Originally published as JCO Early Release 10.1200/JCO.2002.02.082 on June 17 2002

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Phase I Clinical Trial of Recombinant Human Endostatin Administered as a Short Intravenous Infusion Repeated Daily

From the Division of Adult Oncology, Dana-Farber Cancer Institute; Divisions of Hematology/Oncology, Departments of Medicine, Brigham and Women’s Hospital, Massachusetts General Hospital, and Beth Israel Deaconess Medical Center; Department of Surgery, Children’s Hospital; and Dana-Farber/Harvard Cancer Center, Harvard Medical School, Boston, MA; and EntreMed, Rockville, MD.

Address reprint requests to J.P. Eder, Jr, MD, Dana-Farber Cancer Institute, 44 Binney St, Rm M1B34, Boston, MA 02115; email: jeder{at}partners.org

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To perform a phase I trial of recombinant human endostatin (rhEndostatin; EntreMed, Rockville, MD) given as a daily 20-minute intravenous (IV) injection in adult patients with refractory solid tumors.

PATIENTS AND METHODS: The daily dose was increased from 15 to 240 mg/m² by a factor of 100% in cohorts of three patients. In the absence of dose-limiting toxicity, uninterrupted treatment was continued until the tumor burden increased by more than 50% from baseline. Correlative studies included dynamic contrast-enhanced magnetic resonance imaging of tumor blood flow, urinary vascular endothelial growth factor and basic fibroblast growth factor levels, rhEndostatin serum pharmacokinetics, and monitoring of circulating antibodies to rhEndostatin.

RESULTS: There were no notable treatment related toxicities among 15 patients receiving a total of 50 monthly cycles of rhEndostatin. One patient with a pancreatic neuroendocrine tumor had a minor response and two patients showed disease stabilization. Linearity in the pharmacokinetics of rhEndostatin was indicated by dose-proportionate increases in the area under the curve for the first dose and the peak serum concentration at steady state. Daily systemic exposure to rhEndostatin in patients receiving 240 mg/m²/d was approximately 50% lower than that provided by the therapeutically optimal dose in preclinical studies.

CONCLUSION: rhEndostatin administered as a 20-minute daily IV injection at doses up to 240 mg/m² showed no significant toxicities. Evidence of clinical benefit was observed in three patients. Due to high variability between the peak and trough serum concentrations associated with the repeated short IV infusion schedule, daily serum drug levels only briefly exceeded concentrations necessary for in vitro antiangiogenic effects.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
ANGIOGENESIS, THE formation of new microvessels, is fundamental to reproduction, development, and repair. During angiogenesis, such as that required for wound healing, endothelial cells emerge from the quiescent state and undergo rapid proliferation.¹ Tumor growth beyond a size of 1 to 2 mm³ requires the assembly of a vascular network.^2,3 More than a dozen endogenous proteins that act as positive regulators of tumor angiogenesis have been identified. Vascular endothelial growth factor (VEGF)/vascular permeability factor, basic fibroblast growth factor (bFGF), angiopoietins 1 and 2, interleukin-8, and platelet-derived growth factor-beta are angiogenesis-stimulating proteins that have been implicated in tumor development in preclinical systems and in clinical cancer.^4,5

Endogenous mammalian proteins that inhibit endothelial cell growth and that may play a physiologic role in maintaining the normally low replication rate of vascular endothelial cells include angiostatin, endostatin, and tum-5.^6-8 Endostatin was isolated from the culture medium of a murine hemangioendothelioma cell line on the basis of its inhibitory activity against bFGF-stimulated bovine capillary endothelial cell proliferation in vitro.⁶ Structural characterization demonstrated that endostatin is a 20-kd fragment derived from the C-terminal region of mouse collagen XVIII, an extracellular matrix heparin sulfate proteoglycan that is an abundant constituent of blood vessels and most basal laminae in organs distributed throughout the body.^7,9,10 Treatment with recombinant murine endostatin induced the regression of experimental tumors growing in mice to dormant, microscopic lesions.^7,11,12 The antiproliferative activity of endostatin seems to result specifically from effects directed against endothelial cells.¹³ Repeated cycles of recombinant murine endostatin therapy in tumor-bearing mice resulted in prolonged tumor dormancy, without recurrence after discontinuation of treatment and without the development of resistance.¹⁴ Preclinical studies revealed that treatment with recombinant human endostatin (rhEndostatin; EntreMed, Rockville, MD) given subcutaneously (SC) once or twice daily on a continuous basis had promising antimetastatic and growth inhibitory activity against several tumor models with no discernible host toxicity.¹⁵

