Safety and pharmacokinetics of motesanib in combination with gemcitabine for the treatment of patients with solid tumours

The aim of this open-label phase 1b study was to assess the safety and pharmacokinetics of motesanib in combination with gemcitabine in patients with advanced solid tumours. Eligible patients with histologically or cytologically documented solid tumours or lymphoma were enroled in three sequential, dose-escalating cohorts to receive motesanib 50 mg once daily (QD), 75 mg two times daily (BID), or 125 mg QD in combination with gemcitabine (1000 mg m−2). The primary end point was the incidence of dose-limiting toxicities (DLTs). Twenty-six patients were enroled and received motesanib and gemcitabine. No DLTs occurred. The 75 mg BID cohort was discontinued early; therefore, 125 mg QD was the maximum target dose. Sixteen patients (62%) experienced motesanib-related adverse events, most commonly lethargy (n=6), diarrhoea (n=4), fatigue (n=3), headache (n=3), and nausea (n=3). The pharmacokinetics of motesanib and of gemcitabine were not markedly affected after combination therapy. The objective response rate was 4% (1 of 26), and 27% (7 of 26) of patients achieved stable disease. In conclusion, treatment with motesanib plus gemcitabine was well tolerated, with adverse event and pharmacokinetic profiles similar to that observed in monotherapy studies.

Angiogenesis, the process by which new blood vessels develop, plays a critical role in tumour development and metastasis by providing tumours with nutrients and oxygen (Folkman and Shing, 1992). The proangiogenic cytokine vascular endothelial growth factor (VEGF) promotes neovascularisation by binding and activating VEGF receptor 2 (VEGFR2) and VEGFR1 (Ferrara et al, 2003). Solid tumours also express platelet-derived growth factor receptor (PDGFR) and stem cell factor receptor (Kit); both have been shown to play a role in the pathogenesis of several tumour types (Heinrich et al, 2002;Yu et al, 2003). Inhibition of angiogenesis has emerged as an effective treatment strategy in some solid tumours (Yang et al, 2003;Miller et al, 2005;Sandler et al, 2006).
Recent studies have suggested that combination treatment with a VEGF inhibitor plus cytotoxic chemotherapy may result in enhanced efficacy compared with single-agent approaches, whereas maintaining a favourable adverse event profile (Miller et al, 2005(Miller et al, , 2007Giantonio et al, 2007;Herbst et al, 2007;Heymach et al, 2007). Gemcitabine, which has activity in many solid tumours, has been combined with the monoclonal anti-VEGF antibody bevacizumab for the treatment of hepatocellular carcinoma or advanced pancreatic cancer (Kindler et al, 2005;Zhu et al, 2006). The aim of this study was to determine the maximum target dose and the pharmacokinetic profile of motesanib when administered in combination with gemcitabine in patients with advanced solid tumours, and to assess the safety and efficacy of this treatment regimen.

MATERIALS AND METHODS Patients
Eligible adult (X18 years) patients had histologically or cytologically documented solid tumours or lymphoma; Eastern Cooperative Oncology Group score zero to two; systolic/diastolic blood pressure p145/p85 mmHg; adequate cardiac, hepatic, and renal function; and were candidates for gemcitabine treatment as deemed by the investigator. Patients were ineligible if they had received prior treatment with VEGF inhibitors or gemcitabine, had received systemic chemotherapy within 21 days of study day 1 or strong inhibitors of cytochrome P450 (CYP) 3A4 within 7 days of study day 1. Other main exclusion criteria were: non-small cell lung cancer (NSCLC) with squamous cell histology; large central tumour lesions in the thorax (X3 cm and located adjacent to or within the hilum or mediastinum); evidence of active bleeding or bleeding diathesis; and untreated or symptomatic brain metastases. All patients provided written informed consent.

Study design
This was an open-label, phase 1b, dose-finding study conducted at three centres in Australia. All study procedures were approved by the respective independent ethics committee and/or institutional review board. The primary end point was the incidence of dose-limiting toxicities (DLTs). The secondary end point was assessment of the pharmacokinetic profile of motesanib and gemcitabine, respectively, when administered alone or in combination. The safety end points included incidence of adverse events, serious adverse events, and laboratory abnormalities not defined as DLTs. Exploratory end points included the response rate as defined by modified Response Evaluation Criteria in Solid Tumours (RECIST) (Therasse et al, 2000).

