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Research Article: Development
Sequential oral 9-nitrocamptothecin and etoposide: a pharmacodynamic- and pharmacokinetic-based phase I trial
1 H. Lee Moffitt Cancer Center and Research Institute, Departments of Interdisciplinary Oncology and Biochemistry and Molecular Biology, Experimental Therapeutics Program, University of South Florida, Tampa, Florida and 2 University of Miami and Sylvester Cancer Center, Department of Medicine (Hematology/Oncology), Miami, Florida
Requests for reprints: Pamela N. Munster, Comprehensive Breast Program, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, SRB-2, Tampa, FL 33612-9416. Phone: 813-745-8948; Fax: 813-745-1984. E-mail: MunstePN{at}moffitt.usf.edu
Abstract
Purpose: Resistance to topoisomerase (topo) I inhibitors has been related to down-regulation of nuclear target enzyme, whereas sensitization to topo II inhibitors may result from induction of topo II by topo I inhibitors. Here, we evaluated a sequence-specific administration of a topo I inhibitor followed by a topo II inhibitor. Experimental Design: Twenty-five patients with advanced or metastatic malignancies were treated with increasing doses (0.75, 1.0, 1.25, 1.5, 1.75, or 2.0 mg/m2) of 9-nitrocamptothecin (9-NC) on days 1 to 3, followed by etoposide (100 or 150 mg/d) on days 4 and 5. At the maximally tolerated dose, 20 additional patients were enrolled. The median age was 60 years (range, 40–84 years). Endpoints included pharmacokinetic analyses of 9-NC and etoposide, and treatment-induced modulations of topo I and II expression in peripheral blood mononuclear cells. Results: Neutropenia, thrombocytopenia, nausea, vomiting, diarrhea, and fatigue were dose-limiting toxicities and occurred in six patients. Despite a median number of four prior regimens (range 1–12), 2 (4%) patients had an objective response and 13 (29%) patients had stable disease. In contrast to the expected modulation in topo I and II
levels, we observed a decrease in topo II levels, whereas topo I levels were not significantly altered by 9-NC treatment. Conclusions: Sequence-specific administration of 9-NC and etoposide is tolerable and active. However, peripheral blood mononuclear cells may not be a predictive biological surrogate for drug-induced modulation of topo levels in tumor tissues and should be further explored in larger studies. [Mol Cancer Ther 2006;5(8):2130–7]
Introduction
DNA topoisomerases are essential nuclear enzymes that are involved in controlling the topology of supercoiled DNA during cellular functions, including replication, transcription, and cell division. Human topoisomerase I (topo I) functions as a monomer, is ATP independent, is found primarily in the nucleoli, and does not show a cell cycle–dependent variation in amount or activity (1). Topo II generally functions as a homodimer, requires ATP for activity, and shows cell cycle– and proliferation-dependent variations in amount and activity (1–3).
Due to their critical cellular functions, topo enzymes have been successfully targeted for cancer therapy. Topo II inhibitors (etoposide, mitoxantrone, doxorubicin, daunorubicin, and m-AMSA) act by stabilizing a ternary complex of DNA with topo II, thereby blocking the religation of cleaved double-stranded DNA. Drug-stabilized topo II–DNA complexes block replication fork progression, which leads to irreversible DNA damage and cytotoxicity in proliferating cells (2). Inhibitors of topo I [camptothecin, 9-nitrocamptothecin (9-NC), 9-amino-camptothecin, topotecan, and irinotecan (or CPT-11)] may also stabilize cleavable complexes, but these drugs inhibit the religation of single-stranded cleaved DNA (3). Cytotoxicity is believed to result from the collision of a replication fork with drug-stabilized topo-DNA complexes, resulting in irreversible double-strand DNA breaks (4–6). Combinations of topo I and II inhibitors have been studied in vitro (7–14). Antagonistic cytotoxic effects have been shown by many studies in which topo I and II inhibitors were given either simultaneously or in the order of topo II inhibitor first followed by a topo I inhibitor. The sequential treatment of a topo I inhibitor followed by a topo II inhibitor has resulted in a synergistic interaction in several cell lines and xenograft models. The observed synergy was believed to be associated with an up-regulation of cellular topo II in response to treatment with a topo I inhibitor (10, 14). Bonner and Kozelsky (11) reported maximum antiproliferative activity against hamster lung fibroblasts when the topo I inhibitor, topotecan, was given before treatment with the topo II inhibitor, etoposide, compared with the reverse sequence. Additionally, Chen et al. (15) showed that up-regulation of topo II by topotecan increased the sensitivity of the chronic myelogenous leukemia cell line K562 to subsequent etoposide and mitoxantrone.
