Quantitation of tazemetostat in human plasma using liquid chromatography-tandem mass spectrometry.

To support a phase 1 trial in patients with lymphomas, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for tazemetostat quantitation in 20 µL of human plasma. After protein precipitation, chromatographic separation employed a Kinetex C18 column and a gradient of 0.1% formic acid in both water and acetonitrile, during a 3-min run time. Detection was achieved using a SCIEX 6500+ tandem mass spectrometer with electrospray positive-mode ionization. Validation was based on the latest Food and Drug Administration guidance. With a stable isotopic internal standard, the assay was linear within the range of 10-5000 ng/mL and proved to be accurate (91.9%-103.7%) and precise (<4.4% imprecision). Recovery varied between 93.3% and 121.1%, and matrix effect ranged from -25.5% to -4.9%. Hemolysis, lipemia, and dilution did not impact quantitation. Plasma stability was confirmed after three freeze-thaw cycles, 24 h at room temperature, and 4 months at -80°C. Incurred sample reanalysis yielded 94.4% samples within 20% difference (n = 36). External validation showed a mean bias of -11.1%. Pharmacokinetic (PK) data obtained from three patients suggested variable concentration time profiles, warranting collection of further data. The assay proved to be suitable for tazemetostat quantitation in human plasma and will support clinical studies by defining tazemetostat PKs.

Evaluating the indotecan-neutropenia relationship in patients with solid tumors by population pharmacokinetic modeling and sigmoidal Emax regressions.

PURPOSE: This study aimed at characterizing indotecan population pharmacokinetics and explore the indotecan-neutropenia relationship in patients with solid tumors. METHODS: Population pharmacokinetics were assessed using nonlinear mixed-effects modeling of concentration data from two first-in-human phase 1 trials evaluating different dosing schedules of indotecan. Covariates were assessed in a stepwise manner. Final model qualification included bootstrap simulation, visual and quantitative predictive checks, and goodness-of-fit. A sigmoidal Emax model was developed to describe the relationship between average concentration and maximum percent neutrophil reduction. Simulations at fixed doses were conducted to determine the mean predicted decrease in neutrophil count for each schedule. RESULTS: 518 concentrations from 41 patients supported a three-compartment pharmacokinetic model. Body weight and body surface area accounted for inter-individual variability of central/peripheral distribution volume and intercompartmental clearance, respectively. Estimated typical population values were CL 2.75 L/h, Q3 46.0 L/h, and V3 37.9 L. The estimated value of Q2 for a typical patient (BSA = 1.96 m2) was 17.3 L/h, while V1 and V2 for a typical patient (WT = 80 kg) was 33.9 L and 132 L. The final sigmoidal Emax model estimated that half-maximal ANC reduction occurs at an average concentration of 1416 µg/L and 1041 µg/L for the daily and weekly regimens, respectively. Simulations of the weekly regimen demonstrated lower percent reduction in ANC compared to the daily regimen at equivalent cumulative fixed doses. CONCLUSION: The final PK model adequately describes indotecan population pharmacokinetics. Fixed dosing may be justified based on covariate analysis and the weekly dosing regimen may have a reduced neutropenic effect.

Dexamethasone to prevent everolimus-induced stomatitis (Alliance MIST Trial: A221701).

mTOR inhibitors such as everolimus may cause oral stomatitis, often a dose-limiting toxicity. Prior clinical research has suggested that a dexamethasone mouth rinse might help prevent and/or treat this. Alliance A221701 was a randomized phase III trial of patients initiating 10 mg daily oral everolimus that compared dexamethasone mouthwash taken preventively (initial dexamethasone group) versus therapeutically (initial placebo group) to assess two coprimary endpoints: the incidence of mTOR inhibitor-associated stomatitis (mIAS), and the area under the curve (AUC) of mIAS-associated pain over an 8-week treatment period. A Fisher's exact test was used to compare the incidences while a Wilcoxon rank-sum test was used to compare the AUCs. In addition, we performed an exploratory analysis of the association of everolimus trough concentrations and toxicity using a Mann-Whitney U test. Due to slow accrual, this study closed after 39 patients were randomized (19 to upfront placebo and 20 to upfront dexamethasone). There were no significant differences between groups seen in either of the coprimary endpoints; furthermore, we found no association between whole blood everolimus trough concentrations and toxicity. Although limited by poor enrollment, the results of this study do not suggest that prophylactic dexamethasone mouthwash is superior to therapeutic dexamethasone mouthwash (initiated at the first sign of mouth pain) for reducing the incidence or severity of mIAS from everolimus.

