Sep 15, 2005 - Abstract Purpose: To determine whether the preclinical antitumor and antiangiogenic activity of. 2-methoxyestradiol can be translated to the ...
Cancer Therapy: Clinical
A Phase II Multicenter, Randomized, Double-Blind, Safety Trial Assessing the Pharmacokinetics, Pharmacodynamics, and Efficacy of Oral 2-Methoxyestradiol Capsules in Hormone-Refractory Prostate Cancer Christopher Sweeney,1 Glenn Liu,3 Constantin Yiannoutsos,2 Jill Kolesar,3 Dorothea Horvath,3 Mary Jane Staab,3 Karen Fife,1 Victoria Armstrong,1 Anthony Treston,4 Carolyn Sidor,4 and George Wilding3
Abstract
Purpose: To determine whether the preclinical antitumor and antiangiogenic activity of 2-methoxyestradiol can be translated to the clinic. Experimental Design: Men with hormone-refractory prostate cancer were enrolled into this phase II randomized, double-blind trial of two doses of oral 2-methoxyestradiol capsules (400 and 1,200 mg/d) given in 4-week cycles. Pharmacokinetic sampling was done on day 1of cycles 1and 2 and trough samples were obtained weekly. Results: Thirty-three men were accrued between February and September 2001. The notable toxicity related to therapy was one grade 2 and two grade 3 episodes of liver transaminase elevation, which resolved with continued treatment in two patients. There were two cases of deep venous thromboses. The drug had nonlinear pharmacokinetic, rapid conversion to 2-methoxyestrone and f85% conjugation. Trough plasma levels of unconjugated 2-methoxyestradiol and 2-methoxyestrone were f4 and 40 ng/mL, respectively. Prostate-specific antigen declines between 21% and 40% were seen in seven patients in the 1,200 mg group and in one patient in the 400 mg group. The higher-dose group showed significantly decreased prostatespecific antigen velocity (P = 0.037) and compared with the 400 mg dose had a longer median time to prostate-specific antigen progression (109 versus 67 days; P = 0.094) and time on study (126 versus 61 days; P = 0.024). There was a 2.5- and 4-fold increase in sex hormone-binding globulin for the 400 and 1,200 mg dose levels, respectively, at days 28 and 56. Conclusion: 2-Methoxyestradiol is well tolerated and, despite suboptimal plasma levels and limited oral bioavailability with this capsule formulation, still showed some anticancer activity at 1,200 mg/d.
The need for new therapies for prostate cancer is paramount. Prostate cancer is the most common male malignancy in the United States with f200,000 new cases of prostate cancer with f32,000 deaths per year (1). Standard initial therapy for metastatic disease consists of androgen ablation and patients will become refractory to hormonal therapy after a median time of f24 months (2). Only recently has any therapy been shown to prolong overall survival. Specifically, randomized phase III
Authors’ Affiliations: Divisions of 1Hematology-Oncology and 2Biostatistics, Department of Medicine, Indiana University, Indianapolis, Indiana; 3Department of Oncology, University of Wisconsin Comprehensive Cancer Center, Madison, Wisconsin; and 4EntreMed, Rockville, Maryland Received 2/28/05; revised 6/3/05; accepted 6/8/05. Grant support: EntreMed, Rockville, MD. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Christopher Sweeney, Department of Medicine, Indiana University, 535 Barnhill Drive, Room 473, Indianapolis, IN 46202. Phone: 317274-3515; Fax: 317-274-3646; E-mail: chsweene@ @ iupui.edu. F 2005 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-0440
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trials of docetaxel-based chemotherapy have shown a modest survival benefit when compared with mitoxantrone plus prednisone (3, 4). Clearly, new therapies are required. Angiogenesis, the formation of new microvessels from existing vasculature, is fundamental to progressive tumor growth. In the absence of neovascularization, tumor growth is limited to 1 to 2 mm3. Over the last two decades, substantial laboratory and indirect clinical evidence has accumulated to support the central role of angiogenesis in prostate cancer progression. Therefore, the inhibition of angiogenesis may be a viable strategy for the treatment of hormone-refractory prostate cancer. The supporting data include the following. Increased microvessel density in prostate cancer is associated with a shorter time to recurrence and higher stage after a radical prostatectomy as well as a shorter time to recurrence after radiation therapy (5 – 7). Vascular endothelial growth factor (VEGF) is a potent and specific stimulator of endothelial cell proliferation and angiogenesis (8), and VEGF expression, measured in prostate cancer cytosolic extracts, correlates with microvessel density and is associated with relapse after a radical prostatectomy (9). Furthermore, VEGF is found at higher levels in the plasma of patients with metastatic prostate cancer than in the plasma of patients with localized disease or in
