Assessment of efficacy of postinfusion tubing flushing in reducing risk of cytotoxic contamination.

PURPOSE: Infusion of cytotoxic drugs carries the risk of occupational exposure of healthcare workers. Since disconnecting an infusion line is a source of contamination, flushing of tubing after infusion of cytotoxic agents is recommended, but the optimal volume of rinsing solution is unknown. The objective of this study was to assess whether postinfusion line flushing completely eliminates cytotoxics. METHODS: Infusions were simulated with 3 cytotoxics (gemcitabine, cytarabine, and paclitaxel) diluted in 5% dextrose injection or 0.9% sodium chloride injection in 250-mL infusion bags. Infusion lines were flushed using 5% dextrose injection or 0.9% sodium chloride solution at 2 different flow rates. The remaining concentration of cytotoxics in the infusion line was measured by a validated high-performance liquid chromatography (HPLC) method after passage of every 10 mL of flushing volume until a total of 100 mL had been flushed through. RESULTS: All cytotoxics remained detectable even after line flushing with 80 mL of flushing solution (a volume 3-fold greater than the dead space volume within the infusion set). Gemcitabine and cytarabine were still quantifiable via HPLC even after flushing with 100 mL of solution. Efficacy of flushing was influenced by the lipophilicity of drugs but not by either the flushing solvent used or the flushing flow rate. After 2-fold dead space volume flushing, the estimated amount of drug remaining in the infusion set was within 0.19% to 0.56% of the prescribed dose for all 3 cytotoxics evaluated. CONCLUSION: Complete elimination of cytotoxics from an infusion line is an unrealistic objective. Two-fold dead space volume flushing could be considered optimal in terms of administered dose but not from an environmental contamination point of view. Even when flushed, the infusion set should still be considered a source of cytotoxic contamination.

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.

Assessment of graphite electrode on the removal of anticancer drug cytarabine via indirect electrochemical oxidation process: Kinetics & pathway study.

In this paper degradation of cytarabine drug has been studied through electrochemical oxidation process by using graphite electrode. The performance of graphite electrode on the degradation of cytarabine was evaluated by investigating the effects of key parameters: pH (3-9), current density (5-20 mA cm-2) and initial pollutant concentration (5-50 mg L-1) with 0.05 M NaCl as supporting electrolyte. Highest removal efficiency (98%) for 20 mg L-1 of initial cytarabine solution was attained within 60 min electrolysis at 10 mA cm-2. The increase in degradation rate of cytarabine was possibly because of the active chlorine species originated at anode during the electrolysis. Further, efficiency of the graphite electrodes was compared with a metal electrode (copper) and results showed that the cytarabine degradation was facilitated by the in-situ generated OH radicals. However, only 82% of cytarabine was removed after 60 min of reaction time at 15 mA cm-2. The scum of Cu2+ ions deposited on the anode surface inhibit the mass transfer among the cytarabine molecules and generated hydroxyl radicals. The kinetic study also suggests faster reaction rate at graphite (0.12 min-1) than copper (0.05 min-1) electrode. The increase in electrolyte concentration enhanced the degradation rate and decreased the energy consumption from 3.66 to 0.66 kWh m-3. Cytosine was identified as the major transformation product from the UV-Vis spectral analysis and LC-MS analysis. Further, total organic carbon analysis depicts that only 60% of the parent molecule was mineralized. Hence, graphite was found to be an efficient anode material as compared to copper for cytarabine degradation.

Enriching cancer pharmacology with drugs of marine origin.

Marine natural products have proven, over the last half-century, to be effective biological modulators. These molecules have revealed new targets for cancer therapy as well as dissimilar modes of action within typical classes of drugs. In this scenario, innovation from marine-based pharmaceuticals has helped advance cancer chemotherapy in many aspects, as most of these are designated as first-in-class drugs. Here, by examining the path from discovery to development of clinically approved drugs of marine origin for cancer treatment-cytarabine (Cytosar-U®), trabectedin (Yondelis®), eribulin (Halaven®), brentuximab vedotin (Adcetris®), and plitidepsin (Aplidin®)- together with those in late clinical trial phases-lurbinectedin, plinabulin, marizomib, and plocabulin-the present review offers a critical analysis of the contributions given by these new compounds to cancer pharmacotherapy.

Investigation and verification of a bioluminescent biosensor for the quantitation of ara-CTP generation: a biomarker for cytosine arabinoside sensitivity in acute myeloid leukaemia.

