Gamma and UV radiations induced treatment of anti-cancer methotrexate drug in aqueous medium: Effect of process variables on radiation efficiency evaluated using bioassays.

This studystudy focuses on the effect of radiation treatment and hydrogen peroxide (H2O2) on the toxicity of anticancer methotrexate. For cytotoxicity, different bioassays such as Allium cepa, hemolytic, brine shrimp were employed. The Ames test was used for mutagenicity analysis. The solutions having concentrations 5, 10 and 15 ppm were irradiated with UV radiation exposure time 15, 30, 45, 60, 75 and 90 min and gamma radiation absorbed doses 0.3, 0.6, 0.9, 1.2, 2, 3 and 4 kGy in combination with with H2O2. There was a clear difference observed for aqueous solution before and after treatment with reference to cytotoxicity and mutagenicity. In Allium cepa test, a 47.07, 44.36 and 38.23% increase in root length (RL), root count (RC) and mitotic index (MI) was observed, respectively, for UV/H2O2 treatment and in the case of gamma/H2O2 treatment, the RL, RC and MI were increased up to 49.39, 52.63 and 52.38%, respectively. Brine shrimp test has shown 85.95 and 91.30% decrease in toxicity using UV/H2O2 and gamma/H2O2 respectively, while hemolytic test has shown 19.21 and 26.32% hemolysis using UV/H2O2 and gamma/H2O2, respectively. The mutagenicity reduced up to 82.3, 86.46 and 89.59% (TA98) and 85.42, 87.5 and 90.63% (TA100) for UV/H2O2 while 89.59, 90.63 and 93.75% (TA98) and 84.38, 89.59 and 92.71% (TA100) for gamma/H2O2. The UV and gamma radiation along with H2O2 based AOPs are promising approaches to detoxify the wastewater which can be extended to real hospital liquid effluent effectively.

Mechanism and pathways underlying the self-sensitized photodegradation of methotrexate under simulated solar irradiation.

Methotrexate, a chemotherapeutic agent, was found to undergo self-sensitized photodegradation in aqueous environments. As the initial concentration increased, methotrexate was able to enhance its own direct photolysis reaction not only in DI but also in natural waters. The methotrexate degradation rate increased through the production of singlet oxygen (1O2), the triplet excited state of methotrexate (3MTX*), and the triplet excited state of the pteridine structure (3PT*) from the phototransformation byproducts. At low methotrexate concentrations (<20 ppb), 1O2 played an important role, whereas at higher methotrexate concentrations (>2000 ppb), the presence of oxygen decreased the overall methotrexate degradation rate by physically quenching 3MTX* and 3PT*. The cleavage of the CN bond resulted in a significant amount of byproducts: pteridine derivatives and N-(4-aminobenzoyl)-l-glutamic acid (yields: 13.5 ± 0.6% and 32.3 ± 2.2% for 10 ppm and 500 ppb MTX, respectively). The reactivity of the phototransformation byproducts and the substructures of methotrexate were investigated to help elucidate the proposed self-sensitized pathways. The results indicated that methotrexate as well as compounds containing a pteridine structure will generate pteridine byproducts during photodegradation and 3PT* is the primary triplet excited species that can cause self-sensitized photodegradation.

Stability study of methotrexate in 0.9% sodium chloride injection and 5% dextrose injection with limit tests for impurities.