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Cancer Therapy: Clinical
the plasma of healthy controls (10). These data are consistent with findings from other cancers in which VEGF has been elevated in urine and plasma and is typically detected at higher levels in cancer patients than in the healthy volunteers (11). Basic fibroblast growth factor (bFGF) is also produced by prostate cancers and is found at increased levels in the serum of patients with prostate cancer when compared with levels in men without prostate cancer. Inhibition of angiogenesis may therefore be a viable strategy for the treatment of prostate cancer. 2-Methoxyestradiol (2ME2) is an estrogen metabolite that is naturally formed in vivo by the sequential hydroxylation and O-methylation of estradiol at the 2-position. 2ME2 binds very poorly to the estrogen receptor (0.05% of estradiol binding; ref. 12) and does not exhibit direct estrogenic activity in many test systems. Several investigations have provided encouraging results for the use of 2ME2 in the treatment of cancer. Preclinical studies have indicated that 2ME2 is both antiproliferative by acting directly on the tumor cell compartment and antiangiogenic. In vitro, 2ME2 inhibits proliferation of many cancer and endothelial cell lines in the submicromolar to low micromolar range and the activity is independent of the estrogen responsiveness of the cell line. For example, the IC50 value for the hormone-independent prostate cancer cell line, DU-145, was 1.8 Amol/L. The inhibition of proliferation is believed to result primarily from the induction of apoptosis possibly through the activation of p53 or death receptor 5 (13) as well as inhibition of hypoxia-inducible factor-1a, a key angiogenic transcription factor (14). Additionally, the rate of tubulin polymerization or depolymerization is inhibited by 2ME2 in certain cell lines (15). In vivo, oral administration of 2ME2 is effective in xenograft and metastatic disease models with no significant toxicity (16, 17). A concomitant reduction in tumor vasculature has also been observed in 2ME2-treated animals (18). The antiangiogenic activity of 2ME2 has been shown in vivo in corneal micropocket (16), Matrigel plug assays (19), and chick chorioallantoic model systems (20). The doses of 2ME2 assessed in this dose-ranging phase II study were based on the results of the phase I study in patients with locally recurrent or metastatic breast cancer. The phase I study evaluated doses ranging from 200 to 1,000 mg/d (125625 mg/m2/d). This dose range was selected based on results obtained from nonclinical efficacy studies in mice and safety studies in rats and dogs. In vivo studies in murine models have indicated that substantial antitumor and antiangiogenic activities were observed in the range of 75 to 150 mg/kg/d (225-450 mg/m2/d). To obtain efficacy, safety, pharmacokinetic, and pharmacodynamic data on 2ME2 in men, we conducted a randomized, placebo-controlled phase II study.
Patients and Methods This study employed a phase II, multicenter, randomized, doubleblind placebo-controlled design that assessed the safety, pharmacokinetic, and pharmacodynamic characteristics and efficacy of 2ME2. The study was conducted at Indiana University and the University of Wisconsin Comprehensive Cancer Center after local institutional review board approval at both locations. Patients were eligible for this protocol if they had documented hormone-refractory disease with increasing prostate-specific antigen (PSA) by Prostate-Specific Antigen Working Group criteria (21) and/or evidence of metastatic disease and had not previously received chemotherapy. Enrollment required progression on medical or surgical androgen ablation, but castrate
Clin Cancer Res 2005;11(18) September 15, 2005
levels of testosterone were not stipulated. One patient had a total testosterone of 90 ng/dL and all other patients had levels of 10 ng/mL. If a subject was receiving antiandrogen therapy, progression (at least 25% PSA increase) had to be shown after discontinuing this class of agent for at least 4 weeks for flutamide and 6 weeks for bicalutamide. Liver transaminases had to be less than twice the upper limit of normal, bilirubin 100,000/mm3, international normalized ratio of prothrombin time V1.2, and activated partial thromboplastin time within 2 seconds of the upper limit of normal. A Karnofsky performance status of z70 and a stable pain management regimen for at least 2 weeks were required. Patients were also excluded if they had suffered a myocardial infarction within the preceding 3 months, uncontrolled angina in the last 3 months, or uncontrolled congestive heart failure. Major surgery within 21 days of starting 2ME2, administration and coadministration of PC-SPES, or herbal medication containing saw palmetto also precluded enrollment.