Evaluation of the pharmacokinetic drug-drug interaction potential of iohexol, a renal filtration marker.

PURPOSE: Carboplatin dose is calculated based on kidney function, commonly estimated with imperfect creatinine-based formulae. Iohexol is used to measure glomerular filtration rate (GFR) and allows calculation of a more appropriate carboplatin dose. To address potential concerns that iohexol administered during a course of chemotherapy impacts that therapy, we performed in vitro and in vivo pharmacokinetic drug-drug interaction evaluations of iohexol. METHODS: Carboplatin was administered IV to female mice at 60 mg/kg with or without iohexol at 300 mg/kg. Plasma ultrafiltrate, kidney and bone marrow platinum was quantitated by atomic absorption spectrophotometry. Paclitaxel microsomal and gemcitabine cytosolic metabolism as well as metabolism of CYP and UGT probes was assessed with and without iohexol at 300 µg/mL by LC-MS/MS. RESULTS: In vivo carboplatin exposure was not significantly affected by iohexol co-administration (platinum AUC combination vs alone: plasma ultrafiltrate 1,791 vs 1920 µg/mL min; kidney 8367 vs 9757 µg/g min; bone marrow 12.7 vs 12.7 µg/mg-protein min). Paclitaxel microsomal metabolism was not impacted (combination vs alone: 6-α-OH-paclitaxel 38.3 versus 39.4 ng/mL/60 min; 3-p-OH-paclitaxel 26.2 versus 27.7 ng/mL/60 min). Gemcitabine human cytosolic elimination was not impacted (AUC combination vs gemcitabine alone: dFdU 24.1 versus 23.7 µg/mL/30 min). Iohexol displayed no relevant inhibition of the CYP and UGT enzymes in human liver microsomes. CONCLUSIONS: Iohexol is unlikely to affect the clinical pharmacokinetics of carboplatin, paclitaxel, gemcitabine, or other agents used in combination with carboplatin treatment. Measuring GFR with iohexol to better dose carboplatin is unlikely to alter the safety or efficacy of chemotherapy through pharmacokinetic drug-drug interactions.

Quantitation of iohexol, a glomerular filtration marker, in human plasma by LC-MS/MS.

We developed a high-performance liquid chromatography mass spectrometry method for quantitating iohexol in 50 µL human plasma. After acetonitrile protein precipitation, chromatographic separation was achieved with a Shodex Asahipak NH2P-50 2D (5 µm, 2 × 150 mm) column and a gradient of 0.1 % formic acid in acetonitrile and 0.1 % formic acid in water over a 10 min run time. Mass spectrometric detection was performed on a Micromass Quatromicro triple-stage bench-top mass spectrometer with electrospray, positive-mode ionization. The assay was linear from 1 to 500 µg/mL for iohexol, proved to be accurate (101.3-102.1 %) and precise (<3.4 %CV), and fulfilled Food and Drug Administration (FDA) criteria for bioanalytical method validation. Recovery from plasma was 53.1-64.2 % and matrix effect was trivial (-3.4 to -1.3 %). Plasma freeze thaw stability (97.4-99.4 %), stability for 5 months at -80 °C (95.5-103.3 %), and stability for 4 h at room temperature (100.6-103.3 %) were all acceptable. This validated assay using a deuterated internal standard will be an important tool in measuring iohexol clearance and determining glomerular filtration rate (GFR) in patients.

NCI Comparative Oncology Program Testing of Non-Camptothecin Indenoisoquinoline Topoisomerase I Inhibitors in Naturally Occurring Canine Lymphoma.

