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1.  Pharmacokinetics and pharmacodynamics of AZD6244 (ARRY-142886) in tumor-bearing nude mice 
AZD6244 (ARRY-142886) (AstraZeneca, Macclesfield, UK) is a novel small molecule MEK1/2 inhibitor that is currently being tested in Phase II trials. With the recent publication of human pharmacokinetic data from clinical studies, we now know the achievable levels and range of AZD6244 exposure in humans. This study aimed to describe the pharmacokinetic profile of AZD6244 in mice in order to design preclinical studies that recapitulate exposure levels in humans.
Male athymic, nude mice received subcutaneous inoculation of A375 human melanoma cells. Once tumors reached 400–700 mm3, mice were given a single dose of either 5 or 10 mg/kg AZD6244 via oral gavage. Additionally, a subset of mice was dosed once daily for 1 week (10 mg/kg). Mice were killed and plasma and tissues were collected at various time points after the last dose. Samples were analyzed by LC/MS/MS for AZD6244 concentration. Additionally, pharmacodynamic endpoints such as tumor proliferation and ERK phosphorylation were analyzed at various time points after the last dose.
After either a single dose or at steady state, at clinically equivalent exposures, AZD6244 effectively inhibits ERK phosphorylation and suppresses proliferation. Furthermore, we describe a hysteretic relationship between the pharmacokinetics and the pharmacodynamics of AZD6244 and both target and pharmacologic responses.
The information presented herein will drive the rational design of pre-clinical studies that are not only relevant to the clinical setting, but also pave the way to understand the biological response to AZD6244 treatment.
PMCID: PMC4332869  PMID: 20407895
AZD6244; MEK inhibitor; PK/PD relationship; Hysteresis
2.  Initial Testing (Stage 1) of AZD6244 (ARRY-142886) by the Pediatric Preclinical Testing Program 
Pediatric blood & cancer  2010;55(4):668-677.
AZD6244 (ARRY-142886) is a potent small molecule inhibitor of MEK1/2 that is in phase 2 clinical development.
AZD6244 was tested against the PPTP in vitro panel (1 nM-10μM). In vivo AZD6244 was tested at a dose of 100 mg/kg administered orally twice daily five days per week for 6 weeks. Subsequently, AZD6244 was evaluated against two juvenile pilocytic astrocytoma (JPA) xenografts using once and twice daily dosing schedules. Phosphorylation of ERK1/2 was used as a surrogate for in vivo inhibition of MEK1/2 was determined by immunoblotting.
At the highest concentration used in vitro (10 μM) AZD6244 only inhibited growth by 50% in 5 of the 23 cell lines. Against the in vivo tumor panels, AZD6244 induced significant differences in EFS distribution in 10 of 37 (27%) solid tumor models and 0 of 6 acute lymphoblastic leukemia (ALL) models. There were no objective responses. Pharmacodynamic studies indicated at this dose and schedule AZD6244 completely inhibited ERK1/2 phosphorylation. AZD6244 was evaluated against two JPA xenografts, BT-35 (wild type BRAF) and BT-40 (mutant [V600E] BRAF). BT-40 xenografts were highly sensitive to AZD6244, whereas BT-35 xenografts progressed on AZD6244 treatment.
At the dose and schedule of administration used, AZD6244 as a single agent had limited in vitro and in vivo activity against the PPTP tumor panels despite inhibition of MEK1/2 activity. However, AZD6244 was highly active against BT-40 JPA xenografts that harbor constitutively activated BRAF, causing complete regressions.
PMCID: PMC3004092  PMID: 20806365
Preclinical Testing; Developmental Therapeutics; AZD6244
3.  In vitro and in vivo radiosensitization with AZD6244 (ARRY-142886), an inhibitor of Mitogen Activated Protein Kinase/Extracellular Signal-regulated Kinase 1/2 kinase 
The Mitogen-activated Protein Kinase (MAPK) pathway is important for cell proliferation, survival and differentiation and is frequently upregulated in cancers. The MAPK pathway is also activated after exposure to ionizing radiation. We investigated the effects of AZD6244 (ARRY-142886), an inhibitor of MEK1/2, on radiation response.
Experimental Design
The effects of AZD6244 on the in vitro radiosensitivity of human cancer cell lines (A549, MiaPaCa2 and DU145) was evaluated using clonogenic assays. DNA damage repair was evaluated using γH2AX and mitotic catastrophe was measured using nuclear fragmentation. Cell cycle effects were measured with flow cytometry. Growth delay was used to evaluate the effects of AZD6244 on in vivo tumor radiosensitivity.
Exposure of each cell line to AZD6244 prior to irradiation (IR) resulted in an increase in radiosensitivity with dose enhancement factors (DEF) at a surviving fraction of 0.1 ranging from 1.16 to 2.0. No effects of AZD6244 on radiation-induced apoptosis or persistence of γH2AX foci after IR were detected. Cells treated with AZD6244 had an increased mitotic index and decreased Chk1 phosphorylation at 1 and 3 hours after IR. Mitotic catastrophe was increased in cells receiving both AZD6244 and IR compared to the single treatments. In vivo studies revealed that AZD6244 administration to mice bearing A549 tumor xenografts resulted in a greater than additive increase in radiation-induced tumor growth delay (DEF of 3.38).
These results indicate that AZD6244 can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an increase in mitotic catastrophe.
PMCID: PMC2798161  PMID: 19366835
4.  Basal and treatment-induced activation of AKT mediates resistance to cell death by AZD6244 (ARRY-142886) in Braf-mutant human cutaneous melanoma cells 
Cancer research  2010;70(21):8736-8747.
