P450 3A-Catalyzed O-Dealkylation of Lapatinib ... - ACS Publications

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P450 3A Catalyzed O-dealkylation of Lapatinib Induces Mitochondrial Stress and Activates Nrf2 Marsha Rebecca Eno, Bahaa El-Dien M. El-Gendy , and Michael D Cameron Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00524 • Publication Date (Web): 09 Mar 2016 Downloaded from http://pubs.acs.org on March 10, 2016

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Chemical Research in Toxicology

Eno Page 1 P450 3A Catalyzed O-dealkylation of Lapatinib Induces Mitochondrial Stress and Activates Nrf2 Marsha Rebecca Eno§, Bahaa El-Dien M El-Gendyǁ, and Michael D. Cameron§*, §

Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, Florida 33458, United States, ǁChemistry Department, Faculty of Science, Benha University, Benha 13518, Egypt.

Corresponding author: Michael Cameron, Email: [email protected]

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Eno Page 2 Table of Contents Graphic


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Eno Page 3

Abstract Lapatinib (LAP), an oral tyrosine kinase inhibitor for treatment of metastatic breast cancer, has been associated with idiosyncractic hepatotoxicity. Recent investigations have implicated the importance of P450 3A4/5 enzymes in the formation of an electrophilic quinone imine (LAPQI) metabolite generated through further oxidation of O-dealkylated lapatinib (OD-LAP). In the current study, hepatic stress was observed via mitochondrial impairment. OD-LAP caused a time and concentration dependent decrease in oxygen consumption in HepG2 cells while LAP did not alter oxygen consumption rate. Interestingly, however, HepG2 cells transfected with human P450 3A4 did exhibit mitochondrial dysfunction via P450 3A4-mediated metabolism of LAP to OD-LAP. OD-LAP-induced mitochondrial toxicity was enhanced upon depletion of intracellular GSH levels, demonstrating that cellular GSH levels are important in the protection of mitochondrial function against LAPQI. Given the nature of LAPQI and the importance of GSH levels in LAP-induced mitochondrial stress, the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) was evaluated, as this transcription factor induces the expression of NAD(P)H quinone oxidoreductase 1 (NQO-1), glutathione S-transferase (GST), UDPglucuronosyltransferases (UGT), and glutathione synthetase (GS) all of which might be expected to decrease LAP toxicity. Using a FRET-based target gene assay in HepG2 cells, OD-LAP was indeed found to activate Nrf2. Follow-up assays showed increased mRNA levels of Nrf2 target genes after 4 hour treatment with OD-LAP, but not with LAP. LAP activation of Nrf2 was only observed when HepG2 cells were transduced with P450 3A4. The significance of Nrf2 protection was established in vivo in Nrf2-KO mice. Increased transaminase levels were found after a single LAP dose in both Nrf2-KO and control mice, indicating elevated hepatic necrosis, although upon repeat dosing, transaminase levels reverted to baseline levels in the control mice. They continued to rise in Nrf2-KO mice, however, indicating the likelihood that Nrf-2 plays a significant role in combatting the hepatotoxicity triggered by LAP.



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Eno Page 4 Introduction

Drug induced liver injury (DILI) is a significant health problem and can result in acute liver failure, liver transplantation or death.1, 2 Lapatinib (LAP), a dual tyrosine kinase inhibitor of ErbB-2 and EGFR, is approved for the treatment of advanced metastatic breast cancer;3, 4 however, treatment with LAP is associated with an increased incidence of idiosyncratic hepatotoxicity.5 In 2008, the FDA issued a black box warning for LAP following clinical reports of elevated liver enzymes and liver-related deaths during post market surveillance.6, 7 For this reason, LAP prescribing information includes information on liver chemistry monitoring and clinical management of hepatotoxicity.8

Recently it has been reported that a small population of LAP-treated patients had incidences of immune-mediated hepatotoxicity. Patients with HLA (human leukocyte antigen) polymorphism HLA-DQA1*02:01 and HLA-DRB1*07:01 had an increased risk of serious hepatic toxicity (odds ratio, 14.0).8 These findings suggest that LAP-induced hepatotoxicity has an immune component that may be partially due to the covalent modification of proteins by LAP.9, 10 It is important to note that LAP-induced liver injury has been observed in patients without these HLA and that the majority of patients with the indicated HLA do not develop serious liver injury.

The hepatotoxicity of LAP can be attributed to the multifactorial nature of idiosyncratic toxicity, and can be due to a combination of immune–mediated and chemical-induced toxicity that involves direct, acute, and reactive-metabolite mediated toxicity involving drug bioactivation and formation of drug-protein adducts, and oxidative stress that involves mitochondrial dysfunction. Acute chemical-induced toxicity may provide danger associated molecular patterns that are required to produce an immune response.2

LAP was demonstrated to undergo extensive metabolism by P450 3A4/5 enzymes resulting in Odealkylation, N-dealkylation and N-hydroxylation metabolites (Figure 1).11 While the mechanism by which LAP causes hepatotoxicity is unknown, reactive LAP metabolites have been identified such as the O-dealkylated metabolite (OD-LAP). OD-LAP possesses a p-hydroxyanilide group that upon oxidation generates an electrophilic reactive metabolite, lapatinib-quinone imine


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Eno Page 5 (LAPQI), which can be trapped with glutathione (GSH).12 Recently, LAP metabolism and excretion studies have been performed on human volunteers that have highlighted the potential relationship of LAP’s reactive metabolites to its clinical hepatotoxicity. 11 For instance, Chan et al disclosed that LAP may inactivate P450 3A5 by the adduction of LAPQI to the apoprotein.13 Thus, LAP induced hepatotoxicity could be linked to the cytochrome P450-catalyzed formation of LAPQI, which, like other electrophiles that cause hepatotoxicity, such as acetaminophen’s Nacetyl-p-benzoquinone imine, have the potential to covalently modify hepatic proteins and deplete GSH, further exacerbating oxidative stress.14

A number of hepatotoxic drugs that were withdrawn from the market were found to impair mitochondrial function.15, 16 In mammalian cells, the mitochondria are responsible for generating the majority of cellular ATP by the process of oxidative phosphorylation. This is needed for normal cell function, including the ability to respond to endogenous or exogenous stress. Drugs that impair mitochondrial function decrease cell viability, and depending on the severity of the drug insult, can lead to organ or tissue damage.

The liver is exposed to a high level of toxic species, including toxic metabolites, and is thus a major target for tissue-specific toxicity. In response to electrophilic and oxidative stress, activation of the erythroid 2-related factor 2 (Nrf2) pathway induces the expression of several cytoprotective genes, through a cis-acting antioxidant response element (ARE). Nrf2 is regulated through proteosomal degradation. In the presence of electrophiles, Nrf2 ubiquinatin is decreased and it heterodimerizes with a small musculo-aponeurotic factor protein (Maf) and binds to ARE to promote the transcription of numerous cytoprotective genes,17 including, not limited to, NAD(P)H quinone oxidoreductase 1 (NQO-1), heme oxygenase (HO-1), glutathione Stransferase (GST), UDP-glucuronosyltransferase (UGT), glutathione synthetase (GS), and glutamate-cysteine ligase (GCL).18 These important detoxifying and bio transformative enzymes aid in the elimination of potentially harmful chemicals. Of particular relevance with respect to LAP-induced hepatotoxicity is NQO-1 which can reduce the electrophilic metabolite LAPQI to the corresponding hydroquinone, which is not an electrophile. Additionally, GS and GCL are involved in GSH synthesis, and as we demonstrate in this manuscript, cellular GSH levels are



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Eno Page 6 important factors in determining the extent of the mitochondrial toxicity caused by LAP metabolites.

