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Clinical Cancer Research Vol. 11, 8728-8736, December 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Clinical

Phase I and Pharmacokinetic Study of Sequences of the Rebeccamycin Analogue NSC 655649 and Cisplatin in Patients with Advanced Solid Tumors

Alejandro D. Ricart1, Lisa A. Hammond1, John G. Kuhn2, Chris H. Takimoto1, Andrew Goetz1, Bahram Forouzesh1, Leonardo Forero1, Jose L. Ochoa-Bayona1, Kristin Berg1, Anthony W. Tolcher1 and Eric K. Rowinsky1

Authors' Affiliations: 1 Institute for Drug Development, Cancer Therapy and Research Center; 2 Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, Texas

Requests for reprints: Alejandro D. Ricart, Institute for Drug Development, Cancer Therapy and Research Center, 7979 Wurzbach Road, Suite Z414, San Antonio, TX 78229. Phone: 210-616-5860; Fax: 210-692-7502; E-mail: aricart{at}idd.org.


   Abstract
Top
 Abstract
Patients and Methods
 Results
 Discussion
 References
 
Purpose: To evaluate the feasibility of administering NSC 655649, a water-soluble rebeccamycin analogue that inhibits both topoisomerases I and II, in combination with cisplatin (CDDP) in adults with solid malignancies. Major toxicologic and pharmacologic differences between the two sequences of drug administration were also assessed.

Experimental Design: NSC 655649 was administered as a 60-minute i.v. infusion; CDDP was given i.v. before or after NSC 655649 on day 1. Each patient was treated with alternating drug sequences every 3 weeks; doses of each drug were escalated in separate cohorts of new patients. Sequential dose escalation of NSC 655649 or CDDP resulted in three dosage permutations of NSC 655649/CDDP: 440/50, 550/50, and 440/75 mg/m2. After the maximum tolerated dose level was determined, the feasibility of using granulocyte colony-stimulating factor to permit further dose escalation was explored.

Results: Twenty patients were treated with 70 courses of NSC 655649/CDDP. Myelosuppression was the principal toxicity. The incidence of severe neutropenia, often associated with severe thrombocytopenia, was unacceptably high in minimally pretreated patients at the NSC 655649/CDDP dose level of 550/50 mg/m2 without and with granulocyte colony-stimulating factor. Major pharmacokinetic interactions between NSC 655649 and CDDP were not apparent. No relevant sequence-dependent differences in toxicity or pharmacokinetic variables occurred. Three patients had partial responses.

Conclusions: NSC 655649 and CDDP were well tolerated by minimally pretreated subjects at 440 and 50 mg/m2, respectively. Neither pharmacokinetic interactions between the agents nor sequence-dependent toxicologic or pharmacokinetic effects were apparent. The tolerance and preliminary activity observed with this combination suggest that disease-directed evaluations of the regimen are warranted.

Rebeccamycin, a N-glycoside fermentation product originally isolated from the actinomycete strain Saccharothrix aerocolonigenes, showed impressive broad spectrum of antitumor activity against several cancers in vitro, including A549 lung, HCT-116 colon, and KB nasopharyngeal carcinomas; however, poor water solubility precluded its clinical development and led to the evaluation of semisynthetic, water-soluble derivatives (1–4). The rebeccamycin analogue NSC 655649 (1,11-dichloro-6-[2-(diethylamino)ethyl]-12,13-dihydro-12-(4-O-methyl-ß-D-glucopyranosyl)-5H-indolo-[2,3-a]pyrrolo[3,4-c]-carbazole-5,7(6H)-dione) was selected for further development due to its favorable pharmaceutical characteristics and notable antitumor activity (5, 6).

Rebeccamycin and NSC 655649 inhibit the activities of both topoisomerases I and II (2, 3, 7–9). Structure-activity studies have indicated that the linkage of chemical groups with low steric hindrance, such as biethylamine, to the imide nitrogen of the indolocarbazole backbone of rebeccamycin, impart greater topoisomerase II inhibitory activity (2, 4). The small biethylamine group of NSC 655649 allows the molecule to intercalate preferentially and avidly into the minor groove of poly(deoxyadenylate-deoxythymidylic acid) regions of DNA at similar binding sites as doxorubicin and ethidium bromide (6). In contrast to both etoposide and teniposide, which stabilize cleaved DNA by inhibiting religation of the cleaved DNA strands, NSC 655649 markedly inhibits the catalytic steps required for the passage of the intact DNA strand through the single-strand DNA break that leads to the formation of single, rather than double, DNA strand breaks. NSC 655649 is active in etoposide-resistant A549 and HCT-116 cell lines, which have low topoisomerase II levels (5). However, the observation that the teniposide-resistant HCT-116(VM)46 cell line shows slight resistance to rebeccamycin analogue suggests that NSC 655649 is cross-resistant, at least in part, to other natural antitumor agents that induce the multidrug resistance phenotype (5). The in vitro concentrations of NSC 655649 that inhibit growth by 50% (IC50) range from 0.03 to 0.21 µg/mL (5). NSC 655649 also inhibits a broad spectrum of pediatric and adult solid tumors in both in vivo antitumor studies and the tumor-cloning assay (5, 10).

