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].

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The epidermal growth factor receptor (EGFR) is overexpressed in a significant percentage of carcinomas and contributes to the malignant phenotype. CP-358,774 is a directly acting inhibitor of human EGFR tyrosine kinase with an IC50 of 2 nM and reduces EGFR autophosphorylation in intact tumor cells with an IC50 of 20 nM. This inhibition is selective for EGFR tyrosine kinase relative to other tyrosine kinases we have examined, both in assays of isolated kinases and whole cells. At doses of 100 mg/kg, CP-358,774 completely prevents EGF-induced autophosphorylation of EGFR in human HN5 tumors growing as xenografts in athymic mice and of the hepatic EGFR of the treated mice. CP-358,774 inhibits the proliferation of DiFi human colon tumor cells at submicromolar concentrations in cell culture and blocks cell cycle progression at the G1 phase. This inhibitor produces a marked accumulation of retinoblastoma protein in its underphosphorylated form and accumulation of p27KIP1 in DiFi cells, which may contribute to the cell cycle block. Inhibition of the EGFR also triggers apoptosis in these cells as determined by formation of DNA fragments and other criteria. These results indicate that CP-358,774 has potential for the treatment of tumors that are dependent on the EGFR pathway for proliferation or survival. [1]

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Effects of B-DIM and Erlotinib on the Viability of Pancreatic Cancer Cells [2]
It is important to note that, during our pilot studies, as indicated in Materials and Methods, different concentrations of B-DIM and erlotinib were used and are presented in Table 1. Moreover, after analyzing the basal level of expression of EGFR, NF-κB, and COX-2, we chose two cell lines having constitutively activated levels of NF-κB, EGFR, and COX-2 expression (BxPC-3) compared with lower level of NF-κB, EGFR, and COX-2 expression (MIAPaCa). Our results prompted us to select the subsequent concentration of B-DIM and erlotinib as presented below. Cell viability of BxPC-3 and MIAPaCa pancreatic cancer cells treated with B-DIM (20 µmol/L), erlotinib (2 µmol/L), and the combination was determined by the MTT assay, and the data are presented in Fig. 1A and B. Significant inhibition of cell viability was seen in BxPC-3 cells treated with either agent, and this was further enhanced by the combination treatment (P = 0.0001). In addition, we have also tested the effects of treatment on cell viability by clonogenic assay as shown below. Similar treatments of MIAPaCa cells resulted in a significant inhibition of viable cells with B-DIM alone but not when exposed to similar concentrations of B-DIM and erlotinib for the same time, and the effect was not enhanced by the combination treatment (P = 0.0890). The insensitivity of MIAPaCa cells to erlotinib is consistent with a recently published report

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Inhibition of Cell Growth/Survival by Clonogenic Assay [2]
To determine the effect of B-DIM and Erlotinib on cell growth, cells were treated with each of the single agents or their combination and assessed for cell viability by clonogenic assay. The combination of B-DIM and erlotinib resulted in a significant inhibition of colony formation in BxPC-3 cells when compared with either agent alone (Fig. 2A and B). Similar treatment of MIAPaCa cells (Fig. 2C) showed inhibition of colony formation with B-DIM alone and also the combination, but the effect was not enhanced with the combination treatment as was seen in BxPC-3 cells (Fig. 2A and B). These results were similar to those obtained from the soft-agar assay. Overall, the results from clonogenic assay was consistent with the MTT data as shown in Fig. 1A and B, suggesting that B-DIM had a differential effect between BxPC-3 and MIAPaCa pancreatic cancer cells. The mechanisms of such differences were further investigated, and the results are presented in the following sections, but first we have determined the effects of B-DIM, erlotinib, and the combination on apoptotic cell death.

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Induction of Apoptosis by Erlotinib, B-DIM, and the Combination [2]
The underlying mechanism on the inhibition of cell viability was further studied by determining the apoptotic effects of different treatments using the Cell Death Detection ELISA. The combination of B-DIM and erlotinib resulted in a significant induction of apoptosis only in BxPC-3 cells when compared with the apoptotic effect of either agent alone (Fig. 1C). Similar treatment of MIAPaCa cells showed no induction of apoptosis with the combination (Fig. 1D). These results are consistent with cell viability assay by MTT. Subsequently, we sought to find further evidence of apoptosis as presented below.