The current study demonstrates for the first time that the OD-LAP metabolite of the tyrosine kinase inhibitor LAP, causes mitochondrial dysfunction in a time- and concentration-dependent manner. Mitochondrial dysfunction was observed under physiologically relevant concentrations and may be expected to lead to cellular toxicity. Importantly, the expected OD-LAP-mediated toxicity is likely minimized through the activation of Nrf2, which was demonstrated to protect against LAP-induced hepatocellular damage when evaluated in Nrf2-KO and control mice.


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Eno Page 7 Experimental Procedures

Materials. Reagents and solvents were purchased at the highest available grade and purity. Lapatinib ditosylate was purchased from Toronto Research Chemicals (Toronto, Canada) and Biotang Inc., TSZ Chem (Lexington, MA). OD-LAP was chemically synthesized from LAP as described previously (Teng et al., 2010). Stock solutions of LAP and OD-LAP were prepared in DMSO. Midazolam, galactose, formic acid, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Acetonitrile (ACN) was purchased from Fisher Scientific (Fair Lawn, NJ), HEPES from Amresco (Solon, OH), and Pen-strep, blasticidin, and puromycin from Life Technologies (Carlsbad, CA).

Cell Culture Conditions High-glucose Growth Media. The human hepatoma cell line HepG2 was purchased from ATCC (Manassas, VA), and maintained in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies 10569-010) containing 25 mM glucose, GlutaMAX. The cell culture medium was supplemented with 10% (v/v) heat-activated fetal bovine serum, 0.1 mM nonessential amino acids (NEAA), 25 mM HEPES and 100 ug/ml penicillin-streptomycin. The cells were cultured in 5% CO2 at 37°C. The CellSensor® ARE-bla HepG2 cell line (Life Technologies) were maintained in the above growth media with the addition of 5 ug/ml blasticidin. Assay Media. High-glucose growth media without antibiotics. Galactose Media. DMEM deprived of glucose (Sigma Cat.# D5030) supplemented with 10 mM galactose, 1 mM sodium pyruvate, 25 mM HEPES and pH adjusted to 7.4. Unbuffered Media. DMEM base powder (Sigma Cat.# D5030) dissolved in sterile water supplemented with 1.0 mM sodium pyruvate, 25 mM glucose, 2 mM GlutaMAX and pH adjusted to7.4 with NaOH.

Production of Lentivirus and Transduction of HepG2 and CellSensor® ARE-bla HepG2 cells. Lentiviruses, lentiviral plasmids, and reagents were obtained from GeneCopoeia. The P450 3A4 (Ex-Z0804-Lv121), P450 3A5 (I031-Lv121), and control vector with EGFP (green fluorescent protein) (EX-EGFP-Lv121) plasmids were transformed into One Shot® Stbl3TM chemically competent E. coli cells (Life Technologies) and purified using Plasmid Maxi-Prep Kit (Qiagen, Valencia, CA). Two days before transfection, 293TN packaging cells (System



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Eno Page 8 Biosciences) were plated in a 10-cm dish with DMEM and 10% heat-inactivated FBS. Fortyeight hours after seeding, 293TN packaging cells were transfected with lentiviral vectors encoding P450 3A4, P450 3A5, or EGFP control vector, using Lenti-Pac HIV Expression Packaging Kit (HPK-LvTR-20, GeneCopoeia) according to the manufacturer’s instructions. Two days post-transfection, lentivirus-containing culture medium was concentrated using Vivaspin® 20 column (100,000 MWCO PES), sterile filtered by passing through a 0.2 µm filter and added to the target cells (HepG2 cells or CellSensor® ARE-bla Hep G2 cells). Transduced cells were selected with 1.0 µg/ml puromycin.

Functional Characterization of Transduced HepG2 and CellSensor® ARE-bla HepG2 cells To measure P450 3A4 and P450 3A5 activity, assays were performed by incubating 120,000 cells/well of HepG2 cells or CellSensor® ARE-bla HepG2 in 24-well plates with midazolam as the probe substrate. Midazolam was added at 5 µM, 10 µM and 20 µM. The final solvent concentration to the incubation medium did not exceed 0.2% DMSO (v/v). In brief, 250 µl of warmed (37°C) high glucose assay medium containing midazolam was added to each well. After 20 minutes, an aliquot of 100 µl was taken from each well and added to a quenching solution (100 µl of ice-cold acetonitrile with 0.1 µM dextrorphan as an internal standard).

LC-MS/MS Conditions for 1´-hydroxymidazolam. 1´-hydroxymidazolam formed and released into the culture medium was quantified by LC-MS/MS using an ABSciex 6500 QTRAP (ABSciex, Framingham, MA). An aliquot (5 µl) of incubation extract was injected into a Phenomenex Kinetex™ XB-C18, 100 Å (2.6 µm x 50 x 2.1 mm) column that was maintained at 35°C with an aqueous mobile phase containing 0.1% formic acid (solvent-A) and ACN with 0.1% formic acid (solvent-B) run at a flow rate of 0.4 ml/ min. Linear gradients were used as follows: 0–0.3 minutes = 5% B; 2.5–3.0 minutes = 95% B; and 3.1–6.1 = 5% B. 1´hydroxymidazolam and dextrorphan were detected using the mass transitions of m/z 342.2→203.1 and m/z 258.2→157.2, respectively. The optimized declustering potential and collision energy were 80 eV and 58 eV for 1´-hydroxymidazolam, and 95 eV and 38 eV for dextrorphan.


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Eno Page 9 Measurement of NADH/NADPH levels. NADH/NADPH level in HepG2 and HepG2-3A4 cells was measured using the Vybrant® MTT Cell Proliferation kit (Life Technologies). Cells (1 × 105 cells/ml) in 100 µl of phenol red free high glucose growth media were plated in a 96-well plate and the next day were incubated with various concentrations of LAP (10 µM and 50 µM), OD-LAP (10 µM, 30 µM and 100 µM), and vehicle control (0.1% DMSO). After 24 h incubation in the 5% CO2 humidified incubator at 37 °C, the cells were incubated with MTT following the manufacturer’s protocol. Cells were lysed with DMSO and reduction of MTT to insoluble formazan purple crystals was measured at 540 nm absorbance using a Spectra Max M5e microplate reader (Molecular Devices).

ATP Level Measurement. Intracellular ATP levels were measured using the Mitochondrial Tox-Glo Assay (Promega), in accordance with the manufacturer’s instructions. In brief, HepG2 cells were plated at a density of 300,000 cells/well in a 96-well plate in the presence of various concentrations of LAP (10 µM to ~625 nM) and OD-LAP (50 µM to ~750 nM). Cells were grown in standard high glucose medium, but were washed and serially dosed in 100 µl of serumfree, galactose-containing medium. 100 µl of ATP detection substrate was added and luminescence was measured using the EnVision (PerkinElmer).