Several phase I clinical trials of rhEndostatin were recently initiated, beginning with this study at the Dana-Farber/Harvard Cancer Center, to evaluate chronic administration of the protein to patients with refractory solid tumors as a 20-minute intravenous (IV) infusion repeated once daily. Characterizing the clinical toxicity, pharmacokinetic behavior, and biologic activity of rhEndostatin was the primary objective of the study. Establishing whether systemic exposure to the protein at doses effective against tumor models in mice could be achieved in patients after treatment with clinically practical doses was an important end point. In addition, several correlative studies were performed to obtain evidence of a biologic effect resulting from rhEndostatin treatment. The findings of these studies are described in this report.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient Selection and Treatment
The patients were adults (age 18 years) with a radiographically measurable (> 1 cm) solid tumor, other than a primary brain tumor or CNS metastasis, that had either relapsed or failed to respond to all proven therapeutic interventions. Patients were randomly selected for eligibility screening according to an explicitly defined procedure that was established before activation of the trial. A Karnofsky performance status score 80, an estimated life expectancy of at least 6 months, and adequate bone marrow reserve, renal function, and liver function, as indicated by the following laboratory parameters, were required: WBC count greater than 3,000/µL; platelet count 100,000/µL; creatinine 1.5 mg/dL; serum total bilirubin 1.5 mg/dL; and AST less than 2.5 times the upper limit of normal. In addition, an international normalized ratio of prothrombin time less than 1.2 and activated partial thromboplastin time within normal limits were required. All eligibility criteria were documented when patients completed a comprehensive series of screening evaluations within 14 days before starting treatment. The only restrictions on concurrent medications were heparin, warfarin, antiplatelet drugs, and all approved or investigational anticancer agents. Patients were examined weekly while on study. Hematology and clinical chemistry tests, including any relevant serum markers of disease, were performed twice a month.

rhEndostatin Protein was supplied by EntreMed as a sterile solution for injection in glass vials that were stored at -70°C. Each vial contained 5 mL of an 8-mg/mL solution of rhEndostatin in a pH 6.2 buffer composed of citric acid 17 mmol/L, sodium phosphate 66 mmol/L, and sodium chloride 59 mmol/L. Dosing solutions were prepared daily by allowing the vials to thaw at ambient temperature and adjusting the concentration with the citrate-phosphate buffer to deliver the required dosage in a final volume of 75 mL. The solution was infused at a constant rate over 20 minutes IV through a central catheter every 24 hours.

The daily dose was increased in successive cohorts from 15 to 240 mg/m² by a constant factor of 100%. Cohorts of three patients were evaluated at each dose level. Escalation of the dose to the next higher level proceeded after all three patients had received a complete 28-day cycle of therapy without evidence of any severe toxicities, as defined by the current version of the United States National Cancer Institute’s common toxicity criteria (http://www.ctep.nci.nih.gov). Additional cycles of therapy were administered without any interruption in dosing in the absence of severe toxicity or disease progression on continued satisfaction of all eligibility criteria.

Response was measured by the change in area of all bidimensional measurements according to World Health Organization criteria,¹⁶ with the exception that progressive disease was defined in the following manner: greater than 50% increase in the sum of bidimensional areas; the appearance of any new lesions; or greater than 25% increase from the minimum area in the event of a response. Radiographic tumor measurements were performed after the first and second months of therapy, and every 2 months thereafter, in the absence of clinical indications. The study protocol was fully reviewed and approved by the institutional review boards of Dana-Farber/Partners Cancer Care and Beth Israel Deaconess Medical Center (Boston, MA). A signed written informed consent document satisfying all federal and institutional requirements was obtained from each patient as a condition of participating in the study.

Dynamic Contrast-Enhanced Magnetic Resonance Imaging
Dynamic contrast-enhanced magnetic resonance imaging (MRI) studies were performed according to the recommendations resulting from a workshop sponsored by the National Cancer Institute in October 1999.¹⁷ Patients were scanned at 1.5 T using a surface coil according to the following protocol. Precontrast T1-weighted axial images (TurboFLASH; Siemens Medical Systems, Iselin, NJ) were obtained at 5- to 7-mm increments, with selective fat saturation. On the basis of these images, two slices through the tumor, chosen at maximum tumor diameter by M.T.K., and one slice through the aorta or other large vessel to estimate the arterial input function were chosen for the dynamic study. The dynamic contrast injection protocol sequence was as follows: IRTurboFLASH: repetition time/echo time, 5.8 msec/5.2 msec; inversion time, 50 msec; matrix, 128 x 128; read-out bandwidth, 355 Hz/pixel; contrast agent, gadopentetic acid (Gd-DTPA) 0.1 to 0.2 mmol/kg in a volume of 8 to 10 mL infused IV at a rate of 5 mL/sec to ensure a linear relationship of contrast concentration to signal intensity; and acquisition rate, 1.5 image/sec from 0 to 90 seconds. Quantitative parametric data analysis of the tumor regions of interest, before the administration of rhEndostatin and after each of the first three cycles of treatment, was performed on a pixel by pixel basis, by an image processing specialist, according to the Tofts model of perfusion using in-house software. Quantitative parameters relating to tumor vascularity based on a literature-accepted model were derived.^18,19 Parameters including the volume transfer constant or permeability surface area product per unit volume of tissue (K_PSP) and volume of the extracellular space (V_e) were determined after IV injection of Gd-DPTA during a fast gradient echo MRI sequence.

Urinary Angiogenic Proteins
Urine was collected from all patients before the start of rhEndostatin treatment and at the end of each monthly cycle of therapy. An aliquot was removed from a single void and promptly transported over ice to the institutional clinical chemistry laboratory for creatinine determination. Another aliquot from the same specimen was stored at 4°C for not longer than 2 weeks until the concentrations of VEGF and bFGF were determined by sandwich enzyme-linked immunosorbent assay (ELISA) methods (Quantikine R&D Systems, Minneapolis, MN), as previously described.^20,21 The lower limit of quantitation for VEGF and bFGF were 15.6 pg/mL and 1 pg/mL, respectively. Interassay coefficients of variation (CV) were 15% at all concentrations in the standard curves.