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B-DIM Enhances Apoptosis Signaling by Erlotinib [2]
PARP cleavage was determined in BxPC-3 and MIAPaCa cells that were treated with B-DIM (20 µmol/L), erlotinib (2 µmol/L), and the combination (Fig. 3). We found significant amount of PARP (116 kDa) protein cleavage product (85 kDa fragment) after 72-h treatment only in BxPC-3 cells (Fig. 3). In contrast, MIAPaCa cells treated similarly showed only a small cleavage of PARP with B-DIM alone and also in combination but not with erlotinib alone. The induction of apoptosis could be partly due to inactivation of important survival genes; hence, we investigated whether B-DIM, erlotinib, and their combination could affect key survival proteins.

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Effect of B-DIM on Molecules Related to Apoptosis [2]
BxPC-3 and MIAPaCa cells were used to evaluate the effects of B-DIM and/or Erlotinib on the expression of survivin, Bcl-2, Bcl-xL, and c-IAP1/2. Expression of Bcl-2, Bcl-xL, survivin, and c-IAP1/2 proteins was significantly reduced in cells treated with the combination when compared with either agent alone (Fig. 3). There was no influence on antiapoptotic proteins in MIAPaCa cells treated with either agent alone or the combination. These results suggest that B-DIM, erlotinib, and the combination down-regulate key survival proteins and in turn induced apoptotic cell death in BxPC-3 cells but not in MIAPaCa cells. To further determine the molecular mechanism by which B-DIM sensitized BxPC-3 cells to erlotinib-induced inhibition of cell viability and induction of apoptosis, we investigated the role of EGFR and its downstream signaling pathways.

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Effect of B-DIM on the Expression of EGFR Protein [2]
The expression of EGFR was determined by immunoblotting. No baseline expression of EGFR was found in the MIAPaCa cells. EGFR-expressing BxPC-3 cells showed a significant reduction in the expression of EGFR and phosphorylated EGFR levels when exposed to erlotinib plus B-DIM compared with either agent alone (Fig. 3). It is known that the activation of EGFR could in turn regulate an important transcription factor, NF-κB, which is a known regulator of several survival genes such as survivin, c-IAP1/2, Bcl-2, and Bcl-xL. Because we found a greater degree of down-regulation of survivin, c-IAP1/2, Bcl-2, and Bcl-xL in BxPC-3 cells treated with B-DIM and erlotinib compared with either agent alone, and because these genes are transcriptionally regulated by NF-κB, we investigated the effect of each treatment on the DNA-binding activity of NF-κB.

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B-DIM Inhibits NF-κBDNA-Binding Activity [2]
The activation of NF-κB, a nuclear transcriptional factor, was assessed in B-DIM-treated and Erlotinib-treated cells. There was a significant inhibition of NF-κB activation in BxPC-3 cells exposed to both erlotinib and B-DIM compared with erlotinib alone (Fig. 4A). No such inhibition was shown in the MIAPaCa cells (Fig. 4B). These results suggest that the combination of B-DIM and erlotinib causes greater inhibition of cell growth, induction of apoptosis, inhibition of survival factors, inhibition of EGFR, and inactivation of NF-κB.

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Because NF-κB plays important roles in the regulation of prosurvival and antiapoptotic processes, we tested whether overexpression of NF-κB by p65 cDNA transfection could abrogate B-DIM-induced and erlotinib-induced apoptotic processes. Moreover, it is known that NF-κB transcriptionally regulates COX-2, which produces PGE2 and in turn induces cell viability. Thus, we tested whether celecoxib, erlotinib, or B-DIM alone could influence the activity of B-DIM and erlotinib in p65 cDNA transfected cells.