PURPOSE: Only one chemical class of topoisomerase I (TOP1) inhibitors is FDA approved, the camptothecins with irinotecan and topotecan widely used. Because of their limitations (chemical instability, drug efflux-mediated resistance, and diarrhea), novel TOP1 inhibitors are warranted. Indenoisoquinoline non-camptothecin topoisomerase I (TOP1) inhibitors overcome chemical instability and drug resistance that limit camptothecin use. Three indenoisoquinolines, LMP400 (indotecan), LMP776 (indimitecan), and LMP744, were examined in a phase I study for lymphoma-bearing dogs to evaluate differential efficacy, pharmacodynamics, toxicology, and pharmacokinetics. EXPERIMENTAL DESIGN: Eighty-four client-owned dogs with lymphomas were enrolled in dose-escalation cohorts for each indenoisoquinoline, with an expansion phase for LMP744. Efficacy, tolerability, pharmacokinetics, and target engagement were determined. RESULTS: The MTDs were 17.5 mg/m2 for LMP 776 and 100 mg/m2 for LMP744; bone marrow toxicity was dose-limiting; up to 65 mg/m2 LMP400 was well-tolerated and MTD was not reached. None of the drugs induced notable diarrhea. Sustained tumor accumulation was observed for LMP744; γH2AX induction was demonstrated in tumors 2 and 6 hours after treatment; a decrease in TOP1 protein was observed in most lymphoma samples across all compounds and dose levels, which is consistent with the fact that tumor response was also observed at low doses LMP744. Objective responses were documented for all indenoisoquinolines; efficacy (13/19 dogs) was greatest for LMP744. CONCLUSIONS: These results demonstrate proof-of-mechanism for indenoisoquinoline TOP1 inhibitors supporting their further clinical development. They also highlight the value of the NCI Comparative Oncology Program (https://ccr.cancer.gov/Comparative-Oncology-Program) for evaluating novel therapies in immunocompetent pets with cancers.

Rapid Homogeneous Immunoassay to Quantify Gemcitabine in Plasma for Therapeutic Drug Monitoring.

BACKGROUND: Gemcitabine (2',2'-difluoro-2'-deoxycytidine) is a nucleoside analog used as a single agent and in combination regimens for the treatment of a variety of solid tumors. Several studies have shown a relationship between gemcitabine peak plasma concentration (Cmax) and hematological toxicity. An immunoassay for gemcitabine in plasma was developed and validated to facilitate therapeutic drug monitoring (TDM) by providing an economical, robust method for automated chemistry analyzers. METHODS: A monoclonal antibody was coated on nanoparticles to develop a homogenous agglutination inhibition assay. To prevent ex vivo degradation of gemcitabine in blood, tetrahydrouridine was used as a sample stabilizer. Validation was conducted for precision, recovery, cross-reactivity, and linearity on a Beckman Coulter AU480. Verification was performed on an AU5800 in a hospital laboratory. A method comparison was performed with (LC-MS/MS) liquid chromatography tandem mass spectrometry using clinical samples. Selectivity was demonstrated by testing cross-reactivity of the major metabolite, 2',2'-difluorodeoxyuridine. RESULTS: Coefficients of variation for repeatability and within-laboratory precision were <8%. The deviation between measured and assigned values was <3%. Linear range was from 0.40 to 33.02 µ/mL (1.5-125.5 µM). Correlation with validated LC-MS/MS methods was R = 0.977. The assay was specific for gemcitabine: there was no cross-reactivity to 2',2'-difluorodeoxyuridine, chemotherapeutics, concomitant, or common medications tested. Tetrahydrouridine was packaged in single-use syringes. Gemcitabine stability in whole blood was extended to 8 hours (at room temperature) and in plasma to 8 days (2-8°C). CONCLUSIONS: The assay demonstrated the selectivity, test range, precision, and linearity to perform reliable measurements of gemcitabine in plasma. The addition of stabilizer improved the sample handling. Using general clinical chemistry analyzers, gemcitabine could be measured for TDM.

Simultaneous quantitation of abiraterone, enzalutamide, N-desmethyl enzalutamide, and bicalutamide in human plasma by LC-MS/MS.

