New Therapeutic Approach to Reduce Methotrexate Toxicity after High-Dose Chemotherapy in a Child with Acute Lymphocytic Leukemia: Efficacy and Safety of Hemoadsorption with HA-230 Adsorber.

BACKGROUND: High-dose methotrexate (HDMTX) is likely to cause a number of side effects and manifest itself as hepatotoxicity, nephrotoxicity, mucositis, and neurotoxicity. A several studies demonstrated the efficacy of extracorporeal detoxification methods such as plasma exchange, hemodialysis (HD), HD filtration, and hemoperfusion for the treatment of MTX delayed clearance. However, none of the existing methods as effective as expected and limited for general implementation due to a procedure-related complication. CASE REPORT: Here, we report a successful implementation of HA-230 hemoadsorption procedure to remove cumulated MTX from the body and reduce its toxicity in a child with ALL after high-dose chemotherapy. RESULTS AND CONCLUSION: Based on our results, single-hemoadsorption procedure with the HA-230 adsorber in case of delayed methotrexate clearance was safe and well-tolerated in a pediatric patient with ALL and would significantly improve the patient's condition. Further studies need to demonstrate its safety and efficacy in a large number of pediatric patients.

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.

Molecular Networking Reveals Two Distinct Chemotypes in Pyrroloiminoquinone-Producing Tsitsikamma favus Sponges.

The temperate marine sponge, Tsitsikamma favus, produces pyrroloiminoquinone alkaloids with potential as anticancer drug leads. We profiled the secondary metabolite reservoir of T. favus sponges using HR-ESI-LC-MS/MS-based molecular networking analysis followed by preparative purification efforts to map the diversity of new and known pyrroloiminoquinones and related compounds in extracts of seven specimens. Molecular taxonomic identification confirmed all sponges as T. favus and five specimens (chemotype I) were found to produce mainly discorhabdins and tsitsikammamines. Remarkably, however, two specimens (chemotype II) exhibited distinct morphological and chemical characteristics: the absence of discorhabdins, only trace levels of tsitsikammamines and, instead, an abundance of unbranched and halogenated makaluvamines. Targeted chromatographic isolation provided the new makaluvamine Q, the known makaluvamines A and I, tsitsikammamine B, 14-bromo-7,8-dehydro-3-dihydro-discorhabdin C, and the related pyrrolo-ortho-quinones makaluvamine O and makaluvone. Purified compounds displayed different activity profiles in assays for topoisomerase I inhibition, DNA intercalation and antimetabolic activity against human cell lines. This is the first report of makaluvamines from a Tsitsikamma sponge species, and the first description of distinct chemotypes within a species of the Latrunculiidae family. This study sheds new light on the putative pyrroloiminoquinone biosynthetic pathway of latrunculid sponges.

Multianalyte determination of 24 cytostatics and metabolites by liquid chromatography-electrospray-tandem mass spectrometry and study of their stability and optimum storage conditions in aqueous solution.

A multianalyte liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) method for determination of 19 cytostatics and 5 metabolites, from 6 different therapeutic families, has been developed, and the structures of the main characteristic fragment ions have been proposed. Instrumental limits of detection and quantification are in the range 0.1-10.3 and 1.0-34.3 ng mL(-1), respectively. Moreover, the stability of the compounds in aqueous solution was investigated in order to establish the best conditions for preparation and storage of both calibration standards and water samples. Dimethylsulphoxide (DMSO) was selected as solvent for preparation of the stock solutions. At room temperature (25 °C), 11 of the 24 target compounds were shown to be unstable in water (percentage of organic solvent 4%), with concentration losses greater than 20% in less than 24 h. At 4 °C (typical storage temperature for water samples) all compounds, except MTIC and chlorambucil, were stable for 24h, but the number of stable compounds decreased to 10 after 9 days. Freezing of the aqueous solutions improved considerably the stability of various compounds: after 3 months of storage at -20 °C, 10 compounds, namely, 5-fluorouracil, carboplatin, gemcitabine, temozolomide, vincristine, vinorelbine, ifosfamide, cyclophosphamide, etoposide, and capecitabine, remained stable (in contrast to only carboplatin and capecitabine at 4 °C). The addition of acid improved the stability of methotrexate and its metabolite hydroxy-methotrexate but not that of the rest of compounds. The addition of organic solvent (50% methanol or DMSO) prevented the degradation at 4 °C of the otherwise unstable compounds oxaliplatin, methotrexate, erlotinib, doxorubicin, tamoxifen, and paclitaxel. To the authors' knowledge, five of the analytes investigated have never been searched for in the aquatic environment (imatinib, 6α-hydroxypaclitaxel, endoxifen, (Z)4-hydroxytamoxifen, and temozolomide), and for many of them the stability data provided, and even the analytical LC-MS/MS conditions, are the first ever published.

