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Preclinical analyses and phase I evaluation of LY2603618 administered in combination with Pemetrexed and cisplatin in patients with advanced cancer

Article in Investigational New Drugs ? June 2014

DOI: 10.1007/s10637-014-0114-5 ? Source: PubMed

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Invest New Drugs (2014) 32:955?968 DOI 10.1007/s10637-014-0114-5

PHASE I STUDIES

Preclinical analyses and phase I evaluation of LY2603618 administered in combination with Pemetrexed and cisplatin in patients with advanced cancer

Emiliano Calvo & Victor J. Chen & Mark Marshall & Ute Ohnmacht & Scott M. Hynes & Elizabeth Kumm & H. Bruce Diaz & Darlene Barnard & Farhana F. Merzoug & Lysiane Huber & Lisa Kays & Philip Iversen & Antonio Calles & Beatrice Voss & Aimee Bence Lin & Nicolas Dickgreber & Thomas Wehler & Martin Sebastian

Received: 12 March 2014 / Accepted: 12 May 2014 / Published online: 20 June 2014 # Springer Science+Business Media New York 2014

Summary LY2603618 is an inhibitor of checkpoint kinase 1 (CHK1), an important regulator of the DNA damage checkpoints. Preclinical experiments analyzed NCI-H2122 and NCI-H441 NSCLC cell lines and in vitro/in vivo models treated with pemetrexed and LY2603618 to provide rationale for evaluating this combination in a clinical setting. Combination treatment of LY2603618 with pemetrexed arrested DNA synthesis following initiation of S-phase in cells. Experiments with tumor-bearing mice administered the combination of LY2603618 and pemetrexed demonstrated a significant increase of growth inhibition of NCI-H2122 (H2122) and NCI-H441 (H441) xenograft tumors. These data informed the clinical assessment of LY2603618 in a seamless phase I/II study, which administered pemetrexed (500 mg/m2) and

cisplatin (75 mg/m2) and escalating doses of LY2603618: 130?275 mg. Patients were assessed for safety, toxicity, and pharmacokinetics. In phase I, 14 patients were enrolled, and the most frequently reported adverse events included fatigue, nausea, pyrexia, neutropenia, and vomiting. No DLTs were reported at the tested doses. The systemic exposure of LY2603618 increased in a dose-dependent manner. Pharmacokinetic parameters that correlate with the maximal pharmacodynamic effect in nonclinical xenograft models were achieved at doses 240 mg. The pharmacokinetics of LY2603618, pemetrexed, and cisplatin were not altered when used in combination. Two patients achieved a confirmed partial response (both non-small cell lung cancer), and 8 patients had stable disease. LY2603618 administered in

These authors are Thomas Wehler and Martin Sebastian contributed equally to this study. Research support: The study was sponsored by Eli Lilly and Company. Trial Registration ID: NCT01139775

E. Calvo (*) : A. Calles

START Madrid, Clara Campal Comprehensive Cancer Center, Medical Oncology Division, Madrid Norte Sanchinarro University Hospital, Madrid, Spain 28050 e-mail: Emiliano.Calvo@start.

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A. B. Lin Eli Lilly and Company, Indianapolis, IN, USA

E. Kumm inVentiv Health Clinical, LLC, 200 Cottontail Ln, Somerset, NJ, USA

A. Calles Spanish National Cancer Research Centre (CNIO), Madrid, Spain

B. Voss : T. Wehler

Department of Internal Medicine, Hematology and Oncology, Johannes Gutenberg-University Mainz, Mainz, Germany

N. Dickgreber Department of Respiratory Medicine and Thoracic Oncology, Mathias-Spital Rheine, Rheine, Germany

M. Sebastian Department of Hematology/Oncology, Rheumatology, HIV, J.W. Goethe University, Frankfurt, Germany

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combination with pemetrexed and cisplatin demonstrated an acceptable safety profile. The recommended phase II dose of LY2603618 was 275 mg.

