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Complex karyotype, defined as ≥3 cytogenetic abnormalities, is prognostic of survival in patients treated with ibrutinib or venetoclax in relapsed/refractory (RR) chronic lymphocytic leukemia (CLL). Recent studies re-evaluating this dichotomous variable have shown that higher numbers of cytogenetic abnormalities (ie, ≥5) have a worse overall survival in patients treated with chemoimmunotherapy. We sought to determine if increasing karyotypic complexity, treated as a continuous variable, was prognostic of survival for patients treated with ibrutinib for CLL. We conducted a retrospective analysis of all patients with CLL treated with single-agent ibrutinib or in combination with an anti-CD20 antibody at our institution. We included 456 patients with both treatment-naive and RR disease. Median number of prior therapies was 2 (range, 0-13), 30% of patients had presence of del(17p), and 75% expressed unmutated IGHV. Fifty percent had ≥3 cytogenetic abnormalities, including 30% with ≥5. In a multivariable analysis, increasing karyotypic complexity was an independent predictor of shorter progression-free survival (hazard ratio, 1.07; 95% confidence interval, 1.04-1.10; P < .0001) and overall survival (hazard ratio, 1.09; 95% confidence interval, 1.05-1.12; P < .0001). Furthermore, we found that presence of clonal evolution determined by cytogenetic analysis at progression was prognostic of subsequent survival (P = .02). This solidifies karyotypic complexity as an important prognostic factor for patients with CLL treated with ibrutinib. Further research should consider sequential karyotypic analysis as a determination of risk of progression and death in patients with CLL., (© 2021 by The American Society of Hematology.)

Hairy cell leukemia (HCL) is a rare B-cell malignancy, and there is a need for novel treatments for patients who do not benefit from purine analogs. Ibrutinib, an oral agent targeting Bruton tyrosine kinase in the B-cell receptor signaling pathway, is highly effective in several malignancies. Its activity in HCL was unknown, so we conducted a multisite phase 2 study of oral ibrutinib in patients with either relapsed classic or variant hairy cell leukemia. The primary outcome measure was the overall response rate (ORR) at 32 weeks, and we also assessed response at 48 weeks and best response during treatment. Key secondary objectives were characterization of toxicity and determination of progression-free survival (PFS) and overall survival (OS). Thirty-seven patients were enrolled at 2 different doses (24 at 420 mg, 13 at 840 mg). The median duration of follow-up was 3.5 years (range, 0-5.9 years). The ORR at 32 weeks was 24%, which increased to 36% at 48 weeks. The best ORR was 54%. The estimated 36-month PFS was 73% and OS was 85%. The most frequent adverse events were diarrhea (59%), fatigue (54%), myalgia (54%), and nausea (51%). Hematologic adverse events were common: anemia (43%), thrombocytopenia (41%), and neutropenia (35%). Ibrutinib can be safely administered to patients with HCL with objective responses and results in prolonged disease control. Although the initial primary outcome objective of the study was not met, the observation of objective responses in heavily pretreated patients coupled with a favorable PFS suggests that ibrutinib may be beneficial in these patients. This trial was registered at www.clinicaltrials.gov as #NCT01841723.

Patients with chronic lymphocytic leukemia (CLL) who receive chemoimmunotherapy and do not achieve complete remission experience significantly shortened progression-free interval (PFS). Additionally, the majority of patients treated for relapsed disease demonstrate evidence of measurable disease. Eradication of minimal residual disease (MRD) results in improved PFS and overall survival. Maintenance therapy might result in eradication of MRD and improve response duration but might be associated with an increase in incidence of infectious complications. Flavopiridol is a broad cyclin-dependent kinase (CDK) inhibitor with established safety and efficacy in patients with relapsed CLL, particularly patients with high-risk cytogenetic features. A pharmacologically derived schedule was utilized as consolidation therapy in this phase I study to assess the safety and feasibility of outpatient therapy with flavopiridol in patients with low tumor burden. Flavopiridol was administered as a 30-min loading dose of 30 mg/m(2) followed by a 4-h infusion of 30 mg/m(2) once weekly for 3 weeks every 5 weeks (1 cycle) for planned 2 cycles in ten patients. Therapy was extremely well tolerated and no patient developed acute tumor lysis syndrome. The most common toxicities were gastrointestinal. Of the patients, 22 % improved their response from a PR to CR. Eighty-eight percent experienced a reduction in tumor burden as measured by extent of bone marrow involvement including patients with del17p and complex karyotype. The study establishes the safety and efficacy of flavopiridol as consolidation therapy after chemoimmunotherapy for patients with CLL. Further evaluation is required in larger trials for the utility of CDK inhibitors as consolidation or maintenance strategies.Registration number at ClinicalTrials.gov: NCT00377104.