Based on the data discussed above, suggesting maximal antitumor activity when topo inhibitors are sequenced as topo I inhibitor 
topo II inhibitor, we designed a clinical trial that combines the topo I inhibitor, 9-NC, followed by the topo II inhibitor, etoposide. Both agents were given orally. The antitumor activity of 9-NC has been shown in a wide variety of human tumor models (16–21).
A phase I trial with 9-NC as a single agent given orally daily 5 d/wk for 4 weeks identified a phase II dose of 1.5 mg/m2, with myelosuppression being the predominant and only major toxicity for the drug (22). Phase I trials with 9-NC in combination with gemcitabine and capecitabine have been reported recently (23, 24). The dose-limiting toxicity (DLT) for 9-NC in combination with gemcitabine was myelosuppression, whereas the DLT for 9-NC in combination with capecitabine was nausea, despite the use of 5-HT3 antagonists (23, 24).
Here, we report the results of our phase I experience using 9-NC followed by etoposide. Both 9-NC and etoposide were administered orally, allowing patient-convenient dosing of these agents. Patients received 9-NC on days 1 to 3 and etoposide on days 4 and 5 every week. Changes in topo I and topo II expression levels in peripheral blood mononuclear cells induced by 9-NC were measured on day 1 and day 4.
Patients and Methods
Patient Selection
Patients with a histologically confirmed diagnosis of a solid tumor, non–Hodgkin's lymphoma, or Hodgkin's disease who were unresponsive to or had progressed on currently available therapies were eligible. Patients were required to be 18 years or older with an Eastern Cooperative Oncology Group performance status score of 0 to 2 and to have a life expectancy of at least 3 months, and have voluntarily signed a written informed consent approved by the University of South Florida Institutional Review Board. Further eligibility criteria included adequate bone marrow function defined as absolute neutrophil count 
1,500 cells/mm3, platelet count 100,000/mm3, adequate hepatic function defined as total bilirubin <1.5 mg/dL, and alanine aminotransferase and aspartate aminotransferase <2 times and serum creatinine <1.5 times the institutional upper normal limits. An interval of at least 4 weeks since the last chemotherapy (6 weeks for nitrosourea, mitomycin C, and carboplatin), hormonal, immunotherapy, or wide field radiotherapy before enrollment was required. Effective birth control was mandated for both male and female fertile patients and documentation of a negative pregnancy test within 1 week of enrollment. Patients were excluded if they had previous therapy with a topo I inhibitor or were unable to tolerate 3 liters of oral hydration per day during the 3 days of 9-NC administration.
Drug Administration
Patients received 9-NC therapy on days 1, 2, and 3 and etoposide on days 4 and 5 of each week. A 4-week period was defined as one cycle. Both 9-NC and etoposide were administered orally once daily. Patients were advised to refrain from food intake 1 hour before and 2 hours after study drug. On the days of 9-NC administration, patients were asked to drink at least 3 liters of liquids.