Inhibiting the androgen receptor (AR) pathway is an important clinical strategy in metastatic prostate cancer. Novel agents including abiraterone acetate and enzalutamide have been shown to prolong life in men with metastatic, castration-resistant prostate cancer (mCRPC). To evaluate the pharmacokinetics of AR-targeted agents, we developed and validated an LC-MS/MS assay for the quantitation of enzalutamide, N-desmethyl enzalutamide, abiraterone and bicalutamide in 0.05mL human plasma. After protein precipitation, chromatographic separation was achieved with a Phenomenex Synergi Polar-RP column and a linear gradient of 0.1% formic acid in methanol and water. Detection with an ABI 4000Q mass spectrometer utilized electrospray ionization in positive multiple reaction monitoring mode. The assay was linear over the ranges of 1-1000ng/mL for abiraterone and bicalutamide and 100-30,000ng/mL for N-desmethyl enzalutamide and enzalutamide and proved to be accurate (92.8-107.7%) and precise (largest was 15.3% CV at LLOQ for bicalutamide), and fulfilled FDA criteria for bioanalytical method validation. We demonstrated the suitability of this assay in plasma from patients who were administered enzalutamide 160mg, abiraterone 1000mg and bicalutamide 50mg once a day as monotherapy or in combination. The LC-MS/MS assay that has been developed will be an essential tool that further defines the pharmacology of the combinations of androgen synthesis or AR-receptor targeted agents.

LC-MS/MS assay for the quantitation of FdCyd and its metabolites FdUrd and FU in human plasma.

The hypomethylating agent 5-fluoro-2'-deoxycytidine (FdCyd, NSC 48006) is being evaluated clinically both via the intravenous route and via the oral route in combination with 3,4,5,6-tetrahydrouridine (THU), a potent inhibitor of FdCyd catabolism. To determine the pharmacokinetics of FdCyd and downstream metabolites, we developed and validated an LC-MS/MS assay for the quantitation of FdCyd, 5-fluoro-2'-deoxyuridine (FdUrd), and 5-fluorouracil (FU) in 0.2mL human plasma. After acetonitrile protein precipitation, the sample was split and separate chromatography was achieved for FdCyd with a Synergi Polar-RP column and for FdUrd and FU with a Shodex Asahipak NH2P-50 2D column. Gradients of 0.1% acetic acid in acetonitrile and water were used. Detection with a Quattromicro quadrupole mass spectrometer with electrospray ionization in positive-ion (FdCyd) or negative-ion (FdUrd and FU) multiple reaction monitoring (MRM) mode. The assay was linear from 5 to 3000ng/mL for all three analytes and proved to be accurate (96.7-105.5%) and precise (<8.1%CV), and fulfilled FDA criteria for bioanalytical method validation. We demonstrated the suitability of this assay for measuring FdCyd and metabolites FdUrd and FU in plasma from a patient who was administered 120mg PO FdCyd 30min after 3000mg THU. Our LC-MS/MS assay will be an essential tool to further define the pharmacology of FdCyd in ongoing and future studies.

Clinical and pharmacologic evaluation of two dosing schedules of indotecan (LMP400), a novel indenoisoquinoline, in patients with advanced solid tumors.

PURPOSE: Indenoisoquinolines are non-camptothecin topoisomerase I (TopI) inhibitors that overcome the limitations of camptothecins: chemical instability and camptothecin resistance. Two dosing schedules of the novel indenoisoquinoline, indotecan (LMP400), were evaluated in patients with advanced solid tumors. METHODS: The maximum tolerated dose (MTD), toxicities, and pharmacokinetics of two indotecan drug administration schedules (daily for 5 days or weekly) were investigated. Modulation of TopI and the phosphorylation of histone H2AX (γH2AX) were assayed in tumor biopsies; γH2AX levels were also evaluated in circulating tumor cells (CTCs) and hair follicles to assess DNA damage response. RESULTS: An MTD of 60 mg/m(2)/day was established for the daily regimen, compared to 90 mg/m(2) for the weekly regimen. The TopI response to drug showed target engagement in a subset of tumor biopsies. Pharmacokinetics profiles demonstrated a prolonged terminal half-life and tissue accumulation compared to topotecan. Dose-dependent decreases in total CTCs were measured in seven patients. Formation of γH2AX-positive foci in CTCs (day 3) and hair follicles (4-6 h) was observed following treatment. CONCLUSIONS: We established the MTD of two dosing schedules for a novel TopI inhibitor, indotecan. Target engagement was demonstrated as Top1 downregulation and γH2AX response. No objective responses were observed on either schedule in this small patient cohort. The principal toxicity of both schedules was myelosuppression; no significant gastrointestinal problems were observed. Increased DNA damage response was observed in CTCs, hair follicles, and a subset of tumor biopsies.