CellTox™ Green Cytotoxicity Assay. Viability of HepG2 and HepG2-3A4 cells treated with LAP and OD-LAP were measured using the CellTox™ Green Cytotoxicity Assay (Promega). Cells (1 × 105 cells/ml) in 100 µl of phenol red free high glucose growth media were plated in a 96-well clear bottom black plate and the next day the media was exchanged with fresh media containing various concentrations of LAP (10 µM, 30 µM and 50 µM), OD-LAP (10 µM, 30 µM and 100 µM), vehicle control (0.1% DMSO), and toxic control. After 24 hours, CellTox™ Green Reagent (2X) was prepared according to manufacturer’s instructions and 100 µl was added to the cells. The plate was incubated at room temperature for 15 minutes and fluorescence was read at 485Ex and 520Em using the Spectra Max M5e microplate reader (Molecular Devices). Cellular Oxygen Consumption using Seahorse XF24 Analyzer. Measurement of cellular respiration was performed using a Seahorse XF24 analyzer (Seahorse Biosciences, North Billerica, MA). HepG2 and HepG2-3A4 cells were seeded in Seahorse XF 24-well cell culture



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Eno Page 10 microplates at 15,000 cells/well in 100 µl of high glucose growth media and allowed to adhere overnight. Cells were treated with different concentration of drugs for different time points depending on the experiment. Before the experiment, growth medium was replaced with 500 µl unbuffered medium, and cells were equilibrated for 30 min at 37°C in a CO2-free incubator before being transferred to the XF24 analyzer. The oxygen consumption rate (OCR) was measured prior to, during, and after injections with the ATP synthase inhibitor oligomycin, the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and a combined injection of the complex I+II inhibitors antimycin A + rotenone to yield final concentrations in the wells of 1 ug/ul, 1.2 µM, and 1.6 µM+1.6 µM, respectively. Basal mitochondrial respiration was calculated by subtracting non-mitochondrial respiration (OCR following injection with antimycin A + rotenone) from the baseline respiration prior to oligomycin injection. Maximum respiration was calculated by subtracting non-mitochondrial respiration from the maximum respiration measured following FCCP injection. Measurement of Nrf-2 activation by LiveBLAzerTM β-lactamase Reporter Assay. The LiveBLAzerTM ARE-bla β-lactamase reporter assay (Life Technologies) was used to measure activation of antioxidant response element (ARE) regulated genes. The Cell Sensor® ARE-bla HepG2 cell line contains a beta-lactamase reporter gene under the control of ARE stably integrated into HepG2 cells. ARE-bla HepG2, ARE-bla HepG2-3A4 (transduced with hP450 3A4) and ARE-bla HepG2-3A5 (transduced with hP450 3A5) were seeded in clearbottom, black 384-well plates at 3.9×105 cells/ml and cultured in high glucose growth media at 37 °C in a 5% CO2 humidified incubator overnight. The next day test compounds were added and the cells were incubated in the incubator for 15 h. This was followed by addition of the FRET substrate CCF4-AM. The substrate consists of two fluorophores that form a FRET pair, which in the absence of beta-lactamase activity results in a green fluorescence signal. In the presence of beta-lactamase activity, cleavage of the substrate disrupts FRET, resulting in a blue fluorescence signal. Fluorescent measurements were made using the EnVision (PerkinElmer). The β-lactamase activities were measured by the ratio of blue product, monitored at λ=460 nm and the green substrate, monitored at λ=535 nm (See manufacturer’s protocol for detailed instructions).


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Eno Page 11 Quantitative PCR to Measure mRNA Expression Levels of Nrf-2 Activating Genes. Cells were plated in a 6-well plate (7.5 × 105 cells/well) in high glucose growth media and the next day cells were incubated with various concentrations of LAP (dissolved in serum free media) and OD-LAP. RNA was isolated using the RNeasy Plus Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions, and its purity was determined on a spectrophotometer (Thermo Scientific Nanodrop 1000). Pure RNA (2 µg) was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The cDNA was then used with Power SYBR Green PCR master mix (Life Technologies) for real time qPCR quantification using the Applied Biosystems 7900 HT Fast Real-Time PCR system. All samples were analyzed in triplicate. Relative quantities of specifically amplified cDNA were determined using the comparative threshold cycle (CT) method. GAPDH was used as an endogenous reference gene and no-template and no-reverse-transcription controls were used to exclude nonspecific amplification. The cycling conditions used were 95°C for 10 min for initialization followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. Primers for the PCR reactions (purchased from Integrated DNA Technologies) are listed in the Supporting Information section in Table S1.

Animals and Experimental Protocols Male C57BL/6 and Nrf2-KO (C57BL/6 background) mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were 10 weeks old and weighed approximately 23 g. They were housed in the AAALAC accredited Scripps vivarium and kept in a temperature-controlled room with a 12 h light/dark cycle and ad libitum access to food and water. All animal procedures had been reviewed and approved by the Scripps Florida IACUC. The tested LAP dose was 400 mg/kg. This dose represents three times the allometric scaled human LAP dose (1250 mg/day).19 For in vivo studies, 400 mg/kg of LAP was formulated as a 20mg/mL solution in 10/10/80 DMSO/Tween80/Water and injected IP daily for five days. On day 1 and day 5 (8 hours postdose), blood was drawn for the evaluation of ALT and AST and drug level quantitation. A section of liver from each mouse was collected and flash frozen in liquid nitrogen at the end of the experiment on day 5 for quantitation of liver levels of LAP and OD-LAP.



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Eno Page 12 LAP and OD-LAP Quantitation in Plasma and Liver Tissue. The tissue distribution of LAP was evaluated in C57BL/6 and Nrf2-KO mice (n = 4). Tissues were not perfused to reduce the risk that LAP would be eluted from the tissue during the perfusion process. Plasma was generated using standard centrifugation techniques and the plasma and tissues were frozen at 80°C. Plasma and tissues were mixed with ACN (1:5 v:v or 1:5 w:v, respectively). Tissues were sonicated with a probe tip sonicator and clarified by filtration through a 0.2 micron filter plate. Samples were analyzed by LC-MS/MS using an ABSciex 5500 triple quad mass spectrometer. MRM’s were used to detect LAP (m/z 582.1→366.1), OD-LAP (m/z 473.2→349.8), and carbamazepine was used as an internal standard (m/z 238→195.1). Plasma drug levels were determined against standards made in plasma and liver tissue matrix. See Tables S2, S3 and S4 of the supporting information for instrument settings and detailed analytical conditions.

Biomarker Analyses Following collection of blood from anesthetized animals, alanine transaminase (ALT) and aspartate transaminase (AST) levels were determined using a clinical chemistry analyzer, Cobas C311 from Roche Diagnostics.

Statistical Analyses All cell-based experiments were performed in triplicate in one to three independent experiments. The mean and standard deviation (S.D) or standard error of the mean (S.E.M) for each experiment were determined using GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA). Treatment groups were compared with their respective controls using the unpaired t test. P values were calculated by two-tailed analysis, with P < 0.05 considered significant.

Results Effect of OD-LAP on Oxygen Consumption Rate (OCR) in HepG2 Cells To directly examine the response of HepG2 cells to OD-LAP-induced mitochondrial dysfunction, cellular metabolism was assessed using the Seahorse XF24 analyzer, which measures mitochondrial respiration (oxygen consumption) of cells. Addition of oligomycin inhibits the mitochondrial F1-F0-ATPase, resulting in a reduction of electron flow through the electron transport chain, thereby decreasing oxygen consumption. Addition of carbonyl cyanide


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Eno Page 13 4-(trifluoromethoxy) phenylhydrazone (FCCP), on the other hand, accelerates cellular oxygen consumption, allowing the maximal rate of oxygen consumption by the electron transport chain to be observed. Finally, the addition of antimycin/rotenone (inhibits complex III and complex I), allows extra mitochondrial oxygen consumption to be quantitated. Figures 2A and 2B shows the oxygen consumption of HepG2 cells after treatment with vehicle (0.1% DMSO, 24 h) and 30 µM OD-LAP (6 h and 24 h). The oxygen consumption was measured in real-time for 106 minutes, as described in the Experimental Procedures section. The oxygen consumption rate in HepG2 cells, prior to the addition of any of the above-mentioned effectors of the electron transport chain, decreased in the presence of 30 µM OD-LAP for 6 and 24 hours. Once correction for background oxygen consumption was made, the basal mitochondrial oxygen consumption was minimally affected after 6 hours, but the maximal mitochondrial respiratory capacity was significantly reduced. By 24 hours, both basal and maximal mitochondrial respirations were decreased (Figure 2B). Most Seahorse experiments were repeated three to four times. Replicate experiments showed consistent trends but variability was observed in the magnitude of the OCR changes between experiments. OD-LAP incubated at a concentration of 30 µM and 100 µM for 24 hours reduced NAD(P)H levels to ~60% and ~ 40% of the DMSO control, respectively, in HepG2 cells as measured by the MTT assay (Figure 2C). This decrease does not involve cell viability, as demonstrated in Cell-Tox Green viability assay (Figure 2D), suggesting that the observed decrease in the MTT assay was due to mitochondrial impairment and not from reduced cells viability via cellular necrosis.

