Clinical Cancer Research Folkman Stand Up to Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Karp, J. E.
Right arrow Articles by Bauer, K. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Karp, J. E.
Right arrow Articles by Bauer, K. S.
Clinical Cancer Research Vol. 11, 8403-8412, December 1, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Clinical

Phase I and Pharmacokinetic Study of Flavopiridol followed by 1-ß-D-Arabinofuranosylcytosine and Mitoxantrone in Relapsed and Refractory Adult Acute Leukemias

Judith E. Karp1,2, Antonino Passaniti2, Ivana Gojo2, Scott Kaufmann4, Keith Bible4, Tushar S. Garimella2, Jacqueline Greer1,2, Janet Briel1, B. Douglas Smith1, Steven D. Gore1, Michael L. Tidwell2, Douglas D. Ross2,3, John J. Wright5, A. Dimitrios Colevas5 and Kenneth S. Bauer2

Authors' Affiliations: 1 Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins; 2 University of Maryland Greenebaum Cancer Center; and 3 Baltimore Veterans Affairs Medical Center, Baltimore, Maryland; 4 Mayo Clinic, Rochester, Minnesota; and 5 Investigational Drug Branch, Clinical Trials Evaluation Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland

Requests for reprints: Judith E. Karp, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 1650 Orleans Street, CRB Room 289, Baltimore, MD 21231-1000. Phone: 410-503-5399; Fax: 410-614-1005; E-mail: jkarp2{at}jhmi.edu.


   Abstract
Top
 Abstract
Patients, Materials, and Methods
 Results
 Discussion
 References
 
Purpose: The serine/threonine kinase inhibitor flavopiridol targets multiple cyclin-dependent kinases, induces checkpoint arrest, and interrupts transcriptional elongation. We designed a phase I clinical trial using a timed sequential therapy approach where flavopiridol was given for the dual purpose of initial cytoreduction and enhancing cell cycle progression of the remaining leukemia cell cohort followed by cycle-dependent drugs 1-ß-D-arabinofuranosylcytosine (ara-C) and mitoxantrone.

Experimental Design: Flavopiridol was given by 1-hour infusion daily for 3 days beginning day 1 followed by 2 g/m2/72 h ara-C beginning day 6 and 40 mg/m2 mitoxantrone beginning day 9. In vivo correlates included pharmacokinetics, modulation of blast cycle regulators, and serum and marrow supernatant vascular endothelial growth factor levels.

Results: Of 34 adults receiving induction therapy, 16 (47%) evinced direct leukemia cytotoxicity with ≥50% drop in peripheral blast counts and tumor lysis in 9 (26%). Four (12%) died during therapy (two fungal infections and two sudden death). Dose-limiting toxicity occurred at 60 mg/m2/d with profound neutropenia >40 days duration, and maximal tolerated dose was 50 mg/m2/d. Overall response rate was 31% in 26 acute myelogenous leukemia and 12.5% in acute lymphoblastic leukemia. Pharmacokinetics showed that a linear two-compartment model with first-order elimination provided the best fit of the observed concentration versus time data. Flavopiridol down-regulated one or more target proteins in marrow blasts in vivo. Vascular endothelial growth factor was detected in sera and marrow supernatant pretreatment, and sera obtained on day 3 inhibited bovine aortic endothelial cell proliferation by a mean of 32% (range, 10-80%).

Conclusions: Our data suggest that flavopiridol is cytotoxic to leukemic cells and, when followed by ara-C and mitoxantrone, exerts biological and clinical effects in patients with relapsed and refractory acute leukemias. These findings warrant continuing development of flavopiridol at 50 mg/m2/d x 3 days in combination with cytotoxic and biological agents for acute leukemias.

Flavopiridol (L86-8275), a synthetic flavone derivative that was initially isolated from the stem bark of the Indian tree Dysoxylum binectariferum (1, 2), is a potent growth inhibitor of diverse human tumor cell lines and induces apoptosis in hematopoietic cell lines derived from acute myelogenous leukemia (AML), B-cell and T-cell lymphomas, and multiple myeloma (3–5). Flavopiridol-induced apoptosis results at least in part from inhibition of multiple serine/threonine cyclin-dependent kinases (2). Whereas inhibition of cyclin-dependent kinases 2 and 4 contributes to cell cycle arrest in G1 and G2 (6–8), flavopiridol-triggered inactivation of the cyclin-dependent kinase 9/cyclin T complex (also known as PTEF-b) inhibits the activating phosphorylation of RNA polymerase II and diminishes mRNA synthesis (9, 10). Consequently, flavopiridol-treated cells are unable to synthesize transcripts encoding polypeptides, such as cyclin D1 (11), which is expressed in a cell cycle–dependent manner.

Several cellular changes induced by flavopiridol could contribute to its cytotoxicity. Mcl-1 is a short-lived antiapoptotic Bcl-2 family member (12) that is highly expressed in hematopoietic stem cells and essential to promoting stem cell survival (13). Mcl-1 is up-regulated in ~50% of relapsed and refractory leukemias (14) and rapidly down-regulated by flavopiridol (5, 10, 15, 16). Studies in myeloma cells showed that forced Mcl-1 overexpression protects cells from flavopiridol-induced apoptosis (5), suggesting a critical role for Mcl-1 down-regulation in flavopiridol-induced cell death. More recent observations that flavopiridol-induced apoptosis involves caspase-8-independent release of cytochrome c from mitochondria (4) and is enhanced by inhibition of phosphatidylinositol-3 kinase in U937 and primary AML cells (6) are consistent with this model. Additional studies have shown that flavopiridol can decrease production of vascular endothelial growth factor (VEGF; ref. 17), a growth and survival factor for diverse tumor types, including certain acute leukemias (18).

1-ß-D-Arabinofuranosylcytosine (ara-C) and mitoxantrone exhibit significant clinical activity against AML and acute lymphoblastic leukemia (ALL; refs. 19–21). Both drugs induce double-strand breaks in DNA, ara-C by inhibiting DNA replication and repair and mitoxantrone by poisoning topoisomerase II. These insults not only lead to cell cycle arrest in S phase during ara-C exposure (22) and late S/G2 after mitoxantrone (23) but also subsequently trigger apoptosis in susceptible leukemic cells (22–25). These drugs have been combined in adults with AML and ALL (19–21) to induce complete remission (CR) in both older patients and patients with relapsed and refractory disease.

Previous studies from our laboratories have examined the effect of combining flavopiridol with a variety of antineoplastic agents (26, 27). Because flavopiridol induces cell cycle arrest, it antagonizes the effects of S-phase-dependent agents, such as ara-C and topotecan, when administered concomitantly (26). In contrast, when flavopiridol is administered first and then withdrawn in vitro, the surviving cells reenter the cell cycle and are sensitized to S-phase poisons (26). These observations, coupled with the ability of flavopiridol to kill noncycling cells (3), suggested that flavopiridol might be particularly effective when administered first and then followed several days later by ara-C. Consistent with these results, we showed recently that therapeutically achievable flavopiridol concentrations induced apoptotic cell death in bone marrow leukemic blasts in vitro and that flavopiridol-treated blast cultures exhibited increased sensitivity to the subsequent proapoptotic effects of ara-C relative to either agent alone (27).