Three phase I trials of single-agent NSC 655649 have been done in adult patients with solid malignancies. In one study, neutropenia was the principal toxicity when NSC 655649 was administered at doses ranging from 60 to 188 mg/m2/d for 5 days (11). Two partial responses (PR) were noted in patients with cancers of the stomach and unknown primary. In the other two studies, the principal toxicity of NSC 655649 at doses ranging from 20 to 744 mg/m2 as a single i.v. infusion every 3 weeks was also neutropenia, and the doses recommended for phase II studies were 500 and 575 mg/m2 for heavily and minimally pretreated patients, respectively (12, 13). Antitumor activity was noted in two heavily pretreated ovarian cancer patients. In all of these trials, local venous toxicity precluded administration of NSC 655649 through a peripheral vein, and central venous access was required. In subsequent disease-directed studies, notable activity has been shown for NSC 655649 administered for 5 days every 3 weeks in chemotherapy-naive patients with advanced biliary cancers (14).

Supra-additive or synergistic interactions between platinum and topoisomerase inhibitors in preclinical studies as well as the preclinical spectrum and clinical activity of NSC 655649 in tumors sensitive to platinum agents and the minimal overlapping toxicities of NSC 655649 and platinating agents provided the rationale for this evaluation of NSC 655649 and cisplatin (CDDP). The principal objectives of the present study were to determine the maximum tolerated dose (MTD) of sequences of NSC 655649 administered as a 60-minute infusion with CDDP without and with granulocyte colony-stimulating factor (G-CSF) support every 21 days, determine the toxicities of sequences of NSC 655649 and CDDP on this schedule, characterize the pharmacokinetic behavior of NSC 655649 in combination with CDDP, determine possible sequence-dependent pharmacokinetic effects, and seek preliminary evidence of antitumor activity in patients with advanced solid malignancies.


   Patients and Methods
Top
 Abstract
 Patients and Methods
Results
 Discussion
 References
 
Patient selection. Patients with histologically or cytologically confirmed solid malignancies refractory to standard therapy or for whom no standard therapy existed were eligible. Patient entry criteria also included age ≥18 years; life expectancy ≥12 weeks; Southwest Oncology Group performance status ≤2; no prior chemotherapy within 3 weeks (6 weeks for prior mitomycin C or a nitrosourea); adequate hematopoietic [hemoglobin ≥9 g/dL, absolute neutrophil count (ANC) ≥1,500/µL, and platelet count ≥100,000/µL], hepatic (bilirubin <1.5 mg/dL), and renal (serum creatinine <1.5 mg/dL) functions; measurable or evaluable disease; no peripheral neuropathy that interfered with function; and no coexisting medical problem of sufficient severity to limit compliance with the study. All patients gave informed written consent before treatment according to federal and local institutional guidelines.