As shown in Figure 2E, OD-LAP decreased both basal and maximal respiratory capacity of HepG2 cells in a concentration-dependent manner. Since OD-LAP can be further metabolized to LAPQI, and LAPQI can be trapped with GSH12, we tested to see whether depletion of intracellular GSH increased OD-LAP-induced mitochondrial damage. GSH, with or without glutathione S-transferase, quenches electrophiles by coupling them with GSH. BSO (Lbuthionine sulfoximine) depletes intracellular GSH by inhibiting GCL, the enzyme involved in the first step of GSH synthesis. Pre-treatment with BSO (50 µM) for 24 hours was used to deplete GSH in the HepG2 cells. This was followed by the addition of 20 µM OD-LAP for another 24 hours. In control incubations, BSO alone (50 µM) and treatment with OD-LAP (20 µM) only slightly decreased basal and maximal uncoupled respiration compared to vehicle



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Eno Page 14 treated cells (Figure 2F). However, a combination of BSO (50 µM) and OD-LAP (20 µM) drastically diminished the OCR in HepG2 cells. These results indicate that the presence of GSH protects against OD-LAP-induced mitochondrial damage.

Effect of LAP on OCR in HepG2 cells There was no significant difference in oxygen consumption rate between vehicle control HepG2 cells and cells treated with 10 µM LAP (Figure 3A). Note, LAP precipitation was visible in serum-containing assay media at concentrations above 10 µM, precluding matching LAP and OD-LAP concentrations in all assays. However, precipitation was not observed in serum-free media at concentrations up to 50 µM. As seen in Figure 3B, there was also no significant difference in MTT reduction between vehicle treated and 50 µM LAP (dissolved in serum free media) which implies that LAP did not affect cellular NADH/NADPH in HepG2 cells.

Effect of LAP on OCR in transduced 3A4-HepG2 cells Based on the findings described above, we stably expressed HepG2 with human P450 3A4 to investigate the mechanism by which P450 3A4 contributes to the toxicity of LAP. HepG2 cells are largely metabolically incompetent and cannot be used to look at the biotransformation of LAP into OD-LAP,20 so in order to examine P450 3A4-mediated mitochondrial dysfunction caused by LAP, HepG2 cells were virally transduced with enzyme hP450 3A4. The success of the transduction was assessed using midazolam as a P450 3A4 probe to monitor the formation of 1´-OH midazolam in HepG2-3A4 and HepG2-3A5-transduced cell lines. Non-transduced and HepG2 cells transduced with GFP showed no P450 3A4 activity (Figure 4A). The observed rate of midazolam hydroxylation was approximately 3-fold higher than what we have previously reported for human cryopreserved hepatocytes from multiple individual donors.21

To investigate the role of P450 3A4-mediated metabolism as a mechanism of LAP hepatotoxicity, LAP was incubated with transduced HepG2-3A4 cells (note- serum free media was used to allow for higher LAP concentrations). As discussed earlier in the results of Figure 3A, there was no significant change in OCR caused by LAP (10 µM) treatment in the metabolically incompetent HepG2 cells and vehicle treated cells. Treatment of 10 µM LAP for 24 hours caused a significant decrease in OCR when tested in transduced HepG2-3A4 cells, and


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Eno Page 15 treatment with 30 µM for 24 hours drastically reduced the OCR (Figure 4B). As observed in Figure 4C, both 10 µM and 50 µM LAP decreased NAD(P)H levels in the transduced cells but did not affect viability (Figure 4D). These results indicate that P450 3A4 catalyzed Odealkylation of physiologically relevant concentrations of LAP is sufficient to impair mitochondrial function but not viability. Preliminary experiments were conducted with both P450 3A4 and 3A5 transduced cells, and both P450 enzymes appeared to have the same effect on mitochondrial OCR (data not shown). Later experiments utilized only HepG2-3A4 cells because 3A4 expression in humans is ubiquitous, whereas only about 20-30% of patients have functional 3A5.

Effects of LAP and OD-LAP treatment on cellular ATP levels in HepG2-3A4 cells To increase the magnitude of LAP and OD-LAP-induced mitochondrial dysfunction, transduced HepG2 cells were forced to increase their reliance on oxidative phosphorylation for ATP production rather than glycolysis by reducing the concentration of glucose in the growth media and supplementing with galactose. Susceptibility to mitochondrial toxicants increases in galactose grown HepG2 cells.22 Figure 4E shows a concentration-dependent decrease in ATP in the presence of LAP and OD-LAP after 16-hour drug treatment in galactose-containing media. Both LAP and OD-LAP did not affect cell viability at the concentrations tested (Figures 4D and 4F).

SOD1 and SOD2 as markers of cytoplasmic and mitochondrial stress. To test cytoplasmic versus mitochondrial related stress, the mRNA expression of SOD1 and SOD2, which differentially increase in response to cytoplasmic and mitochondrial oxidative stress respectively were measured in HepG2 cells treated for 2 hours and 4 hours with 40 µM LAP, and the same amount of time with 40 µM OD-LAP. As seen in figures 5A and 5B, the mRNA expression of mitochondrial SOD2 increased in cells treated with 40 µM OD-LAP, whereas the expression of the cytoplasmic SOD1 remained unchanged. This further indicated that OD-LAP affects the mitochondria. In transduced HepG2-3A4 cells treated with LAP for 4 hours, there was a significant increase in the mRNA levels of both cytoplasmic SOD1 and



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Eno Page 16 mitochondrial SOD2 (Figures 5C and 5D) indicating increased cytosolic and mitochondrial stress.

Detection of LAPQI Because LAPQI is highly reactive and short-lived, detection of covalent adducts of glutathione with LAPQI (GS-LAP) was taken as evidence of LAPQI formation in cells. GS-LAP was detected when 40 µM OD-LAP was added to HepG2 and HepG2-3A4 cells. However, only HepG2-3A4 cells were capable of generating GS-LAP in incubation containing 40 µM LAP, consistent with earlier data that the toxic metabolite LAPQI can be generated by mitochondria through oxidation of OD-LAP, but that P450 3A enzymes are required for the formation of ODLAP from LAP. Methods and spectra were consistent with earlier published data of GS-LAP formation in HepaRG cells (data not shown).23

Activation of Nrf2 in HepG2 and HepG2-3A4 cells In order to test the hypothesis that the reactive metabolite LAPQI may be involved in the activation of the Nrf2 defense pathway, the ability of the OD-LAP metabolite to induce Nrf2 target genes in HepG2 cells was assessed using the LiveBLAzerTM ARE-bla β-lactamase FRETbased reporter assay. This assay measures Nrf2 activation in CellSensor™ ARE-bla HepG2 cells that contain a beta-lactamase reporter gene under the control of the ARE that has been stably integrated into HepG2 cells. Figure 6A shows Nrf2 activation dose-response curves in the presence of LAP, OD-LAP and a positive control, tert-butylhydroquinone (THBQ). OD-LAP activates the Nrf2 pathways in a dose-dependent manner in HepG2 cells. LAP, on the other hand does not activate Nrf2, likely due to the lack of P450 3A4/5 activity in HepG2 cells. Due to the insolubility of LAP in serum-containing media, LAP was dissolved in serum-free media. In order to observe LAP-induced Nrf-2 activation, the CellSensor™ ARE-bla HepG2 cells were virally transduced with hP450 3A4 and 3A5. As seen in figure 6B, the cells were successfully transduced, as demonstrated by the increased rate of midazolam hydroxylation to 1´-OH midazolam. The ARE-bla β-lactamase reporter assay was carried out again using the transduced cells (Figure 6C). LAP activation of Nrf2 in these cells is consistent with the earlier Seahorse data indicating that LAP needs the metabolic activity of P450 3A4/5 in order to activate the Nrf2 pathway in HepG2 cells.


