Originally published as JCO Early Release 10.1200/JCO.2007.14.4956 on April 7 2008

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Adjei, A. A. Articles by Eckhardt, S. G. Search for Related Content PubMed PubMed Citation Articles by Adjei, A. A. Articles by Eckhardt, S. G. Social Bookmarking                            What's this?

Phase I Pharmacokinetic and Pharmacodynamic Study of the Oral, Small-Molecule Mitogen-Activated Protein Kinase Kinase 1/2 Inhibitor AZD6244 (ARRY-142886) in Patients With Advanced Cancers

Alex A. Adjei, Roger B. Cohen, Wilbur Franklin, Clive Morris, David Wilson, Julian R. Molina, Lorelei J. Hanson, Lia Gore, Laura Chow, Stephen Leong, Lara Maloney, Gilad Gordon, Heidi Simmons, Allison Marlow, Kevin Litwiler, Suzy Brown, Gregory Poch, Katie Kane, Jerry Haney, S. Gail Eckhardt

From the Mayo Clinic, Rochester, MN; Fox Chase Cancer Center, Philadelphia, PA; University of Colorado Health Sciences Center, Denver; Array BioPharma Inc, Boulder, CO; and AstraZeneca, Alderley Park, United Kingdom

Corresponding author: Alex A. Adjei, MD, PhD, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263; e-mail: alex.adjei{at}roswellpark.org

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Purpose To assess the tolerability, pharmacokinetics (PKs), and pharmacodynamics (PDs) of the mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor AZD6244 (ARRY-142886) in patients with advanced cancer.

Patients and Methods In part A, patients received escalating doses to determine the maximum-tolerated dose (MTD). In both parts, blood samples were collected to assess PK and PD parameters. In part B, patients were stratified by cancer type (melanoma v other) and randomly assigned to receive the MTD or 50% MTD. Biopsies were collected to determine inhibition of ERK phosphorylation, Ki-67 expression, and BRAF, KRAS, and NRAS mutations.

Results Fifty-seven patients were enrolled. MTD in part A was 200 mg bid, but this dose was discontinued in part B because of toxicity. The 50% MTD (100 mg bid) was well tolerated. Rash was the most frequent and dose-limiting toxicity. Most other adverse events were grade 1 or 2. The PKs were less than dose proportional, with a median half-life of approximately 8 hours and inhibition of ERK phosphorylation in peripheral-blood mononuclear cells at all dose levels. Paired tumor biopsies demonstrated reduced ERK phosphorylation (geometric mean, 79%). Five of 20 patients demonstrated 50% inhibition of Ki-67 expression, and RAF or RAS mutations were detected in 10 of 26 assessable tumor samples. Nine patients had stable disease (SD) for 5 months, including two patients with SD for 19 (thyroid cancer) and 22 (uveal melanoma plus renal cancer) 28-day cycles.

Conclusion AZD6244 was well tolerated with target inhibition demonstrated at the recommended phase II dose. PK analyses supported twice-daily dosing. Prolonged SD was seen in a variety of advanced cancers. Phase II studies are ongoing.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Mitogen-activated protein kinase kinase (MEK or MAPK/ERK kinase) is a critical enzyme in the RAS/RAF/MEK/ERK pathway that regulates key cellular activities including proliferation, survival, and cell cycle regulation. This pathway is composed of a protein kinase cascade in which RAF, MEK, and ERK are in a sequential order.

MEK1/2 are attractive therapeutic targets because their only known substrates are ERK1/2. MEK inhibitors inhibit growth of human tumors in mouse xenografts^1-7 and leukemia cells in vitro.⁸ Two other MEK inhibitors have been tested in clinical trials. CI-1040 showed insufficient antitumor activity to warrant further development,⁹ and development of a second-generation MEK inhibitor, PD0325901,¹⁰ has recently been discontinued.¹¹ AZD6244 is a potent, selective, adenosine triphosphate–uncompetitive inhibitor of MEK1/2, with an in vitro half maximal inhibitory concentration of 10 to 14 nmol/L against purified enzyme and no inhibition up to 10 µmol/L against numerous other serine/threonine and tyrosine kinases.⁴ AZD6244 has excellent preclinical activity against many different tumors in cell-based growth assays and in human tumor mouse xenograft models, including colorectal,^4,6 pancreatic,⁴ non–small-cell lung,⁶ and hepatocellular cancer⁵ and melanoma.⁷

Given this spectrum of preclinical activity⁴ and the acceptable toxicology profile, a phase I study was undertaken to evaluate the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of AZD6244 in patients with advanced malignancies.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Patient Selection
Eligibility criteria included patients aged 18 years with histologic or cytologic evidence of advanced cancer for which there was no curative or life-prolonging therapy; Eastern Cooperative Oncology Group performance status 2; prior radiation completed 3 weeks before study enrollment; life expectancy of 12 weeks; and adequate bone marrow (platelets 100,000/µL, absolute neutrophil count > 1,500/µL, and hemoglobin 9 g/dL), hepatic (total bilirubin 2.5x the upper limit of normal and AST 2.5x normal), and renal (serum creatinine 1.5x the upper limit of normal) function. In part B, patients were required to have a tumor that was safely accessible for biopsy. All patients gave written informed consent.

