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Journal of Clinical Oncology, Vol 23, No 36 (December 20), 2005: pp. 9179-9186 © 2005 American Society of Clinical Oncology. DOI: 10.1200/JCO.2004.01.0660 Phase I Clinical and Pharmacokinetic Study of Flavopiridol in Children With Refractory Solid Tumors: A Children's Oncology Group StudyFrom the Vanderbilt Children's Hospital, Nashville, TN; University of Southern California Keck School of Medicine, Los Angeles; Children's Oncology Group, Arcadia, CA; Mayo Clinic and Foundation, Rochester, MN; Children's Hospital–King's Daughters, Norfolk, VA; Children's National Medical Center, Washington, DC Address reprint requests to James Whitlock, MD, Vanderbilt Children's Hospital, Pediatric Hematology/Oncology, 2220 Pierce Ave, Room 397 PRB, Nashville TN 37232-6310; e-mail: jim.whitlock{at}vanderbilt.edu; CC: pubs{at}childrensoncologygroup.org
PURPOSE: To determine the dose-limiting toxicities, maximum-tolerated dose, and pharmacokinetics of the cyclin-dependent kinase inhibitor flavopiridol (NSC 649890) when administered as a 1-hour infusion over 3 consecutive days to children with recurrent or refractory solid tumors. PATIENTS AND METHODS: Flavopiridol was administered as a 1-hour intravenous infusion daily for 3 consecutive days every 21 days, or when hematologic toxicity or any grade 2 or greater nonhematologic toxicity resolved. The starting dose was 37.5 mg/m2/d. Dose escalation was in cohorts of three patients in a standard fashion until dose-limiting toxicity and the maximum-tolerated dose were determined. Flavopiridol levels were measured on days 1, 2, and 3. RESULTS: Twenty-five children received flavopiridol at doses of 37.5 to 80 mg/m2/day over 3 consecutive days. The maximum-tolerated dose was 62.5 mg/m2/d. The primary dose-limiting toxicities were neutropenia and diarrhea. No antitumor effect was observed in this population. Mean peak plasma concentrations of 3.71 and 9.11 µmol/L were achieved at the end of the 1-hour infusion, following dose escalation from 37.5 mg/m2 to 80 mg/m2, respectively. The median flavopiridol plasma clearance was 8.0 L/h/m2 (range, 2.6 to 17.1 L/h/m2). CONCLUSION: The maximum-tolerated dose of flavopiridol in children, and the recommended phase II dose for pediatric studies, was 62.5 mg/m2/day when administered as a 1-hour infusion for 3 consecutive days. Dose-limiting toxicities of neutropenia and diarrhea were similar to those in adult studies.
Disruption of the normal regulation of cell cycle progression is a common feature of neoplastic cells.1 Identification of the cyclin-dependent kinases, or CDKs, as central regulators of cell cycle progression has led to interest in CDK inhibition as a potential target for novel antineoplastic agents.2 Flavopiridol is a synthetic flavone with broad antitumor activity in preclinical models.3 Flavopiridol was initially shown to block cellular proliferation by inducing cell cycle arrest, presumably through inhibition of various CDKs, including cdk1, cdk2, cdk4, and cdk6.4-6 The mechanism(s) by which flavopiridol inhibits CDKs may be multiple, including direct competitive inhibition of CDK2 and CDK4 kinase activity6 and transcriptional repression of cyclin D1.7 Subsequent studies demonstrated that flavopiridol induced apoptosis in a variety of leukemic cell lines, including the myeloid cell line HL-60,8,9 lymphoid cell lines SUDHL4, SUDHL6, Jurkat, and MOLT4,9 chronic lymphocytic leukemia (CLL) cell lines 83-CLL and WSU-CLL,10 and myeloma cell lines U266 and RPMI-1640-8226.11 The mechanism of flavopiridol-induced apoptosis is unclear; in some instances apoptosis occurred without evidence of cell cycle arrest.9 In B-CLL cells, flavopiridol led to down-regulation of the antiapoptosis proteins Bcl-2, Mcl-1, XIAP, and BAG-1,12 and induced apoptosis in a p53-independent fashion.13 