Slower degradation rate of cytarabine in blood samples from acute myeloid leukemia by comparison with control samples.

PURPOSE: Cytarabine, a key chemotherapy agent for acute myeloid leukemia (AML) treatment, is deaminated into inactive uracil-arabinoside by cytidine deaminase. This deamination leads to samples stability issues with respect to clinical pharmacokinetic trials. The aim of our study was to study in vitro cytarabine stability in blood samples obtained from AML patients. METHODS: Cytarabine quantification was performed using a fully validated LC/MS/MS method. In vitro cytarabine stability was assessed at room temperature over 24 h in samples coming from 14 AML patients and 7 control patients (CTRL) with no hematological malignancy. In vitro concentrations versus time data were analyzed using a noncompartmental approach. RESULTS: Cytarabine in vitro area under the curve (AUCIVlast) was 22-fold higher in AML samples as compared to CTRL samples (AML mean (standard deviation (SD)), 51,829 (27,004) h ng/mL; CTRL mean (SD), 2356 (1250) h ng/mL, p = 0.00019). This increase was associated with a prolonged in vitro degradation half-life (t1/2IVdeg AML mean (SD), 15 (11.8) h; CTRL mean (SD), 0.36 (0.37) h, p = 0.0033). Multiple linear regression analysis showed that AML diagnosis significantly influenced t1/2IVdeg and AUCIVlas relationship. CONCLUSION: Cytarabine stability is higher in AML than in CTRL samples. The absence of correlation between t1/2IVdeg and AUCIVlast in AML samples suggests that in vitro cytarabine degradation in AML is complex. These results open perspectives including the evaluation of the clinical relevance and the involved molecular mechanisms.

Pharmacokinetics, drug metabolism, and tissue distribution of CPX-351 in animals.

CPX-351, a liposomal encapsulation of cytarabine and daunorubicin at a synergistic 5:1 molar ratio, is indicated for adults with newly diagnosed, therapy-related acute myeloid leukemia or acute myeloid leukemia with myelodysplasia-related changes. In preclinical species, this article demonstrated (1) similar release of cytarabine and daunorubicin by CPX-351 in plasma; (2) similar patterns of metabolism of cytarabine and daunorubicin following administration of CPX-351 versus non-liposomal cytarabine/daunorubicin combination; (3) prolonged tissue exposure to CPX-351; (4) dramatically different tissue distribution of cytarabine and daunorubicin following administration of CPX-351 versus non-liposomal combination (tissue:plasma ratios generally <1 versus >1, respectively); and (5) dramatically lower unbound plasma and tissue concentrations of cytarabine and daunorubicin following administration of CPX-351 versus non-liposomal combination. Together, these results provide insight into the safety profile of CPX-351, as well as mechanisms that drive the improved efficacy observed for CPX-351 versus the conventional 7 + 3 cytarabine/daunorubicin regimen in clinical studies.

Pharmacokinetics of multivesicular liposomal encapsulated cytarabine when administered subcutaneously in dogs.

BACKGROUND: Prolonged cytotoxic concentrations of cytarabine (CA) are required for maximum cytotoxicity. DepoCyt is a human liposomal cytarabine (LC) product that lasts longer in plasma and CSF compared with free CA (FC). The use of LC has not been evaluated in dogs. OBJECTIVES: To perform a LC pharmacokinetic (PK) study when administered SC in dogs. ANIMALS: Five healthy female beagles. METHODS: Three-period, 3-treatment, nonblinded, randomized, and crossover design, including a pilot study. LC was administered at 50 mg/m2 SC and FC was administered at 25 and 50 mg/m2 SC and IV. Plasma CA concentrations were measured until 240, 72, and 8 hours after SC LC, SC FC, and IV FC administration, respectively. CA plasma concentrations were quantitated by ultra-high-performance liquid chromatography with mass spectrometry (MS/MS) detection and concentration-time profiles were evaluated by noncompartmental analysis. RESULTS: Subcutaneous LC administration resulted in a maximum plasma concentration of 26.3 to 59.78 ng/mL, time to reach maximum plasma concentration of 2 hours, area under the concentration-time curve to last measurable concentration of 669.3 to 1126 h × ng/mL, and plasma bioavailability (%F) of 19.6% to 31.3%. The PK profiles of FC after SC and IV administration differed when compared with LC. CONCLUSIONS AND CLINICAL IMPORTANCE: In healthy dogs, SC LC administration at 50 mg/m2 results in measurable plasma CA concentrations, is apparently safe and well tolerated, but does not result in prolonged cytotoxic plasma concentrations. Poor absorption of LC prevented establishment of a complete LC PK profile.