The concentrations of bFGF and VEGF were normalized to the concentration of creatinine measured in each specimen.²¹ The percentage change in the normalized urine concentration of the proteins relative to the pretreatment baseline values was calculated for the serial specimens obtained from each patient. The existence of an overall trend toward increasing or decreasing values in the relative protein concentrations during treatment with rhEndostatin was assessed by calculating the average percent change and by the Wilcoxon signed rank test. The data were categorized as no discernible trend in cases in which the average change was 15%. Independent associations between the sign of the trend in the protein concentrations and treatment with more than two cycles of therapy were evaluated using the Fisher’s exact test.

Pharmacokinetic Sample Collection and Drug Concentration Determinations
Pharmacokinetic blood specimens (5 mL) were obtained immediately before treatment, at the midpoint and end of the first 20-minute infusion of drug, and then at 10 minutes, 30 minutes, and 1, 2, 3, 4, and 6 hours after dosing. On the four subsequent days, samples were acquired before dosing and at the end of the infusion. Thereafter, during administration of the first dose on weeks 2 to 4, sampling was performed before treatment, at the end of the infusion, and 24 hours after dosing. A battery-powered digital timer was used to accurately monitor the beginning and ending times of the drug infusion and sample collection intervals. Blood was drawn into Vacutainer Plus SST serum separation tubes (Becton Dickinson, Franklin Lakes, NJ) from an arm vein. The sample tube was mixed by gentle inversion followed by clotting for 30 minutes and then centrifugation (10 minutes, 1,300 x g, 25°C). The serum derived from each blood sample was divided into four polypropylene cryostorage vials and stored at -70°C until it was assayed.

The concentration of rhEndostatin in serum specimens was measured using the commercially available Accucyte enzyme immunoassay kit for human endostatin (CytImmune Sciences, College Park, MD), as previously described.²² The assay is based on the competitive binding of rhEndostatin and biotinylated rhEndostatin to a polyclonal rabbit antibody. Each study sample was independently assayed in duplicate together with a series of eight serum standards with added rhEndostatin concentrations ranging from 1.95 to 500 ng/mL, and a drug-free sample. Standard curves were analyzed by fitting a four-parameter logistic model to the paired absorbance-serum concentration values by unweighted nonlinear regression using the WinNonlin version 1.1 software package (Scientific Consulting, Apex, NC).²³ Values of the parameters describing the best-fit curve were used to calculate the rhEndostatin concentration in study samples. Specimens with an estimated concentration outside the pseudo log-linear region of the standard curve, which ranged from approximately 4 to 40 ng/mL, were reassayed on appropriate dilution. Study samples were also reassayed in cases where the two initial determinations differed from their average by more than 20%. All assays were performed using an aliquot of the original serum sample that had not been previously thawed. Interday accuracy and precision of the assay were assessed by analyzing the interpolated drug concentrations from 35 standards curves run over a 3-month period. Grand mean (± SD) values of the accuracy and precision were 100.2% ± 2.3% (range, 97.7% to 103.0%) and 9.4% ± 3.2% (range, 5.7% to 14.4%), respectively, for the five standard solutions with concentrations ranging from 3.9 to 62.5 ng/mL, which encompassed the log-linear region of the calibration curve.

Pharmacokinetic Data Analysis
Actual sample times were calculated from the beginning of the first infusion of drug to the midpoint of each sample collection interval. The rhEndostatin serum concentration-time data acquired during the 24 hours after initiating administration of the first dose of the drug was analyzed by noncompartmental methods using routines supplied in the WinNonlin software package.²⁴ The data were corrected by subtracting the rhEndostatin equivalent concentration in the pretreatment serum specimen from the observed concentration in all samples acquired after drug administration. Eliminating the contribution of the endogenous protein was necessary to estimate values of the pharmacokinetic parameters for the exogenously administered compound. The minimum and maximum steady-state concentrations of the drug, C_min^ss and C_max^ss, respectively, were calculated for each patient as the average concentration of all observed values measured after apparent steady state on repeated dosing was achieved, as determined by regression analysis. Estimated values of disposition phase half-lives and the mean residence time at each dose level are reported as the geometric mean ± SD of the values for the individual patients.^25,26 The SD of the geometric mean was estimated by the jackknife technique.²⁷ Mean values for all other pharmacokinetic parameters were calculated as the arithmetic average.