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Erlotinib, B-DIM, and Celecoxib Abrogated Activation of NF-κBActivity Stimulated by p65 cDNA Transfection [2]
Cytoplasmic and nuclear proteins from BxPC-3 and MIAPaCa cells transfected with p65 cDNA and then treated with erlotinib (2 µmol/L), B-DIM (20 µmol/L), or celecoxib (5 µmol/L) or left untreated for 48 h were subjected to analysis for NF-κB activity as measured by Western blot analysis and EMSA. The results showed that erlotinib, B-DIM, and celecoxib inhibited the p65 protein and NF-κB DNA-binding activity more in BxPC-3 cells compared with untreated cells (Fig. 5A and B) and very little effect was seen in MIAPaCa cells. Importantly, NF-κB p65 cDNA transfection enhanced the NF-κB p65 protein and DNA-binding activity only in BxPC-3 cells to a significant level as shown in Fig. 5A and B. On the other hand, no such changes were observed in the MIAPaCa cells. Because the activation of NF-κB induces COX-2 expression leading to the production of PGE2 that is released into the culture medium, we measured the levels of PGE2 in untransfected and transfected cells treated with erlotinib, B-DIM, and the COX-2 inhibitor celecoxib.

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Inhibition of PGE2 Synthesis in p65 cDNA-Transfected Cells [2]
We measured the levels of PGE2 in the conditioned medium collected from both BxPC-3 and MIAPaCa cells as an indicator of COX-2 activity. We found a high level of PGE2 secretion by BxPC-3 cells, whereas MIAPaCa cells showed very low levels of PGE2, which is consistent with its low constitutive expression of COX-2. BxPC-3 and MIAPaCa cells were transfected with p65 cDNA followed by treatment with Erlotinib (10 nmol/L), B-DIM (1 µmol/L), or celecoxib (1 nmol/L) to analyze the levels of PGE2 released into the culture medium (Fig. 5C). No change in PGE2 level was noted when cells were treated with erlotinib alone (P = 0.084). However, a significant reduction in PGE2 level was observed in BxPC-3 cells treated with B-DIM (P = 0.006) and celecoxib (P = 0.005). There was a substantial increase in the PGE2 level in p65 cDNA-transfected BxPC-3 cells compared with untransfected cells (P = 0.009), suggesting that NF-κB could induce COX-2 expression. However, there was no change in PGE2 level in MIAPaCa cells with any of the agents. Collectively, these results suggest that the production of PGE2 is mediated through the NF-κB and COX-2 pathway and that celecoxib could down-regulate both NF-κB and COX-2. These results were subsequently correlated with the degree of apoptosis (Fig. 5D) as presented below.

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Apoptosis through the Inactivation of NF-κB in p65 cDNA-Transfected Cells [1]
p65 cDNA was transfected into BxPC-3 and MIAPaCa cells and then treated with Erlotinib (2 µmol/L), B-DIM (20 µmol/L), or celecoxib (5 µmol/L) for 48 h (Fig. 5D). The degree of apoptosis in p65 cDNA-transfected BxPC-3 cells treated with erlotinib (P = 0.034) was much less compared with untransfected cells treated with erlotinib (P = 0.007). Similar results were observed with both B-DIM and celecoxib treatment in BxPC-3 cells. However, in MIAPaCa cells, no such degree of apoptosis was observed. These results suggest that activation of NF-κB by p65 cDNA transfection could abrogate the apoptosis inducing effect of erlotinib, B-DIM, and celecoxib.

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).

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B-DIM Augments In vivo Therapeutic Effect of Erlotinib on Primary Tumor [2]
Potential therapeutic utility of B-DIM and erlotinib combination in SCID mice bearing orthotopically implanted BxPC-3 pancreatic tumor cells was investigated. A dose of 3.5 mg/d B-DIM per mouse was selected for p.o. administration, whereas erlotinib dose (50 mg/kg body weight i.p.) was based on previously published reports as shown in Fig. 6A. A total of 28 mice were divided into four groups. To ascertain the efficacy of a single-agent treatment compared with combinations, we determined the mean pancreas weight in all treated groups. Under our experimental conditions, administration of B-DIM by gavage treatment and erlotinib alone caused 20% and 35% reduction in tumor weight, respectively, compared with control tumors (Fig. 6C). However, under the experimental conditions, the combination of B-DIM and erlotinib treatment showed significant decrease (P < 0.01) in tumor weight compared with untreated control, B-DIM alone, or erlotinib alone treatment group. These results showed, for the first time, the efficacy of combination of B-DIM and erlotinib in the inhibition of pancreatic tumor growth in an orthotopic model.