Experimental Treatment
This phase I, open-label, multiple-dose study assessed the safety, tolerability, PK, and PD of AZD6244 in patients with advanced solid malignancies. AZD6244 was formulated as an oral powder for reconstitution and supplied in dosing kits in 30-mL amber bottles. Antiemetic prophylaxis was not administered.

Part A was conducted to determine the maximum-tolerated dose (MTD) and used a standard three- to six-patient cohort design¹² evaluating doses of 50, 100, 200, and 300 mg bid. The incidence and severity of adverse events were evaluated and coded according to National Cancer Institute Common Terminology Criteria of Adverse Events (version 3). Response to therapy was monitored by modified Response Evaluation Criteria in Solid Tumors.¹³ AZD6244-related dose-limiting toxicity (DLT) was defined as follows: any grade 4 toxicity (grade 4 neutropenia for > 7 days), grade 3 or 4 neutropenia with fever, grade 3 or 4 thrombocytopenia associated with bleeding (excluding patients receiving systemic anticoagulation), or any grade 3 or 4 nonhematologic toxicity. Grade 2 vomiting on 2 consecutive days despite optimal antiemetic therapy was considered dose limiting, as was any grade 2 toxicity lasting for more than 2 weeks or dosing interruption of more than 2 weeks for drug-related toxicity. The MTD was defined as one dose level below that which induced DLT in more than one third of patients (at least two of a maximum of six patients). Each patient began the study with a single dose of AZD6244 on day 1, with assessment of adverse events on days 1, 2, and 3. If there were no DLTs through day 8, continuous bid dosing commenced. A cycle was defined as 28 days of twice-daily therapy.

In part B, patients were stratified by cancer type (melanoma v other) and randomly assigned to receive the MTD (200 mg bid) or 50% of the MTD dose (100 mg bid) to evaluate the dose that provided the best balance of safety/tolerability and PD effect for future clinical development. Tissue samples (tumor and normal skin) were obtained for PD assessments before dose and after 7 to 21 days of AZD6244 (day 15 ± 7 days). Patients must have taken the assigned dose uninterrupted for 7 days before the postdose biopsy.

Clinical Care of Patients
In the single-dose phase of part A, physical examinations, toxicity assessments, and laboratory analyses were conducted on days 1, 2, and 3. In the bid dosing phase, weekly assessments commenced on day 8 of the first 28-day cycle. ECG and PK assessments were conducted on day 22. In part B, assessments were conducted weekly in cycle 1 and every 28 days in subsequent cycles. Patients could continue on uninterrupted 28-day cycles of AZD6244 provided that there was no disease progression or unacceptable toxicity.

PD Analysis
Blood samples were collected on days 1 and 22 in part A and days 1 and 15 in part B before dose and 1 hour after dose for measurement of pERK levels by fluorescence-activated cell sorting analysis. Samples were treated ex vivo with 12-O-tetradecanoylphorbol-13-acetate for 10 minutes at 37°C within 1 hour of being drawn. ERK phosphorylation was preserved by immediate fixation of the cells with 1.2% methanol-free formaldehyde. Peripheral-blood mononuclear cells (PBMCs) were isolated, washed, and stored at –20°C. For analysis of ERK phosphorylation, cells were treated with an antibody to pERK, followed by a fluorescein isothiocyanate–conjugated secondary detection antibody and pERK quantitation by fluorescence-activated cell sorting analysis.

PK Analysis
Maximum observed plasma concentration (C_max) and median observed time to maximum plasma concentration values for each patient were derived from the plasma concentration-time profile, and the area under the time-concentration curve (AUC_{0-24 hours}) was calculated using the linear trapezoidal rule (for details, see Appendix, online only).

Skin and Tumor Biopsy Sample Collection
Tissue samples (tumor and normal skin) were obtained for PD assessments before dose and after 7 to 21 days of AZD6244 (day 15 ± 7 days). The day 15 (± 7 days) postdose tumor and normal skin biopsies were collected 2 to 4 hours after dose on the same day as PK and PD assessments. Tumor biopsies (18-guage core needle) were taken using computed tomography or ultrasound scan guidance. Samples were fixed and stained with hematoxylin and eosin to confirm the diagnosis and the quality of the biopsy tissue. For optimal comparative biomarker studies, subsequent biopsies were taken from the same site as the screening biopsy. Skin biopsies were taken from the upper arm or buttocks using a 3- to 4-mm punch, using the same fixation method.

Immunohistochemistry
An indirect immunoperoxidase method, with antibodies against pERK1/2 or Ki-67, was used to evaluate pERK status and growth fraction (Ki-67) in situ. Negative and positive controls were included in each immunostained batch of slides. In all cases, these controls stained appropriately. Slides were scored, and representative microscopic fields were photographed. Nuclei and cytoplasm were scored for pERK by estimating the proportion of positive viable tumor cells multiplied by intensity of staining quantified on a 0 to 4+ scale. The proportion of tumor cell nuclei staining for Ki-67 was estimated by microscopic inspection in 10% increments. Only viable tumor was scored, with care taken to avoid necrotic areas of tumor.

Tumor DNA Mutation Analysis
Tumor tissue sections were isolated from paraffin-embedded sample blocks using a 1-mm array punch. Samples were washed and air dried, and DNA was extracted from fixed tissue. Analyses for KRAS, NRAS, and BRAF mutations were performed by established methods (see Appendix).