A pharmacokinetic analysis of cisplatin and 5-fluorouracil in a patient with esophageal cancer on peritoneal dialysis.

BACKGROUND: Very little is known about the pharmacokinetics of chemotherapeutic agents in patients also being treated with continuous ambulatory peritoneal dialysis. We sought to evaluate the pharmacokinetics of cisplatin and 5-fluorouracil in plasma and peritoneal dialysate in a patient being treated for esophageal adenocarcinoma. METHODS: A single patient with esophageal adenocarcinoma and on peritoneal dialysis for end-stage renal disease was treated with cisplatin 25 mg/m(2) on day 1 of weeks 1 and 5 and continuous infusional 5-fluorouracil 1000 mg/m(2)/day on days 1-4 of weeks 1 and 5 along with daily radiation therapy. Intense plasma and dialysate sampling was performed during the week 5 administration, followed by quantitation of platinum by atomic absorption spectrophotometry and 5-fluorouracil by LC-MS/MS. RESULTS: Following systemic administration, clearance of ultrafilterable (active) platinum over the first 6 h was 20.8 L/h, which is lower than previously reported clearance levels of ultrafilterable platinum. Total platinum AUC was 131 µg h/mL, also higher than an AUC previously reported for total platinum in patients with normal renal function. Platinum-related material was detected in the peritoneal cavity, but this is likely inactive. 5-Fluorouracil penetrated the intraperitoneal cavity, but the contribution of peritoneal dialysis to drug clearance was negligible at 0.072 %. CONCLUSIONS: Administration of intravenous cisplatin and 5-fluorouracil chemotherapy to a patient treated with continuous ambulatory peritoneal dialysis is feasible, but clearance in dialysate is nominal, thus suggesting that dose reduction is indicated for cisplatin. Systemic drug administration results in limited intraperitoneal penetration of 5-fluorouracil and inactive platinum species.

Oral and intravenous pharmacokinetics of 5-fluoro-2'-deoxycytidine and THU in cynomolgus monkeys and humans.

INTRODUCTION: 5-Fluoro-2'-deoxycytidine (FdCyd; NSC48006), a fluoropyrimidine nucleoside inhibitor of DNA methylation, is degraded by cytidine deaminase (CD). Pharmacokinetic evaluation was carried out in cynomolgus monkeys in support of an ongoing phase I study of the PO combination of FdCyd and the CD inhibitor tetrahydrouridine (THU; NSC112907). METHODS: Animals were dosed intravenously (IV) or per os (PO). Plasma samples were analyzed by LC-MS/MS for FdCyd, metabolites, and THU. Clinical chemistry and hematology were performed at various times after dosing. A pilot pharmacokinetic study was performed in humans to assess FdCyd bioavailability. RESULTS: After IV FdCyd and THU administration, FdCyd C(max) and AUC increased with dose. FdCyd half-life ranged between 22 and 56 min, and clearance was approximately 15 mL/min/kg. FdCyd PO bioavailability after THU ranged between 9 and 25 % and increased with increasing THU dose. PO bioavailability of THU was less than 5 %, but did result in plasma concentrations associated with inhibition of its target CD. Human pilot studies showed comparable bioavailability for FdCyd (10 %) and THU (4.1 %). CONCLUSION: Administration of THU with FdCyd increased the exposure to FdCyd and improved PO FdCyd bioavailability from <1 to 24 %. Concentrations of THU and FdCyd achieved after PO administration are associated with CD inhibition and hypomethylation, respectively. The schedule currently studied in phase I studies of PO FdCyd and THU is daily times three at the beginning of the first and second weeks of a 28-day cycle.

Plasma pharmacokinetics of the indenoisoquinoline topoisomerase I inhibitor, NSC 743400, in rats and dogs.