Drug dosage and sequencing. The starting doses of NSC 655649 and CDDP were 440 and 50 mg/m2, respectively. These doses produce minimal toxicity when each drug is administered as a single agent (12). Doses of either NSC 655649 or CDDP were escalated in each successive cohort of new patients. The goal was to focus primarily on the dose escalation of NSC 655649, whereas CDDP was dosed in the conventional range of 50 to 75 mg/m2. At least four new patients were treated at each dose level to evaluate more precisely the hematologic toxicity produced by the drug combination and each sequence. Toxicities were graded according to the National Cancer Institute Common Toxicity Criteria version 2.0. Dose-limiting toxicity (DLT) was defined during the first two courses, due to alternating sequences, as neutropenia associated with fever (ANC <1,000/µL and fever ≥38.5°C), ANC <500/µL for >5 days, platelets <25,000/µL, nonhematologic toxicity grade ≥3 (excluding nausea and vomiting), and delay in the administration of subsequent courses due to unresolved hematologic and nonhematologic toxicities lasting longer than 14 and 7 days, respectively. If one DLT occurred, a maximum of six new patients were treated at that dose level. The MTD was defined as the highest dose at which fewer than two of six new patients experienced DLT. If prolonged severe neutropenia was the principal DLT that precluded further dose escalation, G-CSF was added to sequences of NSC 655649 and CDDP and dose escalation was resumed. Intrasubject dose escalation was not allowed. The dose was reduced for patients who received at least two courses of therapy and developed acute or chronic DLTs. The study was designed prospectively to determine the DLT and MTD in minimally and heavily pretreated cohorts. Patients were considered heavily pretreated and ineligible for the first part of this study if they had received radiation therapy to whole pelvis or ≥25% of the bone marrow–containing areas, more than six courses of an alkylating agent–containing chemotherapy regimen, any prior mitomycin C or nitrosourea-containing regimen, or widespread bone metastases with bone marrow involvement. After the MTD of sequences of NSC 655649 and CDDP was determined in minimally pretreated patients, accrual of heavily pretreated patients was to proceed at two dose levels below the MTD that was established for minimally pretreated patients.

The initial patient entered at each dose level received CDDP before the dose of NSC 655649. The second subject at each dose level received CDDP following treatment with NSC 655649. Thereafter, the sequence of drug administration was alternated with each successive patient enrolled at each dose level. The drug sequence was also alternated with each successive course in each individual patient.

Drug administration. NSC 655649 was supplied by the National Cancer Institute as a sterile vial containing 20 mL of a solution of 10 mg/mL NSC 655649 and one equivalent (2.24 mg/mL) of L-tartaric acid in sterile water (USP) for injection. The appropriate dose of the drug was then further diluted in 500 mL of 0.9% NaCl solution and infused i.v. over 60 minutes through a central venous catheter. CDDP (Platinol, Bristol-Myers Squibb, Princeton, NJ) was supplied in 10- and 50-mg vials that were reconstituted with 500 mL of 0.9% NaCl solution along with 12.5 g mannitol and given as a 60-minute i.v. infusion. Patients were hydrated with 1 L of 0.9% NaCl solution with 10 mEq KCl and 1 g MgSO4/L at 150 mL/h for 12 hours before and for at least 6 hours after the CDDP infusion. NSC 655649 was administered either before or after CDDP. Regardless of sequence, the second chemotherapy agent was administered 2 hours after the end of infusion of the first agent. The treatment was repeated every 3 weeks. Antiemetic premedication included ondansetron 8 mg i.v. 30 minutes before CDDP, dexamethasone 20 mg i.v. 30 minutes before CDDP, and 8 mg p.o. every 12 hours for 3 days following CDDP. Lorazepam and prochlorperazine were used at the discretion of the investigator.

Recombinant methionyl human G-CSF (Neupogen, Amgen, Inc., Thousand Oaks, CA) was supplied in vials that contained 300 or 480 µg cytokine. When the feasibility of further dose escalation of NSC 655649 and CDDP with G-CSF was evaluated, G-CSF 5 µg/kg/d s.c. was begun on day 2 (i.e., 24 hours after the last dose of chemotherapy) and administered until the ANC was at least 1,500/µL on two consecutive determinations after the nadir or until day 6 if a nadir was not observed. Patients kept a log that detailed G-CSF administration.

Pretreatment and follow-up studies. Complete medical histories, physical examinations, concurrent medication profiles, assessments of performance status, and routine laboratory studies were done pretreatment and weekly. Routine laboratory studies included a complete blood count, prothrombin and partial thromboplastin times, electrolytes, blood urea nitrogen, serum creatinine, uric acid, glucose, alkaline phosphatase, alanine aminotransaminase, aspartate aminotransaminase, total/direct bilirubin, calcium, phosphorus, magnesium, total protein, albumin, and urinalysis. A serum pregnancy test was done before treatment in women of child-bearing potential. Pretreatment studies also included an electrocardiogram, relevant radiologic studies to evaluate all measurable and assessable sites of disease, and an assessment of relevant tumor markers. Radiologic evaluations for disease status assessment were repeated after every other course. Patients were able to continue treatment if they did not develop progressive disease or experience intolerable toxicity. A complete response was scored if there was disappearance of all measurable and assessable disease on two measurements done at least 4 weeks apart without worsening of disease-related symptomatology or declining performance status. A PR required at least a 50% reduction in the sum of the products of the bidimensional measurements of all lesions documented by two measurements separated by at least 4 weeks. Any concurrent increase in the size of any lesion by ≥25% or the appearance of any new lesion was considered disease progression.

