© 2007 European Society for Medical Oncology
phase I and pharmacokinetics |
Assessment of the biological and pharmacological effects of the 
ß3 and ß5 integrin receptor antagonist, cilengitide (EMD 121974), in patients with advanced solid tumors
1 University of Colorado Cancer Center, Aurora, CO
2 Department of Cancer Biology, M.D. Anderson Cancer Center, Houston, TX
3 ApoCell, Inc., Houston, TX, USA
4 Merck KGaA, Darmstadt, Germany
5 Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, MD, USA
* Correspondence to: Dr S. G. Eckhardt, University of Colorado at Denver and Health Sciences Center, 12801 E. 17th Avenue Campus Box 8117, PO Box 6511, Aurora, CO 80045, USA. Tel: +1-303-724-3850; Fax: +1-303-724-3892; E-mail: gail.eckhardt{at}uchsc.edu
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Abstract |
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Background: Cilengitide, an antiangiogenic agent that inhibits the binding of integrins
ß3 and ß5 to the extracellular matrix, was studied at two dose levels in cancer patients to determine the optimal biological dose. Patients and methods: The doses of cilengitide were 600 or 1200 mg/m2 as a 1-h infusion twice weekly every 28 days. A novel dose escalation scheme was utilized that relied upon the biological activity rate.
Results: Twenty patients received 50 courses of cilengitide with no dose-limiting toxic effects. The pharmacokinetic (PK) profile revealed a short elimination half-life of 4 h, supporting twice weekly dosing. Of the six soluble angiogenic molecules assessed, only E-selectin increased significantly from baseline. Analysis of tumor microvessel density and gene expression was not informative due to intrapatient tumor heterogeneity. Although several patients with evaluable tumor biopsy pairs did reveal posttreatment increases in tumor and endothelial cell apoptosis, these results did not reach statistical significance due to the aforementioned heterogeneity.
Conclusions: Cilengitide is a well-tolerated antiangiogenic agent. The biomarkers chosen in this study underscore the difficulty in assessing the biological activity of antiangiogenic agents in the absence of validated biological assays.
Key words: angiogenesis, cilengitide, integrins, phase I trials
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introduction |
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TopAbstractintroductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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The process of angiogenesis is critical to the growth and progression of tumors. Angiogenesis involves several steps including basal lamina breakdown, extracellular matrix (ECM) remodeling, and endothelial cell proliferation and migration. Tumor cells stimulate angiogenesis through the increased production of proangiogenic factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), resulting in activation of endothelial cells and stromal cells such as pericytes. Endothelial cells penetrate the underlying basement membrane, proliferate, and migrate in the ECM. Integrins are key components in this interaction between activated, proliferating endothelial cells and the surrounding stroma, facilitating the binding of endothelial cells to ECM proteins, such as fibronectin and vitronectin.
Integrins are heterodimeric cell surface adhesion receptors that consist of two noncovalently associated alpha and beta subunits, and function as receptors for ECM proteins such as fibronectin, laminins, collagens, and vitronectin [1]. They are expressed on cell types including proliferating activated endothelial cells, some tumor cells, chondrocytes, leukocytes, myocytes, fibroblasts, and osteoclasts [2–8]. Primary endothelial cells are anchorage dependent and undergo apoptosis when denied integrin-mediated attachment in vitro [9]. Ligand binding to the extracelluar domain of integrin receptors results in receptor activation and the transduction of signals essential for cell adhesion and spreading, migration, proliferation, differentiation, and survival [10, 11].
The integrins ß3 and ß5 appear to be particularly important in the process of angiogenesis and are expressed in a variety of malignancies, including melanoma, breast cancer, prostate cancer, colon cancer, and gliomas [3, 12–14]. The intratumoral expression of integrins has been associated with tumor progression and metastasis in melanoma, breast cancer, and prostate cancer [15–17]. For example, ß3 is highly expressed on malignant breast tumor vasculature, while little is expressed on vessels from normal, fibrocystic, or benign breast tissue lesion [2]. The critical role of integrins in angiogenesis and association with tumor progression make them an attractive target for anticancer therapy.