This approach to maximizing the cytotoxic effects of flavopiridol-containing combinations is reminiscent of timed sequential therapy (TST), a therapeutic strategy that attempts to exploit drug-induced changes in residual leukemia cell growth kinetics to increase the sensitivity of surviving leukemic cells to cycle-dependent antileukemic agents (28, 29). TST has been shown to induce prolonged disease-free survival in certain groups of adults and children with AML (30–32), although there remains a significant proportion for whom TST alone is not curative.

Accordingly, we designed a phase I clinical trial in which flavopiridol, given for the dual purpose of initial cytoreduction and enhancing the cell cycle progression of the remaining leukemic cell cohort, was followed by ara-C and mitoxantrone. The purposes of this trial were to define the dose-limiting toxicities (DLT) of the combination, establish the maximum tolerated dose, evaluate in a preliminary manner the antileukemic effects of flavopiridol and the combination, and determine whether the levels of flavopiridol achieved in vivo were sufficient to inhibit RNA polymerase II phosphorylation and down-regulate cyclin D1, Mcl-1, and VEGF.


   Patients, Materials, and Methods
Top
 Abstract
 Patients, Materials, and Methods
Results
 Discussion
 References
 
Patient eligibility and selection
Adults ages ≥18 years with pathologically confirmed acute leukemia that was unlikely to be cured by existing therapies, including primary refractory (induction failure) or multirefractory (refractory or relapsed after less than three prior induction regimens) AML and ALL; newly diagnosed AML in adults with antecedent hematologic disorder, including myelodysplasia, treatment-related AML, and/or known adverse cytogenetics; and chronic myelogenous leukemia in lymphoid blast crisis (CML-LBC) of either myeloid or lymphoid origin that was resistant to imatinib, were eligible provided they had Zubrod performance status 0 to 2, normal bilirubin, hepatic enzymes less than two times normal, serum creatinine less than 1.5 times the normal, and left ventricular ejection fraction ≥45%. Patients who had undergone allogeneic or autologous stem cell transplantation and had relapsed or were refractory thereafter were eligible for this study. Complete history, physical examination, laboratory, imaging, and cardiac evaluations (electrocardiogram and left ventricular ejection fraction) were done within 3 days of study entry. Recovery from toxicities of previous treatment and intervals of ≥3 weeks from prior chemotherapy and ≥1 week from any growth factor therapy were required before beginning ara-C and mitoxantrone. Patients were ineligible if they had a peripheral blast count ≥50,000/mm3; disseminated intravascular coagulation; active uncontrolled infection; active central nervous system leukemia; history of ara-C-related neurotoxicity; prior radiation of >25% of bone marrow; concomitant radiotherapy, chemotherapy, or immunotherapy; or coexisting medical or psychiatric conditions that could interfere with study procedures. Pregnant or lactating women were ineligible. All patients provided written informed consent according to the University of Maryland at Baltimore and the Johns Hopkins medical institutional review boards and guidelines.

Treatment schema. Flavopiridol was administered over 1 hour daily x 3 beginning day 1 (33, 34). Using a modified dose escalation schema, flavopiridol was dose escalated, with the first four patients receiving 40 mg/m2/d x 3 days. Dose escalation could proceed if no more than one of three patients experienced DLT defined as grade ≥3 nonhematologic toxicity according to the National Cancer Institute Common Toxicity Criteria version 2, bone marrow transplantation criteria, or grade 4 marrow aplasia lasting >40 days. Administration of 2 g/m2 ara-C as a 72-hour continuous infusion (667 mg/m2/24 h; refs. 28, 31, 35, 36) began on day 6. Mitoxantrone (40 mg/m2) was administered as a single i.v. bolus over 30 to 60 minutes on day 9, 12 hours after completion of the ara-C infusion (36). Following completion of the first dose level, cohorts of four to six patients were treated with flavopiridol that was dose escalated by 10 mg/m2/d x 3 days per cohort. The occurrence of any DLT in 33% of patient cohort defined the maximal tolerated dose of flavopiridol. Once maximal tolerated dose was reached, an expanded cohort was treated at the dose level below maximal tolerated dose and observed for DLT. Patients who achieved CR or partial remission after cycle 1 were eligible to receive a second cycle of flavopiridol, ara-C, and mitoxantrone at the identical dose and schedule beginning 30 ± 7 days following hospital discharge from the first cycle.

Supportive care. To decrease the severity of the expected flavopiridol-induced secretory diarrhea (33, 37, 38) without inducing changes in gastrointestinal motility, all patients received 100 µg octreotide (somatostatin analogue) every 8 hours beginning 2 to 4 hours before the first dose of flavopiridol and continuing through day 4. All patients received daily oral allopurinol (300 mg) and aluminum hydroxide (30 mL) every 6 hours until 24 hours after the completion of ara-C and mitoxantrone (days 1-9). Corticosteroid eye drops were used on days 6 to 12 to prevent ara-C-related conjunctivitis. Antiemetics were used according to standard practices. Premenopausal women were placed on hormonal therapy to suppress menstrual bleeding. Norfloxacin (400 mg b.i.d.) for gastrointestinal decontamination and acyclovir or Famvir prophylaxis against herpes simplex virus activation began on day 1 and continued until achievement of an absolute neutrophil count >100/mm3.

Response and toxicity evaluations. To assess response to therapy, bone marrow aspiration and biopsy were done before treatment and on day 3 following completion of the last dose of flavopiridol, day 6 before beginning the ara-C infusion, day 14, and at the time of hematologic recovery or when leukemia regrowth was suspected. Hematologic recovery was defined as absolute neutrophil count ≥500/mm3 and transfusion-independent platelet count 50,000/mm3. CR required a normal bone marrow aspirate with absence of identifiable leukemia, absolute neutrophil count ≥1,000/mm3, platelet count ≥100,000/mm3, and absence of blasts in peripheral blood (39). Clearance of cytogenetic abnormalities was not required for CR but was noted and described separately. Partial remission was defined as the presence of trilineage hematopoiesis in the marrow with normalization of peripheral counts but with 5% to 25% blasts in the marrow (39). NR was defined as persistent leukemia in marrow and/or blood without significant decrease from pretreatment levels. The National Cancer Institute Common Toxicity Criteria version 2.0 was the basis on which all adverse events were described and graded based on the treating physician's assessment. DLT was based on the toxicities incurred during cycle 1.