Sample collections and analytic analysis. Blood samples were collected into heparinized tubes via an indwelling venous catheter placed in the contralateral arm before the NSC 655649 infusion, 30 minutes after beginning of infusion, and immediately before the end of infusion. Samples were also collected at 30 minutes and 1, 2, 3, 4, 6, 8, 12, 24, 48, and 168 hours after the end of the NSC 655649 infusion. Samples for pharmacokinetic analysis were collected during the first two courses of therapy to discern the effects of drug sequencing on the pharmacologic disposition of NSC 655649. The blood samples were centrifuged at 3,000 rpm for 15 minutes immediately after collection. The plasma was transferred to separate tubes and frozen to –20°C until assayed.

The analytic standards for NSC 655649 and rebeccamycin (NSC 359079) were obtained from the Pharmaceutical Management Branch, National Cancer Institute. Liquid-liquid extraction followed by high-performance liquid chromatography was used for the analytic analysis of NSC 655649 as described previously (12). However, the calibration curves were constructed using NSC 655649 areas to known plasma concentrations rather than NSC 655649 to rebeccamycin internal standard ratios. This modification was due to the identification of rebeccamycin as one of the metabolites of NSC 655649 (12). The plasma calibration curves for NSC 655649 were linear (R2 > 0.99) over the range of 0.016 to 16.32 µg/mL. Eighteen separate 11-point standard curves were used in the analysis of the samples. The interday precision for NSC 655649 at the low (0.08 µg/mL), medium (0.82 µg/mL), and high (8.16 µg/mL) concentrations was 8.0%, 6.3%, and 5.2%, respectively.

Pharmacokinetic and pharmacodynamic analyses. Individual NSC 655649 plasma concentration data sets from days 1 to 7 were analyzed by a noncompartmental method using WinNonLin version 4.1 (Pharsight Corp., Mountain View, CA). Elimination rate constants ({lambda}) were estimated by linear regression of at least three terminal plasma concentration-time data points. Terminal half-lives (t1/2) were calculated by dividing 0.693 by the elimination rate constants. The area under curve (AUC) was calculated using the linear trapezoidal rule up to the last measurable data point (Clast) for AUC0-t, then extrapolated to infinity (AUC0-{infty}), using the formula AUC0-t + Clast / {lambda}. The systemic clearance (Cl) was determined by dividing the dose (in mg freebase NSC 655649) by the AUC0-{infty}. Volume of distribution at steady state (Vdss) was estimated multiplying Cl by mean residence time extrapolated to infinity (MRT0-{infty}).

Pharmacokinetic variable data were reported as mean ± SD. The sequence-dependent effects of NSC 655649 and CDDP on pertinent toxicologic variables were assessed using the t test. Probability values (two-sided) of P < 0.05 were regarded as statistically significant. Paired analyses of toxicologic and pharmacologic data for each drug sequence were done using each subject as his own control using Statistica version 6.0 software (StatSoft, Inc., Tulsa, OK). Nonparametric analyses (Wilcoxon's matched pairs test and Mann-Whitney U test) were done when data were not normally distributed.


   Results
Top
 Abstract
 Patients and Methods
 Results
Discussion
 References
 
General
Twenty patients, whose pertinent demographic characteristics are depicted in Table 1, were treated with 70 courses of alternating NSC 655649 and CDDP sequences through four dose levels. All patients had not received previous treatment with a platinum compound. The numbers of new and total patients and total courses administered at each dose level are shown in Table 2. The median number of courses administered per patient was three. Three patients required dose reduction for severe myelosuppression at the highest NSC 655649 dose level (550 mg/m2), two of them despite concomitant use of G-CSF. One heavily pretreated patient, who had received previously four courses of doxorubicin and streptozocin, was inadvertently enrolled.


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  		Table 1. Patient characteristics

 

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  		Table 2. Dose escalation scheme

 
The first patient treated at the NSC 655649/CDDP dose level of 440/50 mg/m2, a 66-year-old female with metastatic endometrial carcinoma, with large retroperitoneal masses invading the inferior vena cava and currently receiving megestrol acetate, experienced a fatal pulmonary embolism after her first course and was not considered fully evaluable for toxicity. The second patient experienced DLT, which prompted the expansion of this cohort to a total of six evaluable patients. No further DLTs were observed during the first two courses, allowing dose escalation to 550/50 mg/m2. At this juncture, there were two DLTs among five patients and additional patient accrual ensued at 550/50 mg/m2 with G-CSF. Nonetheless, an unacceptably high rate of severe hematologic toxicity occurred, with two of six patients experiencing DLT. There were no further efforts to increase the dose of NSC 655649; instead, the dose of CDDP was increased in the fourth cohort. Two patients were treated at the 440/75 mg/m2 dose level, which did not produce significant hematologic toxicity. However, cumulative CDDP-induced toxicity, including mild renal impairment in one patient and moderate ototoxicity in the second, precluded further treatment at this dose level.

