Potential application of pristine and Al-doped graphyne-like BN nanosheet for detection of anticancer fluorouracil drug.

We studied the potential application of the pristine and Al-doped graphyne-like BN nanosheets (Al-BN-yne) in 5-fluorouracil (5-FU) drug detection using DFT calculations. The 5-FU drug preferentially adsorbed via its oxygen atom on the B atom of pristine BN-yne with adsorption energy of - 11.7 kcal/mol and no effect on its electronic properties. Replacing a B atom by an Al atom significantly increased the sensitivity and reactivity of BN-yne to the 5-FU drug. Upon the 5-FU interaction with the Al-BN-yne, an energy of 20.3 kcal/mol is released, and the Eg of Al-BN-yne significantly decreased from 4.83 to 3.80 eV, increasing the electrical conductance. Thus, the Al-BN-yne sheet can generate an electronic signal after the 5-FU drug adsorption, being a promising electronic sensor for 5-FU detection. We predicted that the recovery time for 5-FU drug desorption from the Al-BN-yne sheet surface is 0.07 s, demonstrating that it benefits from a short recovery time.

Senescent Colon and Breast Cancer Cells Induced by Doxorubicin Exhibit Enhanced Sensitivity to Curcumin, Caffeine, and Thymoquinone.

Cellular senescence is a process of physiological growth arrest that can be induced by intrinsic or extrinsic stress signals. Some cancer therapies are associated with senescence of cancer cells with a typical cell cycle arrest. Doxorubicin (Dox) induces senescence by a p53-dependent pathway and telomere dysfunction of numerous cancers. However, cellular senescence induces suppression in proliferation activity, and these cells will remain metabolically active and play an important role in tumor relapse and development of drug resistance. In the current study, we investigated the apoptotic effect of curcumin (Cur), caffeine (Caff), and thymoquinone (TQ) on senescent colon cancer HCT116 and breast cancer MCF7 cell lines treated with Dox. Results showed typical senescence markers including decreased bromodeoxyuridine incorporation, increased accumulation of senescence-associated ß-galactosidase (SA-ß-gal), cell cycle arrest, and upregulation of p53, P-p53, and p21 proteins. Annexin-V analysis by flow cytometry revealed 2- to 6-fold increases in annexin-V-positive cells in Dox-treated MCF7 and HCT116 cells by Cur (15 µM), Caff (10 mM), and TQ (50 µM; P < .001). In comparison between proliferative and senescent of either HCT116 or MCF7 cells, Caff at 15 mM and TQ at 25 µM induced significant increases in apoptosis of Dox-treated cells compared with proliferative cells (P < .001). Data revealed that Cur, Caff, and TQ potentially induced apoptosis of both proliferative and senescent HCT116 and MCF7 cells. In vivo and clinical trials are of great importance to validate this result.

Development of a low-cost electrochemical sensor based on babassu mesocarp (Orbignya phalerata) immobilized on a flexible gold electrode for applications in sensors for 5-fluorouracil chemotherapeutics.

There are increasing concerns regarding the risks arising from the contamination of manipulators of antineoplastic drugs promoted by occupational exposure or even in the dosage of drugs. The present work proposes the use of an electrochemical sensor based on a biopolymer extracted from the babassu coconut (Orbignya phalerata) for the determination of an antineoplastic 5-fluorouracil (5-FU) drug as an alternative for the monitoring of these drugs. In order to reduce the cost of this sensor, a flexible gold electrode (FEAu) is proposed. The surface modification of FEAu was performed with the deposition of a casting film of the biopolymer extracted from the babassu mesocarp (BM) and modified with phthalic anhydride (BMPA). The electrochemical activity of the modified electrode was characterized by cyclic voltammetry (CV), and its morphology was observed by atomic force microscopy (AFM). The FEAu/BMPA showed a high sensitivity (8.8 µA/µmol/L) and low limit of detection (0.34 µmol/L) for the 5-FU drug in an acid medium. Electrochemical sensors developed from the babassu mesocarp may be a viable alternative for the monitoring of the 5-FU antineoplastic in pharmaceutical formulations, because in addition to being sensitive to this drug, they are constructed of a natural polymer, renewable, and abundant in nature. Graphical abstract ᅟ.