Early phase studies of alvocidib showed activity in relapsed CLL including patients with high risk genomic features and those refractory to fludarabine. A multi-center, international, phase II study of alvocidib in fludarabine refractory CLL was undertaken to validate these early results. Patients with fludarabine refractory CLL or prolymphocytic leukemia arising from CLL were treated with single agent alvocidib. The primary outcome measure was overall response rate, with secondary outcomes including survival, toxicity, and response duration. One hundred and sixty five patients were enrolled and 159 patients were treated. The median age was 61 years, the median number of prior therapies was 4, and 96% of patients were fludarabine refractory. The investigator-assessed overall response rate was 25%; the majority of responses were partial. Response rates were lower among patients with del(17p) (14%), but equivalent in patients with del(11q) or bulky lymphadenopathy. Median progression free and overall survival were 7.6 and 14.6 months, respectively. Tumor lysis occurred in 39 patients (25%), and 13 received hemodialysis. Diarrhea, fatigue, and hematologic toxicities were common. Alvocidib has clinical activity in patients with advanced, fludarabine refractory CLL. Future studies should focus on discovery of biomarkers of clinical response and tumor lysis, and enhanced supportive care measures., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Flavopiridol and lenalidomide have activity in refractory CLL without immunosuppression or opportunistic infections seen with other therapies. We hypothesized that flavopiridol treatment could adequately de-bulk disease prior to lenalidomide therapy, decreasing the incidence of tumor flare with higher doses of lenalidomide. In this Phase I study, the maximum tolerated dose was not reached with treatment consisting of flavopiridol 30 mg m(-2) intravenous bolus (IVB) + 30 mg m(-2) continuous intravenous infusion (CIVI) cycle (C) 1 day (D) 1 and 30 mg m(-2) IVB + 50 mg m(-2) CIVI C1 D8,15 and C2-8 D3,10,17 with lenalidomide 15 mg orally daily C2-8 D1-21. There was no unexpected toxicity seen, including no increased tumor lysis, tumor flare (even at higher doses of lenalidomide) or opportunistic infection. Significant clinical activity was demonstrated, with a 51% response rate in this group of heavily pretreated patients. Biomarker testing confirmed association of mitochondrial priming of the BH3 only peptide Puma with response., (© 2015 Wiley Periodicals, Inc.)

Flavopiridol is a small molecule inhibitor of cyclin-dependent kinases (CDK) known to impair global transcription via inactivation of positive transcription elongation factor b. It has been demonstrated to have significant activity predominantly in chronic lymphocytic leukemia and acute myeloid leukemia in phase I/II clinical trials while other similar CDK inhibitors are vigorously being pursued in pre-clinical and clinical studies. Although flavopiridol is a potent therapeutic agent against blood diseases, some patients still have primary or acquired resistance throughout their clinical course. Considering the limited knowledge of resistance mechanisms of flavopiridol, we investigated the potential mechanisms of resistance to flavopiridol in a cell line system, which gradually acquired resistance to flavopiridol in vitro, and then confirmed the mechanism in patient samples. Herein, we present that this resistant cell line developed resistance through up-regulation of phosphorylation of RNA polymerase II C-terminal domain, activation of CDK9 kinase activity, and prolonged Mcl-1 stability to counter flavopiridol's drug actions. Further analyses suggest MAPK/ERK activation-mediated Mcl-1 stabilization contributes to the resistance and knockdown of Mcl-1 in part restores sensitivity to flavopiridol-induced cytotoxicity. Altogether, these findings demonstrate that CDK9 is the most relevant target of flavopiridol and provide avenues to improve the therapeutic strategies in blood malignancies.

