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Phase I Clinical Evaluation of Weekly Administration of the Novel Vascular-Targeting Agent, ZD6126, in Patients With Solid Tumors

Laurens V. Beerepoot, Sandra A. Radema, Els O. Witteveen, Tawnie Thomas, Catherine Wheeler, Sanford Kempin, Emile E. Voest

From the Department of Medical Oncology, University Medical Center Utrecht, Utrecht, the Netherlands; Department of Medical Oncology, St Vincent's Comprehensive Cancer Center, New York, NY; and AstraZeneca, Boston, MA

Address reprint requests to Emile E. Voest, MD, PhD, Department of Medical Oncology (F02.126), University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht, the Netherlands; e-mail: e.e.voest{at}azu.nl

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions REFERENCES

 
PURPOSE: ZD6126 is a novel vascular-targeting agent that induces selective effects on the morphology of endothelial cells by disrupting the tubulin cytoskeleton. This leads to cell detachment and tumor vessel congestion, resulting in extensive central necrosis in a range of tumor xenograft models. Results from a phase I dose-escalation study of ZD6126 are reported.

PATIENTS AND METHODS: Thirty-two patients with advanced cancer received weekly ZD6126 infusion (5 to 28 mg/m²). Assessments for safety and pharmacokinetics were performed. Circulating endothelial cells (CECs) were quantified as a pharmacodynamic marker of vascular damage.

RESULTS: Maximum concentrations of the active species were observed 5 to 25 minutes from the start of infusion, and decayed in a biexponential manner with a half-life of 1 to 3 hours. Maximum serum concentration and area under the time-concentration curve increased with dose in a linear fashion across the dose range of 5 to 28 mg/m². One patient treated at 10 mg/m² with a history of ischemic heart disease experienced acute myocardial infarction 2 weeks after drug discontinuation. Four others had asymptomatic creatine phosphokinase–muscle-brain elevation. Maximum-tolerated dose (MTD) was reached at 20 mg/m²/wk. Dose-limiting toxicities at 28 mg/m² were hypoxia caused by pulmonary embolism and an asymptomatic decrease in left ventricular ejection fraction. No objective antitumor responses were observed. CEC levels increased in the hours after infusion, indicating potential effect of the compound on the vasculature.

CONCLUSION: ZD6126 administered as a weekly infusion was clinically well tolerated. The MTD was reached at 20 mg/m².

	INTRODUCTION

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The integrity of a vascular network is essential for tumor growth and survival. Tumor vessels are less mature than normal blood vessels; tumor vessel characteristics are marked by a high rate of proliferation, lack of pericytes, high tortuosity, increased vascular leakage, and chaotic blood flow. Furthermore, tumor endothelial cells are highly dependent on the tubulin cytoskeleton for their motility, invasion, attachment, alignment, and proliferation.¹

Vascular-targeting agents have been identified through their differential selectivity for tumor endothelial cells and differ from antiangiogenic agents by targeting existing vasculature, rather than preventing the growth of new vessels. Tubulin-interacting agents use the presumed dependency of tumor endothelial cells on tubulin for maintenance of shape.² Most of the commonly used tubulin interacting agents in the clinic (vinca alkaloids, taxanes, and colchicine)^3,4 have a recognized antivascular mechanism, but mostly at drug doses near or above maximum-tolerated dose (MTD), and therefore are associated with high levels of cytotoxicity.⁵ In contrast, a new generation of agents that bind to tubulin, such as combretastatin A4⁶ and ZD6126^7,8 have antivascular effects at lower doses that do not show cytotoxicity. The active form of ZD6126, N-acetyl-colchinol, causes tubulin depolymerization leading to reversible shape changes in immature cells that are reliant on the tubulin cytoskeleton.^7,9 This specific effect on the vasculature was seen in studies with combretastatin, but effects on the normal vasculature and heart were also seen.^10-13 Preclinical studies with ZD6126 in rats and dogs have revealed a heterogeneous effect on the heart. In rats, at high doses, multifocal ventricular myocardial necrosis and/or myocarditis were observed, but in dogs no histologic changes were seen, although postinfusional decreases in heart rate and increased arterial blood pressure were noted. ECG studies showed increases in PR intervals and decreased QTc intervals (data on file, AstraZeneca, Boston, MA). Early clinical experience with the active form of ZD6126, N-acetyl-colchinol, administered as daily or weekly infusions was reported in 1964. Toxicities were observed predominantly after daily administrations and consisted of nausea, vomiting, diarrhea, and myalgia. In two patients acute pulmonary edema was seen, and one patient had acute chest pain.¹⁴ These findings led to extensive cardiovascular monitoring in this study.