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B-DIM Inhibits NF-κBDNA-Binding Activity In vivo [2]
The activation of NF-κB was assessed in B-DIM-treated and Erlotinib-treated tumor tissues. The results show that NF-κB was down-regulated by B-DIM and erlotinib (Fig. 6B). Fig. 6B (bottom) represents results from all seven mice. These in vivo results were similar to our in vitro findings, suggesting that the inactivation of NF-κB is, at least, one of the molecular mechanisms by which B-DIM potentiates erlotinib-induced antitumor activity in our experimental animal model.

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The effects of blocking the epidermal growth factor receptor (EGFR) in acute kidney injury (AKI) are controversial. Here we investigated the renoprotective effect of Erlotinib, a selective tyrosine kinase inhibitor that can block EGFR activity, on cisplatin (CP)-induced AKI. Groups of animals were given either Erlotinib or vehicle from one day before up to Day 3 following induction of CP-nephrotoxicity (CP-N). In addition, we analyzed the effects of erlotinib on signaling pathways involved in CP-N by using human renal proximal tubular cells (HK-2). Compared to controls, rats treated with erlotinib exhibited significant improvement of renal function and attenuation of tubulointerstitial injury, and reduced the number of apoptotic and proliferating cells. Erlotinib-treated rats had a significant reduction of renal cortical mRNA for profibrogenic genes. The Bax/Bcl-2 mRNA and protein ratios were significantly reduced by erlotinib treatment. In vitro, we observed that erlotinib significantly reduced the phosphorylation of MEK1 and Akt, processes that were induced by CP in HK-2. Taken together, these data indicate that erlotinib has renoprotective properties that are likely mediated through decreases in the apoptosis and proliferation of tubular cells, effects that reflect inhibition of downstream signaling pathways of EGFR. These results suggest that erlotinib may be useful for preventing AKI in patients receiving CP chemotherapy[3].

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].

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Cell Viability Assay [2]
To test the viability of cells treated with B-DIM, Erlotinib, or the combination, BxPC-3 and MIAPaCa cells were plated (3,000–5,000 per well) in a 96-well plate and incubated overnight at 37°C. We initially tested a range of concentrations for both B-DIM (10–50 µmol/L) and erlotinib (1–5 µmol/L). Based on the initial results, the concentration of B-DIM (20 µmol/L) and erlotinib (2 µmol/L) were chosen for all assays. The effects of B-DIM (20 µmol/L), erlotinib (2 µmol/L), and the combination on BxPC-3 and MIAPaCa cells were determined by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 72 h and was repeated three times. The color intensity was measured by a Tecan microplate fluorometer at 595 nm. DMSO-treated cells were considered to be the untreated control and assigned a value of 100%. In addition to the above assay, we have also done clonogenic assay for assessing the effects of treatment as shown below.

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Clonogenic Assay [2]
To test the survival of cells treated with B-DIM, Erlotinib, or the combination, BxPC-3 and MIAPaCa cells were plated (50,000–100,000 per well) in a six-well plate and incubated overnight at 37°C. After 72-h exposure to 20 µmol/L B-DIM, 2 µmol/L erlotinib, and the combination, the cells were trypsinized, and the viable cells were counted (trypan blue exclusion) and plated in 100 mm Petri dishes in a range of 100 to 1,000 cells to determine the plating efficiency as well as assess the effects of treatment on clonogenic survival. The cells were then incubated for ~10 to 12 days at 37°C in a 5% CO2/5% O2/90% N2 incubator. The colonies were stained with 2% crystal violet and counted. The surviving fraction was normalized to untreated control cells with respect to clonogenic efficiency, which was 83% for both BxPC-3 and MIAPaCa cells. In addition to this assay, cells were also treated similarly and plated in soft-agar (soft-agar colony assay) and incubated at 37°C. The colonies in the soft agar were also counted in all untreated and treated wells after 12 days.

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Quantification of Apoptosis by ELISA [2]
The Cell Death Detection ELISA kit (Roche Applied Science) was used to detect apoptosis in untreated and treated BxPC-3 and MIAPaCa cells. Cells seeded in six-well plates were treated with B-DIM (20 µmol/L), Erlotinib (2 µmol/L), or the combination. The cells were trypsinized and ~10,000 cells were used as described earlier. Tecan microplate fluorometer was used to measure color intensity at 405 nm. The experiment was repeated three times.