Flavopiridol is a broad cyclin-dependent kinase inhibitor (CDKI) that induces apoptosis of malignant lymphocytes in vitro and in murine lymphoma models. We conducted a Phase I dose-escalation study to determine the maximum tolerated dose (MTD) for single-agent flavopiridol administered on a pharmacokinetically derived hybrid dosing schedule to patients with relapsed and refractory non-Hodgkin's lymphoma. Dose was escalated independently in one of four cohorts: indolent B-cell (Cohort 1), mantle cell (Cohort 2), intermediate-grade B-cell including transformed lymphoma (Cohort 3), and T-/NK-cell excluding primary cutaneous disease (Cohort 4). Forty-six patients were accrued. Grade 3 or 4 leukopenia was observed in the majority of patients (60%), but infection was infrequent. Common nonhematologic toxicities included diarrhea and fatigue. Biochemical tumor lysis was observed in only two patients, and no patients required hemodialysis for its management. Dose escalation was completed in two cohorts (indolent and aggressive B-cell). Dose-limiting toxicities were not observed, and the MTD was not reached in either cohort at the highest dose tested (50 mg/m(2) bolus + 50 mg/m(2) continuous infusion weekly for 4 consecutive weeks of a 6-week cycle). Clinical benefit was observed in 26% of 43 patients evaluable for response, including 14% with partial responses (two mantle cells, three indolent B-cells, and one diffuse large B-cell). The single-agent activity of this first-generation CDKI suggests that other agents in this class merit further study in lymphoid malignancies, both alone and in combination., (Copyright © 2013 Wiley Periodicals, Inc.)

Purpose: Flavopiridol, the first clinically evaluated cyclin-dependent kinase inhibitor, shows activity in patients with refractory chronic lymphocytic leukemia, but prevalent and unpredictable tumor lysis syndrome (TLS) presents a major barrier to its broad clinical use. The purpose of this study was to investigate the relationships between pretreatment risk factors, drug pharmacokinetics, and TLS., Experimental Design: A population pharmacokinetic/pharmacodynamic model linking drug exposure and TLS was developed. Plasma data of flavopiridol and its glucuronide metabolite (flavo-G) were obtained from 111 patients treated in early-phase trials with frequent sampling following initial and/or escalated doses. TLS grading was modeled with logistic regression as a pharmacodynamic endpoint. Demographics, baseline disease status, and blood chemistry variables were evaluated as covariates., Results: Gender was the most significant pharmacokinetic covariate, with females displaying higher flavo-G exposure than males. Glucuronide metabolite exposure was predictive of TLS occurrence, and bulky lymphadenopathy was identified as a significant covariate on TLS probability. The estimated probability of TLS occurrence in patients with baseline bulky lymphadenopathy less than 10 cm or 10 cm or more during the first 2 treatments was 0.111 (SE% 13.0%) and 0.265 (SE% 17.9%), respectively, when flavo-G area under the plasma concentration versus time curve was at its median value in whole-patient group., Conclusions: This is the first population pharmacokinetic/pharmacodynamic model of TLS. Further work is needed to explore potential mechanisms and to determine whether the associations between TLS, gender, and glucuronide metabolites are relevant in patients with chronic lymphocytic leukemia treated with other cyclin-dependent kinase inhibitors., (©2012 AACR.)

Cyclin dependent kinase (CDK) inhibitors, such as flavopiridol, demonstrate significant single-agent activity in chronic lymphocytic leukemia (CLL), but the mechanism of action in these nonproliferating cells is unclear. Here we demonstrate that CLL cells undergo autophagy after treatment with therapeutic agents, including fludarabine, CAL-101, and flavopiridol as well as the endoplasmic reticulum (ER) stress-inducing agent thapsigargin. The addition of chloroquine or siRNA against autophagy components enhanced the cytotoxic effects of flavopiridol and thapsigargin, but not the other agents. Similar to thapsigargin, flavopiridol robustly induces a distinct pattern of ER stress in CLL cells that contributes to cell death through IRE1-mediated activation of ASK1 and possibly downstream caspases. Both autophagy and ER stress were documented in tumor cells from CLL patients receiving flavopiridol. Thus, CLL cells undergo autophagy after multiple stimuli, including therapeutic agents, but only with ER stress mediators and CDK inhibitors is autophagy a mechanism of resistance to cell death. These findings collectively demonstrate, for the first time, a novel mechanism of action (ER stress) and drug resistance (autophagy) for CDK inhibitors, such as flavopiridol in CLL, and provide avenues for new therapeutic combination approaches in this disease.

