Phase I and Pharmacokinetic Study of Flavopiridol followed by1-B-D-Arabinofuranosylcytosine andMitoxantrone in Relapsed and RefractoryAdult Acute Leukemias

Purpose: The serine/threonine kinase inhibitor flavopiridol targets multiple cyclin-dependent kinases, induces checkpoint arrest, and interrupts transcriptional elongation.We designed aphase I clinical trial using a timed sequential therapy approachwhere 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-h-D-arabinofuranosylcytosine (ara-C) and mitoxantrone. Experimental Design:Flavopiridolwas givenby1-hour infusiondaily for 3 daysbeginningday1 followed by 2 g/m/72 h ara-C beginning day 6 and 40 mg/m mitoxantrone beginning day 9. In vivo correlates included pharmacokinetics, modulation of blast cycle regulators, and serum andmarrow supernatant vascular endothelial growth factor levels. Results:Of 34 adults receiving induction therapy,16 (47%) evinced direct leukemia cytotoxicity with z50% 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/m/d with profound neutropenia >40 days duration, and maximal tolerated dose was 50 mg/m/d. Overall response rate was 31% in 26 acute myelogenous leukemia and12.5% in acute lymphoblastic leukemia. Pharmacokinetics showed that a linear two-compartment model with first-order eliminationprovided 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 amean of 32% (range,10-80%). Conclusions:Ourdata 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/m/d 3 days in combinationwith 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 cyclindependent 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 cyclindependent 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 f50% 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 Cancer Therapy: Clinical Authors’Affiliations: Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins; University of Maryland Greenebaum Cancer Center; and Baltimore Veterans Affairs Medical Center, Baltimore, Maryland; Mayo Clinic, Rochester, Minnesota; and Investigational Drug Branch, Clinical Trials Evaluation Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland Received 6/2/05; revised 9/6/05; accepted 9/13/05. Grant support: National Cancer Institute cooperative agreements U01CA69854 (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 with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Judith E. Karp, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 1650 Orleans Street, CRBRoom 289, Baltimore, MD 21231-1000.Phone: 410-503-5399;Fax: 410-614-1005;E-mail: [email protected]. F2005 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-1201 www.aacrjournals.org Clin Cancer Res 2005;11(23) December1, 2005 8403 Research. on April 13, 2017. © 2005 American Association for Cancer clincancerres.aacrjournals.org Downloaded from apoptosis (5), suggesting a critical role for Mcl-1 downregulation 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-h-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, andMethods Patient eligibility and selection Adults ages z18 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 z45%. 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 z3 weeks from prior chemotherapy and z1 week from any growth factor therapy were required before beginning ara-C and mitoxantrone. Patients were ineligible if they had a peripheral blast count z50,000/mm; 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 3 beginning day 1 (33, 34). Using a modified dose escalation schema, flavopiridol was dose escalated, with the first four patients receiving 40 mg/m/d 3 days. Dose escalation could proceed if no more than one of three patients experienced DLT defined as grade z3 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/m ara-C as a 72-hour continuous infusion (667 mg/m/24 h; refs. 28, 31, 35, 36) began on day 6. Mitoxantrone (40 mg/m) 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/m/d 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 F 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 Ag 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 Cancer Therapy: Clinical www.aacrjournals.org Clin Cancer Res 2005;11(23) December1, 2005 8404 Research. on April 13, 2017. © 2005 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Famvir prophylaxis against herpes simplex virus activation began on day 1 and continued until achievement of an absolute neutrophil count >100/mm. 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 z500/mm and transfusion-independent platelet count 50,000/mm. CR required a normal bone marrow aspirate with absence of identifiable leukemia, absolute neutrophil count z1,000/mm, platelet count z100,000/ mm, 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.