In addition, the uncorrected rhEndostatin serum profile for the first dose of the drug and the subsequent nadir and peak concentrations associated with doses 2, 3, 4, 5, and 8 of the repeated daily dosing regimen were simultaneously analyzed by nonlinear regression using the ADAPT II computer program.²⁸ The general equation fit to the experimental data,

equation

was based on the model-independent equation for zero-order IV input and first-order elimination with modifications to accommodate the endogenous protein as measured by the rhEndostatin ELISA, C(0), and multiple dosing.²⁹ The objective function of the maximum likelihood estimator was defined as

equation

where a and b are iterated variance parameters and Y denotes the predicted values.³⁰ For each set of data, the number of exponential terms in the equation, n, was varied from 1 to 3 to identify the best fit of the experimental data, as previously described.²⁴ Values of the parameters corresponding to the equation that best described each plasma profile were used to calculate pharmacokinetic variables according to standard equations.³¹

Serum Antibodies to rhEndostatin
Serum samples to assess the formation of antibodies to rhEndostatin were obtained before treatment, after each week of dosing during the first cycle of therapy, and on a monthly basis as long as the patient remained on the protocol. Blood (7 mL) was collected into serum tubes without additives and allowed to stand for 30 minutes at room temperature to clot, and then the tube was centrifuged (500 x g, 15 minutes, 25°C). Serum was removed for storage at -70°C until end point titers of anti-rhEndostatin immunoglobulins G and M (IgG and IgM) antibodies were determined by ELISA. Immulon 4 plates (Dynex Technologies, Inc, Chantilly, VA) were coated for 2 hours at room temperature with 0.05 mL of rhEndostatin (2 µg/mL) in 0.05 M carbonate-bicarbonate buffer (pH 9.6) (Sigma, St Louis, MO). The coating solution was aspirated and blocked for 30 minutes at room temperature with 0.2 mL of phosphate-buffered saline (PBS) containing 3% nonfat dry milk. The blocking medium was aspirated and 0.05 mL of patient serum was serially diluted beginning from 1/200 in PBS containing 0.1% Tween-20, in triplicate. After a 1-hour incubation at room temperature, the wells were aspirated and washed three times with 0.2 mL of PBS containing 0.1% Tween-20. The plates were tamped dry and the wells were incubated for 30 minutes at room temperature with 0.05 mL of either a 1/4,000 dilution of goat antihuman IgG horseradish peroxidase (Kirkegaard & Perry Laboratory, Inc, Gaithersburg, MD) or goat antihuman IgM horseradish peroxidase (Kirkegaard & Perry Laboratory). The wells were then washed three times with 0.2 mL of PBS containing 0.1% Tween-20 and 0.05 mL of ABTS peroxidase substrate (Kirkegaard & Perry Laboratory). Absorbance at 405 nm was measured after a 30-minute incubation. The data were analyzed to determine an end point titer of immunoreactivity, defined as the inverse dilution resulting in a two-fold increase over baseline absorbance. The serum titer was defined as 0 if a 1/200 dilution of serum resulted in an absorbance that was no greater than the negative controls.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient Selection
The process established for accruing patients into this study was quite different from a typical phase I clinical trial as a consequence of the unprecedented interest resulting from publicity associated with the drug before the study was opened. On recommendation by the Dana-Farber/Partners Cancer Care Human Protection Committee to ensure equitable access of all interested patients to the study, a toll-free telephone number for inquiries regarding the clinical trial was published on the Dana-Farber/Harvard Cancer Center Web site after approval and activation of the protocol. Information about the telephone number was disseminated to the public by the local and national media. A preliminary interview of prospective patients who called this telephone number was conducted by oncology nurses and the study investigators. This initial contact was followed by communications with the physicians of these individuals to confirm their potential eligibility for the study. A confidential database of potentially eligible patients was maintained and continually updated on a daily basis. In advance of opening each successive dose level to accrual, the names in the database were randomly rank ordered for the purpose of selecting patients for the definitive eligibility screening performed at the participating institutions. More than 80% of the patients screened at the study sites proved to be eligible for the study.

Patient Characteristics
Characteristics of the 15 patients evaluated during the course of this study are listed in Table 1. The median age of the study population was 56 years (range, 32 to 72 years); there were 10 women and five men. The median number of prior chemotherapy regimens was three (range, zero to 10), and six patients had prior radiation therapy at least once. The median Karnofsky performance status score was 90. Four patients had breast cancer, three had lung cancer, two had colorectal cancer, and the remainder had other types of solid malignancies.

View larger version (79K): [in this window] [in a new window]   Fig 1. Computed tomography scans of a 52-year-old man with a nonsecretory pancreatic neuroendocrine tumor obtained (A) before dosing, (B) after 2 months of treatment, and (C) after the third cycle of therapy with rhEndostatin 30 mg/m2/d, illustrating a reduction in tumor mass.

Urinary VEGF and bFGF
Urinary levels of VEGF and bFGF were measured before the initiation of treatment and near the completion of every monthly cycle of rhEndostatin. The concentration of bFGF in urine specimens obtained before treatment ranged from 0.4 to 10.1 ng/g creatinine (mean ± SD, 3.9 ± 2.9 ng/g creatinine). Pretreatment urinary VEGF concentrations were also highly variable (range, 9.2 to 389.7 ng/g creatinine), with a mean concentration of 131.0 ± 107.8 ng/g creatinine. The average urinary concentrations of VEGF and bFGF normalized to creatinine and expressed as a percentage of the creatinine normalized baseline value for the individual patients are shown in Fig 2. Urinary bFGF showed a distinct trend toward lower concentrations during therapy in six patients, seven patients showed an increase in urinary concentrations, and there was no difference from the baseline value in two patients. A similar pattern was observed for urinary VEGF levels, with a trend toward lower concentrations in four patients, a distinct increasing trend in six patients, and no difference from baseline for four patients. Data from one patient was not assessable because the baseline VEGF level was below the limit of quantitation of the assay. Regression analysis did not show any evidence of a relationship between the dose of rhEndostatin and average change in the creatinine normalized urinary concentration of bFGF and VEGF during therapy. In addition, there was no association between the duration of therapy and the sign of the trend for either protein. A statistically significant difference in the sign of the trend in urinary VEGF (P = .57; Fisher’s exact test) or bFGF concentrations (P = .99; Fisher’s exact test) was not achieved between patients who received only one or two cycles of therapy and those who received three or more.