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Protein Extraction and Western Blot Analysis [2]
BxPC-3 and MIAPaCa cells treated with B-DIM (20 µmol/L), Erlotinib (2 µmol/L), or the combination for 72 h were used to evaluate the effects of treatment on survivin, Bcl-2, Bcl-xL, EGFR, EGFR-pTyr1173, c-IAP1/2, Src, poly(ADP-ribose) polymerase (PARP), and β-actin expression. The experiment was carried out for a minimum of three times. Cells were harvested as described previously. The samples were loaded on 7% to 12% SDSPAGE for separation and electrophoretically transferred to a nitrocellulose membrane. Each membrane was incubated with monoclonal antibody against survivin, Bcl-2, Bcl-xL, Src, c-IAP1/2, EGFR, EGFR-pTyr1173, PARP, and β-actin. Blots were incubated with secondary antibodies conjugated with peroxidase. The signal intensity was then measured using chemiluminescent detection system.

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Electrophoretic Mobility Shift Assay for NF-κB Activation [2]
To evaluate the effect of B-DIM and Erlotinib on BxPC-3 and MIAPaCa cells, the cells were either untreated or treated with B-DIM (20 µmol/L), erlotinib (2 µmol/L), or the combination with a minimum repeat of experiment at least three times for 72 h. The cells or the minced tumor tissue were homogenized using a Dounce homogenizer in 400 µL ice-cold lysis buffer as described earlier.

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.

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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.

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Mice were randomized into the following treatment groups (n = 7): (a) untreated control; (b) only B-DIM (50 mg/kg body weight), intragastric once every day; (c) Erlotinib (50 mg/kg body weight), everyday i.p. for 15 days; and (d) B-DIM and Erlotinib, following schedule as for individual treatments. All mice were killed on day 3 following last dose of treatment, and their body weight was determined. One part of the tissue was rapidly frozen in liquid nitrogen and stored at −70°C for future use and the other part was fixed in formalin and processed for paraffin block. H&E staining of fixed tissue section was used to confirm the presence of tumor(s) in each pancreas. [2]

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Cisplatin (CP) was freshly prepared in saline at a concentration of 1 mg ml−1 and then injected intraperitoneally in SD rats (n = 28) at a dose of 7 mg/kg on day 0. The dose of CP was selected based on a previous stud. To investigate the effect of Erlotinib, 28 CP-N rats were divided into two groups. Separate groups (n = 14) each of animals were administered with either Erlotinib (20 mg/kg, Cugai Pharmaceutical/F. Hoffmann-La Roche, Basel, Switzerland) (CP+E, n = 14) or vehicle (CP+V, n = 14) daily by oral gavage from day -1 (24 hours prior to the CP injection) to day 3. Vehicle-treated groups received an equivalent volume of saline. Five male SD rats at the age of 6 weeks were used as a normal control group (NC, n = 5). The NC rats were given an equivalent volume of saline daily by oral gavage from day -1 to day 3. At day 4 (96 hours after CP injection), each rat was anesthetized and sacrificed by exsanguination after the cardiac puncture; blood was collected by cardiac puncture and kidneys were collected (Figure 1). Renal tissue was divided; separate portions were snap-frozen in liquid nitrogen or fixed in 2% paraformaldehyde/phosphate-buffered saline (PBS) for later use. All surgery was performed under diethyl ether gas anesthesia, and all efforts were made to minimize suffering.