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Eno Page 17

mRNA expression levels of Nrf2 target genes in HepG2 and HepG2-3A4 cells In order to test the hypothesis that LAPQI the reactive metabolite of LAP, activates Nrf2, the induction of two cytoprotective Nrf2 genes (HO-1 and NQO-1) were measured by quantifying their mRNA levels after treatment with LAP and OD-LAP in HepG2 cells (Figures 7A and 7B). There was no significant increase in mRNA expression of HO-1 or NQO-1 compared to vehicle control after a 2 hour treatment with OD-LAP, but after a four hour treatment, there was a significant increase in mRNA expression of both Nrf2 target genes. LAP, on the other hand, did not activate either gene in the HepG2 cells at either time point.

Based on the results of the previous experiment, where LAP did not activate Nrf2 target genes in HepG2 cells, the transduced P450 3A4 cells were used to evaluate up-regulation of Nrf2 target genes by LAP. Treatment of 3A4-HepG2 cells with 40 µM LAP for two hours did not activate target genes, but the four hour incubation did cause induction of both HO-1 and NQO-1 (Figures 8A and 8B).

In vivo evaluation of LAP-induced hepatic toxicity The experimental dose of LAP used in the mouse studies was set to three-times the humanequivalent dose, as determined by allometric scaling of the AUC values. The mouse equivalent of three times the 1250 mg human dose was determined to be approximately 400 mg/kg. Preliminary experiments demonstrated a high level of variability after oral dosing in non-fasted mice, likely due to LAP solubility and the known effect of food on LAP absorption, so LAP dosing was switched from oral gavage to IP injection in order to reduce variability and to preclude daily fasting of the mice in a multi-day study. As seen in Figure 9A and 9B, LAP (400 mg/kg, IP) administration to Nrf2-KO and C57BL/6 wt male mice caused an increase in the liver enzymes ALT and AST to more than 3-times normal levels in both sets of mice on the first day. With continual dosing, the C57BL/6 looked robust without obvious impairment. The Nrf2-KO mice became dehydrated, had reduced mobility, hunched posture, and reduced grooming. For this reason the multi-day dosing study was ended after five days. ALT and AST levels in the C57BL/6 mice had reverted back to normal levels by day 5. In contrast, the Nrf2-KO mice had further elevation of both enzymes (~8 x normal, ALT and ~ 38 x normal, AST). These results



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Eno Page 18 imply that the liver is protected from LAP-induced toxicity by the Nrf2 detoxification pathway. Plasma levels of LAP and OD-LAP were determined 8 hours after the initial dose on days one and five (Table 1). At the conclusion of the study, liver was collected to determine tissue exposure. While there was not a statistically significant change in plasma levels of the analyte tested between C57BL/6 and Nrf2-KO mice, there was a similar trend of decreasing LAP levels between days 1 and 5 for both strains of mice.


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Eno Page 19 Discussion Even though LAP has a satisfactory safety profile for use in breast cancer treatment, elevation in liver enzymes, ALT > 3 times ULN (upper limit of normal) and total bilirubin >2 times ULN, have been reported in clinical trials in around 1% of patients.5 If Hy’s Rule is correct for LAP, this would result in an expected rate of liver failure resulting in death or liver transplantation in approximately 1:1,000 patients. Ascertaining the true incidence of LAP-induced hepatotoxicity is likely complicated due to co-administered drugs, disease progression, and the likelihood of metastatic breast cancer to metastasize to the liver. Identifying the mechanisms and risk factors that cause treatment-related hepatotoxicity could improve the management of drug-induced toxicity in breast cancer patients. HLA –DQA1*02:01 was found to be associated with an increased incidence of LAP-induced hypersensitivity reactions after pharmacogenetic investigation.9 This might be due to the covalent binding of reactive LAP metabolites to cellular proteins to form haptens that are presented as antigens by DQA1*02:01 and result in an adaptive immune response.24

Reactive metabolites of LAP were previously shown to interact with P4503A enzymes (Figure 1). OD-LAP was demonstrated to be a mechanism-based inhibitor of P450 3A5 via adduction of LAPQI to the apoprotien.13 Hydroxyl amine and N-dealkylated metabolites have been reported but their in vivo concentrations appear to be minimal.11 Additionally, an aldehyde metabolite has been reported. We found an aldehyde contaminant in two commercial sources of LAP, but did not observe the time-dependent formation of the aldehyde in microsomal incubations (data not shown). Induction of P450 3A4 by dexamethasone and rifampicin was found to enhance the cytoxicity caused by LAP and it correlated with the formation of LAPQI cysteine adducts in HepaRG cells.23 As part of our investigation of LAP-induced toxicity, we were able to find LAP adducts to cysteine-containing model peptides containing nucleophilic amino acids, but these were unstable. The intensity of LAP-peptide adducts as detected by LC-MS/MS diminished rapidly at room temperature. Stability was increased when LAPQI was generated from OD-LAP in an electrochemical cell and directly added to peptides, but the addition of denatured cellular extract enhanced the instability and prevented further investigation of lapatinib adducts in cells (data not shown). This phenomenon of instability of protein adducts in cells has been seen before, and it may affect the appearance of toxic effects of LAP in vitro.25



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Eno Page 20

The exact mechanism leading up to the hepatotoxicity of LAP is not well understood, nor is the toxicological implications of the OD-LAP metabolite. While there is an immune component to LAP toxicity, this does not explain the totality of the observed toxicity and we hypothesized that mitochondrial dysfunction might play an additive role to the reported immune-mediated toxicity of LAP. Mitochondrial toxicity has been implemented in drug-induced organ toxicities, especially hepatotoxicity;26 however, mitochondrial toxicity can be difficult to ascertain from in vitro studies because the ultimate toxic insult may be due to metabolites of the parent molecule. For example, the HER2 tyrosine kinase inhibitor CP-724,714 was discontinued from clinical development due to unexpected hepatotoxicity in cancer patients, and it was later found to cause mitochondrial impairment in human hepatocytes that had previously gone undetected in other cell types.27

HepG2 cells were compared to HepG2 cells transduced with hP450 3A4. The cellular oxygen consumption rate, which primarily reflects oxygen utilization by the mitochondrial electron transport chain, was clearly lower in HepG2-3A4 compared to HepG2 cells after treatment with LAP. The involvement of the O-dealkylated metabolite (OD-LAP) was demonstrated by the clear reduction in OCR in HepG2 cells when OD-LAP was directly added to the cells. Further demonstrating impaired mitochondrial function was the dose-dependent decrease in both NAD(P)H and ATP. To ensure that decreased mitochondrial function was not due to a reduction in the number of live cells in the incubations, cytotoxicity was evaluated using a measure of membrane integrity.