Purpose: Based on the promising activity and tolerability of flavopiridol administered with a pharmacokinetically-derived dosing schedule in chronic lymphocytic leukemia (CLL), we conducted a phase I study using this schedule in patients with advanced solid tumors., Experimental Design: Flavopiridol was given IV as a 30-min loading dose followed by a 4-hr infusion weekly for 4 weeks repeated every 6 weeks. Dose-escalation was in cohorts of three patients using the standard 3+3 phase I study design. Blood samples were obtained for pharmacokinetic and pharmacodynamic studies., Results: Thirty-four eligible patients with advanced solid tumors received a total of 208 doses (median 7, range 1-24). Total doses ranged from 40 to 105 mg/m(2). The primary dose limiting toxicity was cytokine release syndrome (CKRS). No antitumor responses were observed. The mean peak plasma concentration across all doses was 1.65 ± 0.86 μM. Area under the concentration-versus-time curve ([Formula: see text]) ranged from 4.31 to 32.2 μM[Symbol: see text]hr with an overall mean of 13.6 ± 7.0 μM[Symbol: see text]hr. Plasma flavopiridol concentrations and AUC increased proportionally with dose. There was no correlation between cytokine levels and clinical outcomes., Conclusions: The maximum-tolerated dose of flavopiridol is 20 mg/m(2) bolus followed by 20 mg/m(2) infusion over 4 h given weekly for 4 weeks on a 6-week cycle in patients with advanced solid tumors. Flavopiridol PK was notably different, and there was a higher frequency of CKRS, despite prophylactic steroids, seen in this patient group compared to previous studies with CLL using a similar dosing schedule.

Older chronic lymphocytic leukemia patients have poor outcomes with standard treatments and are underrepresented in clinical trials. We retrospectively reviewed outcomes of refractory chronic lymphocytic leukemia patients in two age categories (≥70 and <70 years) treated with single-agent flavopiridol, a drug active in genomically high-risk patients, during two trials. No significant difference between older and younger patients was observed in response rates (43 vs. 47%) or progression-free survival (median 8.7 vs. 9.9 months, P>0.80). Although overall survival was worse in older patients (median 2.1 vs. 2.4 years, P=0.02); when adjusted for other factors this difference was no longer significant (P≥0.10). With the exception of infections (older 29% vs. younger 62%) no significant association with toxicity was observed. These data demonstrate that flavopiridol administration to older chronic lymphocytic leukemia patients is feasible, tolerable, and may have similar efficacy to that in younger patients. Development of treatment approaches including flavopiridol should be considered for these older patients.

Background: Flavopiridol is a cyclin-dependent kinase inhibitor in phase II clinical development for treatment of various forms of cancer. When administered with a pharmacokinetically (PK)-directed dosing schedule, flavopiridol exhibited striking activity in patients with refractory chronic lymphocytic leukemia. This study aimed to evaluate pharmacogenetic factors associated with inter-individual variability in pharmacokinetics and outcomes associated with flavopiridol therapy., Methodology/principal Findings: Thirty-five patients who received single-agent flavopiridol via the PK-directed schedule were genotyped for 189 polymorphisms in genes encoding 56 drug metabolizing enzymes and transporters. Genotypes were evaluated in univariate and multivariate analyses as covariates in a population PK model. Transport of flavopiridol and its glucuronide metabolite was evaluated in uptake assays in HEK-293 and MDCK-II cells transiently transfected with SLCO1B1. Polymorphisms in ABCC2, ABCG2, UGT1A1, UGT1A9, and SLCO1B1 were found to significantly correlate with flavopiridol PK in univariate analysis. Transport assay results indicated both flavopiridol and flavopiridol-glucuronide are substrates of the SLCO1B1/OATP1B1 transporter. Covariates incorporated into the final population PK model included bilirubin, SLCO1B1 rs11045819 and ABCC2 rs8187710. Associations were also observed between genotype and response. To validate these findings, a second set of data with 51 patients was evaluated, and overall trends for associations between PK and PGx were found to be consistent., Conclusions/significance: Polymorphisms in transport genes were found to be associated with flavopiridol disposition and outcomes. Observed clinical associations with SLCO1B1 were functionally validated indicating for the first time its relevance as a transporter of flavopiridol and its glucuronide metabolite. A second 51-patient dataset indicated similar trends between genotype in the SLCO1B1 and other candidate genes, thus providing support for these findings. Further study in larger patient populations will be necessary to fully characterize and validate the clinical impact of polymorphisms in SLCO1B1 and other transporter and metabolizing enzyme genes on outcomes from flavopiridol therapy.