The majority of melanomas demonstrate constitutive activation of the RAS-RAF-MEK-MAPK pathway. AZD6244 is a selective MEK1/2 inhibitor which markedly reduces tumor P-MAPK levels, but it produced few clinical responses in melanoma patients. An improved understanding of the determinants of resistance to AZD6244 may lead to improved patient selection and effective combinatorial approaches. The effects of AZD6244 on cell growth and survival were tested in a total of 14 Braf-mutant and 3 wild-type human cutaneous melanoma cell lines. Quantitative assessment of phospho-protein levels in the Braf-mutant cell lines by reverse phase protein array (RPPA) analysis showed no significant association between P-MEK or P-MAPK levels and AZD6244 sensitivity, but activation-specific markers in the PI3K-AKT pathway correlated with resistance. We also identified resistant cell lines without basal activation of the PI3K-AKT pathway. RPPA characterization of the time-dependent changes in signaling pathways revealed that AZD6244 produced durable and potent inhibition of P-MAPK in sensitive and resistant Braf-mutant cell lines, but several resistant lines demonstrated AZD6244-induced activation of AKT. In contrast, sensitive cell lines demonstrated AZD6244 treatment-induced upregulation of PTEN protein and mRNA expression. Inhibition of AKT, TORC1/2, or IGF1R blocked AZD6244-induced activation of AKT and resulted in synergistic cell killing with AZD6244. These findings identify basal and treatment-induced regulation of the PI3K-AKT pathway as a critical regulator of AZD6244 sensitivity in Braf-mutant cutaneous melanoma cells, the novel regulation of PTEN expression by AZD6244 in sensitive cells, and suggest new combinatorial approaches for patients.
PMCID: PMC4286702  PMID: 20959481
AZD6244; AZD8055; MEK; BRAF; MAPK; AKT; PTEN; melanoma
5.  Acute tumour response to the MEK1/2 inhibitor selumetinib (AZD6244, ARRY-142886) evaluated by non-invasive diffusion-weighted MRI 
British Journal of Cancer  2013;109(6):1562-1569.
Non-invasive imaging biomarkers underpin the development of molecularly targeted anti-cancer drugs. This study evaluates tumour apparent diffusion coefficient (ADC), measured by diffusion-weighted magnetic resonance imaging (DW-MRI), as a biomarker of response to the MEK1/2 inhibitor selumetinib (AZD6244, ARRY-142886) in human tumour xenografts.
Nude mice bearing human BRAFV600D WM266.4 melanoma or BRAFV600E Colo205 colon carcinoma xenografts were treated for 4 days with vehicle or selumetinib. DW-MRI was performed before and 2 h after the last dose and excised tumours analysed for levels of phospho-ERK1/2, cleaved caspase 3 (CC3) and necrosis.
Selumetinib treatment induced tumour stasis and reduced ERK1/2 phosphorylation in both WM266.4 and Colo205 tumour xenografts. Relative to day 0, mean tumour ADC was unchanged in the control groups but was significantly increased by up to 1.6-fold in selumetinib-treated WM266.4 and Colo205 tumours. Histological analysis revealed a significant increase in necrosis in selumetinib-treated WM266.4 and Colo205 xenografts and CC3 staining in selumetinib-treated Colo205 tumours relative to controls.
Changes in ADC following treatment with the MEK1/2 inhibitor selumetinib in responsive human tumour xenografts were concomitant with induction of tumour cell death. ADC may provide a useful non-invasive pharmacodynamic biomarker for early clinical assessment of response to selumetinib and other MEK-ERK1/2 signalling-targeted therapies.
PMCID: PMC3776979  PMID: 23942066
MEK; biomarker; non-invasive imaging; diffusion-weighted MRI; human tumour xenografts.
6.  18F-FLT-PET for Response Evaluation of MEK Inhibitor Selumetinib (AZD6244, ARRY-142886) in Patients with Solid Tumors 
Selumetinib (AZD6244, ARRY-142886) is a potent, selective, uncompetitive inhibitor of MEK 1 / 2, part of the RAF/MEK/ERK protein kinase signal cascade, which is responsible for tumor. This pilot study was used to explore if 18F-fluoro-l-thymidine (FLT), a thymidine analogue positron emission tomography (PET) tracer and a surrogate marker for proliferation, can be used as an early predictor of response for patients with solid cancers treated with Selumetinib. FLT-PET scans were performed in four patients at baseline and after 2 weeks of treatment with Selumetinib. FLT uptake in tumors was analyzed qualitatively and quantitatively by measuring standard uptake value (SUV) max in regions of interest (ROI). Results were compared to computed tomography (CT) scans (baseline and after 8 weeks), which were evaluated using the response evaluation criteria in solid tumors (RECIST) 1.0 criteria. One patient with melanoma showed both a qualitative and quantitative decrease in FLT uptake associated with a decrease in sum of longest diameter of 12% RECIST on CT evaluation. Another patient who had colorectal carcinoma (CRC) showed a significant increase in FLT uptake with initially stable, but eventually progressive disease on CT. The other two patients (one with melanoma and one with CRC) showed no significant changes in FLT uptake, whereas CT evaluation showed progressive disease. This is the first report describing changes in FLT-PET in patients receiving Selumetinib. In two patients, changes in FLT uptake as early as after 2 weeks of treatment were consistent with CT results after 8 weeks. Biomarkers to predict and evaluate treatment the outcome of targeted therapies are highly warranted. These initial results need further investigation.
PMCID: PMC3555396  PMID: 23372439
F-18 FLT; PET-CT; Selumetinib; treatment response
7.  Phase I pharmacokinetic and pharmacodynamic study of the prenyl transferase inhibitor AZD3409 in patients with advanced cancer 
British Journal of Cancer  2008;98(12):1951-1958.
AZD3409 is an orally active double prodrug that was developed as a novel dual prenyltransferase inhibitor. The formation of the active metabolite AZD3409 acid is mediated by esterases in plasma and cells. The aim of this phase I study was to determine the maximum tolerated dose, toxicities, pharmacokinetics and pharmacodynamics of AZD3409. AZD3409 was administered orally to patients with advanced solid malignancies using an interpatient dose-escalation scheme starting at 500 mg AZD3409 once daily. Twenty-nine patients were treated at seven dose levels. The MTD of part A was defined as 750 mg b.i.d. in the fasted state. Adverse events were mainly gastrointestinal and the severity was on average mild to moderate and reversible. The dose-limiting toxicities were vomiting, diarrhoea and uncontrolled nausea. Pharmacokinetic studies of the prodrug and the active metabolite indicated dose proportionality. Pharmacodynamic studies showed that farnesyltransferase (FTase) was inhibited at all dose levels. In conclusion, chronic oral dosing with AZD3409 is feasible and results in significant inhibition of FTase activity. Pharmacodynamic studies revealed that the maximal FTase inhibition, estimated at 49±11%, appeared to be reached at AZD3409 acid plasma concentrations at which the occurrence of drug-related toxicity was low. This study supports the rationale to implement biological effect studies in clinical trials with biologically active anticancer drugs to define optimal dosing regimens.