Statistical Evaluation
Safety data were summarized using appropriate descriptive statistics. Baseline scaled ratios were calculated for each assessable pair of biopsies (corresponding to the postdose/predose value). Because the data were treated as being multiplicative, geometric means (gmean) were calculated to give an overall mean level of inhibition, and corresponding CIs were calculated for these mean levels of inhibition.

Correlations of markers between tumor and skin samples were assessed using the Spearman rank correlation coefficient. Differences in time on study between patients who had an oncogene mutation at baseline and those who did not were assessed using a Wilcoxon signed rank test, as were differences in biomarker inhibition between patients with and without the mutation.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Fifty-seven patients (35% malignant melanoma; Table 1) received a total of 184 assessable cycles of therapy across four dose levels. The median number of cycles administered per patient was two (range, one to 22 cycles). Other baseline patient characteristics are listed in Table 1.

Rash. Rash was the most frequent toxicity and DLT, occurring in 74% of all patients, and precluded dose escalation greater than 300 mg bid. The rash was dose dependent, erythematous, and maculopapular, occurring predominantly on the torso. Resolution typically occurred with dosing interruption and/or dose reduction. In part B, an increase in frequency and severity of this rash led to selection of 100 mg bid as the tolerable phase II dose. Of the 43 episodes of skin rash, 34 were of maximum grade 1 or 2, and nine were grade 3 or 4.

GI toxicity. Mild to moderate diarrhea was the principal GI toxicity (56% of patients). Abdominal examination during the diarrhea episodes was benign. Diarrhea resolved promptly with loperamide therapy and/or drug discontinuation. In addition to diarrhea, nausea (n = 25) and vomiting (n = 14) were observed, which resolved quickly and completely with antiemetic therapy.

Edema. Mild to moderate edema occurred in 19 of 57 patients, whereas severe edema occurred in one patient with pre-existing abdominal distension from ascites.

Fatigue. Fatigue was dependent on dose and duration of treatment and mild to moderate in 20 of 22 patients. It was reversible with dose reduction and/or interruption.

Other toxicities. Mild to moderate reversible ALT and AST elevation occurred in 14% and 14% of patients, respectively. Blurred vision, which was transient and reversible, occurred in 12% of patients. These events were all grade 1 or 2. Eight patients (14%) experienced serious adverse events, including hypoxia, pneumonitis, bradycardia, renal insufficiency, and exfoliative dermatitis.

Dose reductions and study discontinuation. Seven patients (12%) required dose reductions for treatment-related toxicity, 24 patients (42%) required drug holidays of up to 2 weeks, and eight patients (14%) discontinued treatment for drug-related toxicity. On the basis of these results, the MTD and recommended dose of AZD6244 as an oral powder for reconstitution formulation for subsequent clinical testing is 100 mg bid.

PK
After a single dose of AZD6244, the median terminal half-life was 8.3 hours. C_max increased with increasing dose (Tables 4 and 5). The mean area under the plasma concentration-time curve (AUC_inf) after single doses of AZD6244 also increased with increasing dose. Similarly, the steady-state AUC over the 12-hour dosing interval (AUC_{0-12 hours}) increased to a maximum at 200 mg bid. In part B, the median observed time to maximum plasma concentration was 1 hour after dose. The mean single-dose C_max values for the 100-mg and 200-mg cohorts were similar to the respective steady-state (day 15) C_max values. In both parts, the single-dose and steady-state AUC values increased with increasing dose in a less than dose-proportional manner (Tables 4 and 5). In part B, however, it is likely that the median terminal half-life (4.7 hours) is an underestimate because of the shorter PK sampling schedule, which ended at 12 hours after dose (before the evening dose).

Inhibition of ERK phosphorylation and Ki-67 labeling index in tumor biopsies. After documenting target inhibition in a surrogate tissue in part A, paired tumor samples were collected before treatment and after at least 7 continuous days of treatment and evaluated for inhibition of ERK phosphorylation by immunohistochemistry. We also evaluated drug effects on downstream signaling events by examining the reduction in the Ki-67 labeling index, a marker of cell proliferation. Figure 1 shows representative immunohistochemistry photomicrographs. Twenty of the 24 paired biopsies were assessable, with 19 having detectable pretreatment pERK expression and all 20 having detectable pretreatment Ki-67 expression. Strong inhibition of ERK phosphorylation was seen with a gmean inhibition of 79% (90% CI, 50% to 91%; Fig 2A). Ki-67 labeling was reduced in post-treatment tumor samples but not as consistently as pERK, the primary proof-of-mechanism biomarker. Nine of 20 samples showed some reduction, with 50% reduction in five samples (Fig 2B). The skin biopsies were generally uninformative because of variable and minimal baseline levels of pERK.

View larger version (96K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Immunostains of pre- and post-treatment melanoma specimens from the same patient. (A) Before dose, tumor cells are reactive to anti-pERK antibody (brown staining; magnification, x100). (B) After dose, cells are unreactive to same anti-pERK antibody (magnification, x100). (C) Before dose, variable nuclear Ki-67 labeling (approximately 30% positive nuclei; magnification, x400). (D) After dose, marked reduction in nuclear Ki-67 labeling (< 1% positive nuclei; magnification, x400).

View larger version (17K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. Percent change from baseline at day 15 (± 7 days) in (A) tumor cell nuclei H-score for pERK and (B) proportion of tumor cell nuclei staining for Ki-67. Patients received 100 mg bid or 200 mg bid (denoted by *).