Toxicity
Hematologic toxicity. Myelosuppression, including severe neutropenia and thrombocytopenia, was the principal DLT for the combination of NSC 655649 and CDDP. Table 3 details the numbers of courses associated with grade 3 and 4 neutropenia and thrombocytopenia as a function of dose level. However, these toxicities were rarely associated with clinical sequelae. Severe neutropenia associated with fever occurred during 7 of 70 (10%) courses and gastrointestinal bleeding associated with severe thrombocytopenia was observed in one patient.


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  		Table 3. Hematologic toxicity

 
The initial NSC 655649/CDDP dose level (440/50 mg/m2) was well tolerated. One of seven new patients experienced DLT characterized by a brief episode of fever and neutropenia. A second patient, who required dose reduction from the 550/50 mg/m2 dose level with G-CSF, experienced uncomplicated grade 4 thrombocytopenia of brief duration. At the first dose level, in which 29 courses were administered, worsening of neutropenia with cumulative treatment was not noted and neutrophils typically recovered to pretreatment levels by day 21. Treatment delays were rarely required. Nevertheless, the rates of hematologic DLTs increased substantially as the dose of NSC 655649 was increased to 550 mg/m2. At the 550/50 mg/m2 dose level, dose-limiting hematologic events were noted in two of five patients. Fever and/or infection associated with grade 4 neutropenia occurred during 2 and 1 of 13 courses, respectively. One of these patients also experienced grade 4 thrombocytopenia.

The addition of G-CSF did not sufficiently improve the tolerance or lessen the magnitude of hematologic toxic effects of the NSC 655649/CDDP regimen. Of 19 total courses administered at the 550/50 mg/m2 dose level with G-CSF, 2 of 6 patients developed DLT after the first course of therapy. One patient experienced sepsis associated with grade 4 neutropenia and thrombocytopenia. The other patient developed febrile neutropenia and grade 3 thrombocytopenia. Grade 3 and 4 thrombocytopenia occurred during 7 of 19 courses, and febrile neutropenia was observed in 3 of 19 courses. Isolated grade 4 neutropenia of brief duration was noted in five other courses. Of note, nadir platelet counts were significantly lower at the 550/50 mg/m2 dose level, with the addition of G-CSF (mean 159,000/µL without G-CSF versus 80,000/µL with G-CSF; P = 0.016).

Treatment-related anemia was typically mild (grade 1) or moderate (grade 2). In general, effects on RBC were cumulative, progressively increasing with repetitive treatment. Severe (grade 3) anemia occurred in 6 of 20 (30%) patients during 7 of 70 (10%) courses. Six of 20 patients required RBC transfusions on at least one occasion.

Drug sequencing. The relevant hematologic toxicities of the NSC 655649/CDDP regimen as a function of drug sequencing are displayed in Table 3. The proportions of events with ANC nadirs <1,000/µL (grade 3 and 4 neutropenia) and platelet counts <50,000/µL (grade 3 and 4 thrombocytopenia) were not sequence dependent, as each sequence resulted in 25 events in 35 total courses (Table 3). In addition, both mean ANC and platelet count nadir values did not differ (P > 0.05, two-tailed t test) at each of the four dose levels, with the exception of the mean nadir neutrophil counts at the NSC 655649/CDDP 550/50 mg/m2 dose level without G-CSF, which was lower when CDDP was administered before NSC 655649 (P = 0.007). Likewise, analysis of paired data sets, in which each patient served as their own control, showed that the percent decrement in ANC and platelet count values was not significantly different.

Nonhematologic toxicity. The nonhematologic toxicities of the NSC 655649/CDDP regimen were similar to the toxicities observed with each individual agent (Table 4). The most common effects were nausea, vomiting, fatigue, anorexia, and hypomagnesemia. There was no nonhematologic DLT. However, one patient with hepatocellular carcinoma, chronic hepatitis, and cirrhosis experienced a transient grade 3 alanine aminotransaminase on day 8 of the first course, which resolved on day 12. This patient received a second course without transaminitis. Because central venous access devices were used for drug administration, drug-related phlebitis, which had been described previously, could not be characterized.


