EGFR (IC50 = 2 nM)

Erlotinib (CP-358774) is also a potent inhibitor, with an IC50 of 1 nM, of the EGFR's recombinant intracellular (kinase) domain. Erlotinib severely inhibits the proliferation of DiFi cells, with an IC50 of 100 nM for an 8-day proliferation assay[1]. When B-DIM and Erlotinib (2 μM) are combined, BxPC-3 cell colony formation is significantly inhibited as opposed to when either agent is used alone. Only in BxPC-3 cells does the combination of B-DIM and Erlotinib (2 μM) significantly induce apoptosis when compared to the apoptotic effect of either agent alone[2].

In comparison to the untreated control, the combination of B-DIM and Erlotinib (50 mg/kg, i.p.) treatment significantly (P <0.01) reduces tumor weight under experimental conditions[2]. In comparison to the CP+vehicle (V) rats, erlotinib (20 mg/kg, p.o.) significantly attenuates the body weight (BW) loss induced by Cisplatin (CP) (P<0.05). Treatment with erlotinib considerably enhances renal function in CP-N (normal control group, NC) rats. Compared to the CP+V rats, the CP+Erlotinib (E) rats exhibit a significant increase in urine volume (UV) (P<0.05) and Cr clearance (Ccr) (P<0.05), as well as a significant decrease in serum creatinine (s-Cr) (P<0.05), blood urea nitrogen (BUN) (P<0.05), and urinary N-acetyl-β-D-glucosaminidase (NAG) index (P<0.05).

The kinase reaction takes place in 50 μL of 50 mM HEPES (pH 7.3), which also contains 15 ng of affinity-purified EGFR from A431 cell membranes, 1.6 μg/mL EGF, 0.1 mM Na₃VO₄, 125 mM NaCl, and 20 μM ATP. To achieve a final DMSO concentration of 2.5%, the compound is added to DMSO. The addition of ATP starts the phosphorylation process, which lasts for 8 mm at room temperature while being constantly shaken. The reaction mixture is aspirated to stop the kinase reaction, and wash buffer is used four times over. Phosphorylated PGT is quantified after 25 microseconds of incubation with 50 microliters of HRP-conjugated PY54 antiphosphotyrosine antibody per well, diluted to 0.2 micrograms/mL in blocking buffer (PBS containing 3% BSA and 0.05% Tween 20). After aspirating out the antibody, the plate is cleaned four times using wash buffer. TMB Microwell Peroxidase Substrate, 50 μL per well, is added to develop the colonmetric signal.0.09 M sulfuric acid, 50 μL per well, is added to stop the signal. The absorbance at 450 nm is used to estimate phosphotyrosine. The signal for controls is proportional to the incubation time for 10 mm and usually ranges from 0.6 to 1.2 absorbance units[1]. In wells devoid of AlP, EGFR, or POT, there is essentially no background signal.

In order to assess the survival of cells treated with B-DIM, Erlotinib, or both, 3,000–5,000 BxPC-3 and MIAPaCa cells are plated per well in a 96-well plate and incubated at 37°C for the entire night. Initially, B-DIM (10-50 µM) and Erlotinib (1-5 µM) are tested at a range of concentrations. The concentrations of B-DIM (20 µM) and Erlotinib (2 µM) are selected for each assay based on the preliminary findings. The standard MTT assay is used to measure the effects of B-DIM (20 µM), Erlotinib (2 µM), and the combination on BxPC-3 and MIAPaCa cells. The assay is performed three times after 72 hours. The Tecan microplate fluorometer measures the color intensity at 595 nm. Cells treated with DMSO are given a value of 100% and are regarded as the untreated control. We have performed clonogenic assay in addition to the aforementioned assay to evaluate the effects of treatment[2].

Mice: The treatment groups consist of seven randomly assigned female ICR-SCID mice, aged 6-7 weeks: (a) control (no treatment); (b) B-DIM (50 mg/kg body weight) administered intragastrically once daily; (c) Erlotinib (50 mg/kg body weight) administered daily intraperitoneally for 15 days; and (d) B-DIM and Erlotinib administered according to the schedule for individual treatments. After receiving their last dose of medication, all mice are killed on day three, and their body weight is recorded. A portion of the tissue is immediately frozen in liquid nitrogen and kept cold (−70°C) for later use, while the remaining portion is fixed in formalin and prepared for paraffin block processing. The presence of a tumor or tumors in each pancreas is verified by staining a fixed tissue section with H&E. Rats: Male Sprague-Dawley (SD) rats six weeks of age, weighing 180–210 g, are utilized. On day 0, SD rats (n=28) receive an intraperitoneal injection of 7 mg/kg of freshly prepared ciprofloxacin (CP) at a concentration of 1 mg/mL. For the purpose of examining Erlotinib's effects, 28 CP-N rats are split into two groups. Animals in two groups (n = 14) are given daily oral gavages of either erlotinib (20 mg/kg) (CP+E, n = 14) or vehicle (CP+V, n = 14) from day -1 (24 hours before the CP injection) to day 3. Groups treated with vehicles are given the same amount of saline. At six weeks of age, a normal control group (NC, n = 5) consists of five male SD rats. From the first to the third day, the NC rats receive an equivalent volume of saline orally via gavage. Day 4 (96 hours post-CP injection): rats are anesthetized, and following a cardiac puncture, they are sacrificed by exsanguination. The kidneys and blood are simultaneously extracted. Renal tissue is sectioned and fixed in 2% paraformaldehyde/phosphate-buffered saline (PBS) for later use, or it can be snap-frozen in liquid nitrogen. In order to reduce suffering as much as possible, diethyl ether gas anesthesia is used during all surgical procedures.

[1]. Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase. Cancer Res. 1997, 57(21), 4838-4848.

[2]. Apoptosis-inducing effect of erlotinib is potentiated by 3,3'-diindolylmethane in vitro and in vivo using an orthotopic model of pancreatic cancer. Mol Cancer Ther, 2008, 7(6), 1708-1719.

[3]. Epidermal growth factor receptor inhibition with erlotinib partially prevents cisplatin-induced nephrotoxicity in rats. PLoS One. 2014 Nov 12;9(11):e111728.

C22H23N3O4

393.44

393.17

C, 67.16; H, 5.89; N, 10.68; O, 16.27

183321-74-6

Erlotinib Hydrochloride;183319-69-9;Erlotinib mesylate;248594-19-6;Erlotinib-d6;1034651-23-4;Erlotinib-13C6;1211107-68-4

White to off-white crystalline powder

COCCOC1=C(C=C2C(=C1)C(=NC=N2)NC3=CC=CC(=C3)C#C)OCCOC

AAKJLRGGTJKAMG-UHFFFAOYSA-N

InChI=1S/C22H23N3O4/c1-4-16-6-5-7-17(12-16)25-22-18-13-20(28-10-8-26-2)21(29-11-9-27-3)14-19(18)23-15-24-22/h1,5-7,12-15H,8-11H2,2-3H3,(H,23,24,25)

N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine

Erlotinib free base; NSC718781; NSC718781; CP358774; OSI-774; OSI 774; NSC 718781; CP-358,774; CP-358774; OSI774

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: 10 mg/mL (25.42 mM) in 50% PEG300 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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: 10 mg/mL (25.42 mM) in 0.5% CMC-Na/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.)