Identification of the Novel Capecitabine Metabolites in Capecitabine-Treated Patients with Hand-Foot Syndrome.

Hand-foot syndrome (HFS), the most common side effect of capecitabine, is a dose-limiting cutaneous toxicity with only rare therapeutic options. The causative mechanisms of HFS are still unclear. Many studies suggested that capecitabine or its metabolites caused the toxicity. This study is attempting to determine if there are any new metabolites that may be present and be linked to toxicity. For this purpose, 25 patients who ingested capecitabine orally were enrolled and divided into HFS positive and negative groups. Urine and plasma samples were collected before administration and five cycles after administration. Eleven phase I and phase II metabolites of capecitabine were detected and identified by ultraperformance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry with a metabolomic approach and MetaboLynxXS. Nine novel metabolites of capecitabine were identified herein, which were not observed in the HFS negative group. Their structures were confirmed by chemical synthesis and nuclear magnetic resonance spectroscopy. The cytotoxities of capecitabine and its metabolites on HaCaT cells were measured. Among them, M9/10 exhibited significant inhibitory activity, and they were produced via acetylation mainly by N-acetyltransferase 2. Our study comprehensively described the metabolism of capecitabine in patients with HFS and detected the novel pathways of capecitabine, which was a positive significance for the mechanism of HFS.

Quantification of 5-FU in skin samples for the development of new delivery systems for topical cancer treatment.

5-Fluorouracil (5-FU) is used for the topical treatment of pre-cancerous skin lesions. However, previous reports focus on 5-FU quantification in plasma samples, failing to provide information about drug quantification in the skin. Therefore, the aim of this work was to develop a simple and reliable analytical method employing HPLC-UV for 5-FU quantification in skin samples. Chromatographic separation was obtained using the commonly available Lichrospher RP-C18 endcapped column. Porcine ear skin matrix-based analytical curves with thymine, as internal standard, were used. 5-FU was eluted at 5.2 min and thymine at 13.0 min. The method was selective, precise and accurate in the linear range from 0.3 to 6 µg/mL. The samples were stable after three cycles of freeze/thawing and extraction efficiency rates above 95% were obtained. In vitro skin penetration studies of an aqueous solution and a commercial cream of 5-FU were performed. The cream improved 5-FU retention into the skin and permeation through the skin compared to the solution. Therefore, the method developed herein can be applied to the study of formulations for topical delivery of 5-FU.

Degradation of the cytostatic 5-Fluorouracil in water by Fenton and photo-assisted oxidation processes.

Cytostatics are part of the forefront research topics due to their high prescription, high toxicity, and the lack of effective solutions to stop their entrance and spread in the environment. Among them, 5-Fluorouracil (5-Fu) has received particular attention because is one of the most prescribed active substances in chemotherapy worldwide. The degradation of 5-Fu by advanced oxidation processes (AOPs) is a poorly addressed topic, and this work brings valuable inputs concerning this matter. Herein, the efficacy of Fenton's process in the degradation of 5-Fu is explored for the first time; the study of the main variables and its successful application to the treatment of real wastewaters is demonstrated. Moreover, hydrogen peroxide-based and photo-assisted techniques (direct photolysis, photodegradation with H2O2 and photo-Fenton) are also investigated for purposes of comparison. Under the best operation conditions obtained (T = 30 °C, [Fe2+]0 = 0.5 mM; [H2O2]0 = 240 mM and pH = 3 for [5-Fu]0 = 0.38 mM), 5-Fu was completely eliminated after 2 h of Fenton's reaction and about 50 % of mineralization was reached after 8 h. The best performance was obtained by the photo-Fenton process, with 5-Fu mineralization level as high as 67 %, using an iron dose within the legal limits required for direct water discharge. Toxicity (towards Vibrio fischeri) of the effluents that resulted from the application of the above-mentioned AOPs was also evaluated; it was found that the degradation products generated from the photo-assisted processes are less toxic than the parent compound, putting into evidence the relevance of such technologies for degradation of cytostatics like 5-Fu.