Keywords CHK1 . Cancer . LY2603618 . Cell cycle . Lung cancer . Pharmacokinetics

Introduction

In 2008, there were an estimated 1.61 million new cases of lung cancer per year in the world; lung cancer is the leading cause of cancer-related mortality, accounting for 1.38 million deaths per year globally [1]. Non-small cell lung cancer (NSCLC) constitutes about 85 % of lung cancer cases, and two-thirds of these patients will present with Stage III/IV disease [2]. For patients with Stage III/IV non squamousNSCLC without EML4-ALK rearrangements or activating EGFR mutations, the first-line standard of care is a platinum-based doublet such as cisplatin/pemetrexed [3, 4]. However, the reported response rates of 39?44 % suggest that improved therapy options are needed for these patients [5, 6].

Checkpoint kinase 1 (CHK1) is a protein kinase critical for the DNA damage checkpoint signal transduction pathway [7]. CHK1 is activated in response to DNA damage, including damage from chemotherapeutic agents that disrupt or block DNA replication [8?11]. Activation of CHK1 in dividing cells produces an arrest in the S and G2/M phases of cell cycle to permit completion of DNA repair prior to mitosis. Inhibition of CHK1 in the presence of DNA damage releases cells from this checkpoint arrest and allows progression into mitosis with damaged DNA and results in apoptosis [12?16]. Several published studies show that inhibition of CHK1 renders cells more susceptible to the cytotoxic effects of gemcitabine, camptothecin, doxorubicin, 5-fluorouracil and radiation, cisplatin, and carboplatin [17?23]. While it was reported that CHK1 inhibition does not potentiate platinum agents in certain tumor cell lines [24], recent articles present evidence that CHK1 inhibition can effectively potentiate the activity of cisplatin to overcome chemoresistance in NSCLC stem cells [25, 26]. Additionally, inhibition of the CHK1 activating kinase (ATR) by caffeine sensitized cells to the antiproliferative activity of pemetrexed [27].

LY2603618, a selective inhibitor of CHK1, enhances the effects of DNA damaging agents in tumors by exploiting the absence of p53 control of the G2/M checkpoint in most cancer cells [28, 29] and has been successfully combined with pemetrexed in a phase I study in patients with advanced cancer [30, 31]. Here, we summarize the results of preclinical studies that evaluated the antitumor effects of pemetrexed and LY2603618, alone and in combination. These experiments explored the mechanism of LY2603618 that may potentiate

the antitumor activity of pemetrexed and provide rationale for exploring this combination in the clinical setting. In vitro and in vivo studies were performed with 2 NSCLC adenocarcinoma models, H2122 and H441. Additionally, we report results of the phase I portion of an open-label seamless phase I/randomized phase II study of LY2603618 administered in combination with pemetrexed and cisplatin. The primary objective was to determine a recommended phase II dose of LY2603618. Secondary objectives included the analyses of safety, pharmacokinetics, and toxicity profile of LY2603618 when administered in combination with pemetrexed and cisplatin.

Patients and methods

Cell culture and reagents

H2122 and H441 NSCLC cells were obtained from American Type Culture Collection, Manassas, Virginia. H2122 cells, lot 3489498, were obtained in April, 2007 and authenticated at SeqWright, Inc (Houston TX) by STR analysis in July, 2007. H441 cells, lot 3984794, were obtained in March, 2006 and authenticated at SeqWright, Inc. by STR analysis in September, 2006. Cells were routinely passaged at 37 ?C, >90 % relative humidity and 5 % CO2. The growth medium was RPMI1640 with 25 mM HEPES and L-Glutamine (Hyclone SH30255.04), (Antibiotic-Antimycotic, Gibco 15240?096) and 10 % of either heat inactivated fetal bovine serum (Gibco 10082) for routine cell passage or dialyzed fetal bovine serum (Gibco 26400) for use with experiments involving drug treatments.