Absorption, Distribution and Excretion
Erlotinib is absorbed approximately 60% after oral administration; food significantly increases its bioavailability to near 100%. Peak plasma concentrations occur 4 hours after administration. Erlotinib solubility is affected by pH; solubility decreases with increasing pH. Smoking also reduces erlotinib exposure. After oral administration of 100 mg, 91% of the dose is recovered, with 83% excreted in feces (1% of the dose in its original form) and 8% excreted in urine (0.3% of the dose in its original form). Apparent volume of distribution = 232 L. Smokers have 24% higher erlotinib clearance. Erlotinib is absorbed approximately 60% after oral administration; food significantly increases its bioavailability to near 100%. Peak plasma concentrations are reached 4 hours after administration. Erlotinib solubility is affected by pH; higher pH results in lower solubility. After absorption, approximately 93% of erlotinib binds to plasma albumin and α1-acid glycoprotein. The apparent volume of distribution of erlotinib is 232 liters. The time required to reach steady-state plasma concentrations is 7–8 days. No significant correlation was observed between clearance and covariates such as patient age, weight, or sex. Erlotinib clearance was 24% higher in smokers.
After oral administration of 100 mg, 91% of the dose is recovered: 83% is excreted in feces (1% of the intact parent drug dose) and 8% is excreted in urine (0.3% of the intact parent drug dose).
For more complete data on absorption, distribution, and excretion of erlotinib (of 10 parameters), please visit the HSDB record page.
Metabolism/Metabolites
Metabolism occurs in the liver. In vitro cytochrome P450 metabolism assays showed that erlotinib is primarily metabolized by CYP3A4, followed by CYP1A2 and the extrahepatic isoenzyme CYP1A1. This study was conducted in healthy male volunteers to investigate the metabolism and excretion of the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. Following a single oral dose of 14C-erlotinib hydrochloride (equivalent to 100 mg of free base, approximately 91 μCi/subject), unmetabolized erlotinib was detected in plasma. The main circulating component was unchanged erlotinib, while the pharmacologically active metabolite M14 accounted for approximately 5% of the total circulating radioactivity. The three main biotransformation pathways of erlotinib were: O-demethylation of the side chain followed by oxidation to carboxylic acid M11 (29.4% of the dose); partial oxidation of acetylene to carboxylic acid M6 (21.0%); and hydroxylation of the aromatic ring to M16 (9.6%). In addition, O-demethylation of M6 to generate M2, O-demethylation of the side chain to generate M13 and M14, and the binding of oxidative metabolites with glucuronic acid (M3, M8, and M18) and sulfate (M9) play minor roles in the metabolism of erlotinib. Identified metabolites account for more than 90% of the total radioactivity recovered in urine and feces. Metabolites observed in humans are similar to those found in toxic animals (rats and dogs). Known metabolites of erlotinib include erlotinib M14. The median half-life is 36.2 hours. A population pharmacokinetic analysis of 591 patients receiving erlotinib hydrochloride monotherapy as second/third-line therapy showed a median half-life of 36.2 hours.

Hepatotoxicity
Elevated serum transaminase levels are common during erlotinib treatment for pancreatic and lung cancer, with at least 10% of patients experiencing ALT levels exceeding five times the upper limit of normal. However, similar ALT elevations can occur with similar antitumor regimens. These abnormalities are usually asymptomatic and resolve spontaneously, but may require dose adjustment or discontinuation (Case 1). In addition, a few cases have been reported as clinically significant liver injury with erlotinib treatment. Liver injury typically occurs within days or weeks of treatment initiation and can be severe, with at least 12 deaths reported in the literature. Liver injury can occur abruptly, and the pattern of serum enzyme elevation is usually hepatocellular (Case 2). Immune allergic reactions (rash, fever, and eosinophilia) are uncommon, and autoantibody formation has not been reported. Routine monitoring of liver function is recommended during treatment. 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: B (Possible but uncommon, a cause of 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 erlotinib during lactation. Because erlotinib binds to plasma proteins at a rate of up to 93%, its concentration in breast milk may be low. However, its half-life is approximately 36 hours, which may allow it to accumulate in the infant. Furthermore, erlotinib is used in combination with gemcitabine to treat pancreatic cancer, which may increase the risk to the infant. The manufacturer recommends discontinuing breastfeeding during erlotinib treatment and for two weeks after the last dose.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found. Protein Binding 93% binds to albumin and α-1 acid glycoprotein (AAG). Interactions Since smoking reduces systemic exposure to erlotinib, patients should be advised to quit smoking. If a patient continues to smoke, an increase in the erlotinib dose may be considered; after quitting smoking, the erlotinib dose should be immediately reduced to the starting dose level. Drugs that increase upper gastrointestinal pH reduce the solubility and bioavailability of erlotinib. 1 Concomitant use with the proton pump inhibitor omeprazole reduced the area under the concentration-time curve (AUC) of erlotinib by 46% and the maximum concentration by 61%. Increasing the erlotinib dose is unlikely to compensate for the loss of exposure, and because the effect of proton pump inhibitors on upper gastrointestinal pH is prolonged, intermittent dosing may not eliminate this interaction. If possible, concomitant use of erlotinib with proton pump inhibitors should be avoided. For patients taking erlotinib, antacids may be considered as an alternative to histamine H2 receptor antagonists or proton pump inhibitors. However, the effects of antacids on the metabolism of erlotinib in vivo have not been studied. If antacids must be used, they should be taken several hours apart from erlotinib. Potential drug interactions (elevated international normalized ratio [INR], and rare reports of bleeding, including gastrointestinal and non-gastrointestinal bleeding). For patients taking warfarin or other coumarin anticoagulants, prothrombin time (PT) or INR should be monitored regularly. For more complete data on erlotinib interactions (7 items in total), please visit the HSDB record page.