PURPOSE: NSC 743400 is a novel synthetic indenoisoquinoline analog under development as an anticancer agent. It is a potent topoisomerase I inhibitor with potential therapeutic advantages over FDA-approved camptothecin derivatives. In preparation for clinical development of NSC 743400, we determined the pharmacokinetics after administration to rats and dogs. METHODS: NSC 743400 was administered intravenously at a dose of 12 or 24 mg/m(2) to rats (single bolus) or 10, 50, 100, 215, 430, or 646 mg/m(2) (intravenous infusion) or 860 or 1720 mg/m(2) (orally) to dogs. RESULTS: Intravenously administered NSC 743400 was eliminated from both species with an estimated t 1/2 of 2-5 h in rat and 6-14 h in dog. Elimination t 1/2 increased with dose in dog. Area under the plasma concentration-versus-time curve (AUC) was comparable in both species, at about 300-400 h ng/mL for the approximately 10 mg/m(2) dose groups. Overall, AUC values increased proportionally with dose for both species but had evidence of more than proportional exposure at the highest doses. Oral dosing resulted in variable drug absorption. CONCLUSIONS: The pharmacokinetic data were used to plan first-in-human clinical trials.

A phase I trial of two sequence-specific schedules of decitabine and vorinostat in patients with acute myeloid leukemia.

This phase I trial evaluated two schedules of escalating vorinostat in combination with decitabine every 28 days: (i) sequential or (ii) concurrent. There were three dose-limiting toxicities: grade 3 fatigue and generalized muscle weakness on the sequential schedule (n = 1) and grade 3 fatigue on the concurrent schedule (n = 2). The maximum tolerated dose was not reached on both planned schedules. The overall response rate (ORR) was 23% (three complete response [CR], two CR with incomplete incomplete blood count recovery [CRi], one partial response [PR] and two morphological leukemic free state [MLFS]). The ORR for all and previously untreated patients in the sequential arm was 13% (one CRi; one MLFS) and 0% compared to 30% (three CR; one CRi; one PR; one MLFS) and 36% in the concurrent arm (p = 0.26 for both), respectively. Decitabine plus vorinostat was safe and has clinical activity in patients with previously untreated acute myeloid leukemia. Responses appear higher with the concurrent dose schedule. Cumulative toxicities may limit long-term usage on the current dose/schedules.

Effect of age on the pharmacokinetics of busulfan in patients undergoing hematopoietic cell transplantation; an alliance study (CALGB 10503, 19808, and 100103).

PURPOSE: Older patients with acute myeloid leukemia (AML) and myelodysplastic syndrome have often been excluded from myeloablative-conditioning regimens containing busulfan because of non-disease-related morbidity and mortality. We hypothesized that busulfan clearance (BuCL) in older patients (>60 years) would be reduced compared to that in younger patients, potentially explaining observed differences in busulfan tolerability. METHODS: AML patients in three CALGB hematopoietic cell transplantation studies were treated with a conditioning regimen using IV busulfan, dosed at 0.8 mg/kg. Plasma busulfan concentrations were determined by LC-MS and analyzed by non-compartmental methods. BuCL was normalized to actual (ABW), ideal (IBW), or corrected (CBW) body weight (kg). Differences in BuCL between age groups were examined using the Wilcoxon rank sum test. RESULTS: One hundred and eighty-five patients were accrued; 174 provided useable pharmacokinetic data. Twenty-nine patients ≥ 60 years old (median 66; range 60-74) had a significantly higher BuCL versus those <60 years old (median 50; range 18-60): BuCL 236 versus 168 mL/min, p = 0.0002; BuCL/ABW 3.0 versus 2.1 mL/min/kg, p = 0.0001; BuCL/IBW 3.8 versus 2.6 mL/min/kg, p = 0.0035; BuCL/CBW 3.4 versus 2.6 mL/min/kg, p = 0.0005. Inter-patient variability in clearance (CV %) was up to 48 % in both age groups. Phenytoin administration, a potential confounder, did not affect BuCL, regardless of weight normalization (p > 0.34). CONCLUSIONS: Contrary to our hypothesis, BuCL was significantly higher in older patients compared to younger patients in these studies and does not explain the previously reported increase in busulfan toxicity observed in older patients.

Phase I study of decitabine in combination with vorinostat in patients with advanced solid tumors and non-Hodgkin's lymphomas.