Background: A pharmacokinetically derived schedule of flavopiridol administered as a 30 min intravenous bolus followed by 4-hour continuous intravenous infusion (IVB/CIVI) is active in fludarabine-refractory chronic lymphocytic leukemia, but no studies examining the feasibility and maximum tolerated dose of this schedule have been reported in acute leukemia., Design and Methods: We conducted a phase I dose escalation trial of single-agent flavopiridol in adults with relapsed/refractory acute leukemias, utilizing a modification of the intravenous bolus/continuous intravenous infusion approach, intensifying treatment for administration on days 1, 2, and 3 of 21-day cycles., Results: Twenty-four adults with relapsed/refractory acute myeloid leukemia (n=19) or acute lymphoblastic leukemia (n=5) were enrolled. The median age was 62 years (range, 23-78). The maximum tolerated dose of flavopiridol was 40 mg/m(2) intravenous bolus plus 60 mg/m(2) continuous intravenous infusion (40/60). The dose limiting toxicity was secretory diarrhea. Life-threatening hyperacute tumor lysis syndrome requiring hemodialysis on day 1 was observed in one patient. Pharmacokinetics were dose-dependent with increased clearance observed at the two highest dose levels. Diarrhea occurrence and severity significantly correlated with flavopiridol concentrations at the end of the 4-hour infusion, volume of distribution, and elimination half-life. Modest anti-leukemic activity was observed, with most patients experiencing dramatic but transient reduction/clearance of circulating blasts lasting for 10-14 days. One refractory acute myeloid leukemia patient had short-lived complete remission with incomplete count recovery., Conclusions: Flavopiridol as a single agent given by intravenous bolus/continuous intravenous infusion causes marked, immediate cytoreduction in relapsed/refractory acute leukemias, but objective clinical responses were uncommon. With this schedule, the dose is limited by secretory diarrhea.

Purpose: Patients with chronic lymphocytic leukemia (CLL) with high-risk genomic features achieve poor outcomes with traditional therapies. A phase I study of a pharmacokinetically derived schedule of flavopiridol suggested promising activity in CLL, irrespective of high-risk features. Given the relevance of these findings to treating genetically high-risk CLL, a prospective confirmatory study was initiated., Patients and Methods: Patients with relapsed CLL were treated with single-agent flavopiridol, with subsequent addition of dexamethasone to suppress cytokine release syndrome (CRS). High-risk genomic features were prospectively assessed for response to therapy., Results: Sixty-four patients were enrolled. Median age was 60 years, median number of prior therapies was four, and all patients had received prior purine analog therapy. If patients tolerated treatment during week 1, dose escalation occurred during week 2. Dose escalation did not occur in four patients, as a result of severe tumor lysis syndrome; three of these patients required hemodialysis. Thirty-four patients (53%) achieved response, including 30 partial responses (PRs; 47%), three nodular PRs (5%), and one complete response (1.6%). A majority of high-risk patients responded; 12 (57%) of 21 patients with del(17p13.1) and 14 (50%) of 28 patients with del(11q22.3) responded irrespective of lymph node size. Median progression-free survival among responders was 10 to 12 months across all cytogenetic risk groups. Reducing the number of weekly treatments per cycle from four to three and adding prophylactic dexamethasone, which abrogated interleukin-6 release and CRS (P < or = .01), resulted in improved tolerability and treatment delivery., Conclusion: Flavopiridol achieves significant clinical activity in patients with relapsed CLL, including those with high-risk genomic features and bulky lymphadenopathy. Subsequent clinical trials should use the amended treatment schedule developed herein and prophylactic corticosteroids.

We previously reported interim results of a phase 1 trial in patients with chronic lymphocytic leukemia (CLL) whereby flavopiridol was administered intravenously as a 30-minute bolus followed by 4-hour infusion. We now report full pharmacokinetic (PK) data, correlations of PK with clinical outcomes, and final response and progression-free survival (PFS). Twenty-one (40%) of 52 patients with relapsed CLL achieved a partial response (PR) with a median PFS of 12 months. Responders included 17 (40%) of 43 fludarabine refractory patients, 7 (39%) of 18 patients with del(17p13), and 14 (74%) of 19 patients with del(11q22). Six responders received repeat therapy at relapse, and 5 responded again with a second median PFS of 10 months. Noncompartmental analysis and nonlinear mixed effects modeling was used to estimate PK parameters and evaluate covariates. Two-compartment population parameter estimates were 31.4 L/h, 65.8 L, 8.49 L/h, and 157 L for CL, V1, Q, and V2, respectively. Flavopiridol area under the plasma concentration-time curve (AUC) correlated with clinical response and cytokine release syndrome, and glucuronide metabolite AUC correlated with tumor lysis syndrome. These composite results confirm high activity of this pharmacokinetically derived schedule in relapsed, genetically high-risk CLL. Furthermore, PK describes some, but not all, variability in response and toxicity.