CAN a P-gp modulator assist in the control of methotrexate concentrations in the rat brain? -inhibitory effects of rhodamine 123, a specific substrate for P-gp, on methotrexate excretion from the rat brain and its optimal route of administration.

Although methotrexate (MTX) is mainly transported by reduced folate carrier, P-gp and MRP1 may also be involved in its transport. In our previous study, a potent P-gp and MRP1 modulator, Cyclosporine A, potentiated MTX concentration in rat brain. Since it is important for MTX therapy for brain tumor to clarify which transporter is dominant, we herein determined whether the specific P-gp substrate, rhodamine123 (Rho123), potentiates the transport and retention of MTX in the brain. Rho123 was injected intravenously or intrathecally into rats immediately after injection of MTX. 6 or 12 hr after the MTX injection, brains were isolated just after the sampling of cerebrospinal fluid (CSF). Blood was also collected intermittently. MTX concentrations were determined in plasma, CSF and the brain using high-performance liquid chromatography with UV detection. When MTX was intravenously injected, Rho123 didn't affect MTX concentrations in the brain. However, Rho123 resulted in significantly higher MTX concentrations in the brain at 12 hr after injection when MTX was intrathecally injected. It is suggested that Rho123 inhibits the excretion of MTX from the brain, but does not potentiate its distribution from the blood into the brain. This reveals that P-gp can be one of the major transporters of MTX in rat brain. Therefore, treatments with P-gp modulators may contribute to intrathecal MTX therapy for brain tumor. Since plasma concentration-time curves of MTX were not affected by Rho123, treatments with P-gp modulators may not potentiate the adverse effects of MTX.

Electrochemical DNA biosensor based on poly(2,6-pyridinedicarboxylic acid) modified glassy carbon electrode for the determination of anticancer drug gemcitabine.

In this study, a simple methodology was used to develop a new electrochemical DNA biosensor based on poly(2,6-pyridinedicarboxylic acid) (P(PDCA)) modified glassy carbon electrode (GCE). This modified electrode was used to monitor for the electrochemical interaction between the dsDNA and gemcitabine (GEM) for the first time. A decrease in oxidation signals of guanine after the interaction of the dsDNA with the GEM was used as an indicator for the selective determination of the GEM via differential pulse voltammetry (DPV). The guanine oxidation peak currents were linearly proportional to the concentrations of the GEM in the range of 1-30mgL(‒1). Limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.276mgL(‒1) and 0.922mgL(‒1), respectively. The reproducibility, repeatability, and applicability of the analysis to pharmaceutical dosage forms and human serum samples were also examined. In addition to DPV method, UV-vis and viscosity measurements were utilized to propose the interaction mechanism between the GEM and the dsDNA. The novel DNA biosensor could serve for sensitive, accurate and rapid determination of the GEM.

Detection of methotrexate in a flow system using electrochemical impedance spectroscopy and multivariate data analysis.

Methotrexate (MTX), a common pharmaceutical drug in cancer therapy and treatment of rheumatic diseases, is known to cause severe adverse side effects at high dose. As the side effect may be life threatening, there is an urgent need for a continuous, bed-side monitoring of the nominal MTX serum level in a patient while the chemical is being administered. This article describes a detection of MTX using a flow system that consists two modified gold electrodes. Interaction of MTX with the antibodies fixed on the electrode surface is detected by electrochemical impedance spectroscopy and evaluated using singular value decomposition (SVD). The key finding of this work is that the change in the electrode capacitance is found to be quantitative with respect to the concentration of MTX. Moreover a calibration curve constructed using the principal component regression method has a linear range of six orders of magnitude and a detection limit of 1.65 × 10(-10) M.