PURPOSE: This phase I study evaluated the safety, tolerability, pharmacokinetics, and preliminary efficacy of the combination of decitabine with vorinostat. PATIENTS AND METHODS: Patients with advanced solid tumors or non-Hodgkin's lymphomas were eligible. Sequential and concurrent schedules were studied. RESULTS: Forty-three patients were studied in 9 different dose levels (6 sequential and 3 concurrent). The maximum tolerated dose (MTD) on the sequential schedule was decitabine 10 mg/m(2)/day on days 1 to 5 and vorinostat 200 mg three times a day on days 6 to 12. The MTD on the concurrent schedule was decitabine 10 mg/m(2)/day on days 1 to 5 with vorinostat 200 mg twice a day on days 3 to 9. However, the sequential schedule of decitabine 10 mg/m(2)/day on days 1 to 5 and vorinostat 200 mg twice a day on days 6 to 12 was more deliverable than both MTDs with fewer delays on repeated dosing and it represents the recommended phase II (RP2D) dose of this combination. Dose-limiting toxicities during the first cycle consisted of myelosuppression, constitutional and gastrointestinal symptoms and occurred in 12 of 42 (29%) patients evaluable for toxicity. The most common grade 3 or higher adverse events were neutropenia (49% of patients), thrombocytopenia (16%), fatigue (16%), lymphopenia (14%), and febrile neutropenia (7%). Disease stabilization for 4 cycles or more was observed in 11 of 38 (29%) evaluable patients. CONCLUSION: The combination of decitabine with vorinostat is tolerable on both concurrent and sequential schedules in previously treated patients with advanced solid tumors or non-Hodgkin's lymphomas. The sequential schedule was easier to deliver. The combination showed activity with prolonged disease stabilization in different tumor types.

Plasma pharmacokinetics and oral bioavailability of the 3,4,5,6-tetrahydrouridine (THU) prodrug, triacetyl-THU (taTHU), in mice.

PURPOSE: Cytidine drugs, such as gemcitabine, undergo rapid catabolism and inactivation by cytidine deaminase (CD). 3,4,5,6-tetrahydrouridine (THU), a potent CD inhibitor, has been applied preclinically and clinically as a modulator of cytidine analogue metabolism. However, THU is only 20% orally bioavailable, which limits its preclinical evaluation and clinical use. Therefore, we characterized THU pharmacokinetics after the administration to mice of the more lipophilic pro-drug triacetyl-THU (taTHU). METHODS: Mice were dosed with 150 mg/kg taTHU i.v. or p.o. Plasma and urine THU concentrations were quantitated with a validated LC-MS/MS assay. Plasma and urine pharmacokinetic parameters were calculated non-compartmentally and compartmentally. RESULTS: taTHU did not inhibit CD. THU, after 150 mg/kg taTHU i.v., had a 235-min terminal half-life and produced plasma THU concentrations >1 µg/mL, the concentration shown to inhibit CD, for 10 h. Renal excretion accounted for 40-55% of the i.v. taTHU dose, 6-12% of the p.o. taTHU dose. A two-compartment model of taTHU generating THU fitted the i.v. taTHU data best. taTHU, at 150 mg/kg p.o., produced a concentration versus time profile with a plateau of approximately 10 µg/mL from 0.5-2 h, followed by a decline with a 122-min half-life. Approximately 68% of i.v. taTHU is converted to THU. Approximately 30% of p.o. taTHU reaches the systemic circulation as THU. CONCLUSIONS: The availability of THU after p.o. taTHU is 30%, when compared to the 20% achieved with p.o. THU. These data will support the clinical studies of taTHU.

A phase I study of vorinostat in combination with idarubicin in relapsed or refractory leukaemia.