Flavopiridol has shown promising activities in hematologic and solid tumor models, as well as in clinical trials in chronic lymphocytic leukemia patients. Flavopiridol has relatively low solubility and high plasma protein-binding. To address these issues and to provide an alternative strategy to achieve clinical efficacy, we encapsulated flavopiridol into a liposomal carrier and characterized its physicochemical and pharmacokinetic properties. The liposomes, comprising hydrogenated soy phosphatidylcholine (HSPC), cholesterol and poly (ethylene glycol) 2000-distearoyl phosphatidylethanolamine (PEG-DSPE), were prepared by polycarbonate membrane extrusion and then loaded with flavopiridol by a pH-gradient driven remote loading procedure. The liposomes had a mean diameter of 120.7 nm and a flavopiridol entrapment efficiency of 70.4%. Pharmacokinetic study in mice after i.v. bolus injection showed that the liposomal flavopiridol had an increased elimination phase half-life (T((1/2)beta), 339.7 min vs. 57.0 min), decreased clearance (CL, 0.012 L/min vs. 0.036 L/min), and increased area under the plasma concentration-time curve (AUC, 10.8 min micromol/L vs. 3.4 min micromol/L) compared to the free drug. This indicates a significant and potentially beneficial change in flavopiridol pharmacokinetics for the liposomal formulation. Further preclinical studies are warranted to define the toxicity and therapeutic efficacy of this novel formulation.

Patients with chronic lymphocytic leukemia (CLL) with high-risk cytogenetic features such as del(17p13) have limited treatment options and decreased overall survival. Dysfunction of p53 leads to resistance to fludarabine-based therapies. Cyclin-dependent kinase inhibitors (CDKi) are a novel class of agents that induce apoptosis in CLL cells independent of p53 mutational status. The synthetic flavone flavopiridol demonstrated promising in vitro activity in CLL. In initial phase I studies using a continuous infusion dosing schedule in a variety of malignancies, no clinical activity was observed. Detailed pharmacokinetic modeling led to the development of a novel dosing schedule designed to achieve target drug concentrations in vivo. In phase I testing, this dosing schedule resulted in acute tumor lysis syndrome (TLS) as the dose-limiting toxicity. With the implementation of a standardized protocol to prevent severe TLS, flavopiridol was administered safely, and responses were observed in heavily pretreated, fludarabine-refractory patients, cytogenetically high-risk patients, and patients with bulky lymphadenopathy. In a pharmacokinetic analysis, flavopiridol area under the plasma concentration-time curve (AUC) correlated with clinical response and cytokine release syndrome. Phase II studies are under way with encouraging preliminary results. Flavopiridol is currently under active investigation in combination with other agents and as a means to eradicate minimal residual disease in patients following cytoreductive chemotherapy. Several other investigational CDKi in preclinical and early clinical development are briefly discussed in this review.

Lenalidomide, an immunomodulatory agent, and flavopiridol, a broad cyclin-dependent kinase inhibitor, are active therapies for clinical use in genomic high-risk chronic lymphocytic leukemia. A high-performance liquid chromatographic assay with tandem mass spectrometric detection has been developed to simultaneously quantify lenalidomide and flavopiridol in human and mouse plasma to facilitate their combined clinical development. Samples were prepared by liquid-liquid extraction with acetonitrile (ACN)-containing internal standard, genistein, followed by evaporation of solvent and reconstitution in 95/5 H2O/ACN. Lenalidomide and internal standard were separated by reversed-phase liquid chromatography on a C-18 column using a gradient of H2O and ACN, each with 0.1% formic acid. Atmospheric pressure chemical ionization in positive ion mode with single reaction monitoring on a triple quadrupole mass spectrometer was applied to detect transitions of lenalidomide (260.06 > 149.10) and flavopiridol (402.09 > 341.02). Lower limits of quantification of lenalidomide and flavopiridol were 1 and 0.3 nM, respectively. Recoveries of lenalidomide and flavopiridol from human plasma ranged from 99% to 116% throughout their linear ranges. Within- and between-run precision and accuracy of replicate samples were all less than 15%. This is the most sensitive analytical method reported to date for both lenalidomide and flavopiridol. This sensitivity will enable late terminal phase concentration measurements and accurate pharmacokinetic parameter estimation in a planned clinical trial with lenalidomide and flavopiridol in patients with chronic lymphocytic leukemia.