PMCID: PMC2441954  PMID: 18506143
AZD3409; prenyl transferase inhibitor; biological effect study
8.  High level of AKT activity is associated with resistance to MEK inhibitor AZD6244 (ARRY-142886) 
Cancer biology & therapy  2009;8(21):2073-2080.
MEK/ERK activities are increased in many primary lung cancers, and MEK inhibitors have been tested clinically for treatment of non-small cell lung cancers. The molecular mechanisms of resistance to MEK inhibitors have not been clearly demonstrated, however, and no molecular biomarker that can predict lung cancer response to MEK inhibitors is available. By determining the dose-responses of 35 human lung cancer cell lines to MEK-specific inhibitor AZD6244, we identified subsets of lung cancer cell lines that are either sensitive or resistant to this agent. Subsequent molecular characterization showed that treatment with AZD6244 suppressed ERK phosphorylation in both sensitive and resistant cells, suggesting that resistance is not mediated by the activities of MEK/ERK themselves. Interestingly, we found that levels of phosphorylated AKT were dramatically higher in the resistant cancer cells than in the sensitive cells. Stable transfection of dominant-negative AKT into resistant cells by retroviral infection restored their susceptibility to AZD6244. These results indicate that phosphorylated AKT may be a biomarker of response to AZD6244 and that modulation of AKT activity may be a useful approach to overcome resistance to MEK inhibitors.
PMCID: PMC2835993  PMID: 19783898
AZD6244; MEK inhibitor; resistance; AKT; lung cancer
9.  A Phase I Dose-Escalation Study of Danusertib (PHA-739358) Administered as a 24-hour Infusion With and Without G-CSF in a 14-day Cycle in Patients with Advanced Solid Tumors 
This study was conducted to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of the intravenous pan-aurora kinase inhibitor PHA-739358, danusertib, in patients with advanced solid tumors.
Experimental Design
In Part 1, patients received escalating doses of danusertib (24-h infusion every 14 days) without filgrastim (G-CSF). Febrile neutropenia was the dose-limiting toxicity without G-CSF. Further dose escalation was performed in part 2 with G-CSF. Blood samples were collected for danusertib pharmacokinetics and pharmacodynamics. Skin biopsies were collected to assess histone H3 phosphorylation (pH3).
Fifty-six patients were treated, 40 in part 1 and 16 in part 2. Febrile neutropenia was the dose limiting toxicity in Part 1 without G-CSF. Most other adverse events were grade 1–2, occurring at doses ≥360 mg/m2 with similar incidence in parts 1 and 2. The MTD without G-CSF is 500 mg/m2. The recommended phase 2 dose (RP2D) in Part 2 with G-CSF is 750 mg/m2. Danusertib demonstrated dose-proportional pharmacokinetics in parts 1 and 2 with a median half-life of 18–26 hours. pH3 modulation in skin biopsies was observed at ≥500 mg/m2. One patient with refractory small cell lung cancer (1000 mg/m2 with G-CSF) had an objective response lasting 23 weeks. One patient with refractory ovarian cancer had 27% tumor regression and 30% CA125 decline.
Danusertib was well tolerated with target inhibition in skin at ≥500 mg/m2. Preliminary evidence of anti-tumor activity, including a PR and several occurrences of prolonged stable disease (SD), was seen across a variety of advanced refractory cancers. Phase II studies are ongoing.
PMCID: PMC2826106  PMID: 19825950
Danusertib; PHA-739358; Aurora Kinase Inhibitor; phase I trial; solid tumors
10.  Phase I Study to Assess the Safety, Tolerability and Pharmacokinetics of AZD4877 in Japanese Patients with Solid Tumors 
Archives of Drug Information  2011;4(2):23-31.
AZD4877 is a potent Eg5 inhibitor that has been shown to have an acceptable tolerability profile in a Phase I study of Western patients with solid tumors. This study was conducted to evaluate the safety, pharmacokinetic (PK) profile, maximum tolerated dose (MTD) and efficacy of AZD4877 in a Japanese population with solid tumors.
In this Phase I, open-label, dose-escalation study, AZD4877 (10, 15, 20 or 25 mg) was administered as a 1-hour intravenous infusion on days 1, 8 and 15 of repeated 28-day cycles to Japanese patients with advanced solid tumors. Adverse events (AEs) were evaluated according to Common Terminology Criteria for Adverse Events (CTCAE) version 3.0. PK variables were assessed pre- and post dosing. The MTD of AZD4877 was determined by evaluating dose-limiting toxicities (DLTs). Efficacy was evaluated by assessing best response according to Response Evaluation Criteria In Solid Tumors version 1.0.
Of the 21 patients enrolled, 18 received at least one dose of AZD4877 (N = 3 in both the 10 and 15 mg cohorts, N = 6 in both the 20 and 25 mg cohorts). The most commonly reported AEs were fatigue and nausea (39% of patients each). One patient in each of the 20 and 25 mg cohorts experienced a DLT (neutropenia and febrile neutropenia). Dose escalation was halted at 25 mg and the MTD was not defined in this population. CTCAE grade ≥3 abnormal laboratory findings/vital signs were reported in 12 patients, with neutropenia (56%) and leukopenia (44%) being the most commonly reported. Exposure to AZD4877 was not fully dose proportional and AZD4877 clearance and elimination half-life appeared independent of dose. The best response to AZD4877 was stable disease in five of 16 evaluable patients.
AZD4877 up to doses of 25 mg was well tolerated in Japanese patients. There was little evidence of clinical efficacy.
PMCID: PMC3130141  PMID: 21765863
AZD4877; Eg5 Inhibitor; Solid Tumors; Japanese
11.  A phase I study to determine the safety, pharmacokinetics and pharmacodynamics of a dual VEGFR and FGFR inhibitor, brivanib, in patients with advanced or metastatic solid tumors 
Annals of Oncology  2010;22(6):1413-1419.