In preclinical leukemia and lymphoma models using human xenografts in SCID mice, flavopiridol induced apoptosis in a dose- and schedule-dependent fashion.14 Continuous infusion of flavopiridol over a period of 72 hours resulted in plasma levels exceeding the in vitro IC50 reported for most human cells lines (20 to 200 nmol/L).14,15 Early clinical trials of flavopiridol explored continuous 72-hour infusion schedules16,17; a dose-limiting toxicity (DLT) of secretory diarrhea in both trials led to the institution of antidiarrheal prophylaxis (ADP), allowing further dose escalation in the National Cancer Institute trial.16 Limited activity in phase II trials with this schedule led to the evaluation of alternative schedules, including a 1-hour daily infusion.18 Preclinical data suggested that flavopiridol-induced apoptosis in hematopoetic cell lines was dependent on peak serum concentrations, with the best antitumor effect in xenografted animals observed following daily bolus intravenous (IV) or intraperitoneal injections leading to peak plasma levels of about 7 µmol/L.14 Tan et al18 evaluated several 1-hour infusion schedules of flavopiridol in a subsequent phase I trial, with the goal of achieving higher serum concentrations and induction of apoptosis. Based on the occurrence of diarrhea as a significant toxicity in previous trials, patients received bismuth salicylate as an antidiarrheal prophylaxis and loperamide at the onset of diarrhea. One-hour infusions daily for 5 days, 3 days, and 1 day once every 3 weeks were evaluated, with a maximum-tolerated dose (MTD) of 37.5 mg/m2/day for 5 days, of 50 mg/m2/day for 3 days, and of 62.5 mg/m2/day for 1 day. Median peak concentrations of flavopiridol of 1.7 µmol/L, 3.2 µmol/L, and 3.9 µmol/L were achieved at the respective MTDs for the 5-day, 3-day, and 1-day schedules, respectively. A DLT of grade 4 neutropenia occurred in two patients treated at 62.5 mg/m2/day for 3 days, as well as episodes of grades 3 to 4 transaminitis and hyperbilirubinemia, which were not dose limiting. Based on promising preclinical data and experience in adult phase I trials, the Children's Oncology Group (COG) conducted a phase I study of flavopiridol as a 1-hour daily infusion for 3 consecutive days in children with refractory solid tumors or lymphomas.
Patients Patients younger than 22 years old with solid tumors or lymphomas refractory to conventional therapies, or with tumors or lymphomas for which there was no accepted conventional therapy, were eligible for the COG ADVL0017 study if they had a Karnofsky performance status  50 (for those > 10 years old) or a Lansky performance status 50 (for those < 10 years old), and a life expectancy of at least 2 months. Organ function requirements included adequate hematologic function (absolute neutrophil count 1,000/mm3, platelet count 75,000/mm3 without transfusion, hemoglobin 8.0 g/dL), hepatic function (bilirubin  1.5x normal, ALT 5x normal, and albumin 2 g/dL), renal function (creatinine 1.5x normal, or creatinine clearance or radioisotope glomerular filtration rate the lower limit of normal), cardiovascular function (shortening fraction 27% or ejection fraction 50%), and CNS function (not on anticonvulsants, CNS toxicity < National Cancer Institute Common Toxicity Criteria grade 2). Patients with leukemia were not eligible. Patients were required to be off cytokines for at least 1 week before study start, to not have received myelosuppressive chemotherapy within 2 weeks of study enrollment (4 weeks for nitrosurea therapy), to not have received allogeneic stem-cell transplantation within the previous 6 months, and to not have received craniospinal radiation or radiation therapy to more than 50% of the pelvis within the previous 6 months. Institutional review board approval and written informed consent were required before entry.