[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.

Therapeutic Uses
Erlotinib hydrochloride monotherapy is indicated for maintenance therapy in patients with locally advanced or metastatic non-small cell lung cancer whose disease has not progressed after four cycles of first-line platinum-based chemotherapy. /US Product Label/ Erlotinib hydrochloride monotherapy is indicated for the treatment of patients with locally advanced or metastatic non-small cell lung cancer who have failed at least one chemotherapy regimen. /US Product Label/ Erlotinib hydrochloride in combination with gemcitabine is indicated for first-line treatment of patients with locally advanced, unresectable, or metastatic pancreatic cancer. /US Product Label/ Drug Warnings The manufacturer states that there are currently no known contraindications to the use of erlotinib. Serious, sometimes fatal, interstitial lung disease-like events have occurred in patients treated with erlotinib. In controlled and uncontrolled studies, approximately 0.7% of approximately 4,900 patients treated with erlotinib reported interstitial lung disease-like events. In the primary efficacy study of non-small cell lung cancer, the incidence of interstitial lung disease-like events was similar (0.8%) in patients treated with erlotinib and those treated with placebo. In the primary efficacy study of pancreatic cancer, 2.5% of patients treated with erlotinib in combination with gemcitabine experienced interstitial lung disease-like events, compared to 0.4% in patients treated with placebo in combination with gemcitabine. These symptoms occurred between 5 days and more than 9 months after the start of erlotinib treatment (median 39 days). Diagnoses reported for suspected interstitial lung disease-like events included pneumonia, radiation pneumonitis, hypersensitivity pneumonitis, interstitial pneumonia, interstitial lung disease, bronchiolitis obliterans, pulmonary fibrosis, acute respiratory distress syndrome, and pulmonary infiltration. In non-small cell lung cancer patients treated with erlotinib, most such cases were associated with confounding or contributing factors, including concurrent or prior chemotherapy, prior radiotherapy, pre-existing parenchymal lung disease, metastatic lung disease, or pulmonary infection. For patients experiencing pulmonary toxicity, erlotinib treatment may need to be interrupted or discontinued. Patients receiving erlotinib have reported hepatorenal syndrome or acute renal failure (sometimes fatal) and renal insufficiency with or without hypokalemia. Factors contributing to these renal adverse reactions include baseline liver function impairment; severe dehydration due to diarrhea, vomiting, and/or anorexia; and concomitant chemotherapy. If dehydration occurs, erlotinib treatment should be paused and rehydration initiated. Regular monitoring of renal function and serum electrolytes is recommended for patients at risk of dehydration. For more complete data on erlotinib warnings (27 in total), please visit the HSDB record page. Epidermal growth factor receptor (EGFR) is overexpressed in a significant proportion of cancers and promotes the formation of malignant phenotypes. CP-358,774 is a direct-acting inhibitor of human EGFR tyrosine kinase with an IC50 of 2 nM and reduces EGFR autophosphorylation in intact tumor cells with an IC50 of 20 nM. Compared to other tyrosine kinases we have studied, this inhibition is selective for EGFR tyrosine kinases, both in isolated kinases and in intact cells. At a dose of 100 mg/kg, CP-358,774 completely inhibited EGF-induced EGFR autophosphorylation in human HN5 tumors growing in xenografts in athymic mice and in EGFR of treated mouse livers. In cell culture, CP-358,774 inhibited the proliferation of DiFi human colon tumor cells at submicromolar concentrations and arrested the cell cycle in the G1 phase. This inhibitor led to a significant accumulation of hypophosphorylated form of retinoblastoma protein (RP) and p27KIP1 in DiFi cells, which may be the cause of cell cycle arrest. EGFR inhibition also induced apoptosis in these cells, which can be determined by DNA fragmentation and other indicators. These results suggest that CP-358,774 has the potential to treat tumors that rely on the EGFR pathway for proliferation or survival. [1]