Monitoring methotrexate in clinical samples from cancer patients during chemotherapy with a LSPR-based competitive sensor.

A competitive binding assay based on localized surface plasmon resonance (LSPR) of folic acid-functionalized gold nanoparticles (FA-AuNPs) and human dihydrofolate reductase enzyme (hDHFR) was developed to detect nanomolar to micromolar concentrations of the widely applied anti-cancer drug, methotrexate (MTX). By the nature of the competitive assay for MTX, the LSPR shift from specific binding between FA-AuNPs and the free enzyme was inversely proportional to the concentration of MTX. In addition, the dynamic range for MTX was tuned from 10(-11) to 10(-6) M by varying the concentration of hDHFR from 1 to 100 nM. Inter-day reproducibility and recovery of MTX spiked in phosphate buffer saline (PBS) were excellent. Potential interferents such as FA, trimethoprim (TMP) and 4-amino-4-deoxy-N-methylpteroic acid (DAMPA) did not occur in the concentration range of interest for MTX. Clinical samples of human serum from patients undergoing MTX chemotherapy were analyzed following a simple solid-phase extraction step to isolate MTX from the serum matrix, with a limit of detection of 155 nM. Validation of the LSPR method was carried out in comparison to Fluorescence Polarization Immunoassay (FPIA), a commonly used method in clinical settings, and LC-MS/MS, a reference technique. The results of the LSPR competitive assay compared well to FPIA and LC-MS/MS, with a slope of 2.4 and 1.1, respectively, for the correlation plots. The method established herein is intended for therapeutic drug monitoring (TDM) of MTX levels in patients undergoing chemotherapy to ensure safety and efficacy of the treatment.

New pemetrexed-peptide conjugates: synthesis, characterization and in vitro cytostatic effect on non-small cell lung carcinoma (NCI-H358) and human leukemia (HL-60) cells.

Pemetrexed (Pem) is a novel antimetabolite type of anticancer drug that demonstrated promising clinical activity in a wide variety of solid tumors, including non-small cell lung carcinoma and malignant pleural mesothelioma. It inhibits enzymes involved in the folate pathway, for which the presence of its free carboxylic groups is necessary. The heteroaromatic ring system of Pem has a modifiable amino group, which opens a possibility to apply a new strategy to conjugate Pem to carrier molecules. Considering this as well as the necessity of untouched carboxylic groups of Pem in the new conjugates, we developed a new synthesis strategy. Here, we describe the synthesis and the characterization of new Pem-peptide conjugates in which cell-penetrating octaarginine or/and lung-targeting H-Ile-Glu-Leu-Leu-Gln-Ala-Arg-NH(2) peptide is attached to the drug by thioether bond. The conjugates characterized by RP-HPLC and MS exhibited cytostatic effect in vitro on non-small cell lung carcinoma as well as on human leukemia cell lines. The IC(50) values of the conjugates were similar, but the conjugates with H-Ile-Glu-Leu-Leu-Gln-Ala-Arg-NH(2) sequence were slightly more effective. Our data show that the in vitro cytostatic effect of the free Pem was essentially maintained after conjugation with cell-penetrating or cell-targeting peptides. Thus, the conjugation strategy reported could lead to the development of a new generation of active Pem conjugates.

Quenched phosphorescence as alternative detection mode in the chiral separation of methotrexate by electrokinetic chromatography.

Quenched phosphorescence was used, for the first time, as detection mode in the chiral separation of methotrexate (MTX) enantiomers by electrokinetic chromatography. The detection is based on dynamic quenching of the strong emission of the phosphorophore 1-bromo-4-naphthalene sulfonic acid (BrNS) by MTX under deoxygenated conditions. The use of a background electrolyte with 3 mg/mL 2-hydroxypropyl-ß-cyclodextrin and 20% MeOH in 25 mM phosphate buffer (pH 7.0) and an applied voltage of 30 kV allowed the separation of L-MTX and its enantiomeric impurity D-MTX with sufficient resolution. In the presence of 1 mM BrNS, a detection limit of 3.2 × 10(-7) M was achieved, about an order of magnitude better than published techniques based on UV absorption. The potential of the method was demonstrated with a degradation study and an enantiomeric purity assessment of L-MTX. Furthermore, L-MTX was determined in a cell culture extract as a proof-of-principle experiment to show the applicability of the method to biological samples.

Simultaneous quantification of 2',2'-difluorodeoxycytidine and 2',2'-difluorodeoxyuridine nucleosides and nucleotides in white blood cells using porous graphitic carbon chromatography coupled with tandem mass spectrometry.