A high-performance liquid chromatographic assay with tandem mass spectrometric detection was developed and validated for quantitation of the broad spectrum kinase inhibitor, flavopiridol, in human plasma. Sample preparation conditions included liquid-liquid extraction in acetonitrile (ACN), drying, and reconstitution in 20/80 water/ACN. Flavopiridol and the internal standard (IS), genistein, were separated by reversed phase chromatography using a C-18 column and a gradient of water with 25 mM ammonium formate and ACN. Electrospray ionization and detection of flavopiridol and genistein were accomplished with single reaction monitoring of m/z 402.09>341.02 and 271.09>152.90, respectively in positive-ion mode [M+H](+) on a triple quadrupole mass spectrometer. Recovery was greater than 90% throughout the linear range of 3-1000 nM. Replicate sample analysis indicated within- and between-run accuracy and precision to be less than 13% throughout the linear range. This method has the lowest lower limit of quantitation (LLOQ) reported to date for flavopiridol, and it allows for more accurate determination of terminal phase concentrations and improved pharmacokinetic parameter estimation in patients receiving an active dosing schedule of flavopiridol.

Effective administration of flavopiridol in advanced-stage chronic lymphocytic leukemia (CLL) is often associated with early biochemical evidence of tumor cell lysis. Previous work using other cell types showed that flavopiridol impacts mitochondria, and in CLL cells flavopiridol down-regulates the mitochondrial protein Mcl-1. We therefore investigated mitochondrial structure and function in flavopiridol-treated CLL patient cells and in the lymphoblastic cell line 697 using concentrations and times at which tumor lysis is observed in treated patients. Mitochondrial membrane depolarization was detected in flavopiridol-treated CLL cells by 6 hours, well before the onset of cell death. Flavopiridol-induced mitochondrial depolarization was not blocked by caspase inhibitors or by the calcium chelator EGTA, but was reduced by Bcl-2 overexpression. Intracellular calcium mobilization was noted at early time points using fluorescence microscopy. Furthermore, electron paramagnetic resonance oximetry showed a gradual but significant reduction in cellular oxygen consumption rate by 6 hours, corresponding with ultrastructural mitochondrial damage detected by electron microscopy. These observations suggest that in CLL and 697 cells, flavopiridol mediates its cytotoxic effects via induction of the mitochondrial permeability transition and changes in intracellular calcium.

Purpose of Review: The synthetic flavone flavopiridol induces apoptosis of chronic lymphocytic leukemia cells in vitro; however, initial studies administering flavopiridol by a 24- to 72-h continuous intravenous infusion demonstrated no clinical activity. This review focuses on a novel dosing regimen that has achieved significant clinical activity in relapsed, poor-risk chronic lymphocytic leukemia., Recent Findings: Binding to human plasma proteins reduces free flavopiridol concentration and makes continuous intravenous infusion dosing ineffective. Pharmacokinetic modeling indicated that administering flavopiridol by a 30-min intravenous bolus followed by a 4-h continuous intravenous infusion would achieve serum concentrations necessary to induce in-vivo apoptosis. Our institution conducted a phase I study in relapsed chronic lymphocytic leukemia. Dose-limiting toxicity was acute tumor lysis syndrome resulting in fatal hyperkalemia. Careful monitoring and aggressive intervention for hyperkalemia, including hemodialysis if necessary, allowed flavopiridol to be given safely. Nineteen of 42 patients responded (45%), including five of 12 patients (42%) with del(17p13) and 13 of 18 patients (72%) with del(11q22)., Summary: Flavopiridol, when administered by a 30-min intravenous bolus followed by a 4-h continuous intravenous infusion, is active in high-risk, refractory chronic lymphocytic leukemia. Careful monitoring and aggressive intervention for tumor lysis syndrome and hyperkalemia is necessary for safe drug administration. Further studies to optimize the dose and schedule of administration, and to study this drug in other hematologic malignancies, are ongoing.