Pharmacokinetics
Sampling. Plasma samples were obtained from the first 18 patients on day 0 before flavopiridol administration and at 0.5, 1, 1.5, 2, 4, 6, 10, 12, 24, 36, 48, and 72 hours, with the 24- and 48-hour samples being drawn before beginning on days 2 and 3 administration of flavopiridol. Blood was immediately centrifuged at 2,500 x g for 10 minutes at 4°C, and plasma was removed and stored at –70°C until the time of assay. Flavopiridol concentrations were determined using a reverse-phase high-performance liquid chromatographic assay as described previously (33, 38). Briefly, 100 µL plasma was precipitated using 500 µL acetonitrile containing the internal standard. The mixture was vortexed and centrifuged at 10,000 x g for 10 minutes at 4°C and the supernatant was evaporated to dryness under a stream of nitrogen. The residue was reconstituted using 100 µL mobile phase and injected into a Waters (Milford, MA) 2690 high-performance liquid chromatography. Separation was achieved by a Waters Xterra RP18 column preceded by a Phenomenex C18 guard column (Torrance, CA) and a mobile phase consisting of a linear gradient of 10 mmol/L ammonium acetate buffer (pH 4) and acetonitrile at a flow rate of 1 mL/min for UV and 0.150 mL/min for mass spectrometry assays. For UV detection, the detector used was a dual-wavelength detector and flavopiridol was detected at 264 nm. For samples in which flavopiridol was undetectable using the UV detector, the flavopiridol was detected by a QuatroMicro liquid chromatography/tandem mass spectrometer detector (Micromass, Manchester, United Kingdom) using a positive electrospray mode with a transition of 401.85 > 221.9 for flavopiridol. The total run time was 25 minutes and the typical retention times were 9.1 minutes for drug and 12.3 minutes for internal standard.

Pharmacokinetic data analysis. Pharmacokinetic models were fit to flavopiridol plasma concentration data using the Nonlinear Mixed Effects Modeling Program version V (Globomax Service Group, Hanover, MD). Two- and three-compartment pharmacokinetic models with first-order elimination were specified using the Nonlinear Mixed Effects Modeling Program PREDP subroutines ADVAN3/TRANS4 and ADVAN11/TRANS4, respectively. The flavopiridol pharmacokinetic variables estimated were the volume of distribution of the central compartment (V1), clearance from the central compartment (CL), volume of distribution of the peripheral compartment (V2), and intercompartmental clearance (Q). Interpatient variability in pharmacokinetic variables was estimated by the exponential error model:

(A)
where {theta} is the population mean value for variable P, Pi is the individual variable estimate, and {eta}i is a random variable with a mean of 0 and a variance of {Omega}2, which describes the deviation of Pi from P. Residual variability for flavopiridol pharmacokinetics was modeled by a proportional error model:

(B)
where Cobs is the observed concentration, Cpred is the model-predicted concentration, and {varepsilon}1 is the proportional error component.

The pharmacokinetic data were fit first. Once a satisfactory model was obtained, the empirical Bayesian estimates of the individual pharmacokinetic variables were plotted against demographic and physiologic variables to study the effect of patient characteristics on CL and V1. This was carried out by plotting the individual partial residues of each variable resulting from the base model against each potential covariate. The covariates tested were bovine serum albumin, sex, height, weight, serum albumin, and serum creatinine. Patterns in these residual plots suggest the addition of the covariate to the model for the variable. Identification of the best structural model was based on the value of the objective function from Nonlinear Mixed Effects Modeling Program output and the diagnostic plots. The maximum concentration after the first dose of flavopiridol (Cmax) was the observed concentration and the area under the plasma concentration curve (AUC) was calculated using noncompartmental modeling.

Laboratory correlates
Flavopiridol target proteins. In preparation for immunoblotting, mononuclear cells were isolated from bone marrow aspirates obtained on day 0 and again on day 3 immediately following completion of flavopiridol by Ficoll-Hypaque density gradient centrifugation. Mononuclear cells were washed twice with PBS and then solubilized in alkylation buffer [6 mol/L guanidine hydrochloride, 250 mmol/L Tris-HCl (pH 8.5 at 21°C), and 10 mmol/L EDTA supplemented immediately before use with 150 mmol/L 2-mercaptoethanol and 1 mmol/L {alpha}-phenylmethylsulfonyl fluoride] after confirmation that isolated cells contained at least 70% leukemic cells. Resulting lysates were then processed for SDS-PAGE and subsequent immunoblotting using techniques described previously in detail (14, 40). Membranes were probed using the following antibodies: Mcl-1 (BD PharMingen, San Diego, CA), Bcl-2 (DAKO, Glostrup, Denmark), cyclin D1 (Calbiochem, San Diego, CA), phospho-RNA polymerase II (Covance, Cumberland, VA), phospho-Tyr705-STAT3 (Cell Signaling, Beverly, MA), and actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Vascular endothelial growth factor and endothelial cell proliferation. The effects of flavopiridol on VEGF expression and secretion were determined initially in U937 monoblastic leukemia cells and bovine aortic endothelial cells (BAEC). U937 cells (1 x 105 per well, six-well plates) were cultured for 24 hours in DMEM plus 2% fetal bovine serum with escalating doses of flavopiridol. Supernatants (0.5 mL) were collected following cell centrifugation and stored at –80°C. BAEC obtained from the Coriell Institute (Camden, NJ) were cultured in 96-well plates (1 x 104 per well) for 16 hours in DMEM plus 2% fetal bovine serum to allow cell attachment and recovery, after which graded doses of flavopiridol were added and cells were incubated for a further 72 hours at 37°C. BAEC proliferation was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Chemical, St. Louis, MO) reduction assay. BAEC monolayers were washed with medium and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye was added. After 4 hours, 10% SDS and 0.01 N HCl were added to solubilize the crystalline formazan product and absorbance at 595 nm was determined after 16 hours.

VEGF levels in conditioned medium, sera, bone marrow cell lysates, and supernatants were measured using the Quantikine human VEGF immunoassay method (R&D, Inc., Minneapolis, MN). All reagents and working standards were prepared at room temperature, microplate strips were removed from the plate frame, and assay diluent (100 µL) was added to each well. After VEGF standard or sample (100 µL/well) was added, wells were covered with adhesive strips and incubated at room temperature for 2 hours. After multiple washings and removal of liquid, the plate was inverted and blotted against a clean paper towel. VEGF conjugate (200 µL) was added to each well for 2-hour incubation at room temperature followed by multiple washings and addition of substrate solution (200 µL) for 25 minutes. The reaction was stopped by 50 µL stop solution and the color change was determined by measuring absorbance of 450 nm using a Bio-Rad (Hercules, CA) ELISA microplate reader. Wavelength correction at 595 nm was used to correct for optical imperfections in the plate.

Pretreatment bone marrow VEGF levels were determined on marrow cells obtained by routine heparinized needle aspiration on day 0 before treatment with flavopiridol and processed immediately. Bone marrow supernatants were obtained by centrifugation of the marrow cell suspension to separate cellular debris. Bone marrow cell lysates were obtained by Ficoll-Hypaque density gradient centrifugation of the marrow cell suspension followed by washing with HBSS and freeze/thaw lysis. Aliquots were stored at –80°C for subsequent ELISA determinations of VEGF levels.

The effects of in vivo flavopiridol administration on net VEGF production were determined in sera obtained longitudinally from nine patients on day 0 before flavopiridol, day 3 at the end of the flavopiridol bolus, and day 6 before the ara-C infusion. The effects of flavopiridol-containing sera on in vitro endothelial cell proliferation were measured using BAEC cultured for 72 hours with plasma samples obtained from six patients on days 0, 3, and 6 as above (10%, v/v). The relationship between inhibition of BAEC proliferation and patient flavopiridol exposure in vivo was assessed using linear regression analysis with Sigma Plot 2000. The percent of control growth versus the Cmax and the AUC of flavopiridol were used for the regression analysis.