Simultaneous determination of cytosine arabinoside and its metabolite uracil arabinoside in human plasma by LC-MS/MS: Application to pharmacokinetics-pharmacogenetics pilot study in AML patients.

Purine analogs like aracytine (AraC) are a mainstay for treating acute myeloid leukemia (AML). There are marked differences in drug dosing and scheduling depending on the protocols when treating AML patients with AraC. Large inter-patient pharmacokinetics variability has been reported, and genetic polymorphisms affecting cytidine deaminase (CDA), the liver enzyme responsible for the conversion of Ara-C to inactive uracil arabinoside (AraU) could be a culprit for either life-threatening toxicities or poor efficacy related to substantial changes in plasma exposure levels among patients. The quantitative determination of Ara-C in plasma is challenging due the required sensitivity because of the short half-life of this drug (i.e., <10 min) and the metabolic instability in biological matrix upon sampling possibly resulting in erratic values. We developed and validated a liquid chromatography tandem mass spectrometry method (UPLC-MS/MS) for the simultaneous determination of Ara-C and Ara-U metabolite in human plasma. After simple and rapid precipitation, analytes were successfully separated and quantitated over a 1-500 ng/ml range for Ara-C and 250-7500 ng/ml range for AraU. The performance and reliability of this method was tested as part of an investigational study in AML patients treated with low dose cytarabine and confirmed marked differences in drug exposure levels and metabolic ratio, depending on the CDA status of the patients. Overall, this new method meets the requirements of current bioanalytical guidelines and could be used to monitor drug levels in AML patients with respect to their CDA phenotypes.

The pharmacokinetics of cytarabine administered subcutaneously, combined with prednisone, in dogs with meningoencephalomyelitis of unknown etiology.

The objective of this study was to describe the pharmacokinetics (PK) of cytarabine (CA) after subcutaneous (SC) administration to dogs with meningoencephalomyelitis of unknown etiology (MUE). Twelve dogs received a single SC dose of CA at 50 mg/m2 as part of treatment of MUE. A sparse sampling technique was used to collect four blood samples from each dog from 0 to 360 min after administration. All dogs were concurrently receiving prednisone (0.5-2 mg kg-1 day-1 ). Plasma CA concentrations were measured by HPLC, and pharmacokinetic parameters were estimated using nonlinear mixed-effects modeling (NLME). Plasma drug concentrations ranged from 0.05 to 2.8 µg/ml. The population estimate (CV%) for elimination half-life and Tmax of cytarabine in dogs was 1.09 (21.93) hr and 0.55 (51.03) hr, respectively. The volume of distribution per fraction absorbed was 976.31 (10.85%) ml/kg. Mean plasma concentration of CA for all dogs was above 1.0 µg/ml at the 30-, 60-, 90-, and 120-min time points. In this study, the pharmacokinetics of CA in dogs with MUE after a single 50 mg/m2 SC injection in dogs was similar to what has been previously reported in healthy beagles; there was moderate variability in the population estimates in this clinical population of dogs.

Exposure-Response Analysis of Alvocidib (Flavopiridol) Treatment by Bolus or Hybrid Administration in Newly Diagnosed or Relapsed/Refractory Acute Leukemia Patients.