Background: This study was designed to determine the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of brivanib in patients with advanced/metastatic solid tumors.
Patients and methods: Ninety patients enrolled in this two-part, phase I open-label study of oral brivanib alaninate. The primary objectives of this study were (in part A) dose-limiting toxicity, maximum tolerated dose (MTD) and the lowest biologically active dose level and (in part B) the optimal dose/dose range. The secondary objectives of this study were preliminary evidence of antitumor activity, PK and PD.
Results: Across part A (open-label dose escalation and MTD) and part B (open-label dose optimization), 68 patients received brivanib alaninate. Brivanib demonstrated a manageable toxicity profile at doses of 180–800 mg. Most toxic effects were mild. Systemic exposure of the active moiety brivanib increased linearly ≤1000 mg/day. The MTD was 800 mg/day. Forty-four patients were treated at the MTD: 20 with 800 mg continuously, 11 with 800 mg intermittently and 13 with 400 mg b.i.d. doses. Partial responses were confirmed in two patients receiving brivanib ≥600 mg. Dynamic contrast-enhanced magnetic resonance imaging demonstrated statistically significant decreases in parameters reflecting tumor vascularity and permeability after multiple doses in the 800-mg continuous q.d. and 400-mg b.i.d. dose cohorts.
Conclusion: In patients with advanced/metastatic cancer, brivanib demonstrates promising antiangiogenic and antitumor activity and manageable toxicity at doses ≤800 mg orally q.d., the recommended phase II study dose.
PMCID: PMC3139984  PMID: 21131369
antiangiogenesis; brivanib; fibroblast growth factor; vascular endothelial growth factor
12.  Antitumor activity of selective MEK1/2 inhibitor AZD6244 in combination with PI3K/mTOR inhibitor BEZ235 in gefitinib-resistant NSCLC xenograft models 
Although the EGF receptor tyrosine kinase inhibitors (EGFR-TKI) gefitinib have shown dramatic effects against EGFR mutant lung cancer, patients become resistant by various mechanisms, including gatekeeper EGFR-T790M mutation, MET amplification, and KRAS mutation, thereafter relapsing. AZD6244 is a potent, selective, and orally available MEK1/2 inhibitor. In this study, we evaluated the therapeutic efficacy of AZD6244 alone or with BEZ235, an orally available potent inhibitor of phosphatidylinositol 3–kinase (PI3K) and mammalian target of rapamycin (mTOR), in gefitinib-resistant non-small cell lung carcinoma (NSCLC) models.
Experimental design
NCI-H1975 with EGFR-T790M mutation, NCI-H1993 with MET amplification and NCI-H460 with KRAS/PIK3CA mutation human NSCLC cells were subcutaneous injected into the athymic nude mice respectively. Mice were randomly assigned to treatment with AZD6244, BEZ235, AZD6244 plus BEZ235, or control for 3 weeks, then all mice were sacrificed and tumor tissues were subjected to western blot analyses and immunohistochemical staining.
AZD6244 could inhibit the tumor growth of NCI-H1993, but slightly inhibit the tumor growth of NCI-1975 and NCI-H460. Combining AZD6244 with BEZ235 markedly enhanced their antitumor effects and without any marked adverse events. Western blot analysis and immunohistochemical staining revealed that AZD6244 alone reduced ERK1/2 phosphorylation, angiogenesis, and tumor cell proliferation. Moreover, MEK1/2 inhibition resulted in decreased AKT phosphorylation in NCI-H1993 tumor model. BEZ235 also inhibited AKT phosphorylation as well as their downstream molecules in all three tumor models. The antiangiogenic effects were substantially enhanced when the agents were combined, which may due to the reduced expression of matrix metallopeptidase-9 in tumor tissues (MMP-9).
In this study, we evaluated therapy directed against MEK and PI3K/mTOR in distinct gefitinib-resistant NSCLC xenograft models. Combining AZD6244 with BEZ235 enhanced their antitumor and antiangiogenic effects. We concluded that the combination of a selective MEK inhibitor and a PI3K/mTOR inhibitor was effective in suppressing the growth of gefitinib-resistant tumors caused by EGFR T790M mutation, MET amplification, and KRAS/PIK3CA mutation. This new therapeutic strategy may be a practical approach in the treatment of these patients.
PMCID: PMC4074836  PMID: 24939055
AZD6244; BEZ235; Tyrosine kinase inhibitor; Non-small cell lung cancer
13.  Genotype-Dependent Sensitivity of Uveal Melanoma Cell Lines to Inhibition of B-Raf, MEK, and Akt Kinases: Rationale for Personalized Therapy 
The response of uveal melanoma cells to inhibition of B-Raf, MEK, and Akt depends on their genotype. These observations support the design of clinical trials of these inhibitors for the treatment of carefully selected UM patients and provide a rationale for personalized therapy.
Inhibitors of B-Raf and MEK kinases hold promise for the management of cutaneous melanomas harboring BRAF mutations. BRAF mutations are rare in uveal melanomas (UMs), but somatic mutations in the G protein α subunits Gαq and Gα11 (encoded by GNAQ and GNA11, respectively) occur in a mutually exclusive pattern in ∼80% of UMs. The impact of B-Raf and MEK inhibitors on Gα-mutant UMs remains unknown.
The impact of the B-Raf inhibitor PLX4720, the MEK inhibitor AZD6244, and the Akt inhibitor MK2206 on UM cell lines was assessed with the use of cell viability, proliferation, and apoptosis assays and immunoblot analysis.
BRAF-mutant UM cells were sensitive to both PLX4720 and AZD6244, undergoing cell cycle arrest but not apoptosis. UM cells with a Gα-protein mutation (GNAQ or GNA11) were mildly sensitive to AZD6244 but completely resistant to PLX4720. In fact, PLX4720 paradoxically increased ERK phosphorylation in Gα-mutant UM cells. The combination of AZD6244 with PLX4720 had synergistic anticancer activity in BRAF-mutant cells but not in Gα-mutant cells. The Akt inhibitor MK2206 sensitized BRAF-mutant cells to both PLX4720 and AZD6244 and sensitized Gα-mutant cells to AZD6244 but did not overcome the resistance of the Gα-mutant cells to PLX4720.