Immortalized cell lines such as HepG2 tend to grow rapidly under hypoxic and high glucose conditions, so they derive most of their ATP from glycolysis rather than mitochondrial oxidative phosphorylation. This condition is termed the Warburg and/or Crabtree effect.28 In order to make the transduced HepG2-3A4 cells more reliant on oxidative phosphorylation to observe the effect LAP has on the mitochondria, we dosed LAP and OD-LAP in galactose-containing media. These conditions reduce the cells ability to get their ATP from glycolysis, and thereby increasing ATP production from oxidative phosphorylation. Such conditions are conducive for assessing druginduced mitochondrial dysfunction, and in our experiments, we were indeed able to see a dose-


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Eno Page 21 dependent decrease in ATP production when HepG2-3A4 cells were dosed in galactosecontaining media with LAP and OD-LAP.

In vitro studies by Teng et al have shown that OD-LAP is further oxidized to LAPQI and can covalently adduct to GSH.12 We tested to see if depletion of GSH in HepG2 cells would increase LAPQI-mediated toxicity and make the cells more susceptible to mitochondrial dysfunction. Treatment of HepG2 cells with BSO depletes GSH. When dosed with OD-LAP at 20µM in addition to BSO, there was a profound decrease in the OCR of HepG2 cells as compared to the control.

LAP metabolism to OD-LAP, and further oxidation to the electrophile LAPQI, appears to be responsible for much of the observed mitochondrial toxicity. However, there may be additional factors yet to be elucidated. This is implicated in the decreased OCR and NAD(P)H levels depicted in Figures 2 and 4, where 30 µM OD-LAP was effective at reducing OCR and NAD(P)H levels, but 10 µM OD-LAP had minimal effect. However, in P450 3A4-transduced HepG2 cells, 10 µM LAP significantly decreased both OCR and NAD(P)H levels and LAP appeared to be a more potent mitochondrial toxin than when the identical concentration of ODLAP was directly added to the cell. The increased expression of both SOD1 and SOD2 in the LAP treated HepG2-3A4 cells implies that there is a cytosolic stress that was not present in HepG2 incubations of OD-LAP. SOD1 and SOD2 are located in the cytoplasm and the mitochondrial matrix, respectively.29

We believe the concentrations of LAP tested in the reported studies are physiologically relevant and that some level of mitochondrial impairment in LAP-treated patients is likely. While LAP concentrations in human liver under the standard dosing schedule are not known, imaging studies using [14C]LAP in male rats demonstrated a high degree of LAP accumulation in the liver and reduced concentrations in the brain when analyzed by whole-body autoradiograph.30 Similarly the P450 3A4 activity in the transduced HepG2 cells used in this study were only three-times the average level seen previously in cryopreserved hepatocytes.21 Given the high degree of variability in P450 3A4 activity between individuals,31 the HepG2-3A4 cells are a reasonable model to study the metabolic activation and associated toxicity of LAP in cells.



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Eno Page 22

Despite evidence of significant mitochondrial impairment and elevated liver enzymes after a single dose in mouse, LAP demonstrated an acceptable safety profile and sufficient therapeutic benefit for approval as a cancer therapy. The potential of LAP or its metabolite (OD-LAP) to upregulate cytoprotective pathways through Nrf2 activation was evaluated. The Nrf2-ARE pathway can sense and respond to chemical and oxidative stress, detoxifying electrophiles and ameliorating their effect. OD-LAP was shown to activate Nrf2 in a cell-based reporter assay and by quantitative RT-PCR, whereas LAP only activated Nrf2 in cells transfected with P450 3A4. Increased mRNA for Nrf2 target genes was observed after 4 hours in the cell-based assays, but not at earlier time points. Additional time would be expected for the translation of Nrf2 targets to take effect. This delay may explain why significant signs of hepatotoxicity were observed after a single LAP dose, but during multi-day dosing in which Nrf2 target genes are likely upregulated prior to the daily dose, LAP did not appear to cause hepatic necrosis, despite very high liver concentrations of both LAP and OD-LAP.

The decreased plasma levels of both LAP and OD-LAP between days one and five suggest the additional possibility of non-Nrf2 mediated enzyme induction. Because the decrease in LAP and LAPQI concentration is similar between days one and five in the Nrf2-KO and in the control mice, but only the control mice exhibit reduced hepatic damage upon repeat dosing, we do not believe that the decreased drug concentrations are significant factors in hepatic protection.

After the initial dose, Nrf2 target genes are likely to decrease cellular damage caused by subsequent doses. This may have an important tie in with immune-mediated hepatotoxicity through reducing the intensity of the danger associated molecular patterns required to produce an immune response. Nrf2 would not be expected to play a role in the development of immune tolerance, which appears to be a driving factor in immune-mediated hepatotoxicity.32-34

The results of the current investigation support the hypothesis that LAP is a mitochondrial toxin and that its hepatocellular toxicity is mainly linked to P450 3A4-catalyzed generation of ODLAP, which is further oxidized to form the reactive electrophile LAPQI. These findings may


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Eno Page 23 explain the liver-related toxicity observed in LAP-treated breast cancer patients. The study illustrates the usefulness of HepG2 cells transduced with P450 3A4 for generating P450 3A4dependent metabolites to investigate metabolism-induced toxicity. In addition, we have highlighted the importance of Nrf2 activation in the protection against LAP-induced toxicity.

Supporting Information PCR primers and a detailed analytical method for quantitation of LAP are included in the supporting information section. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Information Corresponding author: Michael Cameron, Email: [email protected]

Funding The research was partly supported by NIH grant 1S10OD010603 (MC).

Notes The authors declare no competing financial interest.

Acknowledgements The authors would like to thank Li Lin, Susan Khan and Marie Lafitte for technical support on the project.

Abbreviations LAP, lapatinib; OD-LAP, O-dealkylated lapatinib; Nrf2, nuclear factor erythroid 2-related factor c2; NQO-1, NAD(P)H quinone oxidoreductase 1; GST, glutathione S-transferase; UGT, uridine 5'-diphospho-glucuronosyltransferases; GS, glutathione synthetase; GCL, glutamate-cysteine ligase; FRET, fluorescence resonance energy transfer; qRTPCR- Quantitative Reverse-



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Eno Page 24 Transcriptase Polymerase Chain Reaction; FDA, food and drug administration; HLA, human leukocyte antigen; LAPQI, lapatinib-quinone imine; ARE, antioxidant response element; Nrf2KO, Nrf-2 knockout; wild type, wt; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; BSO, L-Buthionine sulfoximine; OCR, oxygen consumption rate; GFP, green fluorescent protein; SOD, superoxide dismutase; ULN, upper limit of normal.


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Eno Page 25

References (1)

Stirnimann, G., Kessebohm, K., and Lauterburg, B. (2010) Liver injury caused by drugs: an update. Swiss Med. Wkly. 140, 18-24.

(2)

Uetrecht, J., and Naisbitt, D. J. (2013) Idiosyncratic adverse drug reactions: current concepts. Pharmacol. Rev. 65, 779-808.

(3)

Rusnak, D. W., Affleck, K., Cockerill, S. G., Stubberfield, C., Harris, R., Page, M., Smith, K. J., Guntrip, S. B., Carter, M. C., Shaw, R. J., Jowett, A., Stables, J., Topley, P., Wood, E. R., Brignola, P. S., Kadwell, S. H., Reep, B. R., Mullin, R. J., Alligood, K. J., Keith, B. R., Crosby, R. M., Murray, D. M., Knight, W. B., Gilmer, T. M., and Lackey, K. (2001) The characterization of novel, dual ErbB-2/EGFR, tyrosine kinase inhibitors: Potential therapy for cancer. Cancer Res. 61, 7196-7203.

(4)

Xia, W. L., Mullin, R. J., Keith, B. R., Liu, L. H., Ma, H., Rusnak, D. W., Owens, G., Alligood, K. J., and Spector, N. L. (2002) Anti-tumor activity of GW572016: a dual tyrosine kinase inhibitor blocks EGF activation of EGFR/erbB2 and downstream Erk1/2 and AKT pathways. Oncogene 21, 6255-6263.