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  		Table 4. Nonhematologic toxicity

 
A 54-year-old male with metastatic esophageal adenocarcinoma was hospitalized due to abdominal pain, dehydration, and weakness on day 15 after his first course of treatment. A computerized tomographic scan showed progressive disease and hazy density changes in the peripancreatic fat, which also suggested pancreatitis.

Pharmacokinetics
An analysis of NSC 655649 pharmacokinetics was done to determine the effect of drug sequencing on the disposition of NSC 655649. All patients had plasma sampling done for pharmacokinetic studies, and plasma sampling until day 6 or 7 was done during 30 courses. For those courses in which the sample was not collected on day 6 or 7, only the observed peak plasma concentration (Cmax) was included in the analysis of sequences. Pharmacokinetic variables determined for one patient who received NSC 655649 at 550 mg/m2 were associated with SEs exceeding 100% and were excluded from the analyses (Fig. 1). Most plasma concentration sets were characterized by the presence of a second NSC 655649 peak in the terminal elimination phase, from 1 to 6 hours after treatment, possibly due to enterohepatic recirculation of drug. It is noteworthy that plasma concentrations of NSC 655649 achieved in the study remained above the in vitro IC50 (range, 0.03-0.21 µg/mL) for 6 to 7 days. All data sets were analyzed by noncompartmental methods. There was significant intersubject variability in NSC 655649 pharmacokinetics, which is shown in the scatter plots of individual Cmax and AUC0-{infty} values as a function of dose (Fig. 2). No relationship between NSC 655649 clearance and body surface area was noted. The mean noncompartmental pharmacokinetic variable estimates at each dose level are listed in Table 5. Analysis of 13 paired NSC 655649 AUC and Cl values for each sequence showed no significant differences in AUC (P = 0.77, t test; P = 0.6, Wilcoxon's matched pairs test) or Cl (P = 0.98, t test; P = 0.97, Wilcoxon's matched pairs test). This suggests that the sequence of drug administration did not significantly influence the disposition of NSC 655649. There were no significant differences (P > 0.05, t test) in relevant pharmacokinetic variables, including Cmax, t1/2, AUC, Vdss, and Cl values, between patients who received NSC 655649 at 550 mg/m2 with and without G-CSF. On the other hand, the Cl of NSC 655649 seemed to decrease with increasing dose from 9.34 L/h/m2 following the 440 mg/m2 dose to 4.98 L/h/m2 following the 550 mg/m2 dose (P = 0.0002, Mann-Whitney U test).



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  		Fig. 1. Mean plasma concentrations of NSC 655649 in patients treated at NSC 655649/CDDP 440/50 and 550/50 mg/m2 dose levels. Sequence: CDDP 50 mg/m2->NSC 655649 550 mg/m2. Points, mean; bars, SE.

 


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  		Fig. 2. Scatter plots showing the distributions of the following noncompartmental pharmacokinetic variables reflecting NSC 655649 exposure: Cmax (A) and AUC0-{infty} (B) as a function of NSC 655649 dose. Sequence: {lozenge}, CDDP->NSC 655649; {blacklozenge}, NSC 655649->CDDP.

 

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  		Table 5. NSC 655649: noncompartmental pharmacokinetic variables as a function of drug sequencing

 
Antitumor activity
Three patients experienced PRs. The first patient, a 60-year-old male with adenocarcinoma of unknown primary with metastases to bone, adrenal gland, peritoneum, and lymph nodes, achieved a PR after the fourth course of NSC 655649/CDDP at the 440/50 mg/m2 dose level, which lasted for two courses. His disease had progressed previously during treatment with the investigational topoisomerase I–targeting agent 9-nitrocamptothecin and the investigational taxane analogue RPR-116258A. The second patient, a previously untreated 65-year-old male with non–small cell lung cancer involving the liver, had a confirmed PR following three courses of NSC 655649/CDDP at 550/50 mg/m2 and progressive disease after five courses. The third patient, a 54-year-old male with pancreatic cancer with neuroendocrine features and hepatic metastases who has been treated previously with regimens consisting of gemcitabine plus irinotecan and capecitabine plus paclitaxel, also experienced a confirmed PR after three courses of NSC 655649/CDDP at the 550/50 mg/m2 dose level with G-CSF. After nine courses, a positron emission tomographic scan did not reveal hypermetabolic hepatic lesions and treatment was terminated. However, progressive disease was documented at 12 months (Fig. 3). In addition, a 52-year-old male with an advanced adenoid cystic carcinoma of the head and neck received nine courses of NSC 655649/CDDP at the 440/50 mg/m2 dose level, which resulted in substantial symptom resolution and stable disease lasting for 12 months.