   Results
Top
 Abstract
 Patients, Materials, and Methods
 Results
Discussion
 References
 
Patient characteristics. Between March 2001 and November 2003, 34 adults with poor-risk, refractory or relapsed acute leukemia were entered in this phase I study of flavopiridol, ara-C, and mitoxantrone. Detailed clinical demographics are presented in Table 1. Four had newly diagnosed secondary AML (myelodysplasia/AML, treatment-related AML), 9 had acute leukemia in first (3 AML and 2 ALL) or second (4 AML) relapse, and 21 had refractory acute leukemia. Of the six patients with primary refractory leukemia, four AML patients failed to achieve CR following a single cycle of TST induction with ara-C, daunorubicin, and etoposide; one AML patient was refractory to standard ara-C plus idarubicin ("3 + 7") followed by one cycle of high-dose ara-C plus etoposide; and one ALL patient failed to achieve CR after two cycles of hyperfractionated cytoxan, vincristine, Adriamycin, and dexamethasone. The vast majority (91%) had received anthracycline or mitoxantrone therapy for acute leukemia or a prior malignancy. Similarly, 91% had received dose-intensive infusional ara-C and/or high-dose ara-C during initial antileukemia induction and/or consolidation regimens. Eleven (32%) had received previous TST with ara-C and anthracycline or mitoxantrone, including 4 with primary refractory AML and 3 in first relapse with CR 1 duration < 6 months.


View this table:
[in this window]
[in a new window]

  		Table 1. Characteristics of 34 adults undergoing TST with flavopiridol

 
Toxicities. Sixteen (47%) of the 34 patients evinced direct leukemia cell cytotoxicity as manifested by a ≥50% drop in peripheral blood blast counts. This effect was seen in AML (12 of 26) and ALL (4 of 8, including the patient with CML-LBC) at median day 4 (range, 2-6) following the initiation of flavopiridol and occurred at all dose levels. Evidence for flavopiridol-induced tumor lysis preceding blast clearance occurred in 9 (26%) patients on median day 2 (range, 1-4) of flavopiridol as manifested primarily as transient hyperphosphatemia (14 patients, peak, 4.9-7.5 mg/dL, median, 6.2) with or without hyperuricemia (3 patients, peak, 7.5-10.2 mg/dL), with resolution within 72 hours. Lactate dehydrogenase elevations occurred before and/or concomitant with blast clearance on median day 3 (range, 2-5) in 15 patients, with peak lactate dehydrogenase being 1.2- to 8.1-fold (median, 1.6-fold) higher than pretreatment levels. Hyperkalemia was not detected, no patient required hemodialysis, and no patient developed an associated coagulopathy.

The predominant nonhematologic toxicities (Table 2) were mucosal, with 3 (9%) patients experiencing grade 3 diarrhea within 48 hours of ending flavopiridol (day 5 or earlier) and 4 (12%) patients experiencing grade ≥2 oral mucositis (days 4, 11, 12, and 24). No patient exhibited the full-blown proinflammatory syndrome that can accompany continuous infusion (2) or bolus (33) administrations of flavopiridol. Drug-induced myelosuppression occurred in all but one patient treated at dose level 1, with the depth and duration of suppression being comparable with that seen in other TST regimens using cytotoxic drugs (30–32). Median day of absolute neutrophil count recovery to >100/mm3 was day 30 (range, 24-55) and recovery to >500/mm3 was day 36 (range, 27-70). Platelet recovery to ≥50,000/mm3 occurred on median day 38 (range, 29-66).


View this table:
[in this window]
[in a new window]

  		Table 2. Nonhematologic toxicities of TST with flavopiridol, ara-C, and mitoxantrone

 
There were no nonhematologic toxicities exceeding grade 1 in the four patients treated at dose level 1 (flavopiridol dose 40 mg/m2/d x 3). One episode of cardiac toxicity (grade 5; discussed below) occurred in the six patients treated at dose level 2 (50 mg/m2/d x 3). DLT was reached at dose level 3 (60 mg/m2/d x 3), where two of the five patients entered at this dose level experienced profound neutropenia >40 days in the absence of detectable residual leukemia in two patients (treatment-related M5 AML following multiple therapies for T-ALL, multirefractory ALL). An additional patient treated on dose level 3 suffered grade 5 cardiac toxicity as described in detail below. After 4 patients entered at dose level 2 (flavopiridol 50 mg/m2/d x 3) as part of an expanded cohort, it was decided in conjunction with Cancer Therapy Evaluation Program investigators to expand this cohort by an additional 15 patients for purposes of cardiac monitoring throughout the first 21 days of therapy. No arrhythmias and no episodes of congestive heart failure were detected in the subsequent 15 patients.

Four (12%) patients died during therapy with flavopiridol, ara-C, and mitoxantrone, two from disseminated fungal infections (days 30 and 37) and two with sudden death from probable cardiac arrhythmias (days 12 and 20). One episode of sudden death occurred on day 20 of therapy (flavopiridol dose level 2) in a 64-year-old male with refractory L3 ALL and with preexisting hypertensive coronary artery disease, fatty liver, diabetes, and a history of deep venous thromboses with multiple pulmonary emboli. The second death occurred in a 27-year-old female with M0 AML on day 12 (flavopiridol dose level 3). Her disease was associated with extramedullary infiltration and she had suffered an acute, nonischemic cardiomyopathy of uncertain etiology following initial induction chemotherapy (left ventricular ejection fraction 25% with left-sided congestive heart failure and mitral regurgitation) that resolved within 4 weeks (left ventricular ejection fraction 60%) and did not recur with consolidation therapy containing mitoxantrone. Her left ventricular ejection fraction before therapy with flavopiridol, ara-C, and mitoxantrone was 60%.

Clinical outcome. Table 2 summarizes the clinical outcome of one cycle of flavopiridol, ara-C, and mitoxantrone. Responses occurred in the first two dose levels, with clear evidence for dose response. For the entire group of 34 patients, 7 (21%) achieved CR and an additional 2 (6%) achieved partial remission for an overall response rate of 27%. Responses occurred in 8 of 26 (31%) AML patients, with the CR rate being 23% (6 of 26) and partial remission rate being 8% (2 of 26). For the ALL cohort (including one with CML-LBC), 1 of 8 (12.5%) achieved CR and there were no partial remissions. When examined according to disease status and prior therapy, 2 of 4 (50%) patients with newly diagnosed AML with poor-risk features achieved CR as did 2 of 7 (29%) with relapsed AML and only 2 of 15 (13%) with refractory AML.

Pharmacokinetics. The observed concentration versus time data were best fit by a linear two-compartment model with first-order elimination compared with a three-compartment model. There was no significant drop in the value of the minimum objective function and the AUC by fitting a three-compartment model to the data. Thus, variables for the two-compartment model are presented and 95% confidence intervals were calculated for each variable based on the asymptotic SEs reported by Nonlinear Mixed Effects Modeling Program. On evaluation of the effect of demographic and physiologic variables on CL and V1, there was no significant effect of any of the variables.