Study design Patients were randomized into one of two cohorts, with each cohort consisting of 16 patients. All patients in both cohorts were evaluated for safety as well as the effect of 2ME2 on changes in tumor response and PSA levels. One cohort received 400 mg 2ME2 and the other 1,200 mg 2ME2 administered as a single daily oral dose in a double-blind manner. 2ME2 was delivered as a capsule formulation, with each unit dose containing 200 mg 2ME2 as a micronized crystalline solid. Randomization was done at each site and drug was dispensed from the representative Investigational Drug Services, with patients instructed to take two capsules from one bottle and four from a second bottle. In the 400 mg dose cohort, the second bottle had four placebo capsules. Each cycle of therapy consisted of 28 days of study drug administration. Patients were treated at the same dose level until the appearance of significant treatment-emergent toxicities or disease progression. Evidence of disease progression was defined as the appearance of new lesions >1 cm in size, unidimensional or bidimensional tumor measurements increasing >50%, or increase in PSA according to Prostate-Specific Antigen Working Group definitions (21) or symptomatic progression. Patients were not required to be removed from study due solely to PSA progression if the investigator felt the patient was potentially benefiting (e.g., slower PSA velocity). Tumor response was evaluated after every cycle by using physical examination and patient interview to monitor for symptomatic progressive disease. Radiographic assessment of tumor response was done after every two cycles for the duration of therapy by using bone scan, chest X-ray, or computed tomography of the abdomen and pelvis (when disease was present at baseline).
Assessment of response Measurable disease. A complete response was defined as the complete disappearance of all clinically detectable disease measured by physical examination and/or radiographic studies for a period of at least 4 weeks. A partial remission was defined as a >50% decrease in the sum of the products of the two longest perpendicular diameters of all measurable lesions for a period of at least 4 weeks without an increase of >25% in the size of any area known to contain malignant disease and without the appearance of any new areas of malignancy. Progressive disease was defined as an increase of at least 25% in the size of measurable lesions. Serologic disease. A partial serologic response was at least a 50% decrease in PSA for three consecutive measurements 4 weeks apart (i.e., a sustained response for 2 months). Serologic progressive disease was defined as a 50% increase in PSA from the lowest level recorded and was confirmed 4 weeks later on the study. Stable serologic disease was scored if neither of these variables were met and the patient in consideration did not have measurable disease progression or
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2-Methoxyestradiol and Prostate Cancer
increase pain as the period 50% increase reference the The date of progression.