Evaluation of Toxicity
Toxicity was evaluated according to the Common Toxicity Criteria version 2.0. All patients were considered evaluable for toxicity if they received any study drug. DLT were defined during cycle 1 as follows. Hematologic DLTs: any grade IV neutropenia (i.e., absolute neutrophil count <500 cells/mm3 for 5 consecutive days), febrile neutropenia (i.e., fever 38.5°C with an absolute neutrophil count <1,000 cells/mm3), grade III thrombocytopenia (<50 x 109/L), or a bleeding episode requiring platelet transfusion. Nonhematologic: grade III or greater nausea and/or vomiting despite the use of 5HT3 antagonists or any grade III or greater nonhematologic toxicity. The maximum administered dose was defined as the dose level at which a DLT was observed in a minimum of two patients (2/6 patients) during the first cycle of therapy. Toxicities beyond cycle 1 were captured throughout the trial, but were not considered DLT. At least three patients were treated at each dose level. In an effort to further define the MTD and the effect of therapy on the biological correlates, one additional dose level escalation within this regimen was conducted to allow dose escalation of etoposide to 150 mg/d for 2 days (in combination with 1/75 mg/m2 of 9-NC on days 1–3, dose level 7). If in any given cohort, one of three patients experienced a DLT, three additional patients were enrolled to that cohort. If two or more patients experienced a DLT, the dose escalation was stopped. Patients were treated until disease progression or until experiencing unacceptable toxicity.
Maximum Tolerated Dose/Dose Recommended for Phase II
The MTD was defined as the dose immediately below the maximum administered dose. At the MTD, an additional 20 patients were enrolled to further characterize the pharmacokinetic and pharmacodynamic end points and assess its suitability for future phase II trials. As prespecified in the protocol, 10 of these patients were ages 65 years.
Pretreatment and Follow-up Studies
Pretreatment evaluation included a complete history and physical examination as well as a complete blood cell count with differential, a comprehensive metabolic panel, chest X-ray, computerized tomography scan, or other imaging techniques (such as focused magnetic resonance imaging scans, bone scans, or ultrasound) as indicated. During therapy, patients were evaluated for toxicity every cycle and for tumor response every two cycles by repeat staging studies. All studies showing disease had to be repeated. Following therapy discontinuation, patients were evaluated every 4 weeks until all study-related toxicities were resolved.
Evaluation of Response
All patients who received a minimum of 2 months of study drug or had progressed before completion of two cycles were evaluable for response. Patients on therapy for at least two cycles of combination treatment had their response classified by standard WHO criteria. Complete response was defined as complete disappearance of all tumor lesions for at least 4 weeks from the date of documentation of complete response. Partial response was defined as a decrease by 50% of the sum of the products of the two largest perpendicular diameters of all measurable lesions as determined by two consecutive observations at least 1 month apart. A decrease by 50% was also required for unidimensional lesions. Progressive disease was defined as a >25% increase in the sum of the products of any measurable lesions compared with best response and/or the appearance of any new lesions, or the occurrence of a malignant pleural effusion or ascites. Stable disease was defined as the absence of an objective response and the absence of disease progression. In the case of serum tumor marker evaluations, progression was defined as >50% increase over any nadir value, and/or in association with the appearance of any new lesions or the occurrence of a malignant pleural effusion or ascites. Nonmeasurable but evaluable disease (e.g., pleural effusion, ascites, and serum tumor marker elevations) was not considered in the assessment of response status except in instances of progressive disease defined above.
Pharmacokinetic Analyses
The plasma pharmacokinetics of 9-NC was determined on day 3 of cycle 1 at 0, 0.5, 1, 1.5, 2, 3, 4, and 8 hours after ingestion of a single oral dose. Additional blood samples were collected on days 4 and 5 for determination of etoposide pharmacokinetics. Plasma samples were analyzed for 9-NC concentrations by an adaptation of the method previously validated for the high-performance liquid chromatography determination of 9-aminocamptothecin (25).