Dose escalation and target dose
Motesanib diphosphate (Amgen Inc., Thousand Oaks, CA, USA) was self-administered continuously by mouth beginning on day 2 of cycle 1. Based on the range of tolerable doses explored in a previous study (Rosen et al, 2007), patients were enroled in three sequential, dose-escalating cohorts (up to six evaluable patients each): motesanib 50 mg once daily (QD) (starting dose), 75 mg two times daily (BID), or 125 mg QD. Enrolment in the higher dose cohorts began only if the initial 50-mg QD dose was well tolerated (four or more patients with no DLTs). Dose escalation within cohorts was not permitted. In the 125-mg QD cohort, if two or fewer patients experienced a DLT in the first 4 weeks of treatment this dose would be considered the target QD dose. Gemcitabine (1000 mg m À2 ) was administered intravenously once weekly for 7 weeks followed by 1 week of rest (cycle 1), then once weekly for 3 weeks followed by 1 week of rest (cycles 2 -11; all 4-week cycles). Combination treatment continued for up to 48 weeks or until disease progression or unacceptable toxicity occurred.
Dose delays and dose reductions were defined in the protocol for both motesanib and gemcitabine according to treatmentrelated toxicities.

Dose-limiting toxicities
DLTs were defined as grade 3 or higher toxicities occurring during cycle 1 that were related to motesanib or to motesanib plus gemcitabine. Fatigue, nausea, diarrhoea, vomiting, neutropenia, febrile neutropenia, thrombocytopenia, anaemia, lymphopenia, alopecia, hypertension, and increased aspartate aminotransferase (AST) or alanine aminotransferase (ALT) were excluded unless the following criteria were met: grade 3 fatigue persisting for X7 days or grade 4 fatigue; grade 3 or 4 nausea, vomiting or diarrhoea despite maximum supportive care; grade 3 neutropenia with fever 438.51C or grade 4 neutropenia; grade 4 thrombocytopenia persisting for 47 days; grade 4 anaemia; grade 4 hypertension; AST or ALT 410 times the upper limit of normal.

Safety
Adverse events were collected throughout the study and classified according to the Medical Dictionary for Regulatory Affairs (2008). Severity was graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 3.0 (2006). Blood pressure and haematology were assessed on each day of a gemcitabine infusion. Blood biochemistry was assessed on day 1 of each cycle. Samples for urinalysis were collected during cycles 1, 2, and 3 and every other cycle thereafter.
Samples were analysed for motesanib (Amgen Inc., Thousand Oaks, CA, USA) and gemcitabine concentrations (Quest Pharmaceutical Services, Newark, DE, USA) using validated liquid chromatography/tandem mass spectrometry methods with a lower limit of quantitation of 0.5 and 10 ng ml À1 , respectively.
Noncompartmental pharmacokinetic analyses were performed using WinNonlin Professional software on Citrix (Version 5.1.1, Pharsight Corporation, Mountain View, CA, USA) to estimate the following pharmacokinetic parameters: maximum observed plasma concentration (C max ), time to C max (t max ), the terminal-phase elimination half-life (t 1/2,z ), observed plasma concentration at 24 h postdose (C 24 ) for motesanib, observed plasma concentration at 2 h postdose (C 2 ) for gemcitabine, and area under the concentration vs time curve from time 0 extrapolated to infinity (AUC 0Àinf ) and 0 -24 h (AUC 0 -24 ) for motesanib or 0 -2 h (AUC 0 -2 ) for gemcitabine.

Tumour response
Computed tomography (CT) or magnetic resonance imaging (MRI) for tumour assessments were performed at baseline and at 12-week intervals, or as clinically indicated. In patients with baseline measurable disease tumour response per modified RECIST (Therasse et al, 2000) was assessed by the investigator. Complete and partial responses were confirmed using CT or MRI scans no less than 28 days after the initial response.