Histone deacetylase inhibitors (HDACi) affect chromatin remodelling and modulate the expression of aberrantly silenced genes. HDACi have single-agent clinical activity in haematological malignancies and have synergistic anti-leukaemia activity when combined with anthracyclines in vitro. We conducted a two-arm, parallel Phase I trial to investigate two schedules of escalating doses of vorinostat (Schedule A: thrice daily (TID) for 14 d; B: TID for 3 d) in combination with a fixed dose of idarubicin in patients with refractory leukaemia. Of the 41 patients enrolled, 90% had acute myeloid leukaemia, with a median of 3 prior therapies. Seven responses (17%) were documented (two complete response (5%), one complete response without platelet recovery (2.5%), and four marrow responses). The 3-d schedule of vorinostat was better tolerated than the 14-d schedule. The maximum tolerated dose for vorinostat was defined as 400 mg TID for 3 d. The most common grade 3 and 4 toxicities included mucositis, fatigue and diarrhoea. Correlative studies demonstrated histone acetylation in patients on therapy and modulation of CDKN1A and TOP2A (topoisomerase II) gene expression. Pharmacokinetic analysis confirmed a dose-related elevation in plasma vorinostat concentrations. The combination of vorinostat and idarubicin is generally tolerable and active in patients with advanced leukaemia and should be studied in the front-line setting.

A phase I, pharmacokinetic, and pharmacodynamic study of two schedules of vorinostat in combination with 5-fluorouracil and leucovorin in patients with refractory solid tumors.

PURPOSE: We conducted a phase I clinical trial to determine the maximum tolerated dose (MTD) of daily or twice daily vorinostat x 3 days when combined with fixed doses of 5-fluorouracil (FU) and leucovorin every 2 weeks. EXPERIMENTAL DESIGN: Vorinostat doses were escalated in a standard 3 x 3 phase I design. FU/leucovorin was started on day 2 of vorinostat and consisted of leucovorin 400 mg/m(2) i.v. over 2 hours followed by FU 400 mg/m(2) i.v. bolus and 2,400 mg/m(2) over 46 hours (sLV5FU2). RESULTS: Forty-three patients were enrolled. Grade 3 fatigue, and hand and foot syndrome were the dose-limiting toxicities (DLT) at the 2,000 mg vorinostat once-daily dose level. Grade 3 fatigue and mucositis were DLTs at the 800 mg vorinostat twice-daily dose level. None of six patients at the 1,700 mg once daily or six patients at the 600 mg twice daily dose levels had a DLT; those dose levels represent the MTD. Twenty-one of 38 patients with FU-refractory colorectal cancer had stable disease, and one had a partial response. Vorinostat maximum serum concentrations at the MTD exceeded concentrations associated with thymidylate synthase downregulation in vitro. No pharmacokinetic interactions were noted between vorinostat and FU. CONCLUSIONS: The MTD of vorinostat in combination with sLV5FU2 is 1,700 mg orally once daily x 3 or 600 mg orally twice daily x 3 days every 2 weeks. Clinical activity in refractory colorectal cancer supports further clinical development of this combination.

Liquid chromatography-tandem mass spectrometric assay for the quantitation in human plasma of the novel indenoisoquinoline topoisomerase I inhibitors, NSC 743400 and NSC 725776.

Topoisomerase I (Topo I) is a recognized target for ovarian, lung, and colorectal cancer therapy. The FDA-approved camptothecin (CPT) Topo I inhibitors, topotecan and irinotecan are labile and their effects are rapidly reversible. The indenoisoquinoline topoisomerase I inhibitors, NSC 743400 and NSC 725776, have been developed as a new generation of Topo I inhibitors and are being advanced to clinical evaluation. To support the clinical development of NSC 743400 and NSC 725776, we developed and validated, according to FDA guidelines, LC-MS/MS assays for the sensitive, accurate and precise quantitation of NSC 743400 and NSC 725776 in 0.2 mL human plasma. After ethyl acetate extraction, separation was achieved with a Synergi Polar RP column and a gradient of 0.1% formic acid in acetonitrile:water. NSC 743400 and NSC 725776 eluted at approximately 3 min, and the total run time was 14 min. Detection consisted of electrospray, positive-mode ionization mass spectrometry. Between 3 and 1000 ng/mL, accuracy was 96.9-108.2% for NSC 743400 and 95.1-106.7% for NSC 725776, and precision was <11.4% for NSC 743400 and <5.9% for NSC 725776. Extraction recovery was >80% for both analytes, and ion suppression ranged from -46.7 to 5.7%. The use of isotopically labeled internal standards and a wash phase at the end of the run were necessary to achieve adequate assay performance. Protein binding in human plasma as assessed by equilibrium dialysis showed both indenoisoquinolines to be more than 98% protein bound.