The pharmacokinetic properties and tubulin-binding kinetics of ZD6126 have been selected to optimize short exposure to the endothelium. Tumor cell necrosis and intravascular thrombosis occurred in the central tumor area, but a viable tumor rim was preserved,^7,8,15 resulting in limited growth delay. Combination therapy (with chemotherapy^16,17 or radiation therapy¹⁸) showed synergistic effects and yielded longer tumor growth delays.

Three phase I dose-escalation studies were performed to investigate dosing schedules of ZD6126 bolus injection: administration once every 3 weeks, one dose daily for 5 days every 3 weeks, and weekly bolus dosing. Here we report a two-center phase I clinical study of escalating doses of ZD6126 given once weekly via a 10-minute intravenous infusion to patients with advanced cancer. The primary objective was to evaluate the toxicity profile and identify the MTD with this dosing schedule.

	PATIENTS AND METHODS

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Eligibility
Adult patients with advanced solid tumors refractory to conventional therapy, a Eastern Cooperative Oncology Group performance status of 0 to 2, and a life expectancy of more than 12 weeks were eligible for inclusion. Those with significant cardiac, hematopoietic, hepatic, or renal dysfunction or known CNS metastases were excluded, as were patients completing radiotherapy or having surgery within 4 weeks of the start of ZD6126 dosing. The study was performed as a two-center, phase I, dose-escalation trial. The University Medical Center Utrecht (Utrecht, the Netherlands) and St Vincent's Comprehensive Cancer Center (New York, NY) institutional ethical review boards approved the study protocol. All patients gave voluntary, written informed consent.

Trial Design
All drug levels consisted of a minimum of three patients. Patients were entered onto the next dose level provided three patients were available for toxicity evaluation at the previous dose level. When a patient did not complete a 4-week treatment schedule for reasons other than a dose-limiting toxicity (DLT), the patient was replaced. Patients who discontinued treatment before receiving four doses of ZD6126 for reasons other than toxicity were not included in the data set used to determine dose escalation but were available for toxicity evaluation after receiving one or more doses of ZD6126. Dose escalation of ZD6126 was done with dose escalation 40% higher than the current dose.^19-21The decision to escalate dose was made once all patients had been observed for a minimum of 28 days. Toxicities were recorded according to the National Cancer Institute Common Toxicity Criteria version 2.0.²²

When a DLT occurred, the cohort was expanded with an additional three patients. A DLT was defined as Common Toxicity Criteria grade 4 neutropenia for more than 5 days; grade 4 thrombocytopenia; any other toxicity grade 2 that was not clearly related to disease progression and could not be controlled by outpatient medical intervention or was not clearly related to concurrent conditions; and any reported elevation of cardiac enzymes, cardiac arrhythmia, change in ejection fraction, hypertension, or hypotension (grade 3) or ECG abnormalities. MTD was defined as one dose level below the dose at which at least two patients (of six) experienced a DLT.

Study Drug, Dosing Schedule, and Study Assessments
ZD6126 was supplied as a 20 mg/mL solution and was diluted under sterile conditions in 100 mL of NaCl 0.9% (range, 100 to 120 mL end volume). The drug was infused using an automated pump during a 10-minute period. The starting dose for clinical evaluation was chosen on the basis of rat and dog toxicity studies. The lowest toxic dose in dogs was 2 mg/kg, which is equivalent to 39 mg/m². The standard recommended starting dose in humans for a cytotoxic compound is one third of the lowest toxic dose in dogs (ie, 13 mg/m²). However, the toxicology studies had shown species, age and sex variation; therefore, the lowest toxic dose in rats was also taken into consideration. A no-effect dose has not been established in the rat. The lowest dosage at which toxicity was seen was 1 mg/kg/d, equivalent to 5 to 7 mg/m²/d. Therefore, the starting dose of ZD6126 was set at 5 mg/m².