Cilengitide (EMD 121974), the inner salt of the cyclized pentapeptide c- [Arg-Gly-Asp-DPhe-(NMeVal)], is a potent and selective inhibitor of the integrins ß3 and ß5 (Figure 1). This agent is a cyclized RGD (Arg-Gly-Asp motif)-containing pentapeptide designed to block integrin ß3- and ß5-mediated endothelial cell attachment and migration. Cilengitide inhibits binding of isolated ß3 and ß5 to vitronectin with an IC50 value of 4 and 79 nm, respectively [18, 19]. In cell adhesion studies assessing the human melanoma M21 or UCLA-P3 human lung carcinoma cell lines, cilengitide inhibited integrin-mediated binding to vitronectin with IC50's of 0.4 and 0.4 µm [18]. The hydrochloride salt of cilengitide, EMD 85189, inhibited the attachment of human umbilical vein endothelial cells to vitronectin with an IC50 of 2 µm [20]. The selectivity of the agent was demonstrated by a negligible effect on cell adhesion to fibronectin (mediated by integrin 5ß1) or to collagen (mediated by integrins 1ß1 and 2ß1). Similarly, fibrinogen binding to platelet receptor glycoprotein IIbIIIa and platelet aggregation was not inhibited by cilengitide [18].
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The in vivo assessment of cilengitide's ability to inhibit angiogenesis was evaluated in the chicken chorioallantoic membrane model where the agent inhibited VEGF and bFGF-induced angiogenesis in a dose-dependent manner by a single intravenous injection. In nude mice bearing M21-L melanoma tumors, cilengitide dosed i.p. at 10, 50, and 250 µg three times per week demonstrated inhibition of tumor growth with a reduction in both tumor volume (55%, 75%, and 89%, respectively) and tumor weight (23%, 38%, and 61%, respectively), when compared to controls [21]. Of note, since M21-L cells lack
integrin receptors, it is presumed that tumor regression was induced by the antiangiogenic effects of EMD 85189 (the hydrochloride salt of cilengitide) on activated endothelial cells in the tumor vascular bed. Medulloblastoma and glioblastoma cell line xenografts in nude mice treated with 100 µg i.p. of cilengitide demonstrated 100% survival at 28 days with almost complete histological disappearance of tumor, compared to the death of 100% of animals treated with a control peptide, EMD 135981 [22]. In an earlier phase I study of cilengitide, the drug was administered i.v. twice weekly for four consecutive weeks [23]. The starting dose of cilengitide in this initial phase I study was 30 mg/m2 with escalations to 60, 120, 180, 240, 400, 600, 850, 1200, and 1600 mg/m2/infusion. Anemia, not exceeding grade 2, was observed at various dose levels [23]. Non-hematological toxicity consisted of nausea, anorexia, vomiting, fatigue, malaise, and headache [23]. Interestingly, no dose-limiting toxic effect (DLT) was observed at doses up to 1600 mg/m2 [23].
Based upon the novel mechanism of action of cilengitide against the ß3 and ß5 integrin receptors and favorable toxicity profile, the current phase I study was conducted using twice weekly intravenous administration of two dose levels in 4-week cycles. The principal objectives of this study were to: (i) determine the optimal biological dose (OBD) of cilengitide, (ii) analyze the effects of cilengitide on various circulating markers of endothelial cell proliferation and angiogenesis, (iii) assess the effects of cilengitide on tumor and endothelial cell apoptosis and gene expression in tumor biopsies, (iv) characterize and quantify the toxic effects of cilengitide administered as a 1-h i.v. infusion twice weekly every 4 weeks in patients with advanced solid malignancies, (v) characterize the PK profile of cilengitide and identify any relationship to its biologic activity or to observed toxicity, and (vi) evaluate the preliminary antitumor efficacy of cilengitide.