Despite promising preclinical studies with the cyclin-dependent kinase inhibitor flavopiridol in chronic lymphocytic leukemia (CLL) and other diseases, previous clinical trials with this agent have been disappointing. The discovery of differential protein binding of flavopiridol in human and bovine serum contributed to an effective pharmacokinetic-derived schedule of administration of this agent. On the basis of pharmacokinetic modeling using our in vitro results and data from a previous trial, we initiated a phase 1 study using a 30-minute loading dose followed by 4 hours of infusion administered weekly for 4 of 6 weeks in patients with refractory CLL. A group of 42 patients were enrolled on 3 cohorts (cohort 1, 30 mg/m2 loading dose followed by 30 mg/m2 4-hour infusion; cohort 2, 40 mg/m2 loading dose followed by 40 mg/m2 4-hour infusion; and cohort 3, cohort 1 dose for treatments 1 to 4, then a 30 mg/m2 loading dose followed by a 50 mg/m2 4-hour infusion). The dose-limiting toxicity using this novel schedule was hyperacute tumor lysis syndrome. Aggressive prophylaxis and exclusion of patients with leukocyte counts greater than 200x10(9)/L have made this drug safe to administer at the cohort 3 dose. Of the 42 patients treated, 19 (45%) achieved a partial response with a median response duration that exceeds 12 months. Responses were noted in patients with genetically high-risk disease, including 5 (42%) of 12 patients with del(17p13.1) and 13 (72%) of 18 patients with del(11q22.3). Flavopiridol administered using this novel schedule has significant clinical activity in refractory CLL. Patients with bulky disease and high-risk genetic features have achieved durable responses, thereby justifying further study of flavopiridol in CLL and other diseases.

Introduction: : Studies with flavopiridol have demonstrated that this agent has in vitro activity in chronic lymphocytic leukemia (CLL) and promotes apoptosis independent of p53 function or prior fludarabine exposure. Based upon this pre-clinical data, a phase I/II study of 24h flavopiridol was performed., Patients and Methods: : Patients with previously treated CLL patients were enrolled on two sequentially performed cohorts of 13 patients. Patients in the first cohort received flavopiridol (80 mg/m(2) as a 24-h continuous infusion [24h CI]) every 2 weeks. Patients in the second cohort received flavopiridol (80 mg/m(2) as a 24h CI) for week 1 and then were dose escalated by 20mg/m(2) every 2 weeks to a maximal dose of 140 mg/m(2) in the absence of symptoms. Patients received up to 12 doses of therapy., Results: : Thirteen patients with fludarabine-refractory or intolerant CLL enrolled in each cohort. Patients received a median of five treatments in each cohort with only two patients completing all 12 courses of therapy. There were no partial or complete responses noted. Toxicity was manageable in most patients and included anemia, thrombocytopenia, infections, diarrhea, and fatigue., Conclusions: : Flavopiridol as a 24-h continuous infusion has no clinical activity in relapsed, fludarabine-refractory CLL.

Purpose: Flavopiridol has in vitro activity in chronic lymphocytic leukemia (CLL) and promotes apoptosis independent of p53 function or prior fludarabine exposure. We sought to determine if flavopiridol has activity in previously treated CLL using two schedules of administration., Patients and Methods: Patients with previously treated CLL were enrolled in two sequentially done phase II studies. Patients in the first trial received flavopiridol (50 mg/m(2)/d) as a continuous infusion (CI) for 72 hours every 2 weeks. Patients in the second trial received flavopiridol 50 mg/m(2) as a 1-hour bolus (IVB) daily for 3 days repeated every 3 weeks. Patients received up to 12 (CI cohort) or 8 (IVB cohort) cycles of therapy., Results: Fifteen patients were enrolled in the 72-hour CI phase II trial; 6 (40%) had intermediate-risk (Rai stage I or II) and 9 (60%) had high-risk (Rai stage III and IV) stages. No responses were noted in this group; 27% had stable disease and 73% had progressive disease. Thirty-six patients were enrolled in the second IVB trial, with 13 (36%) having intermediate and 23 (64%) having high-risk disease. Four patients (11%) had partial responses, 19 (53%) had stable disease, and 13 (36%) had progressive disease. The progression-free survival for responders in the IVB trial was 3, 3, 9, and 19 months. The median progression-free survival was 2 months [95% confidence interval (95% CI), 1.8-3.8] for patients in the CI trial and 3 months (95% CI, 2.5-7.4) for the IVB trial. The median overall survival was 27 months (95% CI, 20-42) for the CI trial and 24 months (95% CI, 18-31) for the IVB trial. Toxicity was manageable and included mainly myelosuppression, infections, diarrhea, and fatigue., Conclusions: Flavopiridol has modest, schedule-dependent clinical activity in relapsed CLL and warrants future investigation utilizing alternative schedules of administration.