A novel whole cell bacterial biosensor, which emits light in response to the active metabolite of cytosine arabinoside (ara-C, cytarabine), ara-CTP, has been investigated and verified. The biosensor has been formulated as an ex vivo assay, designed for peripheral blood or bone marrow cells, which can produce a clinical result within a working day. The nucleoside analogue ara-C is a key agent for treatment of acute myeloid leukaemia (AML); treatment decisions are made rapidly with AML, patients often receiving same-day commencement of chemotherapy. Currently no rapid predictive test is available to select appropriate therapy for patients prior to treatment. Experiments were designed to determine optimal assay conditions using leukaemic cell lines. We observed a significant increase (~15 fold) in bioluminescence signal compared to control after 8-h incubation of the biosensor with ara-C. This corresponded to a >2-log increase in light output per bacterial cell. Interestingly, bioluminescence conferred a survival advantage to the bacteria following ara-C treatment. The assay is sensitive (lower limit of quantitation of 0.05 µM), selective, accurate (≤ 15% RE) and precise (≤ 15% coefficient of variation) over a linear concentration range of ara-CTP (0.05-0.5 µM), and detection is independent of reaction volume. Recovery of added standard was tested using ex vivo patient leukaemic cells (n=5). Stability studies on lyophilized bacterial biosensor were performed to ensure maintenance of performance over 12 months. The biosensor assay could be invaluable to the clinician, assisting with treatment selection, and potentially mitigating the risks of resistance and toxicity observed with this drug.

Removal of highly polar micropollutants from wastewater by powdered activated carbon.

Due to concerns about ecotoxicological effects of pharmaceuticals and other micropollutants released from wastewater treatment plants, activated carbon adsorption is one of the few processes to effectively reduce the concentrations of micropollutants in wastewater. Although aimed mainly at apolar compounds, polar compounds are also simultaneously removed to a certain extent, which has rarely been studied before. In this study, adsorption isotherm and batch kinetic data were collected with two powdered activated carbons (PACs) to assess the removal of the polar pharmaceuticals 5-fluorouracil (5-Fu) and cytarabine (CytR) from ultrapure water and wastewater treatment plant effluent. At pH 7.8, single-solute adsorption isotherm data for the weak acid 5-Fu and the weak base CytR showed that their adsorption capacities were about 1 order of magnitude lower than those of the less polar endocrine disrupting chemicals bisphenol A (BPA) and 17-α-ethinylestradiol (EE2). To remove 90 % of the adsorbate from a single-solute solution 14, 18, 70, and 87 mg L(-1) of HOK Super is required for EE2, BPA, CytR, and 5-Fu, respectively. Effects of solution pH, ionic strength, temperature, and effluent organic matter (EfOM) on 5-Fu and CytR adsorption were evaluated for one PAC. Among the studied factors, the presence of EfOM had the highest effect, due to a strong competition on 5-Fu and CytR adsorption. Adsorption isotherm and kinetic data and their modeling with a homogeneous surface diffusion model showed that removal percentage in the presence of EfOM was independent on the initial concentration of the ionizable compounds 5-Fu and CytR. These results are similar to neutral organic compounds in the presence of natural organic matter. Overall, results showed that PAC doses sufficient to remove >90 % of apolar adsorbates were able to remove no more than 50 % of the polar adsorbates 5-Fu and CytR and that the contact time is a critical parameter.

A historical perspective on the development of the cytarabine (7days) and daunorubicin (3days) treatment regimen for acute myelogenous leukemia: 2013 the 40th anniversary of 7+3.

This paper reviews the development of therapy for acute myelogenous leukemia that in 1973 led to the regimen of 7days of continuous intravenous arabinosylcytosine (cytarabine) and the first 3 concurrent days of intravenous daunorubicin, given the nickname "7+3." The state of leukemia treatment in the 1950s, 1960s and early 1970s is reviewed, the discovery of the two drugs in question described, and the introduction of clinical trials to reach an optimal regimen for their use delineated. During the 1950s, following World War Two and after a period of civil reconstitution, a national effort, facilitated by the U.S. Congress and federal investments in the National Cancer Institute, was initiated to enhance cancer therapy in the United States. The development of mouse models of leukemia and advances in understanding the structure and function of DNA and RNA and the process of cell proliferation provided new targets for drug development and new concepts for their use. The year, 2013, marks the 40th year that this protocol, 7+3, is the method of induction of remission for most patients with acute myelogenous leukemia. Its inadequacies also are made clear. Many patients with the disease die soon after diagnosis, and patients who have more unfavorable oncogenetic subtypes, intrinsically drug resistant cells, and greater intolerance to therapy make up the vast majority of the affected and few are cured. It is evident to all that new paradigms are needed if acute myelogenous leukemia is to be subdued in most patients with the disease.