The average length of time on study for patients carrying mutations (median, 3.5 months; range, 1 to 6 months) was greater than for those without a mutation (median, 2 months; range, 1 to 4 months). There is no statistical evidence of effect (P = .30 by Wilcoxon signed rank test) in this small sample. Four of the 10 patients with a mutation had tumor biopsies assessable for the pERK assay, which showed strong inhibition of ERK phosphorylation (100%, 100%, 83%, and 25%). These four patients, plus one other patient with a mutation, had tissue assessable for Ki-67 labeling and showed a strong labeling index (100%, 97%, 92%, 88%, and 33%). Possibly because of the small numbers in the study, there was no significant difference between biomarker knockdown for those patients with mutation versus those without mutation or with unknown mutation status (pERK: P = .13; Ki-67: P = .13). Of note, three patients showing the strongest reduction in Ki-67 labeling were all mutation positive.

Antitumor Activity
Figure 3 summarizes tumor responses by Response Evaluation Criteria in Solid Tumors. Nineteen patients (33%) had stable disease (SD) at the end of cycle 2, and nine patients (16%) had SD for 5 months. One patient with medullary thyroid cancer experienced SD for 19 cycles, whereas one patient with both uveal melanoma and renal cell carcinoma had SD for 22 cycles.

View larger version (16K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 3. Best percent change from baseline in target lesion size for patients who have at least postbaseline efficacy assessment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

Despite a growing clinical literature on MEK inhibitors, there is only limited evidence to date that MEK can be inhibited consistently in patient tumors at tolerable inhibitor doses. In addition, it is unclear whether such inhibition correlates with clinical outcome and whether MEK inhibition in surrogate tissues corresponds to MEK inhibition in tumors. Accordingly, we determined whether tolerable doses of AZD6244 would inhibit MEK in PBMCs, skin, and patient tumors. Skin biopsies were generally uninformative because of the variable and minimal baseline levels of pERK. We observed a dose-dependent inhibition of ERK phosphorylation in PBMCs, as well as consistent inhibition of ERK phosphorylation when comparing pre- and post-treatment tumor biopsies, but there were insufficient data to suggest a correlation between surrogate tumor tissue PD. We also demonstrated inhibition of Ki-67 in patient tumors, but again, there were insufficient data to conclude whether PBMC samples are suitable surrogate tissues for tumor samples. Because activating mutations in NRAS, KRAS, and BRAF genes correlate in preclinical studies with sensitivity to MEK inhibitors, mutational analysis of these genes was performed in 26 available tumors. In this small sample size, there was a nonsignificant trend towards delayed progression on study in patients with mutations compared with wild-type tumors.

AZD6244 displayed less than dose-proportional PK with increasing C_max and AUC as doses increased from 50 to 300 mg bid. There was a high degree of interpatient variability, which is not surprising for an oral agent. No food effect study was performed, and no guidance for food intake was given except for PK assessments that were performed in the fasting state (1 hour before and 2 hours after dosing). The PK profile supports a bid dosing scheme that results in exposures that adequately inhibit the drug target.

The best clinical response was SD that lasted for 5 or more months in nine patients. Two patients maintained SD for 19 and 22 cycles. One patient with malignant melanoma had a 70% tumor shrinkage after three cycles of AZD6244 but developed symptomatic brain metastases before confirmatory scans could be performed. This patient had an NRAS mutation and showed 100% inhibition of ERK phosphorylation and 97% inhibition of Ki-67. Thus, the present phase I study provides preliminary evidence of antineoplastic activity in humans.

In summary, this study establishes that the MEK inhibitor AZD6244 has a manageable safety and tolerability profile and identifies a suitable dose for subsequent clinical trials (100 mg orally, twice daily continuously) that results in target inhibition. Although this study demonstrates that the MEK1/2 target can be safely inhibited in vivo in humans, our data also suggest that target inhibition may be necessary but not sufficient for antineoplastic activity. These findings support future clinical development of AZD6244, and phase II studies are in progress.

	AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a "U" are those for which no compensation was received; those relationships marked with a "C" were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Employment or Leadership Position: Clive Morris, AstraZeneca (C); David Wilson, AstraZeneca (C); Lara Maloney, Array BioPharma Inc (C); Heidi Simmons, Array BioPharma Inc (C); Allison Marlow, Array BioPharma Inc (C); Kevin Litwiler, Array BioPharma Inc (C); Suzy Brown, Array BioPharma Inc (C); Gregory Poch, Array BioPharma Inc (C); Katie Kane, Array BioPharma Inc (C) Consultant or Advisory Role: Alex A. Adjei, Array BioPharma Inc (C); Roger B. Cohen, Array BioPharma Inc (C); Gilad Gordon, Array BioPharma Inc (C); S. Gail Eckhardt, AstraZeneca (C), Array BioPharma Inc (C) Stock Ownership: Clive Morris, AstraZeneca; David Wilson, AstraZeneca; Lara Maloney, Array BioPharma Inc; Kevin Litwiler, Array BioPharma Inc; Gregory Poch, Array BioPharma Inc Honoraria: None Research Funding: Roger B. Cohen, Array BioPharma Inc, AstraZeneca; S. Gail Eckhardt, AstraZeneca Expert Testimony: None Other Remuneration: S. Gail Eckhardt, AstraZeneca, fellowship funding