Study Design Toxicities were coded according to the National Cancer Institute's Common Toxicity Criteria (version 2.0). Dose-limiting toxicity was defined as any grade 3 or 4 nonhematologic toxicity attributable to the agent, except grade 3 fever; grade 3 diarrhea less than or equal to 72 hours in duration that returned to grade 1 by day 21 of that course; grade 3 nausea, vomiting, or transaminitis that returned to grade 1 or baseline by day 21 of that course; or alopecia. Grade 4 neutropenia or thrombocytopenia that lasted longer than 7 days, thrombocytopenia requiring platelet transfusion support for more than 7 days, or any hematologic toxicity that resulted in a delay between cycles of longer than 7 days were also considered dose limiting. Only DLTs that occurred during the first course were considered in dose escalation. Patients were enrolled in cohorts of three at each dose level, beginning at the 37.5 mg/m2/day for 3 consecutive days dose level. Intrapatient dose escalation was not allowed. If none of the first three patients had DLT, the dose was escalated to the next cohort. If one of three patients in the first cohort had DLT, three more patients were enrolled at that dose level. If none of the next cohort had DLT, the dose was escalated one level. Those patients with grade 3 or greater symptoms of toxicity, which resolved before the next course, and who had evidence of stable or responsive disease, could continue to be treated, but the flavopiridol dose was to be reduced by one level for the subsequent course of therapy. If two or more patients had DLT, the dose was considered not tolerated, and was reduced one level. Enrollment of patients in cohorts of three was continued until the maximum dose at which at least five of six patients did not demonstrate DLT was identified. This dose was considered the MTD.
Response
Follow-Up Pharmacologic studies. Patients who received at least the first week of therapy were considered for the analysis relating pharmacokinetic parameters to dose level. Samples for pharmacokinetic evaluation were obtained on the first 3 days of the first course. Sample collection. Blood samples were drawn during cycle 1 before treatment, 0.9 hours after start of infusion, and 0.08, 0.5, 1, 2, 5, and 10 hours after the end of infusion on day 1. On days 2 and 3, samples were drawn before the infusion and 5 minutes before the end of infusion. Blood samples (4 mL) from a site remote from the flavopiridol infusion line were collected in heparin-containing tubes and immediately cooled in ice water. Plasma was separated by centrifugation (1,000 to 1,200 g for 10 minutes) and transferred into plastic tubes stored at –70°C until analysis. Urine was collected for a 25-hour interval from the beginning of day 1 until blood was drawn before the day 2 infusion.
Assay methods.
Plasma and urine concentrations of flavopiridol were determined by a modification of the reverse-phase high-performance liquid chromatography procedure of Innocenti et al.19 Separation of flavopiridol and the internal standard flavone was achieved on a Discovery RP Amide C16 column (Supelco; Bellefonte, PA) 10 cm x 4.6 mm, 5 µm) with a Discovery RP Amide C16 guard column (Supelco; Bellefonte, PA) 2 cm x 4.0 mm, 5 µm) under gradient elution. The mobile phase consisted of acetonitrile and 50 mmol/L ammonium acetate with 0.1% triethylamine (TEA) adjusted to pH 4.15 with glacial acetic acid. The gradient profile was as follows: elution with 25:75 acetonitrile:ammonium acetate, pH 4.15 with 0.15% TEA for 5 minutes followed by a 5-minute linear gradient to 35:65 acetonitrile:ammonium acetate, pH 4.15 with 0.15% TEA and 10-minute elution with 35:65 acetonitrile:ammonium acetate, and pH 4.15 with 0.15% TEA. After completing the gradient, the columns were re-equilibrated with 25:75 acetonitrile:ammonium acetate, pH 4.15 with 0.15% TEA for 10 minutes before the next injection. The flow rate and detection wavelength were 1.0 mL/min and 263 nm, respectively. Patient and standard curve plasma samples (100 µL) were added to a microcentrifuge tube on ice, followed by the addition of 10 µL flavone (33.8 µmol/L). Plasma proteins were precipitated with 500 µL acetonitrile. After 10 minutes on ice, samples were centrifuged at 14,000 rpm for 2 minutes. The supernatant was dried under nitrogen and reconstituted with 100 µL mobile phase containing 10 µg/mL desipramine and 50 µL was injected onto the HPLC. The assay was linear (r2
Pharmacokinetics.
Flavopiridol plasma concentration data were analyzed by noncompartmental methods using the program WINNonlin (Scientific Consultant, Apex, NC). The apparent terminal elimination rate constants (kz) were determined by linear least-squares regression through the 5- to 25-hour plasma-concentration time points. The apparent elimination half-life (t1/2) was calculated as 0.693/kz. Areas under the plasma concentration-time curves (AUC) were determined using the linear trapezoidal rule from time zero to the time of the last detectable sample (Clast). AUCs through infinite time (AUC0-
Statistical analysis.