Creatine phosphokinase–muscle-brain (CPK-MB), AST, ALT, and lactate dehydrogenase were measured as rapid-return samples predose and 6, 10, and 24 hours after the first two doses (ie, weeks 1 and 2) as medically indicated. In subsequent weeks, CPK-MB levels were measured monthly predose and 4 hours after each dose beginning on week 5. Clinical chemistry was performed on the day of drug administration and 2 more days of each week (ie, 3 per week) for the first 2 weeks of dosing. Thereafter, assessments were made predose on the day of dosing. A 12-lead ECG was performed at screening within 7 days before start of treatment, and was repeated on the day of dosing of week 1 within 1 hour after and again 6 hours after the dose of ZD6126. For weeks 2 to 4, an ECG was performed within 1 hour after the dose of ZD6126. Additional ECGs were performed within 1 hour after the dose of ZD6126 at week 5, and every fourth week thereafter. A multigated angiogram (MUGA) scan for evaluation of left ventricular ejection fraction (LVEF) was done before dosing at weeks 5 and 9. A complete neurologic assessment was performed at screening, on day 1 of week 5, and monthly thereafter. At baseline, and after 3 and 5 months of treatment, tumor size was assessed using computed tomography. Tumor response was recorded according to modified International Union Against Cancer/WHO criteria.²³

Pharmacokinetics
Blood sampling was performed predose and 5 minutes from the start of infusion, at the end of the infusion, and at 10 and 30 minutes and 1, 2, 4, 8, 10, and 48 hours after infusion of ZD6126 during weeks 1 and 2. Validated methods developed by AstraZeneca were used to analyze plasma samples for the concentration of ZD6126 and ZD6126-phenol (ZM445526, N-acetyl-colchinol) by high-performance liquid chromatography with tandem mass spectrometric detection. The assay had a limit of quantification of 10 ng/mL for ZD6126 and 0.6 ng/mL for ZD6126-phenol, a linear range up to 3.2 µg/mL for ZD6126 and up to 1.7 µg/mL for ZD6126-phenol, and precision of 20% across the calibration standard.

Isolation of Circulating Endothelial Cells From Whole Blood
Circulating endothelial cells (CECs) were isolated according to a previously reported and validated methodology using immunomagnetic beads coupled to an antibody directed against CD146^24-27 from blood samples before infusion, and 2, 4, 6, and 8 hours (and in two patients, 24 hours) after infusion of ZD6126 in the first and second week of treatment.

Statistical Analysis
For the analysis of CECs, paired t tests were used at designated time points. Values are reported as mean values ± standard error of the mean.

	RESULTS

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Patient Characteristics
Thirty-two patients were recruited onto the trial; 27 patients were treated at the University Medical Center Utrecht, and five patients were treated at the St Vincent Comprehensive Cancer Center. Table 1 summarizes the demographic data and tumor types of all patients treated with ZD6126. The patients received a median of 4 (range, 1 to 22) weekly bolus infusions with ZD6126. Table 2 lists the dose escalation schedule. All patients had measurable disease when recruited, but five patients discontinued treatment before repeat tumor assessment could be performed.

Cardiovascular Toxicities
Preclinical evaluation of ZD6126 and clinical studies of other vascular-targeting agents have indicated the potential for cardiovascular toxicities, therefore extensive monitoring was conducted during this trial (Table 4). Chest pressure was reported by four patients. One patient (treated at 10 mg/m²/wk) with renal cell carcinoma and a history of coronary artery disease and angioplasty experienced chest pressure on week 1. Fifteen days after the last drug dose (fourth dose, discontinued due to progressive disease), this patient experienced elevated CPK (maximum level, 449 U/L) and CPK-MB (62 U/L is 14%) levels, and ECG abnormalities consistent with acute myocardial infarction. This event was considered a DLT. He recovered without additional complications.

In four other patients (treated at 7, 10, 14, and 20 mg/m², one in each level) limited increases in CPK (with a > 7% CPK-MB fraction suggestive of myocardial damage) were observed. All four patients were asymptomatic and control ECGs did not show signs of myocardial ischemia. In three of four patients with an increase in CPK, levels increased gradually in a linear fashion in the weeks before the time it increased above the upper limit of normal. No correlation of CPK levels and drug exposure was found. Neither the patient that experienced a myocardial infarction nor the other patients treated in this study (who had CPK levels within the normal range) had a gradual increase in CPK during treatment.

In three patients a decrease in LVEF by MUGA scan was noted. One patient (treated at 5 mg/m²) had progressive lung carcinoma and worsening performance; baseline LVEF was 54%, decreasing after four doses to 43%. The other patient (who had ovarian cancer) treated at 5 mg/m² had baseline LVEF of 45% decreasing to 35% after 4 weeks and returning back to baseline at week 8 (42%) with continued treatment. Because of the normalization this was not regarded as a DLT. One patient treated at 28 mg/m², with gastric carcinoma and with progressive disease after four doses, had an LVEF decreasing from 51% before treatment to 37% after four doses. None of the patients showed signs or symptoms of congestive heart failure. Given the mechanism of action of ZD6126, this decline in LVEF may have been drug related.

In this study we observed three thromboembolic events. One patient had an inferior caval vein thrombosis and two patients had pulmonary embolism. All events were observed in patients with progressive disease.