Purpose: To elucidate any differences in the exposure-response of alvocidib (flavopiridol) given by 1-hour bolus or a hybrid schedule (30-minute bolus followed by a 4-hour infusion) using a flavopiridol/cytosine arabinoside/mitoxantrone sequential protocol (FLAM) in patients with acute leukemia. The hybrid schedule was devised to be pharmacologically superior in chronic leukemia based on unbound exposure.Experimental Design: Data from 129 patients in three FLAM studies were used for pharmacokinetic/pharmacodynamic modeling. Newly diagnosed (62%) or relapsed/refractory (38%) patients were treated by bolus (43%) or hybrid schedule (57%). Total and unbound flavopiridol concentrations were fit using nonlinear mixed-effect population pharmacokinetic methodologies. Exposure-response relationships using unbound flavopiridol AUC were explored using recursive partitioning.Results: Flavopiridol pharmacokinetic parameters were estimated using a two-compartment model. No pharmacokinetic covariates were identified. Flavopiridol fraction unbound was 10.9% and not different between schedules. Partitioning found no association between dosing schedule and clinical response. Clinical response was associated with AUC ≥ 780 h*ng/mL for newly diagnosed patients and AUC ≥ 1,690 h*ng/mL for relapsed/refractory patients. Higher exposures were not associated with increases in severe adverse events (≥ grade 3).Conclusions: Pharmacokinetic modeling showed no difference in flavopiridol plasma protein binding for bolus versus hybrid dosing. Further trials in newly diagnosed patients with acute leukemia should utilize the bolus FLAM regimen at the MTD of 50 mg/m2/day. Trials in relapsed/refractory patients should use the hybrid dosing schedule at the MTD (30/60 mg/m2/day) to achieve the higher exposures required for maximal efficacy in this population. Clin Cancer Res; 23(14); 3592-600. ©2017 AACR.

Association of a cytarabine chemosensitivity related gene expression signature with survival in cytogenetically normal acute myeloid leukemia.

The prognosis of cytogenetically normal acute myeloid leukemia (CN-AML) varies greatly among patients. Achievement of complete remission (CR) after chemotherapy is indispensable for a better prognosis. To develop a gene signature predicting overall survival (OS) in CN-AML, we performed data mining procedure based on whole genome expression data of both blood cancer cell lines and AML patients from open access database. A gene expression signature including 42 probes was derived. These probes were significantly associated with both cytarabine half maximal inhibitory concentration values in blood cancer cell lines and OS in CN-AML patients. By using cox regression analysis and linear regression analysis, a chemo-sensitive score calculated algorithm based on mRNA expression levels of the 42 probes was established. The scores were associated with OS in both the training sample (p=5.13 × 10-4, HR=2.040, 95% CI: 1.364-3.051) and the validation sample (p=0.002, HR=2.528, 95% CI: 1.393-4.591) of the GSE12417 dataset from Gene Expression Omnibus. In The Cancer Genome Atlas (TCGA) CN-AML patients, higher scores were found to be associated with both worse OS (p=0.013, HR=2.442, 95% CI: 1.205-4.950) and DFS (p=0.015, HR=2.376, 95% CI: 1.181-4.779). Results of gene ontology (GO) analysis showed that all the significant GO Terms were correlated with cellular component of mitochondrion. In summary, a novel gene set that could predict prognosis of CN-AML was identified presently, which provided a new way to identify genes impacting AML chemo-sensitivity and prognosis.

Simultaneous determination of clofarabine and cytarabine in human plasma by LC-MS/MS.

Combination of cytostatic agents is a basic principle in the treatment of cancer. For the treatment of acute myeloid leukemia (AML), purine analogs, like clofarabine and cytarabine act synergistically. Little is known, however, on their interaction in vivo. We developed a method for the simultaneous determination of clofarabine and cytarabine in human plasma. The substances were extracted from plasma samples by protein precipitation with acetonitrile. Cladribine was the internal standard (IS). The analytes were separated on Synergi HydroRP column (150mm×2.0mm, 4µm) and a triple-quadrupole mass spectrometry with an electrospray ionisation (ESI) source was applied for detection. The mobile phase consisted of acetonitrile, ammonium acetate 2mM and 0.5% formic acid in a gradient mode at a flow rate of 0.5ml/min. The injection volume was 10µl and the total run time was 6.0min. Retention times were 2.46min for clofarabine, 0.97min for cytarabine and 2.43min for the IS. Calibration ranges were 8-1000ng/ml for clofarabine and 20-2500ng/ml for cytarabine. The intra-day and inter-day precision was less than 15% and the relative standard deviation was all within ±15%. This new method allows a rapid and simple determination of both clofarabine and cytarabine in human plasma. It was applied to a pharmacokinetic investigation within a hematological trial in adult patients with AML.

HPLC-MS/MS method for the simultaneous determination of MB07133 and its metabolites, cytarabine and arabinofuranosyluracil, in rat plasma.