	AUTHOR CONTRIBUTIONS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Conception and design: Alex A. Adjei, Clive Morris, David Wilson, Lara Maloney, Gilad Gordon, S. Gail Eckhardt

Administrative support: Alex A. Adjei, Katie Kane

Provision of study materials or patients: Roger B. Cohen, Lorelei J. Hanson, Lia Gore, Laura Chow, Lara Maloney, Gilad Gordon, S. Gail Eckhardt

Collection and assembly of data: Alex A. Adjei, Roger B. Cohen, Wilbur Franklin, Julian R. Molina, Laura Chow, Stephen Leong, Lara Maloney, Gilad Gordon, Heidi Simmons, Allison Marlow, Kevin Litwiler, Suzy Brown, Gregory Poch, Katie Kane, Jerry Haney, S. Gail Eckhardt

Data analysis and interpretation: Roger B. Cohen, Clive Morris, David Wilson, Julian R. Molina, Lia Gore, Laura Chow, Stephen Leong, Lara Maloney, Gilad Gordon, Heidi Simmons, Allison Marlow, Kevin Litwiler, Suzy Brown, Gregory Poch, S. Gail Eckhardt

Manuscript writing: Alex A. Adjei, Roger B. Cohen, Clive Morris, David Wilson, Julian R. Molina, Lara Maloney, Gilad Gordon, Heidi Simmons, Allison Marlow, S. Gail Eckhardt

Final approval of manuscript: Alex A. Adjei, Roger B. Cohen, Clive Morris, Julian R. Molina, Lia Gore, Lara Maloney, Gilad Gordon, Heidi Simmons, Allison Marlow, Kevin Litwiler, Katie Kane, S. Gail Eckhardt

	Appendix

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Patients and Methods
Experimental treatment. Pharmacokinetic (PK) and pharmacodynamic (PD) analyses of AZD6244 in patients with advanced solid malignancies were conducted at three clinical sites in the United States. AZD6244 was formulated as an oral powder for reconstitution and supplied in dosing kits containing 50, 100, 200, or 300 mg in 30-mL amber bottles.

Clinical care of patients. Complete patient histories, physical examinations, CBCs, chemistries, and urinalyses were performed at baseline and before each cycle of treatment. Within 21 days before start of dosing (part B only), and as clinically warranted, patients were also evaluated by 12-lead ECG, echocardiogram, or multiple-gated acquisition scan and oxygen saturation. Laboratory studies were repeated weekly. When appropriate, serologic tumor markers were assessed on day 29 of cycle 1 and day 28 of subsequent cycles. Radiologic studies were performed at baseline and after every two cycles of therapy to assess tumor response.

In the single-dose phase of part A, physical examinations, toxicity assessments, and laboratory analyses were conducted on days 1, 2, and 3. In the bid dosing phase, weekly assessments commenced on day 8 of the first 28-day cycle. ECG and PK assessments were conducted on day 22. Serologic tumor markers were assessed on day 36 in patients with appropriate tumors. In part B, assessments were conducted weekly in cycle 1 and every 28 days in subsequent cycles. When appropriate, serologic tumor markers were assessed on day 29 of cycle 1 and day 28 of subsequent cycles.

PD analysis. Blood samples were collected on days 1 and 22 in part A and on days 1 and 15 in part B before dose and 1 hour after dose for measurement of pERK levels by fluorescence-activated cell sorting analysis. Samples were treated ex vivo with 12-O-tetradecanoylphorbol-13-acetate for 10 minutes at 37°C within 1 hour of being drawn. ERK phosphorylation was preserved by immediate fixation of the cells with 1.2% methanol-free formaldehyde. Peripheral-blood mononuclear cells (PBMCs) were isolated using Accuspin Histopaque 1077 columns (Sigma, St Louis, MO). Cells were collected, washed in phosphate-buffered saline, and permeabilized with ice-cold methanol before storage at –20°C before analysis. On day of analysis, cells were stained with an antibody to pERK, followed by a secondary antibody conjugated to fluorescein isothiocyanate and pERK quantitation by fluorescence-activated cell sorting analysis.

PK analysis. Plasma PK analyses were performed to assess patient exposure to AZD6244 at increasing dose levels and to correlate these levels with observed toxicities. In part A, PK blood samples were collected before dose and at 0.5, 1.0, 1.5, 2, 3, 4, 8, 12, 24, 36, and 48 hours after dose on days 1 to 3. Samples were drawn into K2EDTA tubes and processed to plasma (with no additives) and immediately frozen at –80°C. Trough plasma PK samples were collected on days 8 and 36 with an abbreviated PK assessment on day 22 (0, 1, 3, and 6 hours). In part B, PK blood samples were collected on days 1 and 15 ± 7 days before dose and at 0.5, 1, 1.5, 2, 4, 8, and 12 hours after dose. Samples were drawn into K2EDTA tubes and processed to plasma (with no additives) and immediately frozen at –20°C. Trough plasma PK samples were collected on days 8 and 22. Analytes were quantitated using liquid chromatography/mass spectrometry/mass spectrometry (lower limit of detection = 10 ng/mL for each analyte). Validation of the analytic methods resulted in mean accuracy and precision values for each analyte that met predefined performance criteria (< 15% coefficient of variation or bias for quality control samples above the limit of quantitation and < 20% coefficient of variation or bias at the limit of quantitation). Maximum observed plasma concentration and median observed time to maximum plasma concentration values for each patient were derived from the plasma concentration-time profile, and the area under the time-concentration curve was calculated using the linear trapezoidal rule.