Cmax, AUC0-
General Twenty-five patients were enrolled onto ADVL0017. Four dose levels were evaluated: (1) 37.5 mg/m2; (2) 50 mg/m2; (3) 62.5 mg/m2; and (4) 80 mg/m2. Five patients were inassessable for DLT assessment. Three patients, one at 50 mg/m2, one at 62.5 mg/m2, and one at 80 mg/m2, elected to withdraw before completion of the first course. One patient enrolled at the 50 mg/m2 dose level was not completely evaluated for DLT. One patient enrolled at the 80-mg/m2 dose level experienced disease progression during the first course and was withdrawn before receiving all doses of flavopiridol, without experiencing DLT up to that time. Selected characteristics of all enrolled patients are listed in Table 1.
Thirteen patients received one course of therapy. Two patients with astrocytoma and one patient each with ependymoma, hepatoblastoma, osteosarcoma, and fibrosarcoma received three or more courses of therapy (median, 3; range, 3 to 7). Four patients had stable disease while on protocol therapy and were removed from therapy because their disease progressed. Two patients had stable disease while on protocol and were removed from therapy because of physician or family preference.
DLT
Pharmacokinetics Flavopiridol pharmacokinetics were studied in all patients during cycle 1. A plasma concentration versus time profile for a patient treated with 62.5 mg/m2 flavopiridol is illustrated in Figure 1. At the end of the 1-hour infusion, peak plasma concentrations of greater than 3 µmol/L were achieved in 19 of 20 patients treated with 37.5 mg/m2 to 80 mg/m2 flavopiridol, respectively (Fig 2A). Flavopiridol plasma concentrations remained above 0.1 µmol/L for the full 24-hour dose interval in 6 of 10 patients treated at the 62.5 or 80 mg/m2 dose levels. Flavopiridol pharmacokinetics were determined by noncompartmental analysis. Parameter estimates are summarized in Table 4. Cmax and AUC0-  were significantly related to dose level at the 0.05 level (Fig 2 and Table 4). Flavopiridol plasma clearance values varied over a 10-fold range (3.5 to 39.3 L/h/m2), with a median value of 10.0 L/h/m2 (Fig 3). The relationship between CL per square meter of body-surface area and dose was marginally significant (P = .07). Age was related significantly to clearance (P = .04), with increased age indicating increased CL. This relationship was not apparent when the association between CL per square meter of body-surface area and age was examined. The logarithmic transformation of plasma CL was not significantly related to dose level (P = .0747), age at enrollment (P = .4897), or sex (P = .1377). The mean recovery of flavopiridol in the 24-hour urine was 1.1% (range, 0.27% to 2.12%) of the administered dose.
Flavopiridol is the first inhibitor of cyclin-dependent kinases to enter clinical trials. Its unique mechanism of action and potent preclinical activity heightened interest in it as a novel antineoplastic agent and led to several phase I trials in adults. A trial conducted at the National Cancer Institute16 evaluated 72-hour flavopiridol infusions every 2 weeks, and identified the MTD as 50 mg/m2/day for 3 days, with secretory diarrhea as the DLT. After the addition of antidiarrheal prophylaxis with loperamide and cholestyramine, a second MTD was defined as 78 mg/m2/day for 3 days, with hypotension as the DLT. Antitumor activity was seen in patients with renal, prostate, and colon cancer, and non-Hodgkin's lymphoma. Thomas et al17 also evaluated 72-hour flavopiridol infusions every 2 weeks. The maximum-tolerated dose in that study was 40 mg/m2/24 hours, with a DLT of secretory diarrhea; treatment of established diarrhea with loperamide or cholestyramine was unsuccessful. No hematologic toxicities were seen. A complete response was seen in a patient with gastric cancer. The mean steady-state concentrations attained at the MTDs in these two studies had been shown, in preclinical models, to be sufficient to achieve both CDK inhibition and cell cycle arrest. Phase II