Flow cytometry

Flow cytometry studies of H2122 and H441 used cells seeded in T-flasks (Corning 430641 or 430825) at the density range of 20,000?40,000 cells/cm. Cells were exposed to a pulse of 5-ethynyl?2-deoxyuridine (EdU; Invitrogen cat# A10044) for 30 min (min), collected, washed, and fixed with 1 % formaldehyde in PBS for 5 min. Fixed cells (approximately 1?2?106) were stained with Alexa 647 azide for detecting EdU incorporation, and then with propidium iodide in the presence of RNAse (50 and 100 g/mL, respectively in 1.0 mL) per manufacturer's instructions (Click-it kit, Invitrogen) to measure DNA content. Listmode data were analyzed by WinList Version 6.0 (Verity Software House, Topsham, Maine). Signals due to doublets were discriminated in a plot of propidium iodide peak height versus peak area before quantitation.

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Animals

All animal experiments were conducted in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and with Animal Care and Use Committee guidelines of Eli Lilly and Company. For in vivo drug activity evaluation, female athymic nude mice bearing subcutaneous tumor xenografts were used. Two experiments were performed with H2122 xenografted tumors. Study 1 used Ncr-nude mice from Taconic Farms Inc., Rensselaer, New York. Study 2 used CD-1 nu/nu mice from Charles Rivers Laboratories Inc., Wilmington, Massachusetts. Animals were maintained on PicoLab? Rodent Diet 20 (#5051) standard feed. Due to endogenous levels of folate in mice, folate supplementation was not required with pemetrexed administration. Approximately 5?106 tumor cells suspended in 0.2 mL of a mixture consisting of equal volumes of matrigel and RPMI1640 medium without serum were implanted subcutaneously in the right flank of the mice. Dosing began when tumors reached an average size of 100 mm3. Tumor volumes were measured by a Fowler Electronic Caliber (Model #325?1352).

Two experiments with human H441 lung carcinoma tumor xenografts were performed at Maccine PTE LTD in Singapore. Female BALB/c nu/nu athymic mice were obtained from the Animal Research Centre, Perth, Australia. Animals were maintained either on Harlan Teklad? 18 % Protein Rodent Diet or PMI LabDiet? standard feed. H441 cells were resuspended in PBS 1:1 Matrigel?. Mice were injected subcutaneously in the rear right flank with approximately 5?106 cells in a volume of 0.2 mL for Study 1 and 2.5?106 tumor cells suspended in 0.1 mL for Study 2. Drug dosing began when tumors reached an average size of 100 mm3.

Statistical analysis

Tumor volume data were transformed to a log scale to equalize variance across time and treatment groups. The log volume data were analyzed with a 2-way repeated measures analysis of variance by time and treatment, using the MIXED procedures in SAS software (version 8.2). The correlation model for the repeated measures was spatial power. Treated groups were compared to the control group at each time point. The MIXED procedure was also used separately for each treatment group to calculate adjusted means and standard errors at each time point. Both analyses account for the autocorrelation within each animal and the loss of data that occurs when animals with large tumors are removed from the study early. The adjusted means and standard errors were plotted for each treatment group versus time. The % T/C is 100*(T-T0)/(C-C0) where T and C are the mean tumor volumes in the treatment and control groups, respectively, on the day indicated, and T0 and C0 are the mean tumor volumes at baseline.

Tumor growth delay, the difference in mean crossing time between a treated group and the vehicle control group, was calculated by an individual interpolation statistical method. For each animal, the time to reach a specified tumor volume (threshold) was calculated by interpolating between the last measurement before reaching the threshold and the next measurement. The interpolation is linear using log10(volume) versus time. If an animal never reached the threshold, its crossing time was reported at ">T" where T is the last day measured for that animal. These crossing times were analyzed as "time-to-event" data with right-censoring using a Weibull distribution. Means and standard errors were determined for each treatment group, and p-values were determined by a Kaplan-Meier analysis on the crossing times. Between studies using the same cell line, the threshold tumor volumes were selected based on treatment for an equal number of cycles. This accounts for differences in intrinsic tumor growth rates between replicate studies.