Development and validation of a stability indicating isocratic HPLC method for gemcitabine with application to drug release from poly lactic-co-glycolic acid nanoparticles and enzymatic degradation studies.

OBJECTIVES: Previously reported HPLC methods for gemcitabine determination are time-consuming with complicated mobile phases and gradient elution. Thus, a sensitive and stability-indicating isocratic HPLC method, which provides simple, fast and precise measurements, was developed. This method was applied to study the digestive enzymatic degradation of gemcitabine, for the first time, and the protection afforded following incorporation into poly lactic-co-glycolic acid (PLGA) nanoparticles. METHODS: An analytical HPLC method was developed with an optimized combination of operating conditions. Forced degradation and application of the method to in-vitro drug release studies were conducted. Finally, gemcitabine-loaded nanoparticles were exposed to the digestive enzymes pepsin, trypsin and α-chymotrypsin, and the resulting degradation evaluated. KEY FINDINGS: The analytical method was linear between 1 and 100 µg/ml, with excellent accuracy of 99.91-101.77% and precision of 1.71 or lower, with a 0.014 µg/ml limit of detection (LOD) and a 0.043 µg/ml limit of quantification (LOQ). Following exposure of gemcitabine to stressors, the drug was relatively stable in strong acid (1 N HCl), base (1 N NaOH) and as an aqueous solution exposed to light over 7 days, with less than 10% degradation. However, gemcitabine was more susceptible to degradation at 70°C and oxidative conditions (3% v/v H2 O2 ) with greater than 10% degradation noted after 7 days. In-vitro drug release studies demonstrated a sustained drug release profile from PLGA nanoparticles, which also improved the resistance of gemcitabine to enzymatic degradation. CONCLUSION: These results demonstrate the utility and effectiveness of this simple isocratic HPLC method in evaluating the overall performance of a gemcitabine-loaded formulation.

Fluorescence analysis of 6-mercaptopurine with the use of a nano-composite consisting of BSA-capped Au nano-clusters and core-shell Fe3O4-SiO2 nanoparticles.

A magnetic and fluorescent nano-composite was prepared. It comprised of a core of Fe3O4 nanoparticles (NPs), a silica shell and satellitic Au nano-clusters (AuNCs) capped with bovine serum albumin (BSA). This nano-composite has many desirable properties, e.g. magnetism, red emission, high water solubility, and high resistance to photo-bleaching. On addition of the analyte, 6-mercaptopurine (6-MP) or indeed other similar thiols, AuNCs formed aggregates because the existing cross-links within the Fe3O4 NPs@SiO2 and AuNC structure were broken in favor of the gold-thiol bonds. On suitable irradiation of such aggregates, red fluorescence was emitted at 613 nm. It decreased significantly as a function of the added 6-MP concentration, and the quenching ratio (F0 - F) / F0 was related linearly to the concentration of 6-MP in the range of 0.01 to 0.5 µmol L(-1). The detection limit was 0.004 µmol L(-1) (S/N=3). The method was strongly selective for 6-MP in the presence of oxidants, phenols, heavy-metal ions, and especially bio-thiols.

Anticancer drug detection using a highly sensitive molecularly imprinted electrochemical sensor based on an electropolymerized microporous metal organic framework.

A simple and highly sensitive approach for the detection of the anti-neoplastic drug gemcitabine is presented, based on a one-step electropolymerized molecularly imprinted microporous-metal-organic-framework. The sensitive layer was prepared by electropolymerization of the aniline moieties of p-aminothiophenol- gold nanoparticles on the surface of gold electrodes tethered with p-aminothiophenol, in the presence of gemcitabine as a template molecule. Experimental parameters that control the performance of the sensor were investigated and optimized. Under optimal conditions a calibration curve was obtained in the linear range from 3.8 fM to 38 nM with a limit of detection of 3 fM. The obtained imprinted sensor has the advantages of easy manufacture, high sensitivity and selectivity and good reproducibility. Furthermore the feasibility of the proposed technique has been investigated on spiked serum samples and infusion solution containing gemcitabine.