The response of UM cells to inhibition of B-Raf, MEK, and Akt depends on their genotype. Future use of such targeted therapies in clinical trials of UM patients will require careful design and patient selection based on genotype to provide personalized and effective therapy.
PMCID: PMC3207725  PMID: 21828154
14.  Detection of BRAF mutations in the tumour and serum of patients enrolled in the AZD6244 (ARRY-142886) advanced melanoma phase II study 
British Journal of Cancer  2009;101(10):1724-1730.
This study investigated the potential clinical utility of circulating free DNA (cfDNA) as a source of BRAF mutation detection in patients enrolled into a phase II study of AZD6244, a specific MEK1/2 inhibitor, in patients with advanced melanoma.
BRAF mutations were detected using Amplification Refractory Mutation System allele-specific PCR. BRAF mutation status was assessed in serum-derived cfDNA from 126 patients enrolled into the study and from 94 matched tumour samples.
Of 94 tumour samples, 45 (47.9%) were found to be BRAF mutation positive (BRAF+). Serum-derived cfDNA was BRAF+ in 33 of 126 (26.2%) samples, including in five samples for which tumour data were unavailable. Of BRAF+ tumours, 25 of 45 (55.6%) were BRAF+ in cfDNA. In three cases in which the tumour was negative, cfDNA was BRAF+. Progression-free survival (PFS) of patients with BRAF+ tumour and cfDNA was not significantly different compared with tumour BRAF+ but cfDNA BRAF-negative patients, indicating that cfDNA BRAF detection is not associated with poorer prognosis on PFS in stage III/IV advanced melanoma.
These data demonstrate the feasibility of BRAF mutation detection in cfDNA of patients with advanced melanoma. Future studies should aim to incorporate BRAF mutation testing in cfDNA to further validate this biomarker for patient selection.
PMCID: PMC2778539  PMID: 19861964
AZD6244; amplification refractory mutation system; BRAF; circulating free DNA; cutaneous melanoma
15.  Identification of common predictive markers of in vitro response to the MEK inhibitor selumetinib (AZD6244; ARRY-142886) in human breast cancer and non-small cell lung cancer cell lines 
Molecular cancer therapeutics  2010;9(7):1985-1994.
Selumetinib (AZD6244; ARRY-142886) is a tight-binding, uncompetitive inhibitor of MEK1/2 currently in clinical development. We evaluated the effects of selumetinib in 31 human breast cancer cell lines and 43 human non-small cell lung cancer (NSCLC) cell lines to identify characteristics correlating with in vitro sensitivity to MEK inhibition. IC50 less than 1µM (considered sensitive) was seen in 5 of 31 breast cancer cell lines and 15 of 43 NSCLC cell lines, with a correlation between sensitivity and raf mutations in breast cancer cell lines (p= 0.022) and ras mutations in NSCLC cell lines (p= 0.045). Evaluation of 27 of the NSCLC cell lines with Western blots demonstrated no clear association between MEK and PI3K pathway activation and sensitivity to MEK inhibition. Baseline gene expression profiles were generated for each cell line using Agilent gene expression arrays to identify additional predictive markers. Genes associated with differential sensitivity to selumetinib were seen in both histologies, including a small number of genes in which differential expression was common to both histologies. In total, these results suggest that clinical trials of selumetinib in breast cancer and NSCLC might select patients whose tumors harbor raf and ras mutations respectively.
PMCID: PMC2939826  PMID: 20587667
AZD6244; MEK; Breast cancer; Lung Cancer
16.  Phase I Trial of Oxaliplatin, Infusional 5-Fluorouracil, and Leucovorin (FOLFOX4) With Erlotinib and Bevacizumab in Colorectal Cancer 
Clinical colorectal cancer  2010;9(5):297-304.
This phase I study was conducted to determine the maximum tolerated dose (MTD) of erlotinib, an oral epidermal growth factor receptor tyrosine kinase inhibitor, with 5-fluorouracil/leucovorin/oxaliplatin (FOLFOX4) in patients with advanced colorectal cancer (CRC). Bevacizumab was later included as standard of care at the MTD.
Patients and Methods
Patients received FOLFOX4 with escalating doses of erlotinib: dose level (DL) 1, 50 mg; DL 2, 100 mg; and DL 3, 150 mg once daily continuously. Bevacizumab 5 mg/kg days 1 and 15 was added at the MTD upon Food and Drug Administration approval. Correlative studies included pharmacokinetics, pharmacodynamics was assessed in paired skin biopsies, and fluorodeoxyglucose positron emission tomography scans.
Fifteen patients received 60 cycles (120 FOLFOX treatments). Two dose-limiting toxicities (DLTs) were seen at DL 3: intolerable grade 2 rash (Common Terminology Criteria for Adverse Events version 2) lasting > 1 week, and grade 4 neutropenia. Dose level 2 was expanded to 6 more patients, this time adding bevacizumab, and 1 DLT of grade 3 mucositis occurred. As expected, the primary toxicities were cytopenias, diarrhea, rash, and fatigue. There were 2 occurrences of pneumatosis. One patient experienced an unrelated grade 4 myocardial infarction before starting chemotherapy. No pharmacokinetic drug interactions were observed. The Response Evaluation Criteria in Solid Tumors response rate was 11 of 14 (78%), median progression-free survival was 9.5 months, and median overall survival was 30 months. Three patients are currently alive > 3 years, with 1 having no evidence of disease.
The MTD of erlotinib with FOLFOX4 with or without bevacizumab is 100 mg daily. The regimen appeared to increase toxicity but showed activity in patients with CRC.
PMCID: PMC3033228  PMID: 21208844
Epidermal growth factor receptor; Pharmacokinetics; Tyrosine kinase
17.  MEK inhibition induced downregulation of MRP1 and MRP3 expression in experimental hepatocellular carcinoma 
Hepatocellular carcinoma (HCC) exhibits strong intrinsic and acquired drug resistance which is the main obstacle to chemotherapy. Overexpression of ATP binding cassette (ABC) proteins correlates with activation of mitogen activated protein kinase (MAPK) pathway in HCC. Here, we systematically investigated the inhibition of MAPK pathway and its role in regulating HCC cell growth as well as ABC proteins MRP1 and MRP3 expression.