Patients

Eligible patients were male and female, age 18 years, with histologically or cytologically confirmed solid tumors refractory to standard therapy with measureable or non-measureable disease. Patients had no more than 2 previous lines of chemotherapy or >6 cycles of therapy containing an alkylating agent but were allowed to have 1 extra line of adjuvant/neoadjuvant chemotherapy if completed 1 year prior to enrollment. Patients with prior radiation therapy were allowed to participate if they had recovered from the acute toxic effects of prior treatments [32]. Patients had a performance status of 1 on the Eastern Cooperative Oncology Group (ECOG) scale. Required laboratory tests included adequate hematopoietic function defined as: absolute neutrophil count 1.5?109/L; platelets 100? 109/L; hemoglobin 9 g/dL. Patients had adequate renal and hepatic function defined as: creatinine clearance 45 mL/min; bilirubin 1.5 times upper limits of normal (ULN); alkaline phosphatase, alanine aminotransaminase, and aspartate aminotransferase 3 times ULN (except for patients with liver tumor, which 5 times ULN was acceptable). Patients were excluded from participation for any of the following reasons: radiation to whole pelvis; received treatment with a drug that had not received regulatory approval for any indication within 28 days of initial study drug dose; symptomatic central nervous system malignancy or metastasis; positive results for human immunodeficiency virus, hepatitis B surface antigen, hepatitis C antibodies; previously completed or withdrawn from a study investigating LY2603618 or any other CHK1 inhibitor; could not discontinue concurrent non-steroidal antiinflammatory drugs (NSAIDs) treatment; clinically significant third-space fluid collections that could not be controlled prior to study; received a yellow-fever vaccination within 28 days of enrollment; prior malignancy other than NSCLC,

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cervical carcinoma, or nonmelanoma skin cancer unless diagnosed and definitively treated at least 5 years previously with no recurrence (with the exception of low grade, localized prostate cancer); pregnancy; lactation.

Study design

This was a multi-center, open-label seamless phase I/randomized phase II study of LY2603618 in patients with advanced and/or metastatic cancer. For phase I, eligible patients received LY2603618 as a one-hour intravenous (IV) infusion approximately 24 h after standard doses of pemetrexed (500 mg/m2) and cisplatin (75 mg/m2) on day 1 of a 21-day cycle. A 3+3 dose escalation paradigm was used. Folic acid and vitamin B12 supplementation were administered as premedication for pemetrexed therapy.

Dose escalation was guided by safety assessments during cycle 1 using the standard grading system, Common Terminology Criteria for Adverse Events Version 4.02 (CTCAE v4.02). In addition to adverse events (AEs), available pharmacokinetic data were secondary considerations for dose escalation. The planned duration of treatment was 4?6 cycles of study therapy unless discontinuation criteria were met. It was permissible to administer pemetrexed and LY2603618 treatment beyond 4 to 6 cycles, based on investigator's judgment.

Dose-limiting toxicity (DLT) was defined as an AE likely related to LY2603618 occurring during cycle 1 following the CTCAE v4.02 criteria: Grade 3 thrombocytopenia with Grade 2 bleeding or Grade 4 thrombocytopenia without bleeding; Grade 3 non-hematological toxicity except nausea/ vomiting/diarrhea/electrolyte disturbance all responsive to medical treatment; a period >28 days from day 1 cycle 1 preventing the start of cycle 2; Grade 3 nausea, vomiting, diarrhea or electrolyte disturbance that persists more than 2 days despite outpatient intervention; Grade 3 neutropenia with fever, febrile neutropenia; Grade 4 neutropenia without fever for 7 days. If a single patient experienced DLT within the first cycle of LY2603618, three additional patients were enrolled at that dose level. If a DLT was observed in two or more patients at any dose level, escalation ceased and the previous dose was declared the maximum tolerated dose (MTD).

Drug supply

LY2603618, pemetrexed and cisplatin were provided by Eli Lilly and Company (Indianapolis, IN).

Pharmacokinetic analyses

Pharmacokinetic analyses were performed on patients who received at least 1 dose of LY2603618, pemetrexed, and cisplatin using a validated liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) method for

LY2603618 and pemetrexed and a validated inductively coupled plasma mass spectrometry (ICP-MS) method for total platinum. Pharmacokinetic assessment of LY2603618 was performed following the first LY2603618 dose on day 2 of cycle 1 and 2; blood samples were collected immediately prior to the end of a 1-h infusion ( ................
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