(5)

Moy, B., Rappold, E., Williams, L., Kelly, T., Nicolodi, L., Maltzman, J. D., and Goss, P. E. (2009) Hepatobiliary abnormalities in patients with metastatic cancer treated with lapatinib. J. Clin. Oncol. 27, 15s.

(6)

Gomez, H. L., Doval, D. C., Chavez, M. A., Ang, P. C. S., Aziz, Z., Nag, S., Ng, C., Franco, S. X., Chow, L. W. C., Arbushites, M. C., Casey, M. A., Berger, M. S., Stein, S. H., and Sledge, G. W. (2008) Efficacy and safety of lapatinib as first-line therapy for ErbB2-amplified locally advanced or metastatic breast cancer. J. Clin. Oncol. 26, 29993005.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 47

Eno Page 26 (7)

Cristofanilli, M., Johnston, S. R. D., Manikhas, A., Gomez, H. L., Gladkov, O., Shao, Z. M., Safina, S., Blackwell, K. L., Alvarez, R. H., Rubin, S. D., Ranganathan, S., Redhu, S., and Trudeau, M. E. (2013) A randomized phase II study of lapatinib plus pazopanib versus lapatinib in patients with HER2+ inflammatory breast cancer. Breast Cancer Res. Tr. 137, 471-482.

(8)

Schaid, D. J., Spraggs, C. F., McDonnell, S. K., Parham, L. R., Cox, C. J., Ejlertsen, B., Finkelstein, D. M., Rappold, E., Curran, J., Cardon, L. R., and Goss, P. E. (2014) Prospective validation of HLA-DRB1*07:01 allele carriage as a predictive risk factor for lapatinib-induced liver injury. J. Clin. Oncol. 32, 2296-2303.

(9)

Spraggs, C. F., Budde, L. R., Briley, L. P., Bing, N., Cox, C. J., King, K. S., Whittaker, J. C., Mooser, V. E., Preston, A. J., Stein, S. H., and Cardon, L. R. (2011) HLADQA1*02:01 Is a Major Risk Factor for Lapatinib-Induced Hepatotoxicity in Women With Advanced Breast Cancer. J. Clin. Oncol. 29, 667-673.

(10)

Spraggs, C. F., Budde, L. R., Briley, L. P., Cox, C., Rappold, E., Aktan, G., Williams, L. S., Mooser, V. E., and Cardon, L. R. (2011) HLA-DRB1*07:01-DQA1*02:01 and UGT1A1*28 allele carriage in hepatic serious adverse event cases identified during lapatinib clinical trials. J. Clin. Oncol. 29.

(11)

Castellino, S., O'Mara, M., Koch, K., Borts, D. J., Bowers, G. D., and MacLauchlin, C. (2012) Human Metabolism of Lapatinib, a Dual Kinase Inhibitor: Implications for Hepatotoxicity. Drug Metab. Dispos. 40, 139-150.

(12)

Teng, W. C., Oh, J. W., New, L. S., Wahlin, M. D., Nelson, S. D., Ho, H. K., and Chan, E. C. Y. (2010) Mechanism-Based Inactivation of Cytochrome P450 3A4 by Lapatinib. Mol. Pharmacol. 78, 693-703.


Page 27 of 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Eno Page 27 (13)

Chan, E. C. Y., New, L. S., Chua, T. B., Yap, C. W., Ho, H. K., and Nelson, S. D. (2012) Interaction of Lapatinib with Cytochrome P450 3A5. Drug Metab. Dispos. 40, 14141422.

(14)

Park, B. K., Kitteringham, N. R., Maggs, J. L., Pirmohamed, M., and Williams, D. P. (2005) The role of metabolic activation in drug-induced hepatotoxicity. Annu Rev. Pharmacol. 45, 177-202.

(15)

Dykens, J. A., and Will, Y. (2007) The significance of mitochondrial toxicity testing in drug development. Drug Discov. Today 12, 777-785.

(16)

Dykens, J. A., Marroquin, L. D., and Will, Y. (2007) Strategies to reduce late-stage drug attrition due to mitochondrial toxicity. Expert Rev. Mol. Diagn. 7, 161-175.

(17)

Takakusa, H., Masumoto, H., Mitsuru, A., Okazaki, O., and Sudo, K. (2008) Markers of electrophilic stress caused by chemically reactive metabolites in human hepatocytes. Drug Metab. Dispos. 36, 816-823.

(18)

Baird, L., and Dinkova-Kostova, A. T. (2011) The cytoprotective role of the Keap1-Nrf2 pathway. Arch. Toxicol. 85, 241-272.

(19)

West, G. B., and Brown, J. H. (2005) The origin of allometric scaling laws in biology from genomes to ecosystems: towards a quantitative unifying theory of biological structure and organization. J. Exp. Biol. 208, 1575-1592.

(20)

Wilkening, S., Stahl, F., and Bader, A. (2003) Comparison of primary human hepatocytes and hepatoma cell line HEPG2 with regard to their biotransformation properties. Drug Metab. Dispos. 31, 1035-1042.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 47

Eno Page 28 (21)

Li, X., Jeso, V., Heyward, S., Walker, G. S., Sharma, R., Micalizio, G. C., and Cameron, M. D. (2014) Characterization of T-5 N-oxide formation as the first highly selective measure of CYP3A5 activity. Drug Metab. Dispos. 42, 334-342.

(22)

Marroquin, L. D., Hynes, J., Dykens, J. A., Jamieson, J. D., and Will, Y. (2007) Circumventing the Crabtree effect: replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants. Toxicol. Sci. 97, 539-547.

(23)

Hardy, K. D., Wahlin, M. D., Papageorgiou, I., Unadkat, J. D., Rettie, A. E., and Nelson, S. D. (2014) Studies on the role of metabolic activation in tyrosine kinase inhibitordependent hepatotoxicity: induction of CYP3A4 enhances the cytotoxicity of lapatinib in HepaRG cells. Drug Metab. Dispos. 42, 162-171.

(24)

Andrade, R. J., Lucena, M. I., Alonso, A., Garcia-Cortes, M., Garcia-Ruiz, E., Benitez, R., Fernandez, M. C., Pelaez, G., Romero, M., Corpas, R., Duran, J. A., Jimenez, M., Rodrigo, L., Nogueras, F., Martin-Vivaldi, R., Navarro, J. M., Salmeron, J., de la Cuesta, F. S., and Hidalgo, R. (2004) HLA class II genotype influences the type of liver injury in drug-induced idiosyncratic liver disease. Hepatology 39, 1603-1612.

(25)

Lin, D., Saleh, S., and Liebler, D. C. (2008) Reversibility of Covalent Electrophile Protein Adducts and Chemical Toxicity. Chem. Res. Toxicol. 21, 2361-2369.

(26)

Boelsterli, U. A., and Lim, P. L. (2007) Mitochondrial abnormalities--a link to idiosyncratic drug hepatotoxicity? Toxicol. Appl. Pharmacol. 220, 92-107.

(27)

Feng, B., Xu, J. J., Bi, Y. A., Mireles, R., Davidson, R., Duignan, D. B., Campbell, S., Kostrubsky, V. E., Dunn, M. C., Smith, A. R., and Wang, H. F. (2009) Role of hepatic transporters in the disposition and hepatotoxicity of a HER2 tyrosine kinase inhibitor CP724,714. Toxicol. Sci. 108, 492-500.


Page 29 of 47

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Eno Page 29 (28)

Rodriguez-Enriquez, S., Juarez, O., Rodriguez-Zavala, J. S., and Moreno-Sanchez, R. (2001) Multisite control of the Crabtree effect in ascites hepatoma cells. Eur. J. Biochem. 268, 2512-2519.