trials that used the 72-hour infusion schedule resulted in limited activity in renal cell carcinoma,20 and instances of stable disease, but no objective responses in patients with non–small-cell lung, gastric, colorectal, or prostate cancer.21-24 Preclinical data suggested that flavopiridol-induced apoptosis in hematopoetic cell lines was dependent on peak serum concentrations.18 The utilization of a schedule- and dose-escalation scheme in the COG ADVL0017 study, which closely resembled those of a previous adult study, allows for the direct comparison of flavopiridol pharmacokinetic data between adults and children. The strategy of antidiarrheal prophylaxis with bismuth salicylate adopted by Tan et al was replaced with loperamide, due to concerns regarding salicylate use in children. This study identified the MTD in children to be 62.5 mg/m2/d for 3 consecutive days, a level higher than the one defined in adults by Tan et al. The major DLTs in this pediatric study were diarrhea and neutropenia, both of which occurred at 80 mg/m2/d for 3 consecutive days, and were similar to those in adult phase I flavopiridol studies. Other DLTs were fatigue, hypokalemia, and hypophosphatemia at the 80 mg/m2/d dose level, and were considered to be related to diarrhea. Thus, flavopiridol has a similar spectrum of clinical toxicities in children and adults. Flavopiridol pharmacokinetics were examined in this study to identify peak plasma concentrations and duration of exposure after 1-hour flavopiridol infusion, and to compare pediatric and adult data. Flavopiridol plasma clearance in pediatric patients was constant over the dose range investigated, but was approximately 33% lower than clearance values reported in adult trials that used similar assay methodology.17,18 The lower clearance and higher MTD for 1-hour x 3-day infusions resulted in higher median flavopiridol peak plasma concentrations in children (7.31 µmol/L) than adults (3.18 µmol/L) at the dose recommended for phase II studies, and exceeded the steady-state concentration (500 nmol/L) achieved with a 72-hour continuous infusion in adults.17 Consequently, plasma concentrations of flavopiridol not only reached peak concentrations of 7 to 9 µmol/L that were associated with in vitro apoptotic activity and in vivo tumor regression in preclinical models, but remained above the IC50 for most human tumor cell lines,15 and the Ki for Cdk2 (100 nmol/L) for 24 hours after drug administration. The recommended phase II dose of flavopiridol for pediatric trials utilizing this schedule is 62.5 mg/m2/d for 3 days. However, a novel bolus-infusional schedule of flavopiridol administration was recently reported, which was associated with tumor lysis and clinical responses in patients with fludarabine-refractory chronic lymphocytic leukemia,25 suggesting that this alternative schedule should be considered in future investigations of this novel agent.
The authors indicated no potential conflicts of interest.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
1. Sherr CJ: Cancer cell cycles. Science 274:1672-1677, 1996 2. Senderowicz AM, Sausville EA: Preclinical and clinical development of cyclin-dependent kinase modulators. J Natl Cancer Inst 92:376-387, 2000 3. Kaur G, Stetler-Stevenson M, Sebers S, et al: Growth inhibition with reversible cell cycle arrest of carcinoma cells by flavone L86-8275. J Natl Cancer Inst 84:1736-1740, 1992 4. Worland PJ, Kaur G, Stetler-Stevenson M, et al: Alteration of the phosphorylation state of p34cdc2 kinase by the flavone L86-8275 in breast carcinoma cells: Correlation with decreased H1 kinase activity. Biochem Pharmacol 46:1831-1840, 1993[CrossRef][Medline] 5. Losiewicz MD, Carlson BA, Kaur G, et al: Potent inhibition of CDC2 kinase activity by the flavonoid L86-8275. Biochem Biophys Res Commun 201:589-595, 1994[CrossRef][Medline] 6. Carlson BA, Dubay MM, Sausville EA, et al: Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast carcinoma cells. Cancer Res 56:2973-2978, 