Pharmacokinetic Parameters
In vivo, ZD6126 is rapidly hydrolyzed by alkaline phosphatase to yield ZD6126-phenol, the active species. Reported pharmacokinetic values refer to this bioactive ZD6126-phenol (Table 5, Fig 1). Peak plasma levels of ZD6126-phenol were reached 5 to 25 minutes after the end of infusion and exposure increased with dose in a linear fashion. Body surface area (BSA) of patients varied between 1.54 and 2.20 m² (median, 1.90 m²). Clearance (± standard deviation) was 52.5 ± 24.6 L/h. A relation between BSA and clearance (not normalized for BSA) was analyzed by calculating Pearson and Spearman correlations. Pearson correlation coefficient (r = 0.04; P = .79) and Spearman correlation coefficient ( = 0.106; P = .473) indicated no significant relation.

View larger version (8K): [in this window] [in a new window]   Fig 1. Peak ZD6126-phenol (N-acetyl-colchinol) concentration (Cmax) and ZD6126-phenol area under the curve (AUC) versus dose (in milligrams per square meters) in the patients assessable for pharmacokinetic evaluation. It is presumed that all ZD6126 has been converted to ZD6126-phenol.

Circulating Endothelial Cell Quantification
From 19 patients treated at the UMC Utrecht, longitudinal follow-up of CECs was conducted after the first (one patient) or the first and the second (18 patients) bolus infusions of ZD6126. Average baseline CEC counts were 390 CEC/mL (range, 6 to 1,460 CEC/mL). In 18 patients (95%), CEC numbers increased with a maximum increase over baseline after a median of 4 hours after infusion, either after the first (week 1) or after the second (week 2) dose (Fig 2). At week 1, baseline CEC numbers ([± SEM]) were 372 ± 109 CEC/mL, increasing to 524 ± 136 CEC/mL by 2 to 8 hours after infusion. At week 2, baseline CEC number was 198 ± 53 CEC/mL, increasing to 508 ± 114 CEC/mL by 2 to 8 hours after infusion. In the second week, 4 hours after infusion a significant increase to 303 ± 70 CEC/mL was seen (P = .027 by paired t test). At other time points after infusion, no significant relation could be established. In 12 patients (of the 18 receiving two or more doses), an increase in CEC numbers was found at week 1 as well as at week 2. The ZD6126 dose had no apparent relation with the magnitude of CEC increase and CEC increase did not correlate with peak plasma concentrations or drug exposure (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig 2. Absolute numbers of circulating endothelial cells (CECs) pre- and post-ZD6126 treatment (maximum CEC number between 2 and 8 hours of treatment is shown). To facilitate individual correlations of CEC levels after ZD6126 dose 1, and baseline CEC levels before administration at week 2, data points are linked ( · · · ).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions REFERENCES

At this time it is difficult to explain fully the observed CPK-MB elevations. The lack of signs and symptoms may suggest diffuse small-vessel occlusion, but this cannot be substantiated. The LVEF decrease in two patients and the transient LVEF decrease in one patient could also be due to small vessel occlusion in the myocardium. However, this should then have been accompanied by increased CPK-MB in these patients, and this was not observed. Alternative explanations for the observed decline in LVEF include a general decrease in performance status during progressive end-stage disease or possibly normal variability in the measurement of LVEF by MUGA scan. The observed thromboembolism may be caused by rapid progressive tumor growth as experienced by the patients with this toxicity.²⁸ Alternatively, activation of endothelial cells by direct damage or endothelial shedding after ZD6126 infusion in nontumor vasculature, resulting in thrombosis, may have played a role in the pathogenesis of these events. Overall, the potential for cardiovascular toxicity appears to be a key limiting factor for additional dose escalation. Because there is a concern about the cardiac events of ZD6126, future studies should include not only monitoring of CPK but also monitoring of the more specific cardiac muscle-specific troponins.

In vitro, ZD6126-phenol induces morphologic changes in proliferating human umbilical vein endothelial cells after 40 (ref⁹) or 60 (ref⁷) minutes at a concentration of 0.1 µmol/L, reaching its maximum effect at 1 to 10 µmol/L. Maximum plasma levels in our patient group ranged between 119.89 ng/mL (approximately 0.34 µmol/L) in the lowest dose level and 958.53 ng/mL (approximately 2.7 µmol/L) in the highest evaluated dose level, well above the level needed to induce in vitro endothelial detachment. Although a limited number of patients have been assessed, no relationship between BSA and clearance was apparent, suggesting that dosing could be performed without BSA adjustment. Because in rats the main route of metabolism of the active ZD6126-phenol is through glucuronidation, it is probable that this is also the case in human subjects. Because this can influence the pharmacokinetic profile and possibly significant drug interactions, this should be investigated in future clinical studies.