Skin and tumor biopsy sample collection. Tumor biopsies (18-guage core needle) were taken using computed tomography or ultrasound scan guidance. All samples were fixed immediately after removal in 10% neutral buffered formalin solution for a maximum of 48 hours at room temperature before dehydration and paraffin embedding. The samples were subsequently stained with hematoxylin and eosin to confirm the diagnosis and the quality of the biopsy tissue.

Immunohistochemistry. An indirect immunoperoxidase method was used to evaluate pERK status and growth fraction (Ki-67) in situ. After deparaffinization of tumor specimens, antigen retrieval (EDTA solution), and blocking of nonspecific staining, fixed tissue was stained for 1 hour at room temperature with antibodies against phospho-p44/42 ERK1/2 (Cell Signaling Technologies No. 4376 [rabbit monoclonal] and No. 9109 [mouse monoclonal]; Cell Signaling Technologies, Danvers, MA) or Ki-67 (DAKO M7240; DAKO, Carpinteria, CA) and washed three times in Tris-buffered saline (pH 7.6). Slides were stained for 30 minutes in appropriate secondary antibody (Dako Envision system) and washed three times as previously described. Slides were incubated in diaminobenzidine chromagen for 5 to 7 minutes, washed, counterstained for 25 seconds (Gill II hematoxylin), and cover slipped. Negative and positive controls were included in each immunostained batch of slides. In all cases, these controls stained appropriately. Slides were scored, and representative microscopic fields were photographed using an Olympus DP70 digital photomicroscopic camera (Olympus, Tokyo, Japan). Nuclei and cytoplasm were scored for pERK by estimating the proportion of positive viable tumor cells multiplied by intensity of staining quantified on a 0 to 4+ scale. The proportion of tumor cell nuclei staining for Ki-67 was estimated by microscopic inspection in 10% increments. Only viable tumor was scored, with care taken to avoid necrotic areas of tumor.

Tumor DNA mutation analysis. Sections of tumor tissue were isolated from paraffin-embedded sample blocks using a 1-mm array punch. Punch samples were washed four times in xylene, three times in ethanol, once in 95% ethanol, and once in 70% ethanol (washes were incubated at room temperature for at least 1 hour per wash; the second xylene wash was incubated overnight). Ethanol was removed, and samples were air dried. A DNeasy Kit (Qiagen, Santa Clarita, CA) was used to extract DNA from fixed tissue. The presence or absence of the seven commonly occurring mutations in the KRAS oncogene (amino acid changes in codon 12 [Gly to Ala, Arg, Asp, Cys, Ser, or Val] and codon 13 [Gly to Asp]) in assessable tumor samples was determined using amplification refractory mutation sequencing (allele-specific polymerase chain reaction) (Newton CR, Graham A, Heptinstall LE, et al: Nucleic Acids Res 17:2503-2516, 1989) coupled to Scorpions detection (Whitcombe D, Theaker J, Guy SP, et al. Nat Biotechnol 17:804-807, 1999). Similarly, the presence or absence of the commonly occurring mutations in the BRAF (V600E) and NRAS (Q61R, Q61K) oncogenes were determined initially using amplification refractory mutation sequencing allele-specific amplification (Newton CR, Graham A, Heptinstall LE, et al: Nucleic Acids Res 17:2503-2516, 1989), coupled with real-time fluorescent detection of polymerase chain reaction products specifically to detect point mutations resulting in the amino acid changes. In addition, BRAF exon 15 and NRAS exons 2 and 3 were sequenced using Sanger ABI 3730xl Big Dye Terminator (Applied Biosystems, Foster City, CA) sequencing to identify any additional mutations.

	ACKNOWLEDGMENTS

 
We acknowledge the contributions of Brenda Hippert, R. Kathryn Alpaugh, Patricia Murphy, Edward Temmer, Jackie Heim, Bradley Christensen, Richard Needleman, Colette Toavs, Norma Aumen, Pete Laud, Mireille Cantarini, Lee Booras, Sue Yancik, Tammie Yeh, Michael Doyle, Andrew Hughes, David Whitcombe, Nicola Thelwell, and Harminder Singh.

	NOTES

 
published online ahead of print at www.jco.org on April 7, 2008.

Supported by Array BioPharma Inc (Boulder, CO), and National Cancer Institute Grants No. NCI P30 CA46934 (University of Colorado Health Sciences Center), NCI K24 CA106349 (S.G.E.), and 5P30CA006927 (Fox Chase Cancer Center).