Samples for etoposide concentration were drawn on day 4 of cycle 1 at 0, 0.5, 1, 2, 4, and 8 hours after etoposide dosing. One additional sample was drawn at 24 hours (just before dosing on day 5). Plasma samples were analyzed for etoposide concentrations by a previously published validated high-performance liquid chromatography method (26). Immediately after collection, each blood sample was gently inverted several times for complete mixing with the anticoagulant and then placed in ice. Within 30 minutes of collection, each blood sample was centrifuged for 10 minutes at 
2,500 rpm (1,100 x g) to separate the plasma. The separated plasma was transferred to a screw cap polypropylene tube and placed over crushed dry ice, then stored frozen at –80°C until analysis. All tubes were identified with a study label and sample numbers to assure sample identity.
The following pharmacokinetic variables were estimated for each patient by standard methods; the maximum plasma concentration (Cmax), the time of the maximum plasma concentration (Tmax), the plasma elimination half-life (T1/2), area under the curve (AUC) in plasma, and total body clearance. Pharmacokinetic variables for 9-NC were determined from concentration-time data using a noncompartmental analysis tool in WinNonlin, version 3.0 (Pharsight Corporation, Mountain View, CA).
Topo I and II Expression
To examine the expression of topoisomerase levels, 35 mL whole blood were collected on day 1 before 9-NC administration and on day 4 before etoposide administration. Samples were collected in tubes containing sodium citrate and kept on ice throughout the procedure. Blood was diluted 1:1 with cold sterile PBS, overlayed on 10 mL Ficoll-Paque Plus (Amersham-Pharmacia Biotech, Piscataway, NJ), and centrifuged at 400 x g for 30 minutes. Mononuclear cells were removed from the interface, washed, counted, and finally resuspended in 1 mL aliquots of freezing medium (40% RPMI 1640, 50% fetal bovine serum, and 10% DMSO). Samples were stored at –80°C in a slow freeze container overnight and then transferred to liquid nitrogen until further analysis. For immunoblot analysis of protein expression, cells (1.0–2.0 x 107) were thawed quickly and then placed in 37°C PBS containing 2 mmol/L EDTA and 0.5% bovine serum albumin. After determination of cell count and viability, cells were suspended in SDS sample buffer (2% SDS, 100 mmol/L DTT, 10% glycerol, and 0.01% bromophenol blue). One hundred micrograms (µg) and 75 µg protein were loaded on 7.5% SDS-PAGE gels. Three dilutions of human CCRF-CEM (American Type Culture Collection, Rockland, MD) cells were run on each gel to provide an internal standard for comparing signals from gel to gel (27). Proteins were detected with antibodies against topo II (rabbit polyclonal antibody 454), generated in this laboratory (28), and topo I (C-21), generously donated by Dr. Yung-chi Cheng (Henry Bronson Professor of Pharmacology, Yale School of Medicine, New Haven, CT) and visualized by chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). The immunoblots were analyzed by a GS700 densitometer (Bio-Rad Laboratories, Hercules, CA) and quantified using Bio-Rad Quantity One image analysis software.
Statistical Considerations
The primary purpose of this phase I study was to determine the MTD, DLT, and recommended phase II dose of the combination of 9-NC and etoposide. Descriptive statistics were used in the analyses of all safety and laboratory observations for this study. For each of the pharmacokinetic variables described under the pharmacokinetics section, summary statistics were tabulated by dose, and scatterplots versus dose were examined for relationships to dose. Tumor response rates of patients treated at the MTD were estimated by an exact 95% confidence interval.
The degree of alteration in topo I and II were plotted against the estimated AUC for 9-NC to determine if any possible relationship exists. Regression analyses were used to explore the relationship between the pharmacokinetic variable estimates for 9-NC and alterations in topo I and II expression. Pairwise comparisons were done between pretreatment absolute topo I and topo II levels and toxicity. Pretreatment levels and changes in topo I and topo II (ratio of day 1 to day 4) levels were correlated with 9-NC AUC for each dosage tier and correlated with both hematologic and nonhematologic toxicity. Each patient's sample was run, at a minimum, in duplicate on separate gels for each enzyme.