Table 3 summarizes the results of the final pharmacokinetic model. Mean CL was estimated to be 27.7 L/h with an intersubject variability of 64%. The mean estimate was 57.4 L for V1, 25.3 L/h for Q, and 392 L for V2. The intersubject variabilities for V1 and Q were 44% and 33.8%, respectively. Residual error was estimated at a modest 33%. Fig. 1 shows the population-predicted and individual-predicted concentrations obtained from the final model plotted against the observed concentrations. An example of a representative plasma concentration versus time plot for flavopiridol is depicted in Fig. 2. The Cmax and AUC were calculated using noncompartmental methods. There was a dose-related increase in the dose-normalized Cmax (2.4 ± 1, 2.9 ± 1.8, and 3.2 ± 2 µmol/L) with relatively similar AUC (8.1 ± 1.3, 6.3 ± 4, and 8.3 ± 4.7 µmol/L h) across the three flavopiridol dose levels (40, 50, and 60 mg/m2/d, respectively).


View this table:
[in this window]
[in a new window]

  		Table 3. Clinical outcome of TST with flavopiridol, ara-C, and mitoxantrone according to flavopiridol dose level

 


View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Predicted versus observed concentrations of flavopiridol. Population-predicted (A) and individual-predicted (B) plasma concentrations of flavopiridol plotted as a function of the observed concentrations. {bullet}, individual data; line, unity line.

 


View larger version (11K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Representative plasma concentration versus time plot. Plasma flavopiridol concentration versus time plot for a representative patient receiving 60 mg/m2 flavopiridol as a 1-hour infusion once daily for 3 days. Solid line, model-predicted concentration; {bullet}, measured concentrations.

 
Flavopiridol target proteins. Previous in vitro studies in cells from diverse hematologic malignancies have shown that Mcl-1, cyclin D, Bcl-2, and phospho-RNA polymerase II are down-regulated in response to flavopiridol treatment, whereas p53 and phospho-Tyr705-STAT3 are up-regulated (5, 15, 40, 41). To determine if flavopiridol could exert similar effects in vivo, we examined changes in selected cellular proteins derived from paired samples of leukemic marrow cells from 11 patients (8 AML and 3 ALL, including 1 CML-LBC) obtained on days 0 and 3 after flavopiridol. Figure 3 shows that flavopiridol administration yielded decreases in one or more proteins in 5 of the 11 day 3 marrow blast populations relative to pretreatment levels (obtained from patients P1, P2, P15, P19, and P33). Marrow blasts obtained on day 3 from two (P1 and P19) of four AML patients tested who achieved CR (P1, P19, P20, and P33) showed simultaneous down-regulation of multiple flavopiridol target proteins (Bcl-2, phospho-RNA polymerase II, and Mcl-1) relative to blasts obtained before flavopiridol day 0. Cyclin D1, measurable only in a few of the patients, was decreased in P1. In contrast, day 3 marrow blasts from four nonresponding patients (P2, AML; P9, ALL; P27, AML; and P31, AML) exhibited increases in at least one cycle regulatory protein.



View larger version (47K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Effects of flavopiridol therapy on leukemia cell polypeptides isolated from 11 patients. Relative amounts of cellular polypeptides were assessed by immunoblotting, with total cellular proteins from 2 x 106 patient mononuclear cells loaded in each lane. Analogous results obtained from immunoblotting of HL60 total cellular proteins are shown for comparison purposes. D0, pretherapy; D3, following flavopiridol therapy.

 
Vascular endothelial growth factor and endothelial cell proliferation. To establish that flavopiridol could inhibit the expression and secretion of VEGF from leukemic cells, we quantified changes in VEGF levels in medium conditioned by VEGF-expressing U937 cells following 24-hour exposure to increasing doses of flavopiridol (Fig. 4). As depicted in Fig. 4A, flavopiridol inhibited VEGF secretion by U937 cells in a dose-dependent fashion, with an IC50 of 3 nmol/L. Flavopiridol also inhibited the proliferation of BAEC cells cultured in the presence of escalating doses of drug (Fig. 4B), with an IC50 of 30 nmol/L. Maximal inhibition of BAEC proliferation was achieved at 125 nmol/L.



View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Effect of flavopiridol on the growth of BAEC cells in vitro. BAEC were cultured in 96-well plates (1 x 104 per well) in the presence or absence of flavopiridol (0-500 nmol/L) for 72 hours at 37°C. Growth medium was replaced with medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (0.5 mg/mL) and cultures were incubated for 4 hours. The crystalline formazan product was solubilized with 10% SDS/0.01 N HCl and the absorbance at 595 nm was measured after 16 hours. Dose dependence was calculated from triplicate samples. P < 0.01, 30 nmol/L flavopiridol compared with untreated cells.

 
VEGF levels were determined in marrow cell lysates and supernatants derived from pretreatment marrow aspirates obtained from six individual patients. Cell lysate VEGF levels ranged from 60 to 750 pg/mL and marrow supernatant VEGF levels ranged from 60 to 1,200 pg/mL, without a clear relationship between cell lysate and supernatant levels. To determine the effects of in vivo levels of flavopiridol on BAEC proliferation, sera were obtained longitudinally from nine patients treated at dose levels 1 and 2 on day 0 before flavopiridol, day 3 at the end of flavopiridol, and day 6 before ara-C and assayed for their ability to inhibit BAEC proliferation in vitro (Fig. 5). Sera obtained on day 3 from five patients who eventually achieved CRs inhibited endothelial cell proliferation relative to day 0 plasma, with a mean inhibition of 32% (range, 10-80%; Fig. 5A), whereas sera obtained on day 6 before ara-C administration showed a return toward baseline activity. In contrast, sera obtained from four patients with clinically resistant (progressive) leukemia revealed no inhibitory/recovery pattern (Fig. 5B). We then sought to determine the pharmacokinetic relationship of flavopiridol levels and BAEC growth looking at three different variables of flavopiridol concentration in vivo (Cp at 2 hours, Cmax, and AUC). BAEC proliferation was inversely proportional to the level of flavopiridol in the sera with R2 values of 0.7809 (Cp), 0.7981 (Cmax), and 0.7216 (AUC). This decrease in proliferation was modest, ~30% at the maximum achieved flavopiridol exposure, but statistically significant (P < 0.05), with the change in flavopiridol concentration accounting for 70% of the variability in response.



View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. BAEC growth inhibition pattern. Sera tested for activity using BAEC growth assays. Results from individual patient sera or bone marrow. Points, averages of duplicate samples; bars, range observed. Inhibition of cell growth (mean, 32%; range, 10-80%) was detected on day 3, with resolution of inhibitory activity by day 6 before ara-C administration.