A facile whole-cell biocatalytic approach to regioselective synthesis of monoacylated 1-ß-D-arabinofuranosylcytosine: influence of organic solvents.

The lyophilized Pseudomonas fluorescens cell was an efficient alternative catalyst to enzymes for highly regioselective acylation of a polar nucleoside, 1-ß-D-arabinofuranosylcytosine (ara-C). The cells showed an evident solvent dependence in the reaction. Among the tested solvents except for acetonitrile-pyridine, catalytic activity of the cells clearly increased with increasing the polarity of the organic solvents used. Among all the tested solvents both pure and binary, the best results were observed in isopropyl ether-pyridine system, in which the catalyst also showed good thermal and operational stabilities. For the biocataylsis in isopropyl ether-pyridine, the optimal isopropyl ether concentration, water content, acyl donor/ara-C ratio, biocatalyst dosage and reaction temperature were 30% (v/v), 4%, 45, 50mg/mL and 30 °C, respectively, under which the initial rate, yield and 5'-regioselectivity were 2.93 mM/h, 77.1% and 97.3%, respectively. The bacterial cells exhibited comparable 5'-regioselectivity to the expensive immobilized enzyme, which could also have environmental and cost advantages.

Separation of diastereoisomers of Ara-C phosphotriesters using solid phase extraction and HPLC for the study of their decomposition kinetic in cell extracts.

Separations of the diastereoisomers of three nucleoside 5'-phosphotriester derivatives of Ara-C (tBuSATE, hydroxy tBuSATE and bishydroxy tBuSATE phenylphosphotriester derivatives; pronucleotides) were performed by HPLC using derivatized cellulose and amylose chiral stationary phases. An optimal baseline separation (Rs>1.5) was readily obtained with an amylose based chiral column (AD-H) used in normal phase mode. This stereospecific HPLC method has been associated to a solid phase extraction step using a C18 cartridge and an internal standard for the quantification of one nucleoside 5'-phosphotriester derivative in cell extracts. After optimization, this method was validated in terms of specificity, recovery, linearity, precision and accuracy and detection limit. It was applied to the determination of the apparent rate constants of disappearance and half-lives of each diastereoisomer. This enabled us to conclude that the enzymatic activity involved in the first step of the decomposition pathway of the hydroxyl tBuSATE phenylphosphotriester of Ara-C is stereoselective and is related to the nature of the pyrimidic base.

Supercritical fluid chromatography and high-performance liquid chromatography/tandem mass spectrometric methods for the determination of cytarabine in mouse plasma.

The separation of cytarabine (ara-C) from the endogenous compounds in mouse plasma by packed-column supercritical fluid chromatography (pSFC) was achieved on bare silica stationary phase with an isocratic mobile phase composed of CO2/methanol solvent with addition of ammonium acetate. SFC is commonly assumed to be only applicable to nonpolar and relatively low-polarity compounds. In this work, a broader range of compound polarities amenable to pSFC with appropriate mobile-phase modifiers and additives under normal-phase retention mechanism was demonstrated. The pSFC was integrated with an atmospheric pressure chemical ionization source and a tandem mass spectrometer (MS/MS) to enhance the sensitivity, selectivity, and speed of the assay. The influence of mobile-phase components on chromatographic performance and ionization efficiency of the test compounds was investigated for improving the sensitivity and separation for the analyte and the internal standard. The pSFC-MS/MS approach requiring approximately 2.5 min/sample for the determination of ara-C at nanograms per milliliter in mouse plasma was partially validated with respect to stability, linearity, and reproducibility. The mouse plasma levels of ara-C obtained by the pSFC-MS/MS method were found to be consistent with those determined by various reversed-phase, high-performance liquid chromatography methods using a porous graphite carbon column, a mixed-mode column, or a C18 column in conjunction with an ion-pairing agent coupled to a tandem mass spectrometer.

HPLC separation of some purine and pyrimidine derivatives on Chromolith Performance Si monolithic column.