Results
Patient Characteristics
Forty-five patients (14 females and 31 males) with a median age of 60 years (range, 40–84 years) and a median Eastern Cooperative Oncology Group performance status of 1 (range, 0–2) were enrolled between March 2000 and March 2002 (Table 1
). All patients were assessable for toxicity. Forty-five patients (100%) had received prior chemotherapy, 26 (58%) patients had prior radiation therapy, and 7 (16%) patients had prior immunotherapy or hormonal therapy. The median number of prior chemotherapy regimens was 4 (range 1–12). The predominant tumor type was lung cancer (12 patients, 27%). Other tumor types included pancreatic cancer (5, 11%), non–Hodgkin's lymphoma (4, 9%), sarcoma (3, 7%), hepatoma (3, 7%), head and neck carcinoma (3, 7%), and mesothelioma (3, 7%); other tumor types included carcinoma of unknown primary, renal cell, breast cancer, basal cell, colon, and anal carcinoma, as well as melanoma.
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Nonhematologic Toxicities
Nausea (84%) was the most common nonhematologic toxicity noted. Twenty-six of the 45 patients reported grade 1 or 2 fatigue at baseline before initiation of treatment. Grade 3 fatigue was observed in 11 (25%) patients. No incidence of grade 4 fatigue was observed. Gastrointestinal toxicities, including nausea, vomiting, and diarrhea were the most prominent treatment-related nonhematologic side effect. Grade 1 or 2 nausea and vomiting were observed in the first course in 51% and 37% of patients, respectively. Grade 1 or 2 diarrhea was observed in 22% of the patients during their first course. The occurrences of grade 3 or 4 nausea, vomiting, or diarrhea occurring through all the cycles are listed in Table 3. Although grade 3 nausea, vomiting, and diarrhea were not observed in the first cycle on the dose escalation phase of the trial, these were observed in the dose expansion. However, <33% of the patients in the dose expansion phase were affected; therefore, no further dose modulations were done. Other toxicities included grade 3 dyspnea and electrolyte imbalances. Although it is unclear whether these toxicities were definitely linked to the treatment, were due to the underlying disease, or secondary to the nausea and vomiting observed in some of these patients, grade 3 electrolyte imbalances were only seen at cohorts 6 and 7, and in the dose expansion phase of the trial. The reported grade 3 electrolyte imbalances included hypokalemia and hypomagnesaemia and were listed as DLTs in Table 3.
Response
Response assessment was done in all 45 patients. Two (4%) patients had a partial response and 13 (29%) additional patients had stable disease for >2 months. One of the patients with an objective response had a squamous cell carcinoma of the tongue with supraclavicular lymph node involvement. A second patient had non–Hodgkin's lymphoma with multiple enlarged lymph nodes. In both patients, a partial response was documented as defined by WHO criteria. Of the 13 patients with stable disease, three patients had stable disease for >6 months.
Pharmacokinetic Analyses
Blood samples for pharmacokinetic analyses were available from all patients. The mean pharmacokinetic variable estimates for 9-NC by dose level are shown in Table 4
. The observed mean AUC and Cmax for 9-NC increased with increasing dose. Although the mean Tmax was between 3 and 4 hours at all dose levels, the peak concentration of 9-NC was delayed in many subjects at the higher two doses. This delayed absorption hampered the calculation of the terminal elimination rate and also AUC0-8; therefore, AUC0-8 is presented for all subjects. Although the pharmacokinetics of 9-NC was consistent between subjects at the lower doses, considerable variability was shown in the subjects receiving higher doses.
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Pharmacodynamic Analyses
Thirty-seven of 45 patients had sufficient peripheral blood mononuclear cells for Western analysis. Thirty-one patients had paired (day 1 and day 4) samples available for topo II analyses and 28 patients had paired samples available for topo I analyses.