 

   Discussion
Top
 Abstract
 Patients, Materials, and Methods
 Results
 Discussion
References
 
The design of our phase I trial of flavopiridol followed by ara-C and mitoxantrone for adults with relapsed and refractory acute leukemias was based on findings from our preceding in vitro studies (27, 42); that is, flavopiridol can exert cytotoxic effects on cell lines and cultured human leukemic marrow blasts followed by recruitment and priming of remaining cells for enhanced ara-C sensitivity. Further, the use of flavopiridol as the initial drug in sequence afforded us the opportunity to examine whether flavopiridol could exert direct antileukemic effects in vivo, independent of known antileukemic cytotoxic agents. The clinical results of the trial support the contention that flavopiridol is directly cytotoxic to leukemic cells and, when followed by ara-C and mitoxantrone, exerts measurable biological and clinical effects in patients with relapsed and refractory acute leukemias. This direct cytotoxicity was manifested by a significant drop in peripheral blood blast count in almost half of the patients, with evidence of a preceding flavopiridol-induced tumor lysis syndrome in 26% of those treated. The ability of flavopiridol to induce tumor lysis has been shown in chronic lymphocytic leukemia by Byrd et al. (34) and Donehower et al. (43), raising the possibility that flavopiridol causes cell necrosis as well as apoptosis in selected malignant cell populations.

The DLT for flavopiridol in this regimen occurred at 60 mg/m2/d given as a 1-hour infusion daily for 3 days, with marrow aplasia of >40 days occurring in two patients treated at that dose level. This finding is similar to that of Tan et al. (33), who observed that dose-limiting marrow aplasia occurred at flavopiridol doses ≥52.5 mg/m2/d in solid tumor patients. The depth of myelosuppression in both of the leukemia patients treated at 60 mg/m2/d extended beyond that commonly seen in other TST regimens (30–32, 36) and may relate to the refractory nature of the leukemias or to stem cell toxicity from previous cytotoxic therapies (e.g., treatment-related AML and heavily pretreated ALL). Alternatively, the use of flavopiridol to recruit leukemic blasts into cell cycle may have resulted in recruitment of normal stem cells at the same time, with a resultant prolongation of myelosuppression in patients with preexisting marrow damage.

Pharmacokinetic analyses show that the plasma levels of flavopiridol achieved in our patients are similar to those obtained in earlier studies (2, 33, 34, 38). We were able to describe flavopiridol pharmacokinetics with a two-compartment model using Nonlinear Mixed Effects Modeling Program. As depicted in Table 4, the high intersubject variability in clearance (64%) is a reflection of the high variability in the plasma concentrations. Although no relationship was found between clinical and demographic covariates and clearance (or volume of distribution) to explain the high variability in clearance, the role of the breast cancer resistance protein cannot be ruled out. In this regard, Nakanishi et al. (42) detected resistance to flavopiridol-induced cell death in primary human leukemic marrow blast populations with relatively high (>10,000 copies/pg ß-actin) breast cancer resistance protein mRNA expression as measured by real-time reverse transcription-PCR. Interestingly, no such relationship could be detected between MDR1 mRNA expression and resistance to flavopiridol-induced apoptosis. It is likely that mechanisms in addition to high breast cancer resistance protein expression (either pretreatment or induced by flavopiridol exposure) are involved in flavopiridol resistance, because marked variation in cell apoptosis and survival in the presence of flavopiridol was detected in blast cell populations with relatively low breast cancer resistance protein expression.


View this table:
[in this window]
[in a new window]

  		Table 4. Mean pharmacokinetic variables and estimates of intersubject variability

 
The cytotoxicity of flavopiridol relates to its ability to modulate the activity of diverse proteins that regulate cycle progression and cell survival. We were able to show that the in vivo administration of flavopiridol was associated with changes in one or more cell cycle or survival proteins in leukemic marrow blasts from some of the patients in whom we were able to measure such protein levels. Unfortunately, the data are difficult to analyze quantitatively for several reasons, including disparities in cell numbers and survival among specimens. Quantitation and correlation with clinical outcome would be important to pursue in future correlative clinical laboratory trials involving flavopiridol. In addition, our in vitro studies support the notion that flavopiridol inhibits expression and secretion of VEGF in the VEGF-expressing monoblastic leukemia cell line U937, with inhibition detected at very low drug concentrations. The link between flavopiridol and VEGF suppression is further substantiated by our demonstration that flavopiridol inhibits endothelial cell proliferation at clinically achievable concentrations in vitro. Interestingly, patient sera obtained at the end of the day 3 flavopiridol infusion and containing peak clinically achievable concentrations of flavopiridol had inhibitory effects on endothelial cell proliferation in vitro. Further, pharmacokinetic analyses showed significant dose response with regard to inhibitory effects of day 3 flavopiridol-containing sera on BAEC growth, with the endothelial cell proliferation being inversely proportional to the serum level of flavopiridol. This inhibitory activity could reflect any number of flavopiridol-induced changes in the activities of cyclin-dependent kinases, cyclin D1, or target proteins involved in regulating apoptosis. Nonetheless, it is interesting to speculate that at least part of the propapoptotic effects of flavopiridol might stem from the ability of the drug to decrease VEGF production, which in turn may prevent the observed up-regulation of Mcl-1 expression that has been documented in multiple myeloma cells and has been shown to protect myeloma cells from apoptosis in response to cellular stress (5, 41, 44).

Overall, this clinical trial shows a moderate efficacy of this timed sequential regimen for adults with relapsed and refractory leukemias, particularly AML. We did not examine alternate drug schedules in either our in vitro TST model or our in vivo phase I clinical trials. As such, our data do not preclude the possibility that another sequence of flavopiridol and ara-C might be equally effective in terms of net antileukemic cytotoxicity. In this regard, recent studies in lung cancer cell lines show that flavopiridol causes mitochondrial damage and apoptosis in S phase (45, 46). This induction of apoptosis in S phase is dependent on the ability of flavopiridol to block phosphorylation of E2F1 (45), which in turn stabilizes E2F1/DP-1 heterodimer binding to DNA and thereby represses Mcl-1 transcription (45, 46). Combinations with other cytotoxics may require different schedules depending on the maximal cycle-dependent effects of the specific cytotoxic agent and the growth kinetics of the target cell population (3, 26, 47). Additionally, flavopiridol exhibits synergistic antitumor activities when combined with mechanistically diverse agents, such as the tyrosine kinase inhibitor imatinib (48), the histone deacetylase inhibitor suberoylanilide hydroxamic acid (49), proapoptotic agent, such as tumor necrosis factor–related apoptosis-inducing ligand/Apo2L (50), and phosphatidylinositol 3-kinase inhibitor, such as wortmannin or LY294002 (6). Such synergy may be to some extent independent of cell cycle status (3) and more linked to non-cross-resistant mechanisms of mitochondrial damage (6, 10, 41, 48–50). Finally, alternative schedules of flavopiridol administration are important to consider. Byrd et al. (34) have shed important light on the schedule dependency of flavopiridol in patients with chronic lymphoocytic leukemia related at least in part to the extensive binding of flavopiridol to human plasma proteins. Their data suggest that a combination of a 30-minute i.v. bolus followed by a more sustained (4-hour) infusion might enhance overall flavopiridol cytotoxicity and therefore clinical responses (34, 43). The mechanistic versatility of flavopiridol and its ability to induce leukemic cell death directly support its continuing development in combination with cytotoxic and biological agents for acute leukemias.