MB07133 is an intravenously administered cytarabine mononucleotide (araCMP) prodrug, for the treatment of hepatocellular carcinoma (HCC). A simple, selective and sensitive HPLC-MS/MS method using high pressure liquid chromatography (HPLC) coupled to triple-quadrupole mass spectrometer, was developed and validated for the detection of prodrug MB07133 and its metabolites, cytarabine (araC) and arabinofuranosyluracil (araU) in rat plasma. Protein precipitation using 3% trichloroacetic acid (TCA) was employed to extract analytes from 100µL rat plasma. Adequate separation of araC and araU from their endogenous compounds was achieved on the Synergi(®) fusion-RP column (150mm×4.6mm, 4µm) by a gradient-elution with a mobile phase consisting of ammonium formate (1mM) and methanol at a flow rate of 1mL/min. Multiple reaction monitoring mode (MRM) was applied in the detection of MB07133, araC, araU and Ganciclovir (internal standard) with ion pairs 441.2/330.2, 244.2/112.2, 245.2/113.2 and 256.1/152.2, respectively. The assays were validated with respect to specificity, linearity (100-50000ng/mL for MB07133, 2-1000ng/mL for araC and araU), accuracy and precision, extraction recovery, matrix effect and stability. The validated method has been successfully applied to an intravenous bolus pharmacokinetic study of MB07133 in male Sprague-Dawley rats (18mg/kg i.v.).

Cytarabine-AOT catanionic vesicle-loaded biodegradable thermosensitive hydrogel as an efficient cytarabine delivery system.

Carrier with high drug loading content is one of the most important issues in drug delivery system. In the present work, an ion-pair amphiphilic molecule composed of anticancer drug cation and surfactant anion is used for straightforward fabricating vesicles for cancer therapy. Anticancer drug (cytarabine hydrochloride) and anionic surfactant (AOT) are selected for the fabrication of ion-pair amphiphilic molecule. One amphiphilic molecule contains one drug cation, thus the drug loading content is 50% (mol/mol) in theory. The in vitro drug release study shows that the release time of cytarabine is about 3 times of the pure cytarabine solution and the permeability of cytarabine has been improved about 160 times tested by parallel artificial membrane permeability assay model. However, the hemolytic toxicity is largely decreased in the studied concentration range. The in vitro cytotoxicity results show that cytarabine-AOT amphiphiles have a much lower IC50 (drug concentration resulting in 50% cell death) value and a higher cell inhibition rate comparing with their respective components, indicating its effective therapy for leukemic cells. To obtain a longer and a convenient drug release system, the prepared vesicles are further incorporated into the thermosensitive PLGA-PEG-PLGA hydrogel to prepare a subcutaneous administration. The in vivo drug release results indicate that cytarabine-AOT vesicle-loaded hydrogel is a good injectable delivery system for controlled release of cytarabine for cancer therapy.

Simultaneous determination of 1-ß-d-Arabinofuranosylcytosine and two metabolites, 1-ß-d-Arabinofuranosyluracil and 1-ß-d-Arabinofuranosylcytosine triphosphate in leukemic cell by HPLC-MS/MS and the application to cell pharmacokinetics.

A specific and reliable HPLC-MS/MS method was developed and validated for the simultaneous determination of 1-ß-d-Arabinofuranosylcytosine (ara-C), 1-ß-d-Arabinofuranosyluracil (ara-U) and 1-ß-d-Arabinofuranosylcytosine triphosphate (ara-CTP) in the leukemic cells for the first time. The analytes were separated on a C18 column (100mm×2.1mm, 1.8µm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was used for detection. The ion-pairing reagent, NFPA, was added to the mobile phase to retain the analytes in the column. The cell homogenates sample was prepared by the simple protein precipitation. The calibration curves were linear over a concentration range of 3.45-3450.0ng/mL for ara-C, 1.12-1120.0ng/mL for ara-U and 4.13-4130.0ng/mL for ara-CTP. The intra-day and inter-day precision was less than 15% and the relative error (RE) were all within ±15%. The validated method was successfully applied to assess the disposition characteristics of ara-C and support cell pharmacokinetics after the patients with leukemia were intravenously infused with SDAC and HiDAC. The result of the present study would provide the valuable information for the ara-C therapy.