The Raf1 kinase inhibitor (GW5074) and different MEK inhibitors (U0126 and AZD6244) were used to treat HCC cells to identify their effects on HCC cell growth and ABC proteins expression in vitro. Cell viability tests were performed after the treatment of MAPK pathway inhibitors and in combination with gemcitabine or doxorubicin. Western blot was applied to assess the changes of MAPK pathway and protein expression of MRP1 and MRP3. Flow cytometry was used to measure intracellular doxorubicin accumulation after the treatment of MEK inhibitors.
Both Raf1 inhibitor (GW5074) and MEK inhibitors (U0126 and AZD6244) suppressed HCC cell growth in a dose dependent manner. Pre-treatment of MEK inhibitor U0126 or AZD6244 sensitized HCC cells to gemcitabine or doxorubicin based chemotherapy. Raf1 inhibitor GW5074 had no effect on MRP1 and MRP3 protein expression. Treatment of gemcitabine or doxorubicin activated phosphorylated ERK and induced the upregulation of MRP1 and MRP3. MEK inhibitors U0126 and AZD6244 deactivated phosphorylated ERK, decreased endogenous MRP1 expression, reversed gemcitabine or doxorubicin induced MRP1 and MRP3 upregulation, and increased the intracellular doxorubicin accumulation.
This study provides evidence that MEK inhibitors sensitize HCC cells to chemotherapy by increasing intracellular chemodrug accumulation. MEK inhibirors U0126 and AZD6244 reduced MRP1 as well as MRP3 expression, and may contribute partially to the sensitization. The combination of MEK inhibitor and conventional chemotherapy may offer new therapeutic option for the treatment of resistant HCC.
PMCID: PMC3558388  PMID: 23320839
Hepatocellular carcinoma; MEK; MRP1; MRP3; Multidrug resistance
18.  A phase I safety, pharmacological, and biological study of the farnesyl protein transferase inhibitor, lonafarnib (SCH 663366), in combination with cisplatin and gemcitabine in patients with advanced solid tumors 
This phase I study was conducted to evaluate the safety, tolerability, pharmacological properties and biological activity of the combination of the lonafarnib, a farnesylproteintransferase (FTPase) inhibitor, with gemcitabine and cisplatin in patients with advanced solid malignancies.
Experimental design
This was a single institution study to determine the maximal tolerated dose (MTD) of escalating lonafarnib (75–125 mg po BID) with gemcitabine (750–1,000 mg/m2 on days 1, 8, 15) and fixed cisplatin (75 mg/m2 day 1) every 28 days. Due to dose-limiting toxicities (DLTs) of neutropenia and thrombocytopenia in initial patients, these patients were considered “heavily pretreated” and the protocol was amended to limit prior therapy and re-escalate lonafarnib in “less heavily pre-treated patients” on 28-day and 21-day schedules. Cycle 1 and 2 pharmacokinetics (PK), and farnesylation of the HDJ2 chaperone protein and FPTase activity were analyzed.
Twenty-two patients received 53 courses of therapy. Nausea, vomiting, and fatigue were frequent in all patients. Severe toxicities were observed in 91% of patients: neutropenia (41%), nausea (36%), thrombocytopenia (32%), anemia (23%) and vomiting (23%). Nine patients withdrew from the study due to toxicity. DLTs of neutropenia, febrile neutropenia, thrombocytopenia, and fatigue limited dose-escalation on the 28-day schedule. The MTD was established as lonafarnib 75 mg BID, gemcitabine 750 mg/m2 days 1, 8, 15, and cisplatin 75 mg/m2 in heavily pre-treated patients. The MTD in the less heavily pre-treated patients could not be established on the 28-day schedule as DLTs were observed at the lowest dose level, and dose escalation was not completed on the 21-day schedule due to early study termination by the Sponsor. No PK interactions were observed. FTPase inhibition was not observed at the MTD, however HDJ-2 gel shift was observed in one patient at the 100 mg BID lonafarnib dose. Anti-cancer activity was observed: four patients had stable disease lasting >2 cycles, one subject had a complete response, and another had a partial response, both with metastatic breast cancer.
Lonafarnib 75 mg BID, gemcitabine 750 mg/m2 days 1, 8, 15, and cisplatin 75 mg/m2 day 1 on a 28-day schedule was established as the MTD. Lonafarnib did not demonstrate FTPase inhibition at these doses. Despite the observed efficacy, substantial toxicity and questionable contribution of anti-tumor activity of lonafarnib to gemcitabine and cisplatin limits further exploration of this combination.
PMCID: PMC2813768  PMID: 18058098
Lonafarnib; SCH66336; Cisplatin; Gemcitabine; Farnesyltransferase; Phase I; Pharmacokinetics
19.  Dual blockade of the PI3K/AKT/mTOR (AZD8055) and RAS/MEK/ERK (AZD6244) pathways synergistically inhibits rhabdomyosarcoma cell growth in vitro and in vivo 
To provide rationale for using PI3K and/or MAPK pathway inhibitors to treat rhabdomyosarcomas (RMS), a major cause of pediatric/adolescent cancer deaths.
Experimental design
The prevalence of PI3K/MAPK pathway activation in RMS clinical samples was assessed using immunohistochemistry. Compensatory signaling and crosstalk between PI3K/MAPK pathways was determined in RMS cell lines following p110α shRNA-mediated depletion. Pharmacological inhibition of reprogrammed signaling in stable p110α knockdown lines was used to determine the target-inhibition profile inducing maximal growth inhibition. The in vitro and in vivo efficacy of inhibitors of TORC1/2(AZD8055), MEK(AZD6244) and P13K/mTOR(NVP-BEZ235) were evaluated alone and in pair-wise combinations.