The effects of 9-NC on expression levels of both topo I and topo II were evaluated in peripheral blood mononuclear cells obtained before and after 3 days of exposure to 9-NC by Western blot analyses. Changes in protein expression before and after 9-NC treatment from four select patients are depicted in Fig. 1A
. The values of baseline topo enzyme levels from all patient data are shown as a function of 9-NC dose in Fig. 1B (topo I: 1B-1 and topo II: 1B-2). The ratio of day 4 expression levels and pre-9-NC expression levels of topo I and II were expressed in Fig. 1B (topo I: 1B-3 and topo II: 1B-4-2D). A ratio of >1 reflected an increase in the respective topo level on day 4 compared with levels before 9-NC exposure, whereas a ratio of <1 suggested a decrease in the respective topo level. Although we observed a numerical trend for a decrease in the expression of topo I on pairwise comparison of all samples per cohort, this did not reach statistical significance. In contrast, a significant decrease in topo II was observed when pretreatment and day 4 topo II levels were compared (P = 0.03). There were no statistically significant correlation between pretreatment absolute topo I levels and toxicity, nor between a change in topo I or topo II levels and 9-NC AUC (data not shown).
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Topo I and II inhibitors have activity against a wide range of malignancies, including lung cancers, gastrointestinal cancers, sarcomas, lymphomas, and leukemias. Several phase I/II studies evaluating a specific sequence of topo I inhibitor and topo II inhibitors have now been published. Myelosuppression was the DLT when irinotecan and epirubicin, and topotecan and etoposide were combined (29). Mucositis was the DLT in a third phase I study done in adults exploring a topo II sequencing strategy with topotecan and etoposide in leukemia (30). Timed sequential chemotherapy using topotecan followed by etoposide plus mitoxantrone was reported to be an effective regimen for patients with refractory acute leukemia, and showed a topo II protein level increase after topotecan exposure (15, 31). Crump et al. (32) reported their phase II experience using sequential topotecan and etoposide in relapsed/refractory intermediate grade NHL. Only modest activity was seen despite the presence of marked myelosuppression.
In this phase I study, we evaluated the sequential administration of a topo I inhibitor followed by a topo II inhibitor using two oral compounds. Myelosuppression was relatively mild, but neutropenia was the DLT. At the maximally tolerated dose, 20 additional patients were enrolled, primarily to better define the toxicity profile of this regimen. Overall, this regimen was well tolerated, with only one episode of febrile neutropenia noted in the entire study. Other than fatigue, which was present at baseline in 26 (58%) of the 45 patients, nausea, vomiting, and diarrhea were the most common nonhematologic toxicities. Most of these toxicities were of grade 1 and 2 with only 2 of the 45 patients reporting grade 3 nausea and vomiting despite optimal use of antiemetics. These occurred at the MTD dose expansion. Lower-grade (1 and 2) gastrointestinal symptoms, such as nausea and/or vomiting, were seen in 35 of 45 (78%) patients and occurred at all dose levels. Furthermore, in the dose expansion phase of the trial, 2 of 20 patients experienced grade 3 diarrhea; in one of these patients, a grade 3 anorexia was reported. Other nonhematologic toxicities included weight loss, alopecia, and dyspnea. In the cohorts with concentrations of 9-NC of 2.0 mg/m2, or the 1.75 mg/m2 9-NC dose with the higher etoposide dose (150 mg), several patients were noted to have abnormalities in their electrolytes, including three patients with grade 3 abnormalities. All these patients also experienced other gastrointestinal symptoms; hence, it cannot definitively be determined whether the electrolyte imbalances are independent drug effects or secondary to the observed gastrointestinal effects or both.