Statistical analysis
The safety analysis set included all patients who received X1 dose of motesanib. The pharmacokinetic analysis set included all patients from the safety analysis set who had evaluable plasma samples. The comparative pharmacokinetic analysis subset included patients with evaluable C max values following single-dose administration and multiple-dose administration in combination with gemcitabine.

Dose escalation, dose-limiting toxicities, and target dose
The first cohort (motesanib 50 mg QD starting dose) enroled 11 patients to have six evaluable patients for the safety review (five patients were deemed unevaluable per protocol criteria). No DLTs occurred. The median treatment duration was 83 days (minimum -maximum, 55 -188). Six patients were then enroled in the 125-mg QD cohort. This dose had been previously established as the motesanib maximum-tolerated dose in the first-in-human study (Rosen et al, 2007). One DLT of grade 4 neutropenia occurred that was considered (per investigator) to be related to both motesanib and gemcitabine treatment. The 125-mg QD dose was established as the target once-daily dose per protocol definition (see Materials and Methods). A maximum-tolerated dose was not determined in this study. As the 125-mg QD dose was reasonably well tolerated, doses between 50 mg and 125 mg QD were not tested. Patients in the 125-mg QD cohort received motesanib treatment on a median of 53 days (minimummaximum, 20 -181). The 75-mg BID cohort enroled nine patients. No DLTs occurred. One patient had jugular vein thrombosis that was considered by the study site to be not related to motesanib. However, the cohort was discontinued early because an increased incidence of cholecystitis, compared with the general population of cancer patients, was observed at this dose level in other motesanib studies (Amgen Inc., data on file). The median treatment duration in this cohort was 34 days (minimum -maximum, 14 -112).

Adverse events
Sixteen patients (62%) experienced motesanib treatment-related adverse events, including several events of special interest (Table 2). Hypertension was generally manageable with antihypertensive treatment, although two patients (one in the 50-mg QD cohort and one in the 75-mg BID cohort) had motesanib dose reductions to control hypertension, and treatment was withheld in one patient in the 125-mg QD cohort. Two grade 3 motesanibrelated adverse events (lethargy, considered related to both motesanib and gemcitabine treatment, and deep vein thrombosis; 75-mg BID cohort) and 1 grade 4-related event (neutropenia; 125mg QD cohort) occurred. Serious related events were grade 2 dehydration and lethargy (50-mg QD cohort); and grade 3 deep vein thrombosis (75-mg BID cohort). No cases of cholecystitis occurred. Three patients died during the study. The deaths were attributed to disease progression (50-mg QD cohort), renal failure (50-mg QD cohort), and Pneumocystis jiroveci pneumonia (75-mg BID cohort), respectively. All deaths were unrelated to motesanib treatment.

Pharmacokinetics of motesanib
Mean (s.d.) plasma motesanib concentration vs time profiles and a summary of pharmacokinetic parameters for intensive pharmacokinetic sampling on day 2 of week 1 and day 1 week 2 are shown in Figure 1 and Table 3, respectively. After single-dose oral administration, motesanib was rapidly absorbed, with an overall median t max ranging from 0.5 to 1.5 h for all three dose cohorts. Similar overall median t max values (0.83 -2.0 h) were observed after multiple-dose administration in combination with gemcitabine. The mean t 1/2,z values ranged from 3.8 to 5.7 h after single-dose administration and from 4.3 to 5.0 h after multiple-dose administration in combination with gemcitabine; these values did not appear to be dose-or time-dependent. The mean daily trough plasma concentrations (C 24 ) for the 50-mg QD cohort were below the in vitro IC 50 of human umbilical vein endothelial cell (HUVEC) proliferation (IC 50 ¼ 4 ng/ml) (Polverino et al, 2006) during cycle 1 but were above the IC 50 during cycle 2. Mean C 24 values for both week 1 and week 2 were above the IC 50 for the 125-mg QD cohort, and were above the in vitro 90% inhibitory concentration (IC 90 ) of HUVEC prolifera-tion (IC 90 ¼ 28 ng/ml; Amgen Inc., data on file) for the 75-mg BID cohort.
The C max values (week 1) for the 125-mg QD cohort were comparable to those observed in the monotherapy study (Rosen et al, 2007). However, motesanib C max (except 125 mg QD, week 1) and AUC values for week 1 and 2 in the QD cohorts were slightly lower (o50% decrease) than the values observed in the motesanib monotherapy study.
and AUC values in the same patients were slightly higher (o2-fold) in week 2 compared with week 1.