View larger version (15K): [in this window] [in a new window]   Fig 2. Plot depicting the average creatinine normalized urinary concentration of (A) VEGF and (B) bFGF in individual patients during the first cycle of treatment with rhEndostatin expressed as a percentage of the pretreatment creatinine normalized concentration of the proteins (horizontal line at 100%).

View larger version (20K): [in this window] [in a new window]   Fig 3. Time course of rhEndostatin serum concentration in a patient receiving doses of 240 mg/m2/d during (A) the initial 24 hours after administration of the first dose and (B) the complete first cycle of therapy. Circles indicate observed rhEndostatin serum concentrations; solid line indicates the best-fit curve.

View larger version (15K): [in this window] [in a new window]   Fig 4. Relationship between the (A) Cmaxss and (B) AUC0-24h of rhEndostatin and the daily dose of the drug. Circles indicate observed values in individual patients; horizontal bars indicate average values for each group; solid line is generated from linear regression analysis of the individual patient values.

024h

024h

The body-surface area (BSA) of the patients ranged from 1.43 to 2.22 m² (median, 1.70 m²). The average ± SD CL of rhEndostatin was 43.5 ± 19.9 L/h when calculated without normalizing to BSA. Interpatient variability in the CL was very similar whether normalized to BSA (CV, 42.5%) or not (CV, 45.7%). The relationship between the unnormalized CL values (L/h) and BSA was assessed by examining the Pearson and Spearman correlations. The Pearson correlation coefficient, 0.216, was suggestive of a very weak relationship at best (P = .44). The lack of a significant relationship was confirmed by the Spearman correlation, for which the correlation coefficient was only 0.021 and the slope of the regression line was very close to zero (-0.022, P = .94).

The apparent biologic half-life of rhEndostatin could not be reliably estimated from the terminal region of the serum profiles determined after the first infusion of drug. As shown in Fig 3A, the serum concentration of rhEndostatin declined rapidly upon completion of the 20-minute infusion. At the two initial dose levels, the mean rhEndostatin concentration measured 4 to 6 hours after delivery of the first dose did not differ significantly from the nadir concentration at 24 hours. Even at the two highest dose levels evaluated, endogenous protein detected by the ELISA became a significant component (ie, > 10%) of the total measured serum concentration within 6 hours, which seemed to be before the terminal phase was achieved. Correcting the data by subtraction of the pretreatment rhEndostatin equivalent concentration would introduce a high degree of error as a consequence of calculating the difference of measurements that are comparable in magnitude. Practical limitations on the duration of serial sampling after administration of the first dose, imposed by the necessity to perform the study on an outpatient basis, further complicated the ability to discern whether the true terminal phase was achieved before administration of the next daily dosage.

The time course of the rhEndostatin serum concentration was modeled in an attempt to gain a better understanding of the disposition of the protein. Nonlinear regression analysis would also permit simulations of the concentration time profiles resulting from different doses and administration schedules. As illustrated in Fig 3, a very satisfactory description of the experimental data was realized by simultaneously fitting a multiple dosing model to the complete set of observed drug concentrations over the first 8 days of dosing using the maximum likelihood estimator for the minimization, with simulation thereafter, for the group of patients receiving daily doses of 240 mg/m². An equation with three exponential terms provided the best-fit of the data for this cohort. The endogenous serum concentration of the protein was defined as an iterated parameter in the model. The average CL calculated from estimates of the pharmacokinetic parameters corresponding to the best-fit equations was 21.6 ± 3.3 L/h/m². Mean values of the half-lives for the initial, intermediate, and terminal disposition phases of the serum profile were 0.29 ± 0.05, 1.83 ± 0.45, and 25.0 ± 21.9 hours, respectively. The initial and terminal phases were the principle determinants of rhEndostatin disposition, respectively contributing 37% and 44% on average to the total AUC₀. In contrast to the magnitude of the apparent biologic half-life calculated by direct inspection of the terminal region of the serum profile for the first dose of rhEndostatin (10.3 ± 2.4 hours), the substantially larger value afforded by modeling the data is consistent with the observed increase in C_min values during the first week of treatment and the time required to achieve steady state. Accumulation is not reflected in the C_max values because the residual serum concentration of drug at 24 hours after delivery of the prior dose is insignificant in comparison to the peak concentration provided by the subsequent dose. Similarly, the magnitude of the mean values of the mean residence time (16.4 ± 11.4 hours) and apparent volume of distribution at steady state (412 ± 279 L/m²) estimated by the model were larger than the values determined by noncompartmental analysis. Accordingly, the pharmacokinetic parameters derived from the model fit to the data seem to provide a more accurate representation of the true disposition of rhEndostatin in humans.