Pharmacodynamic monitoring by quantifying CECs was incorporated into the protocol to possibly enable the identification of optimal dosing below the MTD. The limited number of patients studied and the heterogeneous patient population make this study mainly exploratory. CECs currently are under investigation as surrogate markers of treatment efficacy during antiangiogenesis treatment,^29-31 and as a marker of vascular damage.^25-27 The peak level of CECs, which may reflect maximum vascular damage, was seen a median of 4 hours after treatment in our patient group. This time frame is comparable to preclinical observations that show blood vessel congestion after 30 minutes, and endothelial loss and tumor cell degeneration 4 hours after injection.⁸ In another model, a decrease in tumor blood flow evaluated by high-frequency Doppler ultrasound was seen 4 hours after ZD6126 treatment.³² Furthermore, the quantification of CECs suggests increasing vascular damage after repeat dosing: at week 2, a higher CEC increase is seen than at week 1. This finding may be explained by a higher degree of vessel damage after previous endothelial exposure to ZD6126. However, no clear dose-dependent drug effect was present. Although these data do not therefore assist effective dose identification, measurement of CECs is a potential pharmacodynamic tool that warrants additional investigation in larger, more homogenous patient groups. This allows correlative studies of CEC levels, cardiovascular toxicity, and biologic activity.

The MTD was reached at a lower dose (20 mg/m²) in the weekly schedule compared with a trial investigating bolus infusions of ZD6126 by injection every 3 weeks, which resulted in an MTD of 80 mg/m² (data on file, AstraZeneca). In that study, with dynamic contrast-enhanced magnetic resonance imaging, consistent biologic activity 6 hours after infusion was seen at a dose of 80 mg/m².³³

In conclusion, this study shows that ZD6126 administered in a weekly schedule is clinically well tolerated, with an MTD at 20 mg/m². DLT consisted of cardiovascular events. Drug exposure of ZD6126 was reached in the same order of magnitude as seen in preclinical experiments yielding vascular shutdown and tumor necrosis. This study provides a basis for additional studies with ZD6126 in combination with conventional treatment modalities.

	Authors' Disclosures of Potential Conflicts of Interest

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The author or immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. 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.

Authors Employment Leadership Consultant Stock Honoraria Research Funds Testimony Other Catherine Wheeler AstraZeneca (N/R)

Dollar Amount Codes (A) < $10,000 (B) $10,000-99,999 (C) $100,000 (N/R) Not Required

	Author Contributions

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions REFERENCES

 

Conception and design: Laurens V. Beerepoot, Catherine Wheeler, Emile E. Voest Financial support: Catherine Wheeler Administrative support: Laurens V. Beerepoot, Sandra A. Radema, Els O. Witteveen, Catherine Wheeler Provision of study materials or patients: Laurens V. Beerepoot, Sandra A. Radema, Els O. Witteveen, Tawnie Thomas, Sanford Kempin, Emile E. Voest Collection and assembly of data: Laurens V. Beerepoot, Sandra A. Radema, Els O. Witteveen, Tawnie Thomas, Sanford Kempin, Emile E. Voest Data analysis and interpretation: Laurens V. Beerepoot, Sandra A. Radema, Emile E. Voest Manuscript writing: Laurens V. Beerepoot, Emile E. Voest Final approval of manuscript: Laurens V. Beerepoot, Sandra A. Radema, Els O. Witteveen, Catherine Wheeler, Sanford Kempin, Emile E. Voest

 

	Acknowledgment

 
We thank all of the patients who participated in this study, and our research nurses for the assistance in patient care and data management. We also thank Isolde van Alderwegen and Karien Verhulst (AstraZeneca, the Netherlands) for their support.

	NOTES

 
Supported by Grant No. 920-03-090 from the Dutch Society of Scientific Research (L.V.B.). Performed under sponsorship from AstraZeneca, Alderley Park, Macclesfield, United Kingdom, and Boston, MA.

Presented in part at the 38th Annual Meeting of the American Society of Clinical Oncology, May 18-21, 2002, Orlando, FL, and the 12th National Cancer Institute/European Organisation for Research and Treatment of Cancer/American Association of Cancer Research Symposium on Molecular Targets and Cancer Therapeutics, November 15-19, 2003, Boston, MA.

L.V.B. and S.A.R. contributed equally to this work.

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

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Submitted May 18, 2005; accepted November 21, 2005.

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