Elacytarabine in relapsed/refractory acute myeloid leukaemia: an evaluation of clinical efficacy, pharmacokinetics, cardiac safety and effects on lipid profile.

Elacytarabine is the elaidic acid ester derivative of cytarabine, designed to enter cells independently of nucleoside transporters. Effects of elacytarabine on QT interval, serum lipid profile and clinical activity were investigated in 43 relapsed/refractory AML patients. Mean maximum increase in corrected QT interval of 24( ± 29)ms occurred 48 h after elacytarabine infusion without associated arrhythmias or clinical symptoms. A non-clinically significant, elacytarabine exposure-dependent increase in cholesterol was caused by a cholesterol rich lipoprotein depleted of apolipoprotein B formed by infused phospholipids complexing cholesterol. Elacytarabine is clinically active in relapsed/refractory AML: overall response rate (CR + CRi) was 44% (16/36 with 7 non-evaluable patients) and adverse events were manageable. Clinical Trials.gov Identifier: NCT01258816.

Aging of biological matrices and its effect on bioanalytical method performance.

Apart from the well-known matrix effects that can occur in ESI LC-MS, biological matrices may have other effects influencing the quantitative reliability of bioanalytical methods. In this paper, six case studies are presented that show the effect that aging, that is the change in properties and composition of biological matrices over time, can have on the performance of bioanalytical methods. It is shown that selectivity can be affected due to the formation or disappearance of endogenous compounds. Stability can be influenced because of the decrease (or increase) of enzyme activities and recovery can be impacted if the extractability from binding sites in the matrix is enhanced or decreased. A general discussion on the importance of these matrix effects is provided as well as a perspective on how to properly address them in the method-development and validation stages of regulated bioanalysis.

Safety and pharmacokinetics of the antisense oligonucleotide (ASO) LY2181308 as a single-agent or in combination with idarubicin and cytarabine in patients with refractory or relapsed acute myeloid leukemia (AML).

Survivin is expressed in tumor cells, including acute myeloid leukemia (AML), regulates mitosis, and prevents tumor cell death. The antisense oligonucleotide sodium LY2181308 (LY2181308) inhibits survivin expression and may cause cell cycle arrest and restore apoptosis in AML. In this study, the safety, pharmacokinetics, and pharmacodynamics/efficacy of LY2181308 was examined in AML patients, first in a cohort with monotherapy (n = 8) and then post-amendment in a cohort with the combination of cytarabine and idarubicin treatment (n = 16). LY2181308 was administered with a loading dosage of three consecutive daily infusions of 750 mg followed by weekly intravenous (IV) maintenance doses of 750 mg. Cytarabine 1.5 g/m(2) was administered as a 4-hour IV infusion on Days 3, 4, and 5 of Cycle 1, and idarubicin 12 mg/m(2) was administered as a 30-minute IV infusion on Days 3, 4, and 5 of Cycle 1. Cytarabine and idarubicin were administered on Days 1, 2, and 3 of each subsequent 28-day cycle. Reduction of survivin was evaluated in peripheral blasts and bone marrow. Single-agent LY2181308 was well tolerated and survivin was reduced only in patients with a high survivin expression. In combination with chemotherapy, 4/16 patients had complete responses, 1/16 patients had incomplete responses, and 4/16 patients had cytoreduction. Nine patients died on study: 6 (monotherapy), 3 (combination). LY2181308 alone is well tolerated in patients with AML. In combination with cytarabine and idarubicin, LY2181308 does not appear to cause additional toxicity, and has shown some clinical benefit needing confirmation in future clinical trials.

Enhancing the efficacy of Ara-C through conjugation with PAMAM dendrimer and linear PEG: a comparative study.