Serum Antibodies to rhEndostatin
IgG and IgM antibodies specific for rhEndostatin were not detectable in sera obtained from patients before treatment. Low titers ( 1/200 dilution) of both IgG and IgM were detected in serum specimens acquired from all 15 patients after they had received rhEndostatin for 2 weeks. Serial determinations made near the end of each cycle of therapy showed a distinct progressive increase in the IgG titer in all patients, with median dilutions of 1/200 at the end of cycle 1 (n = 15), 1/4,000 (n = 9) after the second cycle, 1/1,600 after cycle 4 (n = 3), and 1/7,200 in the two patients who received 7 months of therapy. One patient who was removed from the study after receiving 12 cycles of therapy for concomitant medical problems without progression developed an IgG titer of 1/16,000 after the 10th cycle. In contrast, the median IgM titer was 1/800 after the first cycle of treatment and it did not show a trend toward further increases in dilution in the majority of patients during continued therapy. There were no significant relationships between the titer of either antibody at the end of the first or second cycles of therapy and the daily dose of rhEndostatin. The presence of these antibodies did not seem to affect the peak or trough serum concentrations of rhEndostatin as determined by the polyclonal ELISA during the first two cycles of therapy.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Tumor angiogenesis offers an attractive target for drug development. There is an extensive body of clinical data associating increased levels of angiogenic proteins, especially VEGF, with cancer progression, advanced stage, and decreased survival.⁴ The various mechanisms by which angiogenic factors exert their biologic effects are also becoming increasingly understood. The first putative antiangiogenic therapy evaluated in humans involved the treatment of pediatric hemangiomas with interferon alfa-2a.³² The realization of dramatic curative regressions of otherwise untreatable advanced lesions evoked considerable interest in identifying other antiangiogenic agents that are more specifically targeted and less toxic than interferon alfa-2a. A number of endogenous proteins, monoclonal antibodies, and synthetic organic molecules with antiangiogenic activity have been discovered during the past decade.³³ The various compounds that are presently undergoing clinical evaluation include several low-molecular-weight tyrosine kinase receptor inhibitors of VEGF receptor 2, platelet-derived growth factor, and bFGF, as well as monoclonal antibodies to VEGF and the integrin alpha(v)beta(3). Preliminary findings indicate that none seem to be free of potentially serious toxicities.

Endostatin is an endogenous mammalian protein that inhibits the migration and proliferation of endothelial cells and angiogenesis.³⁴ These biologic effects have been demonstrated in experimental systems using various preparations of mouse and human endostatin that exhibit distinct species and source-dependent differences in the primary and tertiary structure of the protein that modulate solubility, metal-binding properties, and biologic activity.^7,15,35,36 In vivo efficacy studies showed that the growth of Lewis lung carcinoma in mice was inhibited by 80% to 90% after treatment with rhEndostatin given SC at a dose of 50 mg/kg repeated twice daily.¹⁵ The development of pulmonary lesions in animals inoculated with the B16-BL6 murine melanoma was prevented by treatment with rhEndostatin 4.5 mg/kg given SC once daily. The optimally effective dosage of 50 mg/kg against syngeneic murine tumor models produced an AUC₀ of 700 µg·min/mL in mice when given as a single SC injection.¹⁵ Dosing regimens that maintained this level of systemic exposure to the protein on a daily basis for at least 1 month were not associated with any significant drug-related toxicities.

The dosing regimen of rhEndostatin selected for the initial phase I clinical trials was a short IV infusion repeated once every day. Repeated daily treatment with rhEndostatin at doses ranging from 15 to 240 mg/m² given as a 20-minute IV infusion for as long as 11 months failed to show any significant toxicity. The maximum dose evaluated in this study was established in advance as the largest practical daily dose of the protein that could be delivered chronically on the basis of the supply available at the time and economic considerations. Minor (grade 1) rashes developed in two patients that did not require either intervention or an interruption in rhEndostatin administration. The drug was delivered through a central catheter and there were only two episodes of infusion-line associated sepsis during the course of more than 1,400 IV administrations, amounting to an incidence of less than 0.2%.

There were no objective responses to therapy among any of the 15 patients evaluated during the course of this study. However, objective therapeutic activity is infrequently observed during phase I clinical trials of cytotoxic chemotherapeutic agents.³⁷ Nevertheless, there was evidence of therapeutic benefit with rhEndostatin treatment in three of the patients. One patient achieved a minor response with a maximum tumor volume reduction of 17% lasting for more than 11 months. Two other patients showed stable disease, by conventional definition (< 25% progression), for periods of 3 and 4 months.

Since tumor dormancy without shrinkage has been recognized as a possible outcome after effective antiangiogenic treatment, a noninvasive radiographic technique to detect alterations in tumor blood flow, tumor vascular volume, or vascular permeability could provide mechanism-based indications of a biologic effect of these agents.³⁸ In this study, the application of dynamic contrast-enhanced MRI using Gd-DTPA was assessed. A positive correlation between tumor enhancement and microvessel density has been demonstrated in previous clinical studies that have used this technique to monitor tumors at various locations, including the breast, cervix, and liver.^18,19,39-42 Other important advantages of Gd-DTPA–based imaging methods include the availability of the contrast media, its relative lack of side effects, and extensive clinical experience with the agent. However, the relatively low molecular weight of the compound represents a potential disadvantage, due to rapid extravasation from normal vessels, which occurs at even greater rates from more porous tumor vessels. Thus, quantitative determinations of intravascular volume and vascular permeability based on the rate of contrast extravasation are derived functions and not authentic biologic measurements.