Presented at the American Association for Cancer Research–National Cancer Institute–European Organisation for Research and Treatment of Cancer International Conference on Molecular Targets and Cancer Therapeutics, November 14-18, 2005, Philadelphia, PA.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
1. Sebolt-Leopold JS, Dudley DT, Herrera R, et al: Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo. Nat Med 5:810-816, 1999[CrossRef][Medline]

2. Tecle H: The development of kinase inhibitors with unusual mechanism of action: CI-1040, a selective, non-ATP competitive MEK inhibitor in the clinic. Presented at the IBC 2nd International Conference of Protein Kinases: Target Validation, Drug Discovery and Clinical Development of Kinase Therapeutics, Boston, MA, September 9-10, 2002

3. Trachet E, Przybranowski S, Howard C: In vivo evaluation of MEK inhibitor, CI-1040 (PD 0184352), against a panel of human pancreatic tumor xenografts. Proc Am Assoc Cancer Res 43:2096, 2002 (abstr 5426)

4. Yeh TC, Marsh V, Bernat BA, et al: Biological characterization of ARRY-142886 (AZD6244), a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor. Clin Cancer Res 13:1576-1583, 2007[Abstract/Free Full Text]

5. Huynh H, Soo KC, Chow PK, et al: Targeted inhibition of the extracellular signal-regulated kinase kinase pathway with AZD6244 (ARRY-142886) in the treatment of hepatocellular carcinoma. Mol Cancer Ther 6:138-146, 2007[Abstract/Free Full Text]

6. Davies B, Logie A, McKay J, et al: AZD6244 (ARRY-142886), a potent inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 kinases: Mechanism of action in vivo, pharmacokinetic/pharmacodynamic relationship, and potential for combination in preclinical models. Mol Cancer Ther 6:2209-2219, 2007[Abstract/Free Full Text]

7. Haass NK, Sproessor K, Nguyen TK, et al: The mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor AZD6244 (ARRY-142886) induces growth arrest in melanoma cells and tumor regression when combined with docetaxel. Clin Cancer Res 14:230-239, 2008[Abstract/Free Full Text]

8. Milella M, Kornblau SM, Estrov Z, et al: Therapeutic targeting of the MEK/MAPK signal transduction module in acute myeloid leukemia. J Clin Invest 108:851-859, 2001[CrossRef][Medline]

9. Rinehart J, Adjei AA, Lorusso PM, et al: Multicenter phase II study of the oral MEK inhibitor, CI-1040, in patients with advanced non-small-cell lung, breast, colon, and pancreatic cancer. J Clin Oncol 22:4456-4462, 2004[Abstract/Free Full Text]

10. Lorusso P, Krishnamurthi S, Rinehart JR, et al: A phase 1-2 clinical study of a second generation oral MEK inhibitor, PD 0325901, in patients with advanced cancer. J Clin Oncol 23:194S, 2005 (suppl, abstr 3011)

11. Pfizer Inc: Pfizer pipeline as of July 31, 2007. http://www.pfizer.com/files/research/pipeline/2007_0731/pipeline_2007_0731.pdf

12. Ratain MJ, Mick R, Schilsky RL, et al: Statistical and ethical issues in the design and conduct of phase I and II clinical trials of new anticancer agents. J Natl Cancer Inst 85:1637-1643, 1993[Free Full Text]

13. Therasse P, Arbuck SG, Eisenhauer EA, et al: New guidelines to evaluate the response to treatment in solid tumors: European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 92:205-216, 2000[Abstract/Free Full Text]

14. Waterhouse D, Rinehart J, Adjei AA, et al: A phase 2 study of an oral MEK inhibitor, CI-1040, in patients with advanced non-small-cell lung, breast, colon, or pancreatic cancer. Proc Am Soc Clin Oncol 22:204, 2003 (abstr 816)

15. Solit DB, Garraway LA, Pratilas CA, et al: BRAF mutation predicts sensitivity to MEK inhibition. Nature 439:358-362, 2006[CrossRef][Medline]

16. LoRusso P, Krishnamurthi S, Rinehart J, et al: Clinical aspects of a phase I study of PD0325901, a selective oral MEK inhibitor in patients with advanced cancer. American Association for Cancer Research–National Cancer Institute–European Organisation for Research and Treatment of Cancer Molecular Targets and Cancer Therapeutics Meeting, San Francisco, CA, October 22-26, 2007 (abstr B113)

17. Wang D, Boerner SA, Winkler JD, et al: Clinical experience of MEK inhibitors in cancer therapy. Biochim Biophys Acta 1773:1248-1255, 2007[Medline]

Submitted October 16, 2007; accepted December 21, 2007.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				D.-L. Ou, Y.-C. Shen, J.-D. Liang, J.-Y. Liou, S.-L. Yu, H.-H. Fan, D.-S. Wang, Y.-S. Lu, C. Hsu, and A.-L. Cheng Induction of Bim Expression Contributes to the Antitumor Synergy Between Sorafenib and Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase Inhibitor CI-1040 in Hepatocellular Carcinoma Clin. Cancer Res., September 15, 2009; 15(18): 5820 - 5828. [Abstract] [Full Text] [PDF]

				N. Normanno, A. Morabito, A. De Luca, M. C. Piccirillo, M. Gallo, M. R Maiello, and F. Perrone Target-based therapies in breast cancer: current status and future perspectives Endocr. Relat. Cancer, September 1, 2009; 16(3): 675 - 702. [Abstract] [Full Text] [PDF]

				Y.-K. Yoon, H.-P. Kim, S.-W. Han, H.-S. Hur, D. Y. Oh, S.-A. Im, Y.-J. Bang, and T.-Y. Kim Combination of EGFR and MEK1/2 inhibitor shows synergistic effects by suppressing EGFR/HER3-dependent AKT activation in human gastric cancer cells Mol. Cancer Ther., September 1, 2009; 8(9): 2526 - 2536. [Abstract] [Full Text] [PDF]

				S. Ocak, M. L. Sos, R. K. Thomas, and P. P. Massion High-throughput molecular analysis in lung cancer: insights into biology and potential clinical applications Eur. Respir. J., August 1, 2009; 34(2): 489 - 506. [Abstract] [Full Text] [PDF]

