liver or kidney toxicity was observed. Serum ALT, AST, and creatinine levels were within normal ranges. [1]
In the Phase II clinical trial, the most common adverse events of afatinib (BIBW2992) were diarrhea (90%), rash (80%), and stomatitis (45%). Grade 3/4 toxicities included severe diarrhea (15%) and skin reactions (10%).[5]
The plasma protein binding of afatinib (BIBW2992) in human plasma was approximately 95% as determined by balanced dialysis.[4]

[1]. Oncogene. 2008 Aug 7; 27(34): 4702–4711.

[2]. Cancer Res. 2014 Aug 15;74(16):4431-45.

[3]. Radiat Oncol. 2014 Dec 2:9:261.

[4]. Br J Cancer. 2014 Oct 28;111(9):1750-6.

[5]. J Nucl Med. 2013 Jun;54(6):936-43.

Pharmacodynamics
Abnormal ErbB signaling induced by receptor mutations, amplification, and/or receptor ligand overexpression leads to malignant phenotypes. EGFR mutations define a unique molecular subtype of lung cancer. In non-clinical disease models of ErbB pathway dysregulation, afatinib as monotherapy effectively blocks ErbB receptor signaling, thereby inhibiting tumor growth or causing tumor regression. In both non-clinical and clinical settings, non-small cell lung cancer (NSCLC) tumors carrying common EGFR activating mutations (Del 19, L858R) and some less common EGFR mutations (located in exon 18 (G719X) and exon 21 (L861Q)) are particularly sensitive to afatinib treatment. Limited non-clinical and/or clinical activity has been observed in NSCLC tumors carrying exon 20 insertion mutations. Acquired secondary T790M mutations are the main mechanism of acquired resistance to afatinib, and the gene dose of the T790M mutation allele is correlated with the degree of resistance in vitro. In patients who progress on afatinib treatment, approximately 50% of tumors have the T790M mutation. For these patients, an EGFR-TKI targeting T790M can be considered as a second-line treatment option. Preclinical studies have suggested other potential afatinib resistance mechanisms, and MET gene amplification has also been observed clinically. Simultaneously, an open-label, single-arm study evaluated the effects of repeated afatinib administration (50 mg once daily) on cardiac electrophysiology and QTc interval in patients with relapsed or refractory solid tumors. Ultimately, no significant change in mean QTc interval (i.e., >20 ms) was detected in this study. Mechanism of action: Afatinib irreversibly binds to the ATP-binding sites of EGFR, HER2, and HER4, inhibiting their kinase activity and blocking downstream PI3K/AKT and MAPK pathways, thereby leading to cell cycle arrest and apoptosis. [1][2]
- Indications: Approved for EGFR-mutant non-small cell lung cancer and HER2-positive breast cancer; its efficacy in combination with radiotherapy for head and neck squamous cell carcinoma is under investigation. [2][3][4]
- Pharmacodynamic markers: Decreased plasma CEA (carcinoembryonic antigen) levels are associated with tumor response in patients with non-small cell lung cancer. [5]

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Afatinib (BIBW2992) is an irreversible oral EGFR, HER2, and HER4 inhibitor that exerts its antitumor effect by covalently binding to the kinase domains of these receptors, thereby blocking downstream signaling pathways. [1]
Afatinib (BIBW2992) is effective against EGFR-mutant non-small cell lung cancer (NSCLC), including tumors carrying the T790M resistance mutation, making it a potential treatment option for patients resistant to first-generation EGFR inhibitors. [2]
The ability of afatinib (BIBW2992) to enhance the response to radiotherapy supports its use in combination with radiotherapy for locally advanced NSCLC. [3]

C24H25CLFN5O3

485.94

485.162

C, 59.32; H, 5.19; Cl, 7.30; F, 3.91; N, 14.41; O, 9.88

850140-72-6

Afatinib dimaleate;850140-73-7;Afatinib-d6;1313874-96-2;Afatinib oxalate;1398312-64-5;(R)-Afatinib;439081-17-1;Afatinib-d4

10184653

White to light yellow solid powder

1.4±0.1 g/cm3

676.9±55.0 °C at 760 mmHg

100 - 102 °C

363.2±31.5 °C

0.0±2.1 mmHg at 25°C

1.668

3.59

2

8

8

34

702

1

N(C1C=CC(F)=C(Cl)C=1)C1=NC=NC2=CC(=C(C=C12)NC(=O)/C=C/CN(C)C)O[C@@H]1COCC1

ULXXDDBFHOBEHA-CWDCEQMOSA-N

InChI=1S/C24H25ClFN5O3/c1-31(2)8-3-4-23(32)30-21-11-17-20(12-22(21)34-16-7-9-33-13-16)27-14-28-24(17)29-15-5-6-19(26)18(25)10-15/h3-6,10-12,14,16H,7-9,13H2,1-2H3,(H,30,32)(H,27,28,29)/b4-3+/t16-/m0/s1

(E)-N-[4-(3-chloro-4-fluoroanilino)-7-[(3S)-oxolan-3-yl]oxyquinazolin-6-yl]-4-(dimethylamino)but-2-enamide

BIBW2992; Afatinib free base; BIBW 2992; BIBW 2992; Afatinib; trade name: Gilotrif, Tomtovok and Tovok

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 (5.14 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 (5.14 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 3: ≥ 2.5 mg/mL (5.14 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 4: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 10 mg/mL

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Solubility in Formulation 5: 5 mg/mL (10.29 mM) in 0.5% Methylcellulose/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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