   Acknowledgments
 
We thank Yean Kit Lee, Crescent R. Isham, Ngo Dai, and David Loegering for assistance with the immunoblotting.


   Footnotes
 
Grant support: National Cancer Institute cooperative agreements U01 CA69854 (J.E. Karp and K.S. Bauer) and CA70095 (J.E. Karp), NIH grant CA97129 (K. Bible), and American Cancer Society grant CCE-98842 (K. Bible).

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 6/ 2/05; revised 9/ 6/05; accepted 9/13/05.


   References
Top
 Abstract
 Patients, Materials, and Methods
 Results
 Discussion
 References
 

  1. Maik RG, Kattice S, Bhat SV, Alreja B, de Souza NJ, Rupp RH. An antiinflammatory cum immunomodulatory piperidinylbenzopyranone from Dysoxylum binectariferum: isolation, structure and total synthesis. Tetrahedron 1998;44:2081–6.[CrossRef]
  2. Senderowicz AM, Sausville EA. Preclinical and clinical development of cyclin-dependent kinase modulators. J Natl Cancer Inst 2000;92:376–87.[Abstract/Free Full Text]
  3. Bible KC, Kaufmann SH. Flavopiridol: a cytotoxic flavone that induces cell death in noncycling A549 human lung carcinoma cells. Cancer Res 1996;56:4856–61.[Abstract/Free Full Text]
  4. Decker RH, Dai Y, Grant S. The cyclin-dependent kinase inhibitor flavopiridol induces apoptosis in human leukemia cells (U937) through the mitochondrial rather than the receptor-mediated pathway. Cell Death Differ 2001;8:715–24.[CrossRef][Medline]
  5. Gojo I, Zhang B, Fenton RG. The cyclin-dependent kinase inhibitor flavopiridol induces apoptosis in multiple myeloma cells through transcriptional repression and down-regulation of Mcl-1. Clin Cancer Res 2002;8:3527–38.[Abstract/Free Full Text]
  6. Yu C, Rahmani M, Dai Y, et al. The lethal effects of pharmacological cyclin-dependent kinase inhibitors in human leukemia cells proceed through a phosphatidylinositol 3-kinase/Akt-dependent pathway. Cancer Res 2003;63:1822–33.[Abstract/Free Full Text]
  7. Carlson BA, Dubay MM, Sausville EA, Brizuela L, Worland PJ. Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK)2 and CDK 4 in human breast carcinoma cells. Cancer Res 1996;56:2973–8.[Abstract/Free Full Text]
  8. Worland PJ, Kaur G, Stetler-Stevenson M, Sebers S, Sartor O, Sausville EA. Alteration of the phosphorylation state of p34cdc2 kinase by the flavone L86-8275 in breast carcinoma cells. Biochem Pharmacol 1993;46:1831–40.[CrossRef][Medline]
  9. Chao SH, Price DH. Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo. J Biol Chem 2001;276:31793–9.[Abstract/Free Full Text]
  10. Lam LT, Pickeral OK, Peng AC, et al. Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol. Genome Biol 2001;2:1–11.
  11. Carlson B, Lahusen T, Loaiza-Perez A, et al. Down-regulation of cyclin D1 by transcriptional repression in MCF-7 human breast carcinoma cells induced by flavopiridol. Cancer Res 1999;59:4634–43.[Abstract/Free Full Text]
  12. Craig RW. MCL1 provides a window on the role of the BCL2 family in cell proliferation, differentiation and tumorigenesis. Leukemia 2002;16:444–54.[CrossRef][Medline]
  13. Opferman JT, Iwasaki H, Ong CC, et al. Obligate role of anti-apoptotic MCL-1 in the survival of hematopoietic stem cells. Science 2005;307:1101–4.[Abstract/Free Full Text]
  14. Kaufmann SH, Karp JE, Svingen PA, et al. Elevated expression of the apoptotic regulator Mcl-1 at the time of leukemic relapse. Blood 1998;91:991–1000.[Abstract/Free Full Text]
  15. Kitada S, Zapata JM, Andreeff M, Reed JC. Protein kinase inhibitors flavopiridol and 7-hydroxy-staurosporine down-regulate antiapoptosis proteins in B-cell chronic lymphocytic leukemia. Blood 2000;96:393–7.[Abstract/Free Full Text]
  16. Taniai M, Grambihler A, Higuchi H, et al. Mcl-1 mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in human cholangiocarcinoma cells. Cancer Res 2004;64:3517–24.[Abstract/Free Full Text]
  17. Mellilo G, Sausville EA, Cloud K, Lahusen T, Varresio L, Senderowicz A. Flavopiridol, a protein kinase inhibitor, down-regulates hypoxic induction of vascular endothelial growth factor expression in human monocytes. Cancer Res 1999;59:5433–7.[Abstract/Free Full Text]
  18. Ferrara N, Gerber H-P, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003;9:669–76.[CrossRef][Medline]
  19. Buchner T, Hiddeman W, Wormann B, et al. Double induction strategy for acute myeloid leukemia: the effect of high-dose cytarabine with mitoxantrone instead of standard-dose cytarabine with daunorubicin and 6-thioguanine: a randomized trial by the German AML Cooperative Group. Blood 1999;93:4116–24.[Abstract/Free Full Text]
  20. Feldman EJ, Alberts DS, Arlin Z, et al. Phase I clinical and pharmacokinetic evaluation of high-dose mitoxantrone in combination with cytarabine in patients with acute leukemia. J Clin Oncol 1993;11:2002–9.[Abstract/Free Full Text]
  21. Weiss M, Maslak P, Feldman E, et al. Cytarabine with high-dose mitoxantrone induces rapid complete remissions in adult acute lymphoblastic leukemia without the use of vincristine or prednisone. J Clin Oncol 1996;14:2480–5.[Abstract]
  22. Datta R, Banach D, Kojima H, et al. Activation of the CPP32 protease in apoptosis induced by 1-ß-D-arabinofuranosylcytosine and other DNA-damaging agents. Blood 1996;88:1936–43.[Abstract/Free Full Text]
  23. Ho AD, Seither E, Ma DDF, et al. Mitoxantrone-induced toxicity and DNA strand breaks in leukemic cells. Br J Haematol 1987;65:51–5.[Medline]
  24. Bhalla K, Ibrado AM, Tourkina E, et al. High-dose mitoxantrone induces programmed cell death or apoptosis in human myeloid leukemia cells. Blood 1993;82:3133–40.[Abstract/Free Full Text]
  25. Rowe TC, Chen GL, Hsiang YH, Liu LF. DNA damage by antitumor acridines mediated by mammalian DNA topoisomerase II. Cancer Res 1986;46:2021–6.[Medline]
  26. Bible KC, Kaufmann SH. Cytotoxic synergy between Flavopiridol (NSC 649890, L86-8278275) and various antineoplastic agents: the importance of sequence of administration. Cancer Res 1997;57:3375–80.[Abstract/Free Full Text]