1ß-d-Arabinofuranosylcytosine (Cytarabine, Ara-C) is a key drug in the treatment of acute myeloid leukemia. Ara-C has a number of limitations such as a rapid deactivation by cytidine deaminase leading to the formation of a biologically inactive metabolite, Ara-U (1ß-d-arabinofuranosyluracil), a low lipophilicity, and fast clearance from the body. To address these problems, we developed a conjugate in which hydroxyl-terminated PAMAM dendrimer, G4-OH ["D"] and PEG were used as carriers for the drug (Ara-C). The conjugates were synthesized using an efficient multistep protection/deprotection method resulting in the formation of a covalent bond between the primary hydroxyl group of Ara-C and dendrimer/PEG. The structure, physicochemical properties, and drug release kinetics were characterized extensively. (1)H NMR and MALDI-TOF mass spectrometry suggested covalent attachment of 10 Ara-C molecules to the dendrimer. The release profile of Ara-C in human plasma and in PBS buffer (pH 7.4) showed that the conjugates released the drug over 14 days in PBS, with the release sped up in plasma. In PBS, while most of the drug is released from PEG-Ara-C, the dendrimer continues to release the drug in a sustained fashion. The results also suggested that the formation of the inactive form of Ara-C (Ara-U) was delayed upon conjugation of Ara-C to the polymers. The inhibition of cancer growth by the dendrimer-Ara-C and PEG-Ara-C conjugates was evaluated in A549 human adenocarcinoma epithelial cells. Both dendrimer- and PEG-Ara-C conjugates were 4-fold more effective in inhibition of A549 cells compared to free Ara-C after 72 h of treatment.

Simultaneous determination of cytosine arabinoside and its metabolite, uracil arabinoside, in human plasma using hydrophilic interaction liquid chromatography with UV detection.

A practical high-performance liquid chromatography using a Cosmosil HILIC column and UV detection was developed for determining the concentrations of cytosine arabinoside (Ara-C) and uracil arabinoside (Ara-U), which is a major metabolite of Ara-C, in human plasma. This method was used to determine the plasma concentrations of Ara-C and Ara-U in a patient treated with high-dose Ara-C therapy for end-stage renal failure.

The pharmacokinetics of cytarabine in dogs when administered via subcutaneous and continuous intravenous infusion routes.

This crossover study compared the pharmacokinetics of cytarabine in six healthy dogs following intravenous constant rate infusion (CRI) and subcutaneous (SC) administrations, as these are two routes of administration commonly employed in the treatment of meningoencephalitis of unknown etiology. Each dog received a SC cytarabine injection of 50 mg/m(2) or an 8 h CRI of 25 mg/m(2) per hour, with a 7-day washout before receiving the alternative treatment. Blood samples were collected for 16 h after CRI initiation and for 8 h after SC injection. Plasma concentrations were measured by high-pressure liquid chromatography (HPLC). Pharmacokinetic parameters were estimated using the best-fit compartmental analysis for both CRI and SC routes. Terminal half-life (T(1/2) ) of cytarabine was 1.35 ± 0.3 and 1.15 ± 0.13 h after SC administration and CRI, respectively. Mean peak concentration (Cmax ) was 2.88 and 2.80 µg/mL for SC and CRI administration, respectively. Volume of distribution was 0.66 ± 0.07 l/kg. The 8-h CRI produced steady-state plasma concentrations as determined by consecutive measurement that did not decline until the end of the infusion. The SC administration did not achieve steady-state concentrations because cytarabine administered by this route was rapidly absorbed and eliminated quickly. The steady state achieved with the cytarabine CRI may produce a more prolonged exposure of cytarabine at cytotoxic levels in plasma compared to the concentrations after SC administration.

Long circulating PEGylated PLGA nanoparticles of cytarabine for targeting leukemia.

The present investigation was aimed at developing PEGylated PLGA nanoparticles of cytarabine. PLGA Nanoparticles were prepared by modified nanoprecipitation method, optimized for mean particle size (152 ± 6 nm) and entrapment efficiency (41.1 ± 0.8%) by a 3² factorial design. The PEGylated PLGA nanoparticles of cytarabine had a zeta potential of -7.5 ± 1.3 mV and sustained the release of cytarabine for 48 h by Fickian diffusion. The IC50 values for L1210 cells were 6.5, 5.3, and 2.2 µM for cytarabine, cytarabine loaded PLGA nanoparticles and cytarabine loaded PLGA-mPEG nanoparticles respectively. Confocal microscopy and flow cytometry showed that the nanoparticles were internalized by the L1210 cells and not simply bound to their surface. Biodistribution studies showed that the PEGylated nanoparticles of cytarabine were present in significantly higher concentrations in blood circulation as well as in brain and bones and avoided RES uptake as compared to the free drug.