In recognition of these problems, a series of recommendations were recently made to standardize the acquisition of dynamic MRI data for the purpose of integrating results from different institutions and to ensure that the acquired data reflect the underlying physiology as accurately as possible.¹⁷ Perfusion imaging studies performed in accordance with these guidelines in a group of 10 patients during the course of this clinical trial failed to identify any consistent blood flow change in the evaluated tumors as a result of treatment with rhEndostatin. In particular, lung nodules and liver metastases proved difficult to image reproducibly because of motion artifact. This made it difficult to scan the exact same location (region of interest) within a lesion on different days, thereby precluding meaningful comparisons of serial determinations. Phase I trials in patients with multiple types of solid tumors and widespread metastatic disease may not represent the ideal situation for assessing the application of this technique. High-molecular-weight contrast media, which will not extravasate within a porous tumor vasculature, are in development and, although not available for clinical use at present, may offer potential for superior performance characteristics based on biophysical principles rather than artificially derived values.

The potential significance of the angiogenic proteins VEGF and bFGF as prognostic indicators in cancer is increasingly being recognized. There is considerable controversy regarding the value of serial determinations of these proteins during therapy. Nevertheless, urinary bFGF is recognized as an indicator of disease activity in pediatric hemangiomas and its response to treatment with interferon alfa.^43,44 VEGF levels have been shown to decrease after surgical tumor resection or successful cytotoxic therapy.⁴ However, the response to antiangiogenic therapy may differ depending on the mechanism of action of a given agent, such as a decrease after treatment with anti-VEGF antibody or a trend toward increasing levels, at least transiently, for a receptor tyrosine kinase inhibitor directed at VEGF receptor 2. Thus, the anticipated effect of rhEndostatin therapy on urinary levels of VEGF and bFGF could not be predicted.¹³ Urine levels of both VEGF and bFGF were measured during the course of this study. Approximately equivalent groups of patients demonstrated either a decrease or increase in the creatinine normalized concentration of VEGF or bFGF in urine, without any evidence of a dose-dependent or duration-of-therapy–dependent association between the sign of the trend. The majority of patients in this study had urinary levels within the normal range at enrollment. Despite the generally advanced stage of disease, the overall excellent performance status may also be either a reflection of the normal levels or a contributing factor. Thus, monitoring urinary VEGF and bFGF levels provided no significant information. This may reflect the relatively low levels present at the outset of the trial, perhaps because bFGF and VEGF were unimportant in tumor progression in these patients or because rhEndostatin does not decrease the levels of these proteins, either biologically or when administered by this schedule in this patient population.

The pharmacokinetics of rhEndostatin was characterized during this study with a commercially available ELISA to measure the concentration of the protein in serum. Because this assay utilizes a polyclonal antibody for rhEndostatin,²² there is some concern about the proper interpretation of the pharmacokinetic data, due to the potential nonspecificity for detecting the administered form of the protein. rhEndostatin is actually a mixture of approximately equivalent amounts of two proteins that differ by the presence or absence of the last four amino acids in the N terminus.¹⁵ The rhEndostatin ELISA seems to be 100% cross-reactive for both components in the mixture, which suggests the possibility of significant cross-reactivity with products resulting from biotransformations directed at the N-terminal region of the protein, such as hydrolytic degradation or glycolysis, that could conceivably inactivate the protein.

The mean background concentration of endostatin in serum specimens obtained from the 15 patients evaluated in this study shortly before initiation of rhEndostatin therapy was 18 ± 9 ng/mL. This is very similar to the mean concentrations of circulating endostatin found in the sera of patients with hepatocellular carcinomas (18 ng/mL) and healthy volunteers (10 to 15 ng/mL).^22,45,46 Substantially higher concentrations of endogenous endostatin have also been reported.^47,48 Although the same assay was used for each of these studies, experience gained during the course of validating the methodology in our laboratory revealed that erroneous results can be readily obtained. Several procedural modifications to the assay were implemented that resulted in a considerable improvement in both the accuracy and precision of the method. These included increasing the number of calibration standards with concentrations within the region of pseudo log-linear response, which ranged from approximately 4 to 40 ng/mL, restricting quantitation to this region of the standard curve, and limiting each analytic run to the wells in one half of the 96-well microtiter plate because of the kinetics of the color development reaction.