With the exception of cohort 2, the AUCs of 9-NC were linear with an increase in 9-NC dose. The AUC means ranged from 188 to 515 ng h/mL and the Cmax mean values range from 31 to 82 ng/mL across the cohorts (Table 4). These values were comparable with another study evaluating 9-NC as a single agent in patients with advanced solid tumors, which focused on the effects of food on pharmacokinetic profile of 9-NC. Patients in the described study were treated with 1.5 mg/m2 for 5 days. The mean AUC for 9-NC with food were 330 ± 182 ng h/mL. Values for fasting patients were 558 ± 379 ng h/mL (33). The high AUC for 9-NC in cohort 2 may be explained by the considerable variation (up to 10-fold) in the plasma concentrations of 9-NC between patients in the same cohort and the limited number of patients per cohort. This notable variation in 9-NC plasma concentration has also been observed by others (33, 34).
Etoposide pharmacokinetic, i.e., the plasma half-life, the AUC, and clearance of etoposide were not affected by increasing doses of 9-NC or increasing 9-NC AUC.
Other investigators, using the same sequencing strategy, showed that topo I levels are down-regulated after treatment with topo I inhibitors, whereas topo II levels increase in preclinical models (35–39). Crump et al. (30) tested the topo I inhibitor, topotecan, daily for days 1 to 5, and etoposide on days 6 to 8 in adult patients with acute myeloid leukemia. During the first 72 hours of topotecan infusion, an increase in topo II was reported that returned to baseline by the end of the topotecan infusion, suggesting that the timing of the topo evaluation may affect the finding of the studies. Future studies may be needed to define the optimal time point for analysis of topo levels and the potential relevance and mechanism of this time-sensitive variation. Licitra et al. (40) evaluated a 72-hour continuous infusion of topotecan followed by etoposide on days 8 to 10. They found some responses and an occasional increase in topo II levels in tumor tissue, but also encountered severe myelosuppression. In our study, we saw a decrease in topo II levels after exposure to 9-NC at 72 hours. Although this decrease was statistically robust, this study was preformed in peripheral blood mononuclear cells and not in tumor cells. This decrease in topo II levels may not reflect the effects of a topo I inhibitor on topo II levels in tumor cells. Furthermore, although we observed a decrease in topo II levels overall, in some patients the topo II were increased. However, further statistical analysis did not support a correlation with response or clinical benefit. As discussed above, the time point of analysis at 72 hours in our study may have missed an early increase in topo II levels and this trial does not allow any speculation on whether the decrease in topo II levels is transient and would return to baseline at a later time point. Although these finding may differ from our reports suggesting an increase in topo II levels after exposure to a topo I inhibitor, these results should be evaluated with consideration of several limitations. Because only two patients had a partial response, the small sample size would not allow any correlation with response and we found no correlation between observed changes in topo II levels in patients with stable disease versus nonresponders. These studies may be further limited by the range of different tumor types enrolled onto this trial. It is possible that 72 hours is not the optimal time to detect maximal changes in topo expression in some or all of the patients due to individual differences in pharmacokinetics or tumor types. The logistics of the specimen sampling may lead to variations in quality that may be difficult to assess given the small sample size. However, foremost, the analyses of the peripheral blood mononuclear cells may not reflect any changes expected in tumor cells.
In conclusion, our phase I study of the sequential use of 9-NC and etoposide, both given orally, was found to be a well-tolerated regimen and showed activity in this phase I trial. Further disease-specific phase II testing is therefore warranted. The recommended phase II doses are 1.75 mg/m2 for 9-NC on days 1 to 3 and 100 mg for etoposide on days 4 and 5 given every week. Of the patients on the dose expansion part, 10 of 20 patients were older than 65 years. This regimen may provide a relatively well-tolerated alternative in previously treated patients with a variety of malignancies or in an elderly population.
Acknowledgments
We thank the Clinical Pharmacology core for contributions, the nurses and medical staff of the Clinical Research Unit for support, all the patients and their families for their courageous contribution to research, and Anita Bruce for editorial assistance.
Footnotes
Grant support: Grant CA082533 MOPP P01.
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.
Received 1/23/06; revised 5/ 4/06; accepted 5/31/06.
References
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