PI3K pathway activation was seen in 82.5% rhabdomyosarcomas with co-activated MAPK in 36% and 46% of alveolar and embryonal sub-types respectively. p110α knockdown in cell lines over the short and long term was associated with compensatory expression of other p110 isoforms, activation of the MAPK pathway and cross-talk to reactivate the PI3K pathway. Combinations of PI3K pathway and MEK inhibitors synergistically inhibited cell growth in vitro. Treatment of RD cells with AZD8055 plus AZD6244 blocked reciprocal pathway activation, as evidenced by reduced AKT/ERK/S6 phosphorylation. In vivo, the synergistic effect on growth and changes in pharmacodynamic biomarkers was recapitulated using the AZD8055/AZD6244 combination but not NVP-BEZ235/AZD6244. Pharmacokinetic analysis provided evidence of drug-drug interaction with both combinations.
Dual PI3K/MAPK pathway activation and compensatory signaling in both rhabdomyosarcoma subtypes predicts a lack of clinical efficacy for single agents targeting either pathway, supporting a therapeutic strategy combining a TORC1/2 with a MEK inhibitor.
PMCID: PMC3818134  PMID: 23918606
Rhabdomyosarcoma; PI3K pathway inhibitors; MAPK pathway inhibitor; synergistic inhibition; pharmacodynamics/pharmacokinetics
20.  Phase I and pharmacological study of the broad-spectrum tyrosine kinase inhibitor JNJ-26483327 in patients with advanced solid tumours 
British Journal of Cancer  2010;103(7):987-992.
JNJ-26483327 is an oral, potent, multi-targeted tyrosine kinase inhibitor, inhibiting kinases of epidermal growth factor receptor (EGFR)-1, -2 and -4, rearranged during transfection (RET) receptor, vascular endothelial growth factor receptor (VEGFR)-3 and Src family (Lyn, Fyn, Yes) at low nanomolar concentrations. This phase I, accelerated titration study assessed maximum tolerated dose, safety, pharmacokinetics and pharmacodynamic effects of JNJ-26483327.
Nineteen patients with advanced cancers received JNJ-26483327 continuous twice daily (BID) in escalating dose cohorts ranging from 100 to 2100 mg. Pharmacodynamic effects were assessed in paired skin biopsies and blood.
JNJ-26483327 was well tolerated in doses up to 1500 mg BID, with target-inhibition-related toxicity such as diarrhoea and skin rash, and other common reported toxicities being nausea, vomiting, anorexia and fatigue. At 2100 mg, two episodes of dose-limiting toxicity were observed, consisting of grade 3 anorexia and a combination of grade 3 anorexia and fatigue, respectively. Pharmacokinetics were dose proportional up to 1500 mg in which plasma levels were obtained showing anti-tumour activity in xenograft mouse models. Pharmacodynamic analysis did not show a substantial effect on expression of Ki-67, p27kip1, phosphorylated mitogen-activated protein kinase, phosphorylated Akt and EGFR, and serum levels of sVEGFR-2, VEGF-C and VEGF-D remained unchanged. Stable disease was noted in six patients (32%).
JNJ-26483327 is well tolerated and shows a predictable pharmacokinetic profile; the recommended dose for further studies is 1500 mg BID.
PMCID: PMC2965873  PMID: 20823884
phase I; JNJ-26483327; pharmacokinetics; pharmacodynamics; tyrosine kinase inhibitor
21.  Phase 1 study of lenvatinib combined with carboplatin and paclitaxel in patients with non-small-cell lung cancer 
British Journal of Cancer  2013;109(3):538-544.
This dose-finding study evaluated lenvatinib, an oral multitargeted receptor tyrosine kinase inhibitor, in combination with carboplatin/paclitaxel in chemotherapy-naïve non-small-cell lung cancer (NSCLC) patients.
Patients and Methods:
Patients received lenvatinib twice daily (BID) with carboplatin (area under the curve 6 mg ml−1 min−1, day 1)/paclitaxel (200 mg m−2, day 1) every 3 weeks. The initial dose of lenvatinib was 6 mg BID. The primary end point was maximum tolerated dose (MTD) of lenvatinib. At the MTD, the cohort was expanded by 16 patients. Safety, pharmacokinetics, pharmacodynamics, and antitumor effects were evaluated.
Twenty-eight patients were treated. At 6 mg BID, dose-limiting toxicities (DLTs) included febrile neutropenia/gingival infection (n=2). No DLTs occurred with 4 mg BID, the recommended MTD for the expansion. Common grade 3/4 toxicities included neutropenia, leukopenia, hypertension, and thrombocytopenia. The combination had no significant impact on individual drug pharmacokinetics. Response rate and median progression-free survival were 68% and 9.0 months, respectively, with 4 mg BID. In the plasma biomarker analysis, stromal cell-derived factor 1α, stem cell factor, and granulocyte colony-stimulating factor correlated with antitumor activity.
The MTD for lenvatinib with carboplatin/paclitaxel is 4 mg BID in advanced NSCLC patients. This regimen demonstrated manageable tolerability and encouraging antitumor activity.
PMCID: PMC3738144  PMID: 23860537
angiogenesis inhibitor; carboplatin; lenvatinib; lung cancer; paclitaxel; tyrosine kinase inhibitor
22.  A phase I trial of MK-0731, a Kinesin Spindle Protein (KSP) inhibitor, in patients with solid tumors 
Investigational New Drugs  2011;30(3):1088-1095.
The kinesin spindle protein (KSP) is essential for separation of spindle poles during mitosis. Its inhibition results in mitotic arrest. This phase I trial examined safety, tolerability, dose-limiting toxicity (DLT), maximum tolerated dose (MTD), pharmacokinetic parameters, and anti-tumor activity of MK-0731, a potent inhibitor of KSP.
Experimental design
In part 1, patients with advanced solid tumors received MK-0731 intravenously over 24 h every 21 days starting at 6 mg/m2, escalating until MTD was reached. In part 2, patients with taxane-resistant tumors received the MTD. Plasma samples were collected to analyze the pharmacokinetics of MK-0731. Tumor response was evaluated using Response Evaluation Criteria in Solid Tumors (RECIST) v1.0.
In part 1, 21 patients (median age 63 years) were treated with MK-0731 at doses ranging from 6 to 48 mg/m2/24 h for median four cycles. The dose-limiting toxicity was neutropenia and the MTD was 17 mg/m2/24 h. At the MTD, AUC (±SD) was 10.5 (±7.3) μM × hour, clearance (±SD) was 153 mL/min (±84), and t1/2 was 5.9 h. In part 2, 22 patients received the MTD and there were no DLTs. Although there were no objective tumor responses, four patients (with cervical, non-small cell lung, and ovarian cancers) had prolonged stable disease.