The pharmacokinetics of rhEndostatin in humans was found to be very similar to its behavior in preclinical animal models.¹⁵ In addition, its pharmacokinetic behavior was generally comparable to that of other endogenous human proteins, such as interleukin-11 and interferons alfa, beta, and chi, that have been exogenously administered.^49,50 Apparent linear pharmacokinetics in cancer patients was indicated by dose-proportionate increases in both the AUC_024h for the first dose of the protein and the C_max^ss. There was also good agreement between the predicted value of 30.3 L/h/m² for the CL in humans obtained from interspecies allometric scaling and the overall mean value of 24.5 ± 10.4 L/h/m² determined in this study. The administered doses of rhEndostatin in this study were adjusted to the BSA of each patient according to the generally accepted practice for scaling doses of anticancer chemotherapeutic agents from preclinical toxicology studies to establish a safe starting dose for phase I clinical trials.^51,52 The continued use of this convention is based on the presumed existence of a relationship between the CL of a drug and BSA, thereby minimizing interpatient variability in the AUC, which would presumably enhance safety. However, in this study, the CL of rhEndostatin was found to be independent of BSA, suggesting that BSA dose adjustment may not be warranted. In consideration of the relatively small number of patients evaluated in this clinical trial, this finding should be substantiated in a larger population, such as by comparing the magnitude of interpatient variability in the AUC of rhEndostatin in groups treated with a fixed amount of the drug and a comparable constant dose normalized to BSA.

At the highest daily dose of 240 mg/m² evaluated, the average C_max^ss of 10,900 ± 1,620 ng/mL exceeded the upper range of 50% inhibitory concentration (IC₅₀) values that have been reported for the in vitro antiangiogenic effects of rhEndostatin (ie, 7.5 to 35 µg/mL).^15,53 In addition, the average AUC_024h achieved at this dose level, 688 ± 98 µg·min/mL, was very similar to the AUC₀ in mice treated with a single dose of 50 mg/kg.¹⁵ However, it should be noted that optimal efficacy of rhEndostatin against murine tumor models was afforded by twice-daily administration of this dose.¹⁵ Serum concentrations of the protein rapidly declined after the end of the infusion, to a level that was 37 times less on average than the peak concentration within 4 hours in the group of patients evaluated at the 240-mg/m² dose level. The mean C_min^ss at 24 hours after administration of the previous 240-mg/m² dose, 202 ± 65 ng/mL, was less than the lower range of IC₅₀ values for in vitro antiangiogenic activity.

In conclusion, there were no clinically significant toxicities associated with the administration of rhEndostatin as a daily 20-minute IV injection among a group of 15 patients who received a total of 50 monthly cycles of therapy at five dose levels ranging from 15 to 240 mg/m²/d. Indications of clinical benefit to treatment with the protein consisted of a minor tumor reduction in one patient and disease stabilization in two others. The daily systemic exposure to endostatin proteins in patients treated with rhEndostatin 240 mg/m²/d was approximately 50% lower than that provided by the dosing regimen that afforded maximum tumor growth inhibition in preclinical studies. After the administration of each daily dose, the duration of time that serum levels of the protein exceeded the range of concentrations necessary for in vitro antiangiogenic effects with rhEndostatin was brief. It is uncertain whether the protein remains in a biologically active form 6 to 24 hours after dosing when the apparent endostatin serum concentrations decay to levels below the 5.0 to 7.5 µg/mL IC₅₀ of rhEndostatin in the in vitro angiogenesis assays described in the literature.⁵³ Administering rhEndostatin by continuous IV infusion could optimize the potential for achieving clinical antitumor activity by avoiding the high variability between the peak and trough serum concentrations as well as providing a constant input of the biologically active protein. Subsequent to the initiation of this clinical trial, it was shown that the efficacy of cytostatic agents could be notably enhanced by delivering them in a manner that provides continuous systemic exposure.^54-56 Of particular interest, the activity of rhEndostatin against a human pancreatic cancer xenograft model in mice when given by continuous intraperitoneal infusion at a rate of 2 mg/kg/d was similar to a 10-fold higher dose administered as a daily intraperitoneal injection.⁵⁶ The mean steady-state serum concentration of rhEndostatin in mice provided by the continuous intraperitoneal infusion was 230 ng/mL. This steady-state concentration should be achieved in humans on delivery of the drug by continuous IV infusion at a rate of 125 mg/m²/d, based on the mean CL determined in this study. With the development of a formulation of rhEndostatin that is stable for at least 48 hours, we recently initiated another clinical study to evaluate the administration of the protein by continuous IV infusion at rates of 60 and 240 mg/m²/d in expanded cohorts of patients. The results of this ongoing study will be reported in a subsequent communication.

	ACKNOWLEDGMENTS

 
This study was performed under the sponsorship of EntreMed, Inc, Rockville, MD, and partially funded by grant nos. U01-CA-62490 (D.W.F.) and 2 P30 CA0516 (E. Benz) in addition to limited research support from the sponsor.

We thank Marybeth Gallant, Robert Matthews, Karla Ronaczeki, Michelle Kneissl, Lisa Gaines, Caroline Harvey, Catherine Butterfield, Kristin Roper, the nursing staff of Dana 1 Adult Infusion Unit, Dana-Farber Cancer Institute; Massachusetts General Hospital Cancer Center; and General Clinical Research Center, Beth Israel Deaconess Medical Center, and Guangxin Xu for expert technical assistance.

	NOTES

 
Preliminary results of the study were presented at the eleventh National Cancer Institute/European Organization for Research and Treatment of Cancer/American Association of Cancer Research symposium on new drugs in cancer therapy, Amsterdam, the Netherlands, November 7-10, 2000, and the Thirty-Seventh Annual Meeting of the American Society of Clinical Oncology, San Francisco, CA, May 12-15, 2001.

The first two authors contributed equally to this study.

This article was published ahead of print at www.jco.org.

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Submitted February 15, 2002; accepted May 20, 2002.

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