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Due to the existence of independently activated survival pathways, the blockade of epidermal growth factor receptor (EGFR) by EGFR tyrosine kinase inhibitors is insufficient to produce effective antitumor activity. Therefore, a multi-target strategy may improve the efficacy of anti-EGFR therapy. In this study, we used an orthotopic animal tumor model to investigate the effects of 3,3'-bisindolylmethane (Bioresponse BR-DIM, or B-DIM for short)—a formulation of bisindolylmethane with higher bioavailability—on cell viability and apoptosis in combination with erlotinib in vitro and in vivo. BxPC-3 and MIAPaCa cells with different levels of EGFR and nuclear factor-κB (NF-κB) DNA binding activity were treated with B-DIM (20 μmol/L), erlotinib (2 μmol/L), and their combination, respectively. Cell viability and apoptosis were detected by MTT assay and histone-DNA ELISA. NF-κB DNA binding activity was assessed by electrophoretic mobility shift assay. We found that, compared with erlotinib or B-DIM alone, erlotinib combined with B-DIM significantly reduced cell viability in BxPC-3 cells (as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide staining and colony formation assay), induced apoptosis, downregulated EGFR phosphorylation, NF-κB DNA binding activity, and expression of anti-apoptotic genes. In contrast, no such effects were observed in MIAPaCa cells using similar methods. More importantly, these in vitro results were validated in animal models, indicating that the efficacy of B-DIM combined with erlotinib as an anti-tumor drug is far superior to that of either drug alone. These results suggest that B-DIM may be an effective treatment strategy for patients with EGFR and NF-κB activation in tumors, which may help improve treatment outcomes. [2] Regarding the limitations of this study, we must consider the following issues. First, the food intake of the three groups of animals was not controlled in this study. Since the animals were not paired for feeding, it was difficult to determine whether the weight loss was due to low intake or CP-induced AKI itself. Secondly, this study did not investigate renal tubular dysfunction, including loss of salt and magnesium, which is one of the most common physiological abnormalities in CP-N. Thirdly, this study did not examine the therapeutic effect of erlotinib on the recovery phase of CP-induced AKI. Clinically, the therapeutic effect of erlotinib on the recovery phase and its preventive effect on early AKI are crucial for patients receiving CP chemotherapy. Finally, this study did not confirm the effect of erlotinib on the antitumor effect of CP. Further research is needed to evaluate whether the inhibitory effect of erlotinib on CP-induced cell death is kidney-specific using different tumor cell lines (as previously studied). In summary, our in vivo and in vitro studies have shown that erlotinib has a renal protective effect against CP-N, which may be attributed to its inhibition of apoptosis and proliferation of proximal tubular cells. The protective effect of erlotinib appears to be achieved by inhibiting downstream signaling pathways of EGFR, including the MAPK and PI3K-Akt pathways. These results suggest that erlotinib may help prevent acute kidney injury in patients receiving CP chemotherapy. [3]

C22H23N3O4

393.44

393.168

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

176870

White to off-white crystalline powder

1.2±0.1 g/cm3

553.6±50.0 °C at 760 mmHg

223 - 228ºC

288.6±30.1 °C

0.0±1.5 mmHg at 25°C

1.615

2.39

1

7

11

29

525

0

O(C([H])([H])C([H])([H])OC([H])([H])[H])C1C([H])=C2C(=NC([H])=NC2=C([H])C=1OC([H])([H])C([H])([H])OC([H])([H])[H])N([H])C1=C([H])C([H])=C([H])C(C#C[H])=C1[H]

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.)