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  		Fig. 3. Computed tomographic scan of the abdomen in a 54-year-old male with metastatic pancreatic cancer. A, pretreatment; B, significant PR after five courses; C, after nine courses of therapy.

 

   Discussion
Top
 Abstract
 Patients and Methods
 Results
 Discussion
References
 
DNA topoisomerases, which are responsible for relaxing the torsional stress that accumulates when the DNA double helix unwinds, are the targets of many therapeutic agents. Their actions are required for chromosome condensation and sister chromosome disjunction during mitosis and for the processes of transcription and replication (15, 16). The mechanism of action and resistance of rebeccamycin analogues are different from those of the epipodophyllotoxins, anthracyclines, and anthracenediones (5, 9, 17). NSC 655649 principally inhibits the catalytic step of topoisomerase function, disrupts normal DNA strand passage, and induces single-strand DNA breaks (5, 9). NSC 655649 is active against cancer cells with acquired resistance to etoposide and teniposide (5). In phase I studies, NSC 655649 has shown antitumor activity in gastrointestinal tumors as well as in taxane- and platinum-resistant ovarian cancer (11, 12). The enhanced clinical benefit observed with NSC 655649 in a phase II trial served in part as the impetus for an ongoing phase III trial in advanced biliary cancers (14). The activity of NSC 655649 is also being evaluated in patients with a wide range of malignancies, including relapsed or refractory neuroblastoma and extensive-stage small cell lung cancer. Because CDDP is among the most active agents that are commonly used to treat these malignancies, this feasibility study of NSC 655649 combined with CDDP is an important step in the development of NSC 655649. From a mechanistic standpoint, because topoisomerase I inhibitors interfere in the repair of CDDP-induced DNA interstrand cross-links, there is also considerable interest in evaluating this regimen (18–20). Finally, the lack of overlapping principal toxicities also supports a rationale for combining NSC 655649 and CDDP. Some previous studies of topoisomerase inhibitors in combination with CDDP have yielded significant different hematologic toxicities depending on the schedule used (21, 22). This study was done to assess the overall feasibility of combining these two agents. Specifically, the study sought to determine the MTDs and toxicities of the combination with an exploration of the effect of sequence on outcome.

Both neutropenia and thrombocytopenia were the DLTs of sequences of NSC 655649 and CDDP. Although toxicity at the initial NSC 655649/CDDP dose level (440/50 mg/m2) was tolerable, with DLT occurring in only one of seven new patients, unacceptable rates of severe toxic effects resulted from attempts to marginally increase the dose of NSC 655649. A high rate of severe hematologic toxicity also thwarted efforts to use G-CSF to permit further dose escalation. The clinical result, which indicated that thrombocytopenia was worse following the addition of G-CSF, is intriguing. Patients who received G-CSF were nearly identical with respect to the range and distribution of performance status, age, sex and prior myeloablative therapy. The administration of hematopoeitic colony-stimulating factors in either close proximity to or concurrent with cytotoxic chemotherapy has been shown to worsen myelosuppression (23–25). One possible explanation for the accentuated neutropenia and thrombocytopenia is that the administration of G-CSF on day 2 of treatment primed more primitive hematopoietic progenitor cells, thereby increasing their susceptibility to the chemotherapy on a cytokinetic basis.

Because of significant toxicity in minimally pretreated patients, the combination was not evaluated further in heavily pretreated patients. Due to the appearance of mild to moderate cumulative CDDP toxicity, dose escalation of CDDP was not completely explored. Therefore, NSC 655649/CDDP doses of 440/50 mg/m2 are the MTDs and recommended doses for future phase II trials, with subsequent escalation of the CDDP dose if the initial dose level is well tolerated. Moreover, the use of NSC 655649 and CDDP at these doses should be limited to patients similar to those enrolled in this study (i.e., untreated or minimally pretreated patients with an acceptable performance status). Given the available data, a sequence-dependent effect on antitumor activity cannot be ruled out. The efficacy of both sequences should be evaluated in a randomized phase II study, with a limited pharmacokinetic sampling strategy.