An in vitro liver model on microfluidic device for analysis of capecitabine metabolite using mass spectrometer as detector.

In this work, an in vitro liver model in a microfluidic device to imitate and detect prodrug metabolism was developed. A widely used prodrug capecitabine (CAP), which needs to be metabolized into active intermediate in the liver and then transformed into final effective drug in tumor cells, was selected as a model compound. The microfluidic device we exploited consists of a cell co-culture section, in which HepG2 cells were cultured to represent liver while MCF-7 cells were used to represent the tumor tissue, and an on-line solid phase extraction (SPE) section connecting to the ionization source of the ESI-Q-TOF mass spectrometer. The prodrug metabolism was realized and confirmed within this in vitro liver model as the intermediate product of the prodrug 5'-deoxy-5-fluorouridine (DFUR) was successfully detected with MS after the conditioning of HepG2 cells, and the anti-tumor effect of the active metabolite was observed through cell vitality assays of MCF-7 cells. The limit of detection (LOD) using on-chip SPE was at 10nM and semi-quantitative analysis could be realized. This system has been proved useful and practical, showing a potential to replace conventional drug screening methods.

Quantitative determination of decitabine incorporation into DNA and its effect on mutation rates in human cancer cells.

Decitabine (5-aza-2'-deoxycytidine) is a DNA methyltransferase inhibitor and an archetypal epigenetic drug for the therapy of myeloid leukemias. The mode of action of decitabine strictly depends on the incorporation of the drug into DNA. However, DNA incorporation and ensuing genotoxic effects of decitabine have not yet been investigated in human cancer cell lines or in models related to the approved indication of the drug. Here we describe a robust assay for the quantitative determination of decitabine incorporation rates into DNA from human cancer cells. Using a panel of human myeloid leukemia cell lines we show appreciable amounts of decitabine incorporation that closely correlated with cellular drug uptake. Decitabine incorporation was also detectable in primary cells from myeloid leukemia patients, indicating that the assay is suitable for biomarker analyses to predict drug responses in patients. Finally, we also used next-generation sequencing to comprehensively analyze the effects of decitabine incorporation on the DNA sequence level. Interestingly, this approach failed to reveal significant changes in the rates of point mutations and genome rearrangements in myeloid leukemia cell lines. These results indicate that standard rates of decitabine incorporation are not genotoxic in myeloid leukemia cells.

[Field survey of the anticancer drug contamination in the preparation environment. Usefulness of the 5-FU monitoring by the coupon method].

For medical professionals involved in cancer chemotherapy, occupational exposure of anticancer agents is considered a health risk. Education about the handling of anticancer drugs and proper use of protective equipment are important for reducing occupational exposure of anticancer drugs. Furthermore, monitoring of the contamination level of anticancer drugs is important for determining the propriety of anti-contamination methods. Cyclophosphamide (CPA) has been used as the standard drug of the contamination level; however, it is rarely detected because of the disparity between drug preparation frequency and consumption, and use of a closed system. Therefore, we chose 5-fluorouracil (5-FU) as the standard drug and attempted to monitor its contamination levels by sampling using drug absorption sheets (the coupon method). We measured contamination levels inside a biological safety cabinet (BSC) and at its lower floor, and at a preparation worktable, nurses' station worktable, its lower floor and floor of the hospital room in a chemotherapy room for outpatients of the Iwate Medical University Hospital for 3 time periods. 5-FU was detected in 72% of the coupons (n=108), while CPA was detected in only 7% of the coupons (n=108). Monitoring of 5-FU contamination levels by using the coupon method was considered useful for evaluating anti-contamination method and the contamination process.

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.

A sensitive LC-MS/MS method for quantifying clofarabine triphosphate concentrations in human peripheral blood mononuclear cells.