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patients and methods |
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patient selection
Patients with histologically documented solid malignancies refractory to standard therapy or for whom no effective therapy existed were eligible for this study. Other relevant eligibility criteria included as follows: (i) age at least 18 years, (ii) Karnofsky performance status (KPS) at least 60%, (iii) no chemotherapy or investigational agents within 4 weeks (6 weeks for nitrosureas or mitomycin C and 2 weeks for palliative radiotherapy to bone or brain metastases), (iv) adequate hematopoietic (absolute neutrophil count,
1500/µl; hemoglobin, 9.0 g/dl; platelet count, 100 000/µl), hepatic (total bilirubin, within normal institutional limits; aspartate aminotransferase (AST) and alanine aminotransferase (ALT) 
2.5 times the upper limit of normal), and renal (creatinine level, within normal institutional limits) functions, (v) no coexisting medical problem of sufficient severity to limit full compliance with the study, and (vi) life expectancy of at least 12 weeks. Relevant exclusion criteria included as follows: (i) uncontrolled brain metastases, including symptomatic lesions and lesions requiring therapy to suppress symptoms (including glucocorticoids and/or anticonvulsants), and patients with signs and symptoms indicative of brain metastases were required to have had radiological imaging of the brain that was negative for the presence of metastases before study entry, (ii) any serious concomitant systemic disorders incompatible with the study (at the investigator's discretion); these included, but were not limited to, ongoing or active infection, symptomatic congestive heart failure, unstable angina pectoris, and significant central nervous system or psychiatric illness/social situations limiting compliance with study requirements, (iii) pregnancy, and (iv) humanimmunodeficiency virus positive patients receiving combination antiretroviral therapy (due to possible PK interactions with cilengitide). Informed consent was obtained according to federal and institutional guidelines.
drug administration
The initial doses of cilengitide that were chosen were 600, 1200, and 2400 mg/m2 administered as a 1-h i.v. infusion twice weekly, where one course was defined as 28 days. These doses were chosen as ones that would be expected to reach and maintain adequate serum concentrations required for biological activity (>2 µM) without excessive toxicity. The primary objective of this study was determination of the OBD, which was defined as the lowest tolerable dose at which at least five or six patients demonstrated biologic activity. This corresponded to a biological activity rate (BAR) of at least 83%. BAR was defined as the percentage of patients who, at a given dose level, met at least two of the following criteria: (i) fifty percent or greater increase in tumor cell apoptotic rate as measured by TUNEL assay (discussed below), (ii) fifty percent or greater increase in endothelial cell apoptotic rate as measured by TUNEL assay, or (iii) fifty percent or greater decrease in tumor microvessel density (MVD) as measured by CD31 immunohistochemistry (discussed below).
In order to allow appropriate measures of safety, patients were initially enrolled sequentially to one of three cohorts: 600, 1200, or 2400 mg/m2. The dose was escalated as follows: (i) in cohorts of 3 patients demonstrated biological activity, (ii) expansion of the cohort to six patients when at least two patients demonstrated biological activity, (iii) continued dose escalation if no more than four of six patients demonstrated biological activity, and (iv) OBD would be declared at the dose at which at least five patients of six demonstrated biological activity. The OBD dose level would then accrue six additional patients to better characterize the BAR of cilengitide and be recommended for further testing in phase II studies. If, at any point, DLT was observed, the standard 3 + 3 phase 1 dose escalation schema and maximum tolerated dose definition was utilized. DLT was defined as grade 3 or greater non-hematologic toxicity excluding diarrhea, nausea, and vomiting in the absence of adequate antiemetic and antidiarrheal medication, or grade 4 thrombocytopenia or neutropenia. Toxic effects were graded according to the National Cancer Institute's (NCI) Common Toxicity Criteria Version 2.0.
Cilengitide was manufactured by Merck (Darmstadt, Germany) and supplied by the NCI, as an isotonic solution containing 450 mg of lyophilized drug dissolved in 30 ml of sodium chloride and water for injection (at a concentration of 15 mg/ml). Cilengitide was administered as an i.v. infusion either without further dilution or further diluted with isotonic (0.9%) sodium chloride or 5% dextrose in water in 250 or 500 ml standard infusion bags.
pretreatment and follow-up studies
Informed consent, histories, physical examinations, concomitant medication histories, assessment of KPS, routine laboratory studies, electrocardiogram, and adverse event evaluations were carried out within 2 weeks of start of study. Routine laboratory studies included a complete blood cell count (CBC), differential white blood cell (WBC) count, electrolytes, urea, creatinine, albumin, calcium, phosphate, glucose, total protein, alkaline phosphatase, total bilirubin, lactate dehydrogenase, AST, and ALT. A CBC with differential WBC count was carried out weekly for cycles 1–2 and during week 1 of cycle 3 onwards. Serum chemistries were carried out during weeks 1 and 3 of cycle 1 and 2 and during week 1 from cycle 3 onwards. Physical examinations, assessment of KPS, adverse event evaluations, and concomitant medications were carried out during week 1 of each cycle. Evaluations of measurable or assessable disease were carried out within 2 weeks of start of study and after every 8 weeks using Response Evaluation Criteria in Solid Tumors [24]. Patients were allowed to continue treatment in the absence of disease progression or intolerable toxicity.