MK-0731 at the MTD of 17 mg/m2/day every 21 days in patients with solid tumors had few grade 3 and 4 toxicities with the major DLTs at higher doses being myelosuppression. Anti-tumor efficacy was suggested by the length of stable disease in selected patients with taxane-resistant tumors.
PMCID: PMC3394096  PMID: 21424701
Kinesin spindle protein; Oncology; Neutropenia
23.  Safety, tolerability, pharmacokinetics and pharmacodynamics of AZD8055 in advanced solid tumours and lymphoma 
British Journal of Cancer  2012;107(7):1093-1099.
This study assessed the safety, tolerability, pharmacokinetics and pharmacodynamics of the first-in-class dual mammalian target of rapamycin complex (mTORC)1/mTORC2 inhibitor, AZD8055.
Patients with advanced solid malignancies or lymphomas were recruited into this phase I, open-label, dose-escalation study of AZD8055 starting at 10 mg twice-daily oral dosing (BID).
Forty-nine patients received AZD8055. Dose-limiting toxicities were reported at 40 mg (n=1), 90 mg (n=1) and 120 mg (n=3) BID; all were grade 3 rises in transaminases, reversible in all patients, apart from one who had liver metastases. The maximum tolerated dose was defined as 90 mg BID. The most frequent adverse events assessed to be related to AZD8055 were increased alanine aminotransferase (22%), increased aspartate aminotransferase (22%) and fatigue (16%). AZD8055 was rapidly absorbed (median tmax ∼0.5 h) and exposure increased with increasing doses. Seven patients had stable disease for ⩾4 months. Partial metabolic responses, assessed by fluorodeoxyglucose positron emission tomography, were observed at ⩾40 mg BID (n=8 at day 35).
The maximum tolerated dose for AZD8055 is 90 mg BID. Apart from elevated transaminases, which occurred at most dose levels, the drug had an acceptable toxicity profile; however, no RECIST responses were seen.
PMCID: PMC3461162  PMID: 22935583
AZD8055; mTOR inhibitors; phase I
24.  Apoptosis Induction by MEK Inhibition in Human Lung Cancer Cells Is Mediated by Bim 
PLoS ONE  2010;5(9):e13026.
AZD6244 (ARRY-142886) is an inhibitor of MEK1/2 and can inhibit cell proliferation or induce apoptosis in a cell-type dependent manner. The precise molecular mechanism of AZD6244-induced apoptosis is not clear. To investigate mechanisms of AZD6244 induced apoptosis in human lung cancer, we determined the molecular changes of two subgroups of human lung cancer cell lines that are either sensitive or resistant to AZD6244 treatment. We found that AZD6244 elicited a large increase of Bim proteins and a smaller increase of PUMA and NOXA proteins, and induced cell death in sensitive lung cancer cell lines, but had no effect on other Bcl-2 related proteins in those cell lines. Knockdown of Bim by siRNA greatly increased the IC50 and reduced apoptosis for AZD6244 treated cells. We also found that levels of endogenous p-Thr32-FOXO3a and p-Ser253-FOXO3a were lower in AZD6244-sensitive cells than in AZD6244-resistant cells. In the sensitive cells, AZD6244 induced FOXO3a nuclear translocation required for Bim activation. Moreover, the silencing of FOXO3a by siRNA abrogated AZD6244-induced cell apoptosis. In addition, we found that transfection of constitutively active AKT up-regulated p-Thr32-FOXO3a and p-Ser253-FOXO3a expression and inhibited AZD6244-induced Bim expression in sensitive cells. These results show that Bim plays an important role in AZD6244-induced apoptosis in lung cancer cells and that the PI3K/AKT/FOXO3a pathway is involved in Bim regulation and susceptibility of lung cancer cells to AZD6244. These results have implications in the development of strategies to overcome resistance to MEK inhibitors.
PMCID: PMC2946397  PMID: 20885957
25.  A Phase I study of the Heat Shock Protein 90 inhibitor alvespimycin (17-DMAG) given intravenously to patients with advanced solid tumors 
A Phase I study to define toxicity and recommend a Phase II dose of the HSP90 inhibitor alvespimycin (17-DMAG; 17-dimethylaminoethylamino-17-demethoxygeldanamycin). Secondary endpoints included evaluation of pharmacokinetic profile, tumor response and definition of a biologically effective dose (BED).
Patients and Methods
Patients with advanced solid cancers were treated with weekly, intravenous (IV) 17-DMAG. An accelerated titration dose escalation design was used. The maximum tolerated dose (MTD) was the highest dose at which ≤ 1/6 patients experienced dose limiting toxicity (DLT). Dose de-escalation from the MTD was planned with mandatory, sequential tumor biopsies to determine a BED. Pharmacokinetic and pharmacodynamic assays were validated prior to patient accrual.
Twenty five patients received 17-DMAG (range 2.5 to 106 mg/m2). At 106mg/m2 of 17-DMAG 2/4 patients experienced DLT, including one treatment related death. No DLT occurred at 80mg/m2. Common adverse events were gastrointestinal, liver function changes and ocular. AUC and Cmax increased proportionally with 17-DMAG doses ≤ 80mg/m2. In peripheral blood mononuclear cells significant (p <0.05) HSP72 induction was detected (≥ 20mg/m2) and sustained for 96 hours (≥ 40mg/m2). Plasma HSP72 levels were greatest in the two patients who experienced DLT. At 80mg/m2 client protein (CDK4, LCK) depletion was detected and tumor samples from 3/5 patients confirmed HSP90 inhibition. Clinical activity included complete response (castration refractory prostate cancer, CRPC 124 weeks), partial response (melanoma, 159 weeks) and stable disease (chondrosarcoma, CRPC and renal cancer for 28, 59 and 76 weeks respectively).
The recommended Phase II dose of 17-DMAG is 80mg/m2 weekly, IV.
PMCID: PMC3060938  PMID: 21278242

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