The chromatographic behavior of some purines and pyrimidines on a monolithic Chromolith Performance Si column under normal-phase high-performance liquid chromatography mode has been studied. Column pressure, column efficiency and selectivity of Chromolith Performance Si column were compared to those of conventional spherical 5 microm silica packed columns Econosphere Silica and Zorbax Rx-SIL. The investigation has shown that application of Chromolith Performance Si column for analysis of polar solutes can reduce the separation time without sacrificing column efficiency and selectivity. Improvement of the monolithic silica column efficiency for polar solutes is observed when ternary mobile phases (mixtures of hexane-isopropanol with ethylene glycol, water or acetonitrile) are applied.

Micellar electrokinetic capillary chromatography quantification of cytosine arabinoside and its metabolite, uracil arabinoside, in human serum.

Cytosine arabinoside (Ara-C) is widely used to induce remission in adult granulocytic leukemia. High doses can be infused in refractory leukemia or in relapse. After injection, Ara-C is quickly metabolized to uracil arabinoside (Ara-U), the main inactive metabolite. We here described a micellar electrokinetic capillary chromatography (MECC) method to simultaneously determine Ara-C/Ara-U in human serum using 6-O-methylguanine as an internal standard. The assay was linear from 6.25 to 200 microg/ml with a quantification limit between 3 and 6 microg/ml. The analytical precision was satisfactory between 2 and 4.3% (within-run) and 3.7 and 7.3% (between-runs). This assay was applied to the analysis of serum from acute granulocytic leukemia patient treated by high doses cytarabine (3 g/m2 body surface).

Separation of aracytidine and cytidine by capillary electrophoretic techniques.

Aracytidine (cytarabine, 1-beta-D-arabinofuranosylcytosine) is a synthetic analog of cytidine in which ribose is substituted by arabinose; it is used as a drug for the treatment of leukemia. A fast and reliable capillary electrophoretic method for the analysis of cytarabine and cytidine is described. The procedure utilizes the interactions with sodium dodecyl sulfate (SDS) micelles and borate, present in the background electrolyte, for the mobilization and selective separation of the analytes. The detection is carried out by UV absorbance at 275 nm. The method was applied both to pharmaceutical preparations and human serum. Analysis of an untreated serum requires 15 min; the detection limit is 0.8 microgram/mL and the relative standard deviation (RSD) is 5.3%.

Formation of beta-galactosyl compounds of arabinosylcytosine in growing culture of Sporobolomyces singularis.

Two new compounds of 1-beta-D-arabinofuranosylcytosine (ara-C) were found to be produced in a high yield in a culture filtrate of Sporobolomyces singularis, when grown on a medium containing lactose and ara-C. The compounds I and II were obtained as white needle crystals from the culture filtrate by preparative paper chromatography, gel-filtration on Sephadex G-10 and Toyopearl HW-40S, lyophilization, and ethanol treatment. The compounds I and II were identified as 3'-O-(beta-D-galactopyranosyl)-ara-C and 3'-O-[beta-D-galactopyranosyl-(1-->4)-O-beta-D-galactopyranosyl]-ara-C, respectively, on the basis of the various experimental results, viz., elementary analyses, UV, IR, 1H-, and 13C-NMR spectra, and products by hydrolysis with alpha- and beta-galactosidases. Also, the yeast produced a large amount of 3'-O-beta-galactosyl compounds of adenosine and inosine in the culture filtrate when grown on a medium containing lactose and their ribonucleosides.

Evidence for the conversion of adenosine to 2'-deoxycoformycin by Streptomyces antibioticus.

The incorporation and distribution of 14C in 2'-deoxycoformycin, elaborated by Streptomyces antibioticus, were studied with [U-14C]glycine, [U-14C]adenosine and [U-14C]adenine. Similar ratios of 14C in the aglycon and carbohydrate portions of 2'-deoxycoformycin, ara-A, and adenosine isolated from the RNA indicated that [U-14C]adenosine was incorporated into 2'-deoxycoformycin without cleavage of the N-glycosylic bond. Following the addition of [U-14C]adenine, 98% of the 14C isolated from [14C]-2'-deoxycoformycin resided in the aglycon. 2'-Deoxycoformycin biosynthesis may not require the de novo purine biosynthetic pathway as evidenced by the failure to detect incorporation of [U-14C]glycine into 2'-deoxycoformycin. These data suggest that the biosynthesis of 2'-deoxycoformycin involves the incorporation of the carbon-nitrogen skeleton of an intact purine nucleoside or nucleotide, thereby implying that a purine ring is opened enzymatically between C-6 and N-1 and a one-carbon unit is added to form the 1,3-diazepine ring of 2'-deoxycoformycin.