PK sampling and assay
To study the PKs of cilengitide, whole blood samples were obtained from an indwelling venous catheter placed in the arm. Samples were collected before the infusion, at the end of infusion, and at 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 8.0, and 24.0 h after the end of the infusion on day 1 of cycle 1. Blood samples were drawn into a 7-ml red-top or serum-separator vacutainer tube. They were placed immediately into an ice bath and allowed to clot over 30 min. Specimens were then centrifuged for 30 min at 3000 x g, aspirated and cryopreserved at –20°C in two 1 ml aliquots.
EMD 121974 concentrations in PK serum samples were measured using a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method previously validated for human plasma and cross validated for human serum during this study [25]. After addition of the internal standard EMD 66203 to the samples, hydrophilic serum constituents were separated by online extraction on a Waters Oasis Hydrophilic-Lipophilic Balance column in the presence of 0.1% formic acid. Following chromatographic separation on a reverse phase C8 column, analyte and Internal Standard were detected at characteristic mass transitions using an API 3000 MS/MS instrument with a turbo ion spray interface working in the positive ionization mode. Depending on their expected concentrations, samples were run within one of the two calibration ranges (1–1000 and 500–500 000 ng/ml) which were established by seven calibrators each. Batch acceptance criteria were proven by two corresponding quality control sets at four concentrations each. Serum concentration versus time data were analyzed using a two-compartment open model with zero-order input and first-order elimination from the central compartment. Correlation coefficients for curve fits were >0.99 for all individual curves using 1/y2 weighting. All curve fitting and parameter calculations were done using WinNonlin Professional version 4.1 (Pharsight Corporation, Mountain View, CA) software on a PC-based computer.
correlative studies
circulating soluble cell adhesion and angiogenic protein analysis.
Cell adhesion molecules important in capillary morphogenesis can also be inhibited as a secondary effect of cilengitide. The following cell adhesion molecules were assessed in serum pretreatment and at the end of cycles 1 and 2: VEGF, bFGF, E-selectin, P-selectin, intracellular cellular adhesion molecule (ICAM-1), and vascular cellular adhesion molecule (VCAM-1) using defined enzyme-linked immunosorbent assay methods (R&D Systems, Minneapolis, MN). Five milliliters of whole blood samples (EDTA) were drawn at these time points. Serum from these samples was aliquoted into 500 µl volumes and frozen at –70°C until analysis. Samples were run in triplicate and all results were required to exhibit a coefficient of variation (CV)% <20% to be considered valid. Interday assays were carried out to establish the reproducibility of the assays (CV% <20%). The correlative study assays were carried out according to the manufacturer's instructions, and were carried out in the phase 1/Translational Research Laboratory at University of Colorado Health Sciences Center (UCHSC).
tumor biopsies.
Patients with accessible tumor tissue had biopsies of tumor at baseline and within ±7 days of the end of cycle 1. Biopsies were carried out by core needle biopsy or 8-mm diameter punch biopsy blades after sterilization of the biopsy site with iodine and the subcutaneous administration of approximately 1–2 ml of 2% lidocaine with 1 : 100 000 epinephrine (Abbott Laboratories, Abbott Park, IL) for local anesthesia and control of bleeding. Specimens were divided and placed in formalin (for subsequent paraffin embedment) or snap frozen with liquid nitrogen (for subsequent RNA isolation). Due to difficulties with RNA integrity, samples collected later in the study were immediately placed into RNAlater (Sigma-Aldrich, St. Louis, MO).
To quantify MVD by CD31 immunohistochemistry, a CD31 antibody was utilized on 2 µm paraffin sections using the alkaline phosphatase/antialkaline phosphatase procedure (APAAP) [26]. Sections were then dewaxed, rehydrated, and predigested with protease type XXIV for 20 min at 37°C. The primary, secondary, and APAAP complex were added and developed with New Fuschin solution. The specimens were then scanned at low optical power and microvessel counting was carried out on x250 fields. Three areas per case were chosen for microvessel counting and the microvessel score represented the mean value of all three fields. These studies were carried out in the Pathology Core Laboratory within the University of Colorado Cancer Center.