(29)

Pereira, C. V., Nadanaciva, S., Oliveira, P. J., and Will, Y. (2012) The contribution of oxidative stress to drug-induced organ toxicity and its detection in vitro and in vivo. Expert Opin. Drug Metab. Toxicol. 8, 219-237.

(30)

Polli, J. W., Humphreys, J. E., Harmon, K. A., Castellino, S., O'Mara, M. J., Olson, K. L., John-Williams, L. S., Koch, K. M., and Serabjit-Singh, C. J. (2008) The role of efflux and uptake transporters in N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5({[2(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (GW572016, Lapatinib) disposition and drug interactions. Drug Metab. Dispos. 36, 695-701.

(31)

Parkinson, A., Mudra, D. R., Johnson, C., Dwyer, A., and Carroll, K. M. (2004) The effects of gender, age, ethnicity, and liver cirrhosis on cytochrome P450 enzyme activity in human liver microsomes and inducibility in cultured human hepatocytes. Toxicol. Appl. Pharm. 199, 193-209.

(32)

Mak, A., and Uetrecht, J. (2015) The Combination of Anti-CTLA-4 and PD1-/- Mice Unmasks the Potential of Isoniazid and Nevirapine To Cause Liver Injury. Chem. Res. Toxicol. 28, 2287-2291.

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Metushi, I. G., Cai, P., Dervovic, D., Liu, F., Lobach, A., Nakagawa, T., and Uetrecht, J. (2015) Development of a novel mouse model of amodiaquine-induced liver injury with a delayed onset. J. Immunotoxicol. 12, 247-260.

(34)

Uetrecht, J., and Kaplowitz, N. (2015) Inhibition of immune tolerance unmasks druginduced allergic hepatitis. Hepatology 62, 346-348.



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Table 1: LAP distribution in C57Bl/6 and Nrf-2 KO mice in plasma and liver on Day 1 and Day 5a Mice

C57Bl/6

Analyte

LAP OD-LAP

Nrf-2 KO

LAP OD-LAP

Plasma concentration (µM)

Liver (µM)

Day 1

Day 5

Day 5

27.3 ± 12.3

19.1 ± 5.6

2620 ± 260

5.6 ± 2.1

4.3 ± 1.0

463 ± 49

33.7 ± 11.0

14.6 ± 2.6

3290 ± 550

6.6 ± 1.9

3.7 ± 0.3

594 ± 103

a

Eight hours after a single 400 mg/kg IP dose in C57Bl/6 and Nrf-2 KO mice. Tissues were not perfused to assure that LAP was not washed out during perfusion.


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Eno Page 31 Legends Figure1. Reactive metabolites of lapatinib.

Figure 2. Effect of OD-LAP on OCR in HepG2 cells. (A) OCR after 6 h and 24 h exposure of 30 µM OD-LAP. (B) Basal respiration and maximum respiration (FCCP) after 6 h and 24 h exposure of 30 µM OD-LAP. (C) Concentration dependent decrease in NAD(P)H levels after 24 hour OD-LAP treatment relative to DMSO control (set as 100%). (D) Cell viability after 24 hour exposure of OD-LAP as measured by membrane integrity. (E) Concentration dependence of ODLAP on OCR after 24 hour treatment. (F) Effect of a 24 h pre-treatment of HepG2 cells with 50 µM BSO followed by a 24 h treatment with 20 µM OD-LAP. Data represent the mean ±SD of triplicate values. *P < 0.05; **P < 0.01; ***P < 0.001 compared with vehicle (unpaired t test, two-tailed P values).

Figure 3. Effect of LAP on OCR in HepG2 cells. (A) OCR after 24 h exposure of 10 µM LAP. (b) NAD(P)H levels after 24 hour LAP treatment relative to DMSO control.

Figure 4. Effects of LAP and OD-LAP treatment in HepG2-3A4 cells. (A) 1´-OH midazolam formation in HepG2 with or without viral transduction with GFP, P450 3A4, or P450 3A5. (B) OCR after 24 hour exposure of 10 µM and 30 µM OD-LAP in HepG2-3A4 cells. (C) Concentration dependent decrease in NAD(P)H levels after 24 hour LAP treatment in P450 3A4 transduced HepG2 cells relative to DMSO control. (D) Cell viability of HepG2-3A4 cells after 24 hour exposure of LAP. (E) Cellular ATP depletion in the presence of various concentrations of LAP (10 µM, 5 µM, 2.5 µM, 1.25 µM and 625 nM) and OD-LAP (50 µM, 25 µM, 12.5 µM, 6.25 µM, 3.125 µM and 781 nM) in HepG2-3A4 cells in galactose containing media. (F) Cell viability of HepG2-3A4 cells after 24 hour exposure of OD-LAP. Data represent the mean ±SD of triplicate values. *P < 0.05; **P < 0.01; ***P < 0.001 compared with vehicle (unpaired t test, two-tailed P values).

Figure 5. Quantitative RT-PCR of SOD1 and SOD2. SOD1 and SOD2 are associated with the cytoplasm and the mitochondrial matrix, respectively. Relative expression levels of SOD1 and SOD2 mRNA in HepG2 cells after exposure to (A) LAP and (B) OD-LAP and in HepG2-3A4



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Eno Page 32 cells, (C) LAP and (D) OD-LAP, for 2 h and 4 h is depicted. Data represent the mean ±SD of triplicate values. *P < 0.05; **P < 0.01; compared with vehicle (unpaired t test, two-tailed P values).

Figure 6. Nrf2 activation in HepG2 and transduced HepG2 cells with or without hP450 3A4 or 3A5 measured by the FRET-based LiveBLAzerTM ARE-bla β-lactamase reporter assay. Cells were treated with compounds for 15 h and the response ratios were plotted against drug concentration. (A) ARE-bla β-lactamase reporter assay measuring Nrf2 activity in HepG2 cells treated with THBQ (+ control), OD-LAP, and LAP. (B) 1-OH-Midazolam formation in ARE-bla HepG2 with or without viral transduction with GFP, P450 3A4, or P450 3A5. (C) ARE-bla βlactamase reporter assay measuring Nrf2 activity in HepG2 cells transduced with P450 3A4 or 3A5 that were treated with THBQ (+) control, OD-LAP or LAP.

Figure 7. Quantitative RT-PCR - HepG2 cells. mRNA expression of (A) HO-1 (B) NQO-1 in HepG2 cells after exposure to LAP and OD-LAP for 2 h and 4 h. Data represent the mean ±SD of triplicate values. *P < 0.05; **P < 0.01; compared with vehicle (unpaired t test, two-tailed P values).

Figure 8. Quantitative RT-PCR - HepG2-3A4 cells. mRNA expression of (A) HO-1 and (B) NQO-1 in HepG2 cells transduced with hP450 3A4 after exposure to LAP for 4 h. Data represent the mean ±SD of triplicate values. *P < 0.05; **P < 0.01 compared with vehicle (unpaired t test, two-tailed P values).

Figure 9. Multi-day evaluation of hepatic toxicity in C57BL/6 and Nrf2 knockout mice. ALT (A) and AST (B) levels in wild-type C57Bl6 and Nrf2-KO mice were measured after 1 and 5 days dosing (400 mg/kg LAP, IP). Values are representative of the mean (n = 4) +/- SEM. *P < 0.05; **P < 0.01; comparison made between mouse strains (unpaired t test, two-tailed P values).


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Eno Page 33 Figure 1



Eno Page 34

h) (2 4 D -L A P

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Eno Page 35

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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Eno Page 39



Eno Page 40

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Eno Page 41

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Eno Page 42

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Eno Page 44


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Figure 8


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rf 2K N

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P450 3A-Catalyzed O-Dealkylation of Lapatinib ... - ACS Publications

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