A novel assay for the simultaneous quantification of the widely used anticancer agent 2',2'-difluorodeoxycytidine (gemcitabine; dFdC), its deaminated metabolite 2',2'-difluorodeoxyuridine (dFdU) and their mono-, di- and triphosphates (dFdCMP, dFdCDP, dFdCTP, dFdUMP, dFdUDP and dFdUTP) in peripheral blood mononuclear cells (PBMCs) is described. Separation of all eight compounds was achieved within 15 min using a porous graphitic carbon column (Hypercarb) with a gradient from 0 to 25 mM ammonium bicarbonate in acetonitrile/water (15:85, v/v). Calibration ranges in PBMC lysate from 4.29 to 429, 29.0 to 2900, 31.4 to 3140 and 36.9 to 3690 nM for dFdC, dFdCMP, dFdCDP and dFdCTP and from 42.1 to 4210, 25.4 to 2540, 43.2 to 4320 and 52.7 to 5270 nM for dFdU, dFdUMP, dFdUDP and dFdUTP, respectively, were validated. Accuracies were within 82.3-119% at the lower limit of quantification (LLOQ) and the precisions were less than 20.0%. At the other tested levels accuracies were within 91.4-114% and precisions less than 14.9%. Mixtures of (13)C,(15)N(2)-labeled dFdC and dFdU nucleotides were synthesized and used as internal standards. Whole blood samples showed extensive ongoing dFdC metabolism when stored at room temperature, but not on ice-water, which made the addition of enzyme inhibitors unnecessary. Stock solutions and samples were stable under all analytically relevant conditions. The method was successfully applied to clinical samples.

10-Oximeguanacone, the first nitrogenated acetogenin derivative found to be a potent inhibitor of mitochondrial complex I.

A new 10-keto bis-tetrahydrofuran acetogenin, guanacone (1), has been isolated from a cytotoxic extract of Annona aff. spraguei seeds. The 10-oximeguanacone derivative 1f is the first bioactive nitrogenated acetogenin found to be a very potent inhibitor of complex I. In addition, a SAR study of guanacone analogues is reported based on the titration of the NADH oxidase and NADH:ubiquinone oxidoreductase activities.

Overexpression and large-scale production of recombinant L-methionine-alpha-deamino-gamma-mercaptomethane-lyase for novel anticancer therapy.

The goal of the next generation of cancer chemotherapy is effective tumor-selectivity. A tumor-selective target with high therapeutic potential is the elevated methionine requirement of tumor cells relative to normal cells. We have termed the elevated requirement for methionine in tumors methionine dependence. To selectively target the methionine dependence of tumors for treatment on a large-scale preclinical and clinical basis, the L-methionine alpha-deamino-gamma-mercaptomethane-lyase (methioninase, METase) gene from Pseudomonas putida has been cloned in Escherichia coli using the polymerase chain reaction (PCR). The METase gene was then ligated into the pT7-7 overexpression plasmid containing the T7 RNA polymerase promoter and recloned in E. coli strain BL21(DE3). The pAC-1 clone was isolated by its yellow-orange color which is due to high enrichment of the pyridoxal phosphate-containing recombinant methioninase (rMETase) and distinguished rMETase-overproducer from rMETase-negative colonies. A scale-up production protocol which contained a heat step, two DEAE Sepharose FF ion-exchange, and one ActiClean Etox endotoxin-affinity chromatography columns has been established. The pAC-1 clone produces rMETase at approximately 10% of the total soluble protein and up to 1 g/liter in shake-flask culture. The protocol can produce therapeutic rMETase at the multi-gram level per batch with high yield (> 60%), high purity (> 98%), high stability, and low endotoxin. Purified rMETase is stable to lyophilization. The t1/2 of rMETase was 2 h when rMETase was administered by i.v. injection in mice. Studies of the antitumor efficacy of rMETase in vitro and in vivo on human tumors xenografted in nude mice demonstrated that all types of human tumors tested including those from lung, colon, kidney, brain, prostate, and melanoma were sensitive to rMETase. In contrast, normal cells were insensitive to rMETase in vitro and correspondingly, no toxicity was detected in vivo at the effective doses. In conclusion, the overexpression clone and large-scale production protocols for rMETase have enabled rMETase to be used as a tumor-selective therapeutic with broad indication and high promise for effective, low-toxicity human cancer therapy.

7-Hydroxyguanine, a novel antimetabolite from a strain of Streptomyces purpurascens. I. Taxonomy of producing organism, fermentation, isolation and biological activity.

Strain A-347, an actinomycete isolated from a soil sample, was found to produce a new antimetabolite, 7-hydroxyguanine. The aerial mycelium formed spiral spore chains with spiny spore surface. The chemical composition of strain A-347 indicated that it was an actinomycete of cell wall Type I. From its morphological, cultural, physiological characteristics and direct comparison with the type culture, this strain was classified as Streptomyces purpurascens. 7-Hydroxyguanine was purified from the broth filtrate by ion exchange chromatography and crystallized from hot 2 M NH4OH. 7-Hydroxyguanine inhibited the growth of experimental tumors such as L1210 leukemia.