				K. P. Hoeflich, C. O'Brien, Z. Boyd, G. Cavet, S. Guerrero, K. Jung, T. Januario, H. Savage, E. Punnoose, T. Truong, et al. In vivo Antitumor Activity of MEK and Phosphatidylinositol 3-Kinase Inhibitors in Basal-Like Breast Cancer Models Clin. Cancer Res., July 15, 2009; 15(14): 4649 - 4664. [Abstract] [Full Text] [PDF]

				B. N. Rexer, R. Ghosh, and C. L. Arteaga Inhibition of PI3K and MEK: It Is All about Combinations and Biomarkers Clin. Cancer Res., July 15, 2009; 15(14): 4518 - 4520. [Abstract] [Full Text] [PDF]

				F. Honecker, H. Wermann, F. Mayer, A. J.M. Gillis, H. Stoop, R. J.L.M. van Gurp, K. Oechsle, E. Steyerberg, J. Th. Hartmann, W. N.M. Dinjens, et al. Microsatellite Instability, Mismatch Repair Deficiency, and BRAF Mutation in Treatment-Resistant Germ Cell Tumors J. Clin. Oncol., May 1, 2009; 27(13): 2129 - 2136. [Abstract] [Full Text] [PDF]

				E. J. Chung, A. P. Brown, H. Asano, M. Mandler, W. E. Burgan, D. Carter, K. Camphausen, and D. Citrin In vitro and In vivo Radiosensitization with AZD6244 (ARRY-142886), an Inhibitor of Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase 1/2 Kinase Clin. Cancer Res., May 1, 2009; 15(9): 3050 - 3057. [Abstract] [Full Text] [PDF]

				K. S.M. Smalley, K. L. Nathanson, and K. T. Flaherty Genetic Subgrouping of Melanoma Reveals New Opportunities for Targeted Therapy Cancer Res., April 15, 2009; 69(8): 3241 - 3244. [Abstract] [Full Text] [PDF]

				C. A. Pratilas, B. S. Taylor, Q. Ye, A. Viale, C. Sander, D. B. Solit, and N. Rosen V600EBRAF is associated with disabled feedback inhibition of RAF-MEK signaling and elevated transcriptional output of the pathway PNAS, March 17, 2009; 106(11): 4519 - 4524. [Abstract] [Full Text] [PDF]

				A. A. Adjei, M. Christian, and P. Ivy Novel Designs and End Points for Phase II Clinical Trials Clin. Cancer Res., March 15, 2009; 15(6): 1866 - 1872. [Abstract] [Full Text] [PDF]

				A. Jimeno, W. A. Messersmith, F. R. Hirsch, W. A. Franklin, and S. G. Eckhardt KRAS Mutations and Sensitivity to Epidermal Growth Factor Receptor Inhibitors in Colorectal Cancer: Practical Application of Patient Selection J. Clin. Oncol., March 1, 2009; 27(7): 1130 - 1136. [Abstract] [Full Text] [PDF]

				S. Daouti, H. Wang, W.-h. Li, B. Higgins, K. Kolinsky, K. Packman, A. Specian Jr., N. Kong, N. Huby, Y. Wen, et al. Characterization of a Novel Mitogen-Activated Protein Kinase Kinase 1/2 Inhibitor with a Unique Mechanism of Action for Cancer Therapy Cancer Res., March 1, 2009; 69(5): 1924 - 1932. [Abstract] [Full Text] [PDF]

				M. W. VanBrocklin, M. Verhaegen, M. S. Soengas, and S. L. Holmen Mitogen-Activated Protein Kinase Inhibition Induces Translocation of Bmf to Promote Apoptosis in Melanoma Cancer Res., March 1, 2009; 69(5): 1985 - 1994. [Abstract] [Full Text] [PDF]

				O. K. Mirzoeva, D. Das, L. M. Heiser, S. Bhattacharya, D. Siwak, R. Gendelman, N. Bayani, N. J. Wang, R. M. Neve, Y. Guan, et al. Basal Subtype and MAPK/ERK Kinase (MEK)-Phosphoinositide 3-Kinase Feedback Signaling Determine Susceptibility of Breast Cancer Cells to MEK Inhibition Cancer Res., January 15, 2009; 69(2): 565 - 572. [Abstract] [Full Text] [PDF]

				A. Muchir, J. Shan, G. Bonne, S. E. Lehnart, and H. J. Worman Inhibition of extracellular signal-regulated kinase signaling to prevent cardiomyopathy caused by mutation in the gene encoding A-type lamins Hum. Mol. Genet., January 15, 2009; 18(2): 241 - 247. [Abstract] [Full Text] [PDF]

				J. Leyton, G. Smith, M. Lees, M. Perumal, Q.-d. Nguyen, F. I. Aigbirhio, O. Golovko, Q. He, P. Workman, and E. O. Aboagye Noninvasive imaging of cell proliferation following mitogenic extracellular kinase inhibition by PD0325901 Mol. Cancer Ther., September 1, 2008; 7(9): 3112 - 3121. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Adjei, A. A. Articles by Eckhardt, S. G. Search for Related Content PubMed PubMed Citation Articles by Adjei, A. A. Articles by Eckhardt, S. G. Social Bookmarking                            What's this?