Clofarabine triphosphate is an intracellular active metabolite of clofarabine. In the present study, we developed and validated a rapid, sensitive, and selective liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for quantifying clofarabine triphosphate concentrations in human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from blood using the Ficoll gradient centrifugation method. Chromatographic separation was performed on a CN column using an isocratic mobile phase comprising acetonitrile/5mM ammonium acetate with 0.001% ammonium hydroxide (20/80, v/v) at a flow rate of 0.60 mL/min. Detection was carried out by MS/MS in the multiple reaction monitoring mode using a negative electrospray ionization interface. The method was validated in concentration ranges of 1.25-100 ng/10(7) cells with acceptable accuracy and precision using 50 µL of cell extract. Clofarabine triphosphate was stable in a series of stability studies with bench-top, auto-sampler, and repeated freeze-thaw cycles. The validated method was successfully used to measure the concentrations of clofarabine triphosphate in PBMCs from cancer patients treated with clofarabine.

Quantitative determination of azacitidine triphosphate in peripheral blood mononuclear cells using liquid chromatography coupled with high-resolution mass spectrometry.

Azacitidine is a cytidine analog used in the treatment of myelodysplastic syndromes, chronic myelomonocytic leukemia and acute myeloid leukemia. The pharmacological effect of azacitidine arises after incorporation into the DNA and RNA. To this end, the drug first has to be converted into its triphosphate forms. This paper describes the development of an assay for quantitative determination of azacitidine triphosphate (aza-CTP) in peripheral blood mononuclear cells (PBMCs). To quantify aza-CTP, separation from the endogenous nucleotides cytidine triphosphate (CTP) and uridine triphosphate (UTP) is required. This was a challenge as the structures of these nucleotides are highly similar and the monoisotopic molecular masses of aza-CTP, UTP and the naturally occurring [(13)C]- and [(15)N]-isotopes of CTP differ less than 0.02 Da. Efforts to select a specific MS(2)-fragment for aza-CTP using a triple quadrupole mass spectrometer remained without success. Therefore, we investigated the feasibility to separate these highly resembling nucleotides based on accurate mass spectrometry using a linear trap quadrupole (LTQ) coupled with an Orbitrap. The LTQ-Orbitrap was able to differentiate between aza-CTP and the endogenous nucleotides UTP and [(13)C]-CTP. There was no baseline resolution between aza-CTP and [(15)N]-CTP, but the [(15)N]-CTP interference was low. For quantification, extracted ion chromatograms were obtained for the accurate m/z window of the aza-CTP product ion. The assay was able to determine aza-CTP concentrations in PBMC lysate from 40.7 to 281 nM. Assuming that an average cell suspension extracted from 16 mL blood contains 10 to 42 million PBMCs per mL, this range corresponds with 2.58/10.9-17.8/74.9 pmol aza-CTP per million PBMCs. Intra-assay accuracies were between -1.1 and 9.5% deviation and coefficient of variation values were ≤13.2%. The assay was successfully applied to quantify aza-CTP in samples from two patients treated with azacitidine. Aza-CTP concentrations up to 19.0 pmol per million PBMCs were measured. This is the first time that aza-CTP concentrations were quantified in PBMCs from patients treated with azacitidine.

A validated LC-MS/MS method for rapid determination of methotrexate in human saliva and its application to an excretion evaluation study.

A sensitive and simple method for the methotrexate quantification was developed using aminopterin as internal standard. Methotrexate is an anticancer agent that is widely used in a variety of human cancers including primary central nervous system lymphoma. The compound was quantified by liquid-chromatography coupled to electrospray ionization (positive ion-mode) low-energy collision dissociation-tandem mass spectrometry. Quantitative detection was by multiple reaction monitoring of the transitions of the [M+H]+ ion of MTX to its common product ion at m/z 308.4 and of aminopterin at m/z 441.2→m/z 294.0. The method demonstrated linearity over at least three orders of magnitude and had a detection limit of 1ng/ml for methotrexate. A run time of less than 8.0min for each sample made it possible to analyze a large number of human saliva samples per day. Application of this procedure was demonstrated to a saliva excretion study of methotrexate on the samples obtained after an intravenously administration of 1mg/kg/dose of methotrexate to six patients with acute lymphoblastic leukemia.