For apoptotic endothelial cells, a double-staining technique was utilized according to previously published procedures [27]. After the tissue was incubated in a blocking solution for 20 min at room temperature, the samples were incubated with a 1 : 400 dilution of a rat monoclonal antibody to CD31 (Pharmingen, San Diego, CA) followed by incubation with a 1 : 200 dilution of a Texas Red-conjugated secondary antibody. A TUNEL assay was then carried out using a commercial kit (Promega, Madison, WI) with fluorescein-deoxyuridine triphosphate. Immunofluorescent microscopy was carried out: CD31+ endothelial cells were identified by red fluorescence and apoptosis (DNA fragmentation) was detected by localized green and yellow fluorescence within the nucleus (visualized by a Hoecsht stain). Quantification of endothelial apoptosis was expressed as the average TUNEL-positive endothelial cells in five random fields at x40 magnification. Apoptosis of tumor cells was quantified based upon the number of non-CD31 TUNEL-positive cells in five random fields. These studies were carried out in the laboratory of DM at the M.D. Anderson Cancer Center.
Total RNA was extracted from biopsy samples utilizing the RNeasy kit (Qiagen, Valencia, CA) and used in real-time quantitative PCR reactions to measure the expression of ICAM-1, VCAM-1, E-selectin, and VE-cadherin relative to baseline values using the 
Ct method. These assays were carried out in the phase 1/Translational Research Laboratory at the UCHSC.
Total RNA extracted from patient biopsy samples was also used to generate complementary RNA probes for use in gene array experiments. The goal was to identify a cohort of genes that appeared to be stimulated or suppressed by cilengitide treatment. These experiments were carried out on an Affymetrix gene chip analyzer system in the Microarray Core Lab within the University of Colorado Cancer Center.
statistical analysis.
Cell adhesion molecules obtained at baseline and at various time points during cycles 1–3 were analyzed by scatter plots of biomarker expression versus time for each patient. Mean curves of biomarker expression at each time point for the two dose levels were constructed. Analysis of variance for repeated measures was carried out to examine the main effect of dose and time and dose by time interaction for each biomarker using SAS version 9.1. The results of endothelial and tumor cell apoptosis were reported as a percentage. A arcsine square root transformation for percent apoptosis followed by two-sample paired t-tests to examine whether the change of percent apoptosis pre- and posttreatment was different from zero was carried out.
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results |
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Twenty patients received 50 total courses (400 infusions) of cilengitide at doses of 600 or 1200 mg/m2 twice weekly for 28 days. All patients were assessable for toxicity. Nineteen of the 20 patients had previously received chemotherapy, radiotherapy, or both. The patient characteristics, dose levels, as well as the number of patients and courses administered, as a function of dose level, are depicted in Table 1. A total of 10 patients each were treated at the 600 and 1200 mg/m2 dose levels. No patients required dose reduction. One patient at the 1200 mg/m2 dose was delayed 2 weeks due to anemia. The median number of courses (eight infusions) administered per patient was 2.5 (range, 1–8), and the median number of infusions administered was 20 (range, 8–64). Dose escalation to 2400 mg/m2 was not pursued due to the fact that there was no emerging biological data to indicate that dose escalation was warranted along with concerns that the volume required (>250 ml/h) was not feasible.
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toxic effects
The toxic effects of cilengitide observed in this study were all mild to moderate in severity. Among 50 treatment courses administered, the most common grade 1 or 2 adverse events possibly attributable to cilengitide included fatigue (16 courses, 32%), nausea (eight courses, 16%), and headache (four courses, 8%). Other grade 1 or 2 toxic effects seen in >5% of treatment courses administered were rash (6%), vomiting (6%), and anorexia (6%). None of these toxic effects appeared to be dose-dependent and no patient required dose reduction. One patient with metastatic melanoma was delayed by 2 weeks between course 1 and 2 due to anemia, which was thought to be related to extensive prior chemotherapy treatment, and unlikely related to cilengitide. One patient with metastatic melanoma experienced sudden death due to respiratory failure that was attributed to a long-standing history of chronic obstructive pulmonary disease.
PK studies
Serum sampling for PK studies was carried out in all 20 patients. The maximum concentration (Cmax) of cilengitide at the two doses used was 72.5 ± 19.7 and 132.8 ± 23.3 µg/ml, respectively. The volume of distribution was 10.7 ± 3.2 and 11.9 ± 3.0 l/m2, respectively. Individual serum cilengitide concentration data were well fit by a two-compartment model with zero-order input. Representative concentration data versus time data for both dose levels are displayed in Figure 2. PK parameter estimates for patients treated with cilengitide at both dose levels are listed in Table 2.