Although nausea, vomiting, and fatigue occurred frequently, severe nonhematologic toxicity was uncommon. Hypomagnesemia was mild and manageable. Of note, the three patients who experienced hyperbilirubinemia after the first course had locally advanced or metastatic hepatic lesions. Two of them developed hematologic DLT by day 10 during course 1. One of these patients had received treatment with an investigational topoisomerase I inhibitor (exatecan) linked to a biodegradable carrier (DE-310) 7 weeks previously (26). It was subsequently discovered that the conjugated and free plasma drug concentrations of this compound, which is a substrate and a potential inhibitor of CYP3A4, are sustained for several weeks. This patient had the lowest NSC 655649 clearance of the 20 patients enrolled into the trial; consequently, this patient's half life, Cmax, and AUC were the highest (AUC = 500.7 µg/mL h; Fig. 1). At the lowest dose level, the third patient had grade 4 neutropenia that lasted <5 days. One patient experienced a clinical course compatible with acute pancreatitis. Although transient transaminase elevation has been observed after NSC 655649 therapy, acute pancreatitis has not been reported previously.

NSC 655649 had a long terminal elimination half-life and a large volume of distribution. Significant interpatient variability was noted. Pharmacokinetic data from previous phase I trials showed that NSC 655649 exhibited dose-proportional kinetics over the dose range tested (11, 12). Interestingly, Cl values for NSC 655649 were lower at the 550 mg/m2 dose level in the present trial, which may explain the substantial increase in both severity and rate of toxicities at this NSC 655649 dose. Nonetheless, this interpretation is confounded by the low numbers of patients treated, the substantial interindividual variability in the pharmacokinetic behavior of NSC 655649, and the fact that similar Cl values have been observed at lower NSC 655649 doses in previous studies (12, 13). In addition, some patients who had relatively low NSC 655649 Cl values and severe toxicity at the 550 mg/m2 dose level also developed severe toxicity at the 440 mg/m2 dose level, which indicates that the low Cl values noted at the 550 mg/m2 dose level may be due to inherent intersubject variability and not to nonlinear pharmacokinetics. A higher proportion of patients had hepatic compromise at the NSC 655649 550 mg/m2 dose level (9 of 11 total patients) compared with the 440 mg/m2 cohort patients (3 of 7), although there were no statistical differences in the liver function tests. The principal mechanisms of NSC 655649 elimination are hepatic metabolism and biliary excretion (11, 12). Renal clearance of the parent compound accounted for ~2% of overall drug disposition (12). The metabolism of NSC 655649 has also been investigated using in vitro systems (27). Incubation of NSC 655649 with human liver microsomes converted NSC 655649 into several metabolites in a NADPH-dependent manner. Eight metabolites (M1-M8) were chromatographically resolved by UV and fluorescent detection. M3 was identified as the major metabolite. Troleandomycin, a well-known CYP3A4 inhibitor, blocked the formation of most of the metabolites by 90% to 97%. In contrast, CYP2 inhibitors had no effect on the metabolism of NSC 655649. Moreover, microsomes expressing human CYP3A4 generated all NSC 655649 metabolites and specific antibodies inhibited their formation by 67% to 99%. As noted by the authors, the specific biliary transporter for NSC 655649 has yet to be determined (27). Considering all these data, caution should be observed in patients with significant liver disease and/or taking concomitant drugs metabolized by CYP3A4. There was no correlation between body surface area and NSC 655649 clearance. This finding suggests that NSC 655649 dosing could be more appropriately based on determinants of NSC 655649 clearance, such as measures of hepatic function. Larger studies should be designed to properly test this hypothesis.

In conclusion, myelosuppression was the principal DLT observed in this phase I trial. The addition of G-CSF did not sufficiently improve the tolerance of the treatment. Moreover, the timing of G-CSF administration could produce significant thrombocytopenia. The observation of antitumor activity with this combination in patients with various malignancies, including adenocarcinoma of unknown primary, non–small cell lung cancer, and pancreatic cancer, is encouraging and provides a rational for further phase II disease-directed evaluations of the regimen. Furthermore, the relatively slow clearance of NSC 655649 and the ability to attain plasma concentrations that greatly exceed the IC50 in preclinical models support the development of NSC 655649 on a three-weekly schedule.


   Footnotes
 
Grant support: NIH grants UO1 CA69853 and CA54174 and Frederic C. Bartter Clinical Research Unit of the Audie Murphy Veterans Administration Hospital through NIH grant M01 RR01346.

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.

Note: Presented in part at the 38th Annual Meeting of the American Society of Clinical Oncology, Orlando, FL, May 18-21, 2002.

Received 7/20/05; revised 9/22/05; accepted 10/ 5/05.


   References
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 Abstract
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 References
 

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