1996 7. Carlson B, Lahusen T, Singh S, et al: Down-regulation of cyclin D1 by transcriptional repression in MCF-7 human breast carcinoma cells induced by flavopiridol. Cancer Res 59:4634-4641, 1999 8. Bible KC, Kaufmann SH: Flavopiridol: A cytotoxic flavone that induces cell death in noncycling A549 human lung carcinoma cells. Cancer Res 56:4856-4861, 1996 9. Parker BW, Kaur G, Nieves-Neira W, et al: Early induction of apoptosis in hematopoietic cell lines after exposure to flavopiridol. Blood 91:458-465, 1998 10. Konig A, Schwartz GK, Mohammad RM, et al: The novel cyclin-dependent kinase inhibitor flavopiridol downregulates Bcl-2 and induces growth arrest and apoptosis in chronic B-cell leukemia lines. Blood 90:4307-4312, 1997 11. Semenov I, Akyuz C, Roginskaya V, et al: Growth inhibition and apoptosis of myeloma cells by the CDK inhibitor flavopiridol. Leuk Res 26:271-280, 2002[CrossRef][Medline] 12. Kitada S, Zapata JM, Andreeff M, et al: Protein kinase inhibitors flavopiridol and 7-hydroxy-staurosporine down-regulate antiapoptosis proteins in B-cell chronic lymphocytic leukemia. Blood 96:393-397, 2000 13. Byrd JC, Shinn C, Waselenko JK, et al: Flavopiridol induces apoptosis in chronic lymphocytic leukemia cells via activation of caspase-3 without evidence of bcl-2 modulation or dependence on functional p53. Blood 92:3804-3816, 1998 14. Arguello F, Alexander M, Sterry JA, et al: Flavopiridol induces apoptosis of normal lymphoid cells, causes immunosuppression, and has potent antitumor activity in vivo against human leukemia and lymphoma xenografts. Blood 91:2482-2490, 1998 15. Sedlacek HH, Czech J, Naik R, et al: Flavopiridol (L86 8275; NSC 649890), a new kinase inhibitor for tumor therapy. Int J Oncol 9:1143-1168, 1996 16. Senderowicz AM, Headlee D, Stinson SF, et al: Phase I trial of continuous infusion flavopiridol, a novel cyclin-dependent kinase inhibitor, in patients with refractory neoplasms. J Clin Oncol 16:2986-2999, 1998 17. Thomas JP, Tutsch KD, Cleary JF, et al: Phase I clinical and pharmacokinetic trial of the cyclin-dependent kinase inhibitor flavopiridol. Cancer Chemother Pharmacol 50:465-472, 2002[CrossRef][Medline] 18. Tan AR, Headlee D, Messmann R, et al: Phase I clinical and pharmacokinetic study of flavopiridol administered as a daily 1-hour infusion in patients with advanced neoplasms. J Clin Oncol 20:4074-4082, 2002 19. Innocenti F, Stadler WM, Iyer L, et al: Flavopiridol metabolism in cancer patients is associated with the occurrence of diarrhea. Clin Cancer Res 6:3400-3405, 2000 20. Stadler WM, Vogelzang NJ, Amato R, et al: Flavopiridol, a novel cyclin-dependent kinase inhibitor, in metastatic renal cancer: A University of Chicago Phase II Consortium study. J Clin Oncol 18:371-375, 2000 21. Shapiro GI, Supko JG, Patterson A, et al: A phase II trial of the cyclin-dependent kinase inhibitor flavopiridol in patients with previously untreated stage IV non-small cell lung cancer. Clin Cancer Res 7:1590-1599, 2001 22. Schwartz GK, Ilson D, Saltz L, et al: Phase II study of the cyclin-dependent kinase inhibitor flavopiridol administered to patients with advanced gastric carcinoma. J Clin Oncol 19:1985-1992, 2001 23. Aklilu M, Kindler HL, Donehower RC, et al: Phase II study of flavopiridol in patients with advanced colorectal cancer. Ann Oncol 14:1270-1273, 2003 24. Liu G, Gandara DR, Lara PN Jr, et al: A Phase II trial of flavopiridol (NSC #649890) in patients with previously untreated metastatic androgen-independent prostate cancer. Clin Cancer Res 10:924-928, 2004 25. Lin TS, Dalton JT, Wu D, et al: Flavopiridol given as a 30-min intravenous (IV) bolus followed by 4-hr continuous IV infusion (CIVI) results in clinical activity and tumor lysis in refractory chronic lymphocytic leukemia (CLL). J Clin Oncol 22:573s, 2004 (suppl; abstr 6564) Submitted December 29, 2004; accepted September 21, 2005.
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Copyright © 2005 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