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) represent the 1200-mg/m2 dose level. The horizontal line represents the serum concentration of cilengitide that resulted in optimal tumor growth inhibition in human melanoma xenografts.
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pharmacodynamic analyses
Serum from pretreatment and during cycles 1–3 was studied to assess inhibition of various cell adhesion and angiogenic molecules as an effect of cilengitide. When either individual patient values or mean circulating levels of VEGF, bFGF, P-selectin, ICAM, or VCAM were analyzed, treatment with cilengitide was not associated with significant dose- or time-dependent changes (data not shown). The only statistically significant changes were modest increases in E-selectin over time, after exclusion of one patient with outlying values (P = 0.0052), as depicted in Figure 3. Additionally, there were no significant changes in these markers among the three patients with disease stabilization lasting >4 months.
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Although assessment of MVD was originally intended to be one of the biological end points in this study, it was eliminated after the first three biopsy sets were analyzed, due to concerns regarding intrapatient heterogeneity. For example, in one patient with metastatic melanoma, the baseline level of MVD varied such that the extent of reduction in MVD in two adjacent biopsies ranged from 15% to 55%. This finding, along with concerns regarding the use of MVD as a pharmacodynamic end point in human studies, prompted the elimination of this end point in subsequent patient samples [28]. Likewise, the use of RT-PCR for changes in gene expression and gene array analysis was fraught with sample integrity issues and inter- and intrapatient heterogeneity, making meaningful interpretation difficult.
Twelve patients (six at each dose level) did have evaluable pre- and post treatment tumor biopsies for assessment of tumor and endothelial apoptosis, using a semiautomated technique. Although there did appear to be evidence of posttreatment increases in tumor and endothelial cell apoptosis in selected patients, again, the small sample numbers and variability precluded any statistical significance. Figures 4 and 5 depict the individual results for the 12 patients and a representative example of a stained slide, respectively.
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antitumor activity
There were no partial or complete responses. Stable disease [24] was observed in one patient with colon cancer for 6 months, in a patient with ocular melanoma for 4 months, and in another patient with adenoid cystic cancer for 4 months. One patient with metastatic melanoma maintained stable disease for 15 courses of cilengitide.
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discussion |
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TopAbstractintroductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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Cilengitide is a novel compound targeting angiogenesis, the cornerstone of tumor growth and metastasis. Activated endothelial cells express integrins
ß3 and ß5, which is the target of cilengitide [20]. Integrin-mediated attachment to ECM proteins is necessary for endothelial cells to migrate and form new blood vessels, thereby promoting tumor cell dissemination and metastasis. Increased expression of integrins is seen in the vasculature of a variety of tumors. This study was aimed at defining the OBD of cilengitide by calculating BARs based upon MVD and tumor and endothelial cell apoptosis in tumor tissue before and after treatment. Markers of angiogenesis and endothelial cell adhesion and migration, including VEGF, ICAM, bFGF, VCAM, P-selectin, and E-selectin, were also assessed in an attempt to establish dose–response relationships. In addition, toxic effects were further evaluated at the two dose levels studied. Unfortunately, despite the attempt at a novel biologically based trial design, no reliable correlation was found among the various biological markers assessed in this study and treatment with cilengitide. Although there was a very modest increase in E-selectin at certain time points, these results are difficult to interpret since one would hypothesize a decrease with treatment and such decreases have been observed in regimens containing bevacizumab [29]. Nonetheless, overall, these results are similar to those observed in the initial phase I study of cilengitide, where no consistent treatment effect on circulating biomarkers was established [23]. Likewise, despite considerable efforts to obtain evaluable tumor tissue, neither tumor nor endothelial cells reliably demonstrated induction of apoptosis after cilengitide treatment. To be fair, at the time of this study, there was a lot of optimism and enthusiasm regarding incorporation of biomarkers in early studies of angiogenesis inhibitors, without sufficient, or indeed any, level of preclinical validation of such biomarkers. These targets were chosen based upon the proposed mechanism of action of cilengitide and in vitro and in vivo studies of other proteins involved in angiogenesis. Since the design and completion of this study, several VEGF-targeted agents have been approved for commercial use, including bevacizumab, sorafenib, and sunitinib, all of which have utilized traditional clinical end points in dose selection [30–32]. Interestingly, the VEGF-targeted agents are associated with mechanism-based toxicity (hypertension, proteinuria) that has facilitated dose selection. In retrospect, biomarkers more specific to the effects of cilengitide could have been explored, such as phosphorylation of focal adhesion kinase or paxillin and downstream effectors of integrin signaling [33, 34].