Tumour response
Of the 26 patients enroled, one patient in the 125-mg QD dose cohort with stage IV bladder cancer had a confirmed partial response (

DISCUSSION
Inhibition of angiogenesis has emerged as an effective therapy with acceptable tolerability in a variety of different solid tumours, including colorectal cancer (Hurwitz et al, 2004), NSCLC (Sandler AUC 0 -inf ¼ area under the plasma concentration versus time curve from time 0 to infinity; AUC 0 -24 ¼ area under the plasma concentration versus time curve from time 0 -24 h postdose; BID ¼ two times daily; CL/F ¼ apparent clearance; C max ¼ maximum observed concentration after dosing; C 12 ¼ observed concentration at 12 h postdose; C 24 ¼ observed concentration at 24 h postdose; NR ¼ not reported; QD ¼ once daily; t max ¼ time of C max ; t 1/2,z ¼ estimated terminal elimination half-life. a For the 75-mg BID dose cohort, t 1/2,z values were estimated based on the terminal slope after the first dose on week 1 or 2, AUC 0 -24 values were estimated using 2 Â AUC 0 -12 , and CL/F was estimated by total daily dose/(2 Â AUC 0 -12 ).  et al, 2006), and breast cancer (Miller et al, 2007). Recent studies have suggested that treatment with an antiangiogenic agent in combination with cytotoxic chemotherapy can result in increased antitumour efficacy, compared with chemotherapy alone, while maintaining a tolerable safety profile (Miller et al, 2005;Giantonio et al, 2007;Herbst et al, 2007). Motesanib is a highly specific inhibitor of VEGFR1, 2, and 3; of PDGFR, and Kit (Polverino et al, 2006), all of which have been implicated as key regulators of angiogenesis, lymphangiogenesis, and tumour cell proliferation (Heinrich et al, 2002;Ferrara et al, 2003;Yu et al, 2003). In the first-in-human monotherapy study, motesanib showed encouraging efficacy and acceptable toxicity in patients with advanced solid tumours (Rosen et al, 2007). In the study reported here, combination therapy with motesanib 125 mg QD plus gemcitabine (1000 mg m À2 ) was well tolerated and 125 mg QD was established as the maximum target dose. Adverse events considered related to motesanib were generally of mild to moderate severity. The overall safety profile was similar to that observed in a previous monotherapy study (Rosen et al, 2007), and was comparable to results reported from monotherapy studies of other VEGF inhibitors (Yang et al, 2003;Herbst et al, 2005;Motzer et al, 2006) and those from chemotherapy combination studies (Kindler et al, 2005;Richly et al, 2006;Heymach et al, 2007). Similarly, specific adverse events typically associated with VEGF inhibition, such as hypertension, bleeding, or thromboembolic events, did not occur more frequently in the study described here than in monotherapy studies of other VEGF inhibitors (Herbst et al, 2005) or in gemcitabine combination studies, such as a phase 2 trial of bevacizumab plus gemcitabine in advanced pancreatic cancer (Kindler et al, 2005). Of note, other toxicities, for example, dermatologic toxicities, frequently reported with certain VEGFR and PDGFR inhibitors both as single-agent treatment (Escudier et al, 2007) or in combination with cytotoxic chemotherapy (Richly et al, 2006) did not occur in this study. Gemcitabine coadministration did not affect the tolerability profile of motesanib in this clinical trial. This finding is consistent with prior studies, which demonstrated that the addition of cytotoxic chemotherapy did not significantly alter the safety profile of other tyrosine kinase inhibitors including sorafenib (Richly et al, 2006) and vandetanib (Heymach et al, 2007). Although no cases of cholecystitis occurred in this study, the 75-mg BID dose cohort was discontinued early because of an increased risk of cholecystitis in patients who received this dose in other motesanib studies. The cause and relation to motesanib is unknown and is currently under investigation. Acute cholecystitis related to treatment with multikinase inhibitors was earlier reported only once in a patient receiving sunitinib (Motzer et al, 2006). The pharmacokinetics of motesanib and gemcitabine were not markedly affected by coadministration