or palliative radiation. Time to progression was defined measured from the initiation of therapy until at least a in PSA level confirmed at least 4 weeks later, taking as lowest PSA level recorded since the treatment started. the first recorded increase was deemed the date of
Statistical analyses Patients were enrolled until 16 patients were treated in each arm. If two or more of these 16 patients showed a tumor response (including PSA response), then an amendment was to be made to allow an additional 16 patients to be enrolled in that arm. If six or more of the 32 patients treated at one dose level had a tumor response, it was considered that this treatment arm would warrant further study. This study had a 95% probability of stopping early for any given arm if the treatment was ineffective (tumor response rate of V5%). With a two-stage design, each treatment arm of this study would have at least 80% power to accept the alternative hypotheses and reject the null hypothesis at a 0.05 a level. Prostate-specific antigen velocity before and after treatment with 2-methoxyestradiol. Regression analyses were done post hoc to estimate the PSA rate of increase (velocity) for each patient as stabilization of disease was noted on study. There were patients who had PSA declines, although there were no PSA declines meeting Prostate-Specific Antigen Working Group definition of ‘‘response.’’ A regression line was fitted through the PSA measurements obtained on each subject before and after initiation of therapy. A ‘‘change point’’ regression model was employed to test whether the slope of the regression before and after initiation of therapy (possible change point) changed for each subject. A random-effects model was used where the initial PSA level (intercept) and the rate of change, the slope (i.e., PSA velocity), before and after therapy were allowed to vary by subject. The dose (400 or 1,200 mg qd) was also incorporated into the model as was an interaction term between PSA slope and dose level to test whether dose was related to rate of PSA increase. The mathematical equation of the regression model is:
where yij is the log PSA level of subject i at time point tjj (measured as weeks from enrollment), t 0i is the week when treatment with 2ME2 was initiated for this subject, (tij t 0j ) = max[0,(t ij t 0j )] counts the number of weeks before treatment initiation, (tij t 0j ) = min[0,(tij t 0j )] counts the number of weeks after treatment initiation, and Xi is the dose level assignment for subject i (1: 1,200 mg, 0: 400 mg). The following questions were tested through this model: Is the slope before and after treatment different from 0? Slopes that are substantially (i.e., significantly in the statistical sense) higher than 0 indicated PSA increases compared with baseline, whereas negative slopes are associated with PSA decreases. The second question was: Is there a statistically significant treatment effect (measured by the doseby-week interaction)? A positive interaction effect would imply an increase in PSA velocity in the higher compared with the lower dose, whereas a negative interaction effect would indicate that the PSA velocity is lower in the higher dose compared with the lower dose. Both questions were tested via the t test of whether the regression line slope is equal to 0. The third question was: Are the slopes of PSA increases within each treatment arm different before and after treatment initiation? Appropriate contrast equations were constructed and tested via the F test.
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Analysis of duration of therapy with 2-methoxyestradiol. We focused our analyses on the first two cycles of therapy because almost all subjects received at least 8 weeks of treatment. Analyses beyond this point become tenuous as subjects were withdrawn from the study for reasons related to the study outcome in a nonrandom fashion. This has potentially serious and unpredictable effects on the study outcome. For example, if the slope of PSA increase is steeper in the less effective treatment group, subjects may be removed earlier and at lower PSA levels compared with subjects treated with the more effective treatment (which may result in apparently slower PSA increases). Thus, the difference in PSA levels between the two treatments may be attenuated or even reversed in favor of the less effective therapy, inducing serious bias in the analyses. To assess whether the duration of therapy and time to PSA progression with the low and high doses of 2ME2 were equal between the two groups, we carried out a Kaplan-Meier estimation of the distribution of therapy durations. We compared these between the two groups using the log-rank test. All analyses were carried out according to the intent-to-treat principle, so all patient results were analyzed according to initial treatment (dose) group randomization status despite treatment adjustments or interruptions for toxicity reasons. Statistical tests were carried out at the 95% significance level.
Pharmacodynamics Blood samples were collected at baseline and on day 1 of all cycles to measure changes in the levels of PSA, sex hormone-binding globulin (SHBG), dihydroepiandrosterone, dihydroepiandrosterone sulfate, dihydrotestosterone, and total and free testosterone levels. The measurements were done in Clinical Laboratory Improvement Amendments – certified laboratories at Indiana University and the University of Wisconsin Comprehensive Cancer Center. Blood and urine samples were also collected to monitor changes in the levels of the angiogenic proteins VEGF (plasma and urine), bFGF (plasma and urine), and vascular cell adhesion molecule (serum only). VEGF in EDTA plasma was measured by a commercially available 96well plate quantitative sandwich immunoassay (Quantikine human VEGF, R&D Systems, Minneapolis, MN) with a standard curve ranging from 31.2 to 500 pg/mL. At the time of assaying, all samples and standards were brought to room temperature and prepared on the plate as recommended by the manufacturer. The plate was read at 450 nm using a Molecular Devices (Sunnyvale, CA) SpectraMax 190 plate reader. The standard curve was constructed by plotting VEGF concentration versus absorbance. A linear scale gave the best fit for this assay. The reported limit of sensitivity is 9.0 pg/mL in plasma. The assay was validated in the laboratories at the University of Wisconsin Comprehensive Cancer Center, showing a mean r 2 of 0.999 (range, 0.997-1.00) over 5 months. Within-day variability was assessed with triplicate determinations of each standard curve, with a coefficient of variation (CV) of