As expected, cilengitide was well tolerated, with the most common adverse events being fatigue, nausea, headache, rash, vomiting, and anorexia. In the prior phase I study with this compound, nausea, anorexia, vomiting, fatigue, and malaise were the most common adverse events [23]. Other drugs of the integrin antagonist class, such as Vitaxin, have been studied in phase I trials. Vitaxin is a humanized monoclonal antibody directed against integrin ß3. Adverse events seen with this drug were mainly infusion reactions, such as fever, chills, nausea, and flushing [35]. Overall for this class of agents, there do not appear to be mechanism-based toxic effects and certainly no DLT; thus, underscoring the difficulty in choosing an appropriate dose for disease-directed studies.
Serum PKs were shown to be dose-dependent with the area under the curve (AUC)/dose values being 0.41 ± 0.11 and 0.39 ± 0.08 for the 600 and 1200 mg/m2 doses, respectively. Cmax values were also dose-dependent with similar Cmax/dose values at both dose levels. This data are consistent with the data from the prior phase I study [23] which showed a linear relationship between both AUC and Cmax with respect to dose. The terminal half-life (t1/2ß) of 4 h is relatively short, thus supporting a twice-weekly dosing schedule, although the concentrations achieved did exceed those associated with antitumor activity in sensitive melanoma xenograft models [21]. Studies are ongoing exploring cilengitide administered by a continuous intravenous infusion.
There were no complete or partial responses in this study. One patient with metastatic melanoma maintained stable disease for 15 months. This patient was initially diagnosed with a stage III melanoma treated with surgical resection and recurred 1 year later, with metastatic disease. This patient had not received prior radiation or chemotherapy. In addition, prolonged disease stabilization of at least 4 months was observed in three other patients. In the prior phase I study of cilengitide, prolonged disease stabilization was seen in two patients with renal cell and one with colon cancer [23]. Similar to other antiangiogenic agents, such as bevicizumab, the predominant effect of single-agent use is not disease regression, making it very difficult to discriminate between inherent tumor growth kinetics and drug effects, in the absence of randomized studies.
Cilengitide is the first low molecular weight peptide drug of its class targeting the integrins vß3 and vß5. Thus far, antiangiogenic agents such as sorafenib, bevacizumab, or sunitinib have been most effective as single agents in tumors not responsive to chemotherapy, such as renal cell cancer and gastrointestinal stromal cancer, where varying degrees of tumor regression have been associated with prolonged disease stability and survival [31, 32, 36, 37]. Bevacizumab, which demonstrates single-agent response rates of 5%–10%, has been most effective against breast, lung, and colorectal carcinomas when given in combination with traditional chemotherapy agents [30, 38, 39]. The tolerability of cilengitide indicates that it would be an ideal drug to combine with other chemotherapy agents or with radiotherapy. A recently published randomized phase II study assessed the effects of cilengitide 600 mg/2 twice weekly with gemcitabine in advanced pancreatic cancer and concluded that there was no survival benefit of the combination [40]. Although disappointing, these results are consistent with the largely negative randomized studies of antiangiogenic agents in combination with gemcitabine in this disease [41, 42]. Interestingly, in this randomized study of cilengitide, there were no significant changes noted in serum or plasma VEGF, bFGF, or urinary bFGF [40]. Currently, there are several other phase I/II studies under way (including one with radiotherapy) in glioblastoma multiforme, where promising evidence of activity was noted in phase I, and in lymphoma, prostate cancer, and melanoma. Lastly, with regards to future studies of such novel compounds, it is going to be imperative that preclinical and clinical scientists work together to establish validated biomarkers of drug effect that can be based upon a testable hypothesis in early clinical trials. In such a scenario, this type of biologically based clinical trial design with predetermined end points may be useful at establishing optimal dose-schedules for disease-directed studies.
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Study support: NCI U01 CA099176, K24 CA106349.
Received for publication January 14, 2007. Revision received March 3, 2007. Accepted for publication March 23, 2007.
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References |
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