of the other compound. The C max and AUC values for motesanib on week 2 compared with week 1 varied across dose groups. Moreover, there was no apparent trend of increasing or decreasing motesanib exposure  Patients with measurable disease at baseline, n (%) 11 (100) 6 (100) 9 (100) 26 (100) Response assessment, n (%) Confirmed complete response a 0 (0) 0 (0) 0 (0) 0 (0) Confirmed partial response a 0 (0) 1 (17) 0 (0) 1 (4) Stable disease 4 (36) 2 (33) 1 (11) 7 (27) Progressive disease 7 (64) 3 (50) 2 (22) 12 (46) Not done 0 (0) 0 (0) 6 (67) 6 (23) Confirmed objective response b , % (95% CI) 0 (0.0 -28.5) 17 (0.4 -64.1) 0 (0.0 -33.6) 4 (0.1 -19.6) a Patients with a response assessment of complete response or partial response that was not confirmed at least 4 weeks later are reported as stable disease. b Objective response is defined as a tumour response assessment of either confirmed complete response or confirmed partial response.
Safety and pharmacokinetics of motesanib plus gemcitabine TJ Price et al during coadministration with gemcitabine with increasing doses of motesanib. An effect was not expected because, unlike motesanib, gemcitabine is not metabolised by CYP3A4 (Shipley et al, 1992;Mini et al, 2006). Exposure to motesanib in patients in the 50-mg QD and 125-mg QD dose cohorts was similar (although slightly lower) to that observed in a previous monotherapy study (Rosen et al, 2007). The 75-mg BID dose has not been tested in monotherapy studies and, therefore, no comparison of pharmacokinetic parameters and trough concentrations can be made. Although mean gemcitabine concentrations on week 2 were slightly higher than those on week 1, exposure to gemcitabine, both alone and in combination with motesanib, was no greater than the exposure observed in previous studies investigating gemcitabine monotherapy (Cattel et al, 2006;Gietema et al, 2006). Of the 26 patients enroled in this study, one achieved a confirmed partial response and seven achieved stable disease as their best tumour response. Although interpretation of the efficacy results is limited by the small sample size and the exploratory nature of this end point, the data support further exploration in larger studies, which is also encouraged by the recent experience with other combination treatments. Specifically, the addition of VEGF inhibitors to cytotoxic chemotherapy has been shown to enhance antitumour activity, compared with single-agent chemotherapy treatment (Miller et al, 2005(Miller et al, , 2007Giantonio et al, 2007;Herbst et al, 2007;Heymach et al, 2007). In particular, preliminary results from a phase 3 study in patients with advanced NSCLC have suggested that improved efficacy can be achieved when combining a VEGF inhibitor with a chemotherapy regimen that includes gemcitabine and cisplatin (Manegold et al, 2005). Progression-free survival was longer among patients who received bevacizumab plus gemcitabine/oxaliplatin compared with patients treated with gemcitabine/oxaliplatin plus placebo (6.7 vs 6.1 months) (Mane-gold et al, 2005). On the other hand, preliminary results from another phase 3 study suggest that treatment of advanced pancreatic cancer with bevacizumab and gemcitabine does not improve survival, although the proportion of patients with an objective response or stable disease was slightly greater (54 vs 47%) (Kindler et al, 2007). Interestingly, preclinical evidence has suggested that inhibition of PDGFR, which regulates vascular survival and is expressed in most common solid tumours, may play an important role in enhancing the therapeutic effect of cytotoxic chemotherapy (Pietras et al, 2002). The potential efficacy of motesanib, which inhibits both VEGFR and PDGFR, in combination with gemcitabine or chemotherapy in general, warrants further investigation in larger studies.
In conclusion, treatment with motesanib 125 mg QD plus gemcitabine was well tolerated, with adverse event and pharmacokinetic profiles similar to those observed in motesanib monotherapy studies. The 125-mg QD dose in combination with cytotoxic chemotherapy is being investigated in larger studies to further evaluate safety and efficacy.