  27. Karp JE, Ross DD, Yang W, et al. Timed sequential therapy of acute leukemia with flavopiridol: in vitro model for a phase I clinical trial. Clin Cancer Res 2003;9:307–15.[Abstract/Free Full Text]
  28. Burke PJ, Karp JE, Braine HG, Vaughan WP. Timed sequential therapy of human leukemia based upon the response of leukemic cells to humoral growth factors. Cancer Res 1997;37:2138–46.
  29. Karp JE, Donehower RC, Enterline J, Dole GB, Fox MG, Burke PJ. In vivo cell growth and pharmacologic determinants of clinical response in acute myelogenous leukemia. Blood 1989;73:24–30.[Abstract/Free Full Text]
  30. Archimbaud E, Thomas X, Leblond V, et al. Timed sequential chemotherapy for previously treated patients with acute myeloid leukemia: long-term follow-up of the etoposide, mitoxantrone, and cytarabine-86 trial. J Clin Oncol 1995;13:11–8.[Abstract/Free Full Text]
  31. Geller RB, Burke PJ, Karp JE, et al. S. A two-step timed sequential treatment for acute myelocytic leukemia. Blood 1989;p74:1499–506.[Abstract/Free Full Text]
  32. Woods WG, Alonzo TA, Lange BJ, Jeha S, Estey EH. Acute myeloid leukemia (AML) in adolescents and young adults (AYAs): comparison of outcomes between patients treated on childhood or adult protocols. Blood 2001;98:462a.
  33. Tan AR, Headlee D, Messman 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 2002;20:4074–82.[Abstract/Free Full Text]
  34. Byrd JC, Peterson BL, Gabrilove J, et al. Treatment of relapsed chronic lymphocytic leukemia by 72-hour continuous infusion or 1-hour bolus infusion of flavopiridol: results from Cancer and Acute Leukemia Group B study 19805. Clin Cancer Res 2005;11:4176–81.[Abstract/Free Full Text]
  35. Donehower RC, Karp JE, Burke PJ. Pharmacology and toxicity of high-dose cytarabine by 72-hour continuous infusion. Cancer Treat Rep 1986;70:1059–65.[Medline]
  36. Karp JE, Gojo I, Pili R, et al. Targeting vascular endothelial growth factor (VEGF) for relapsed and refractory adult acute myelogenous leukemias (AMLs): therapy with sequential ara-C, mitoxantrone and bevacizumab. Clin Cancer Res 2004;10:3577–85.[Abstract/Free Full Text]
  37. Kahn ME, Senderowicz A, Sausville EA, Barrett KE. Possible mechanisms of diarrheal side effects associated with the use of a novel chemotherapeutic agent, flavopiridol. Clin Cancer Res 2001;7:343–9.[Abstract/Free Full Text]
  38. Rudek MA, Bauer KS, Jr., Lush RM III, et al. Clinical pharmacology of flavopiridol following a 72-hour continuous infusion. Ann Pharmacother 2003;37:1369–74.[Abstract/Free Full Text]
  39. Cheson BD, Bennett JM, Kopecky KJ, et al. Revised recommendations of the international working group for diagnosis, standardization of response criteria, treatment outcomes and reporting standards for therapeutic trials in acute myeloid leukemia. J Clin Oncol 2003;21:4642–9.[Abstract/Free Full Text]
  40. Bible KC, Bible RH, Kottke TJ, et al. Flavopiridol binds to duplex DNA. Cancer Res 2000;60:2419–28.[Abstract/Free Full Text]
  41. Zhang B, Gojo I, Fenton RG. MCL-1 is a critical survival factor for multiple myeloma. Blood 2002;99:1885–93.[Abstract/Free Full Text]
  42. Nakanishi T, Karp JE, Tan M, et al. Quantitative analysis of breast cancer resistance protein and cellular resistance to flavopiridol in acute leukemia patients. Clin Cancer Res 2003;9:3320–8.[Abstract/Free Full Text]
  43. Byrd JC, Lin T, Dalton J, et al. Flavopiridol administered as a pharmacologically derived schedule demonstrates marked clinical activity in refractory, genetically high risk, chronic lymphocytic leukemia. Blood 2004;104:101a.
  44. Le Gouill S, Podar K, Amiot M, et al. VEGF induces Mcl-1 up-regulation and protects multiple myeloma cells against apoptosis. Blood 2004;104:2886–92.[Abstract/Free Full Text]
  45. Litz J, Carlson P, Warshamana-Greene GS, Grant S, Krystal GW. Flavopiridol potently induces small cell lung cancer apoptosis during S phase in a manner that involves early mitochondrial dysfunction. Clin Cancer Res 2003;9:4586–94.[Abstract/Free Full Text]
  46. Ma Y, Cress WD, Haura EB. Flavopiridol-induced apoptosis is mediated through up-regulation of E2F1 and repression of Mcl-1. Mol Cancer Ther 2003;2:73–81.[Abstract/Free Full Text]
  47. Schwartz GK, O'Reilly E, Ilson D, et al. Phase I study of the cyclin-dependent kinase inhibitor flavopiridol in combination with paclitaxel in patients with advanced solid tumors. J Clin Oncol 2002;20:2157–70.[Abstract/Free Full Text]
  48. Yu C, Krystal G, Dent P, Grant S. Flavopiridol potentiates STI571-induced mitochondrial damage and apoptosis in BCR-ABL-positive human leukemia cells. Clin Cancer Res 2002;8:2976–84.[Abstract/Free Full Text]
  49. Almenara J, Rosato R, Grant S. Synergistic induction of mitochondrial damage and apoptosis in human leukemia cells by flavopiridol and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). Leukemia 2002;16:1331–43.[CrossRef][Medline]
  50. Rosato RR, Dai Y, Almenara JA, Maggio SC, Grant S. Potent antileukemic interactions between flavopiridol and TRAIL/Apo2L involve flavopiridol-mediated XIAP downregulation. Leukemia 2004;18:1780–8.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Molecular Cancer TherapeuticsHome page
C. Mitchell, M. A. Park, G. Zhang, A. Yacoub, D. T. Curiel, P. B. Fisher, J. D. Roberts, S. Grant, and P. Dent
Extrinsic pathway- and cathepsin-dependent induction of mitochondrial dysfunction are essential for synergistic flavopiridol and vorinostat lethality in breast cancer cells
Mol. Cancer Ther., December 1, 2007; 6(12): 3101 - 3112.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
J. E. Karp, B. D. Smith, M. J. Levis, S. D. Gore, J. Greer, C. Hattenburg, J. Briel, R. J. Jones, J. J. Wright, and A. D. Colevas
Sequential Flavopiridol, Cytosine Arabinoside, and Mitoxantrone: A Phase II Trial in Adults with Poor-Risk Acute Myelogenous Leukemia
Clin. Cancer Res., August 1, 2007; 13(15): 4467 - 4473.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Karp, J. E.
Right arrow Articles by Bauer, K. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Karp, J. E.
Right arrow Articles by Bauer, K. S.