PURPOSE: Results of an evaluation of the stability of methotrexate in 0.9% sodium chloride injection and 5% dextrose injection are presented. METHODS: Methotrexate concentrated solution (100 mg/mL) was diluted to nominal concentrations of 0.2 and 20 mg/mL in infusion bags containing 0.9% sodium chloride injection or 5% dextrose injection. The filled bags were stored for 28 days at 25 °C and 60% relative humidity and protected from light. Samples were withdrawn for analysis on the day of preparation and after 3, 7, 14, 21, and 28 days. The test program included visual inspections, measurements of pH and infusion bag weight loss, and high-performance liquid chromatography assays to determine methotrexate content and characterize degradation products. RESULTS: At both evaluated concentrations, methotrexate in 0.9% sodium chloride injection was stable for 28 days; only minor (<0.05%) increases in amounts of known and unknown degradation products were detected. In 5% dextrose injection, methotrexate at the higher concentration was stable for 28 days, with minor formation of degradation products; in the 0.2-mg/mL solution, however, methotrexate was stable for only 3 days. At later time points, an unknown impurity present at a concentration higher than 0.1% was observed. CONCLUSION: At concentrations of 0.2 and 20 mg/mL, methotrexate in 0.9% sodium chloride injection was found to be stable for 28 days when stored at 25 °C and protected from light. Under the same storage conditions, methotrexate in a 20-mg/mL solution prepared with 5% dextrose injection was stable for 28 days, whereas a 0.2-mg/mL solution in the same diluent was stable for only 3 days.

Association of ABCC2 -24C>T polymorphism with high-dose methotrexate plasma concentrations and toxicities in childhood acute lymphoblastic leukemia.

Methotrexate (MTX) is a key agent for the treatment of childhood acute lymphoblastic leukemia (ALL). Increased MTX plasma concentrations are associated with a higher risk of adverse drug effects. ATP-binding cassette subfamily C member 2 (ABCC2) is important for excretion of MTX and its toxic metabolite. The ABCC2 -24C>T polymorphism (rs717620) reportedly contributes to variability of MTX kinetics. In the present study, we assessed the association between the ABCC2 -24C>T polymorphism and methotrexate (MTX) toxicities in childhood ALL patients treated with high-dose MTX. A total of 112 Han Chinese ALL patients were treated with high-dose MTX according to the ALL-Berlin-Frankfurt-Muenster 2000 protocol. Our results showed that presence of the -24T allele in ABCC2 gene led to significantly higher MTX plasma concentrations at 48 hours after the start of infusion, which would strengthen over repeated MTX infusion. The -24T allele in ABCC2 gene was significantly associated with higher risks of high-grade hematologic (leucopenia, anemia, and thrombocytopenia) and non-hematologic (gastrointestinal and mucosal damage/oral mucositis) MTX toxicities. This study provides the first evidence that the -24T allele in ABCC2 gene is associated with the severity of MTX toxicities, which add fresh insights into clinical application of high-dose MTX and individualization of MTX treatment.

In vitro pro-oxidant action of Methotrexate in presence of white light.

Methotrexate (MTX) an anti-cancer drug as well as a photosensitizer is able to generate reactive oxygen species (ROS). Cu (II) is present associated with chromatin in cancer cells and has been shown to be capable of mediating the action of several anti-cancer drugs through production of ROS. The objective of the present study is to determine Cu (II) mediated anti-cancer mechanism of MTX under photoilluminated condition as well as alone, using alkaline single cell gel electrophoresis (comet assay). We have shown that cellular DNA breakage was enhanced when Cu (II) is used with MTX as compared to MTX alone. It is also shown that MTX alone as well as in combination with Cu (II) is able to generate oxidative stress in lymphocyte which is inhibited by scavengers of ROS but the pattern of inhibition was differential as was also demonstrated by plasmid nicking assay. Thus, we can say that MTX exhibit pro-oxidant action in presence of white light which gets elevated in presence of Cu (II). Hence, we propose that the mobilization of endogenous copper is possibly involved in killing of cancer cells by MTX during chemo-radio therapy besides acting as antifolate.

Determination of dissociation constants of folic acid, methotrexate, and other photolabile pteridines by pressure-assisted capillary electrophoresis.

Pressure-assisted CE (PACE) was applied to determine the previously inaccessible complete set of pK values for folic acid and eight related multiprotic compounds. PACE allowed the determination of all acidity macroconstants at low (

Methotrexate as a photosensitiser.

The absorption, excitation and emission spectra of methotrexate (MTX) solutions in natural saline water and sodium hydroxide at 10(-4)-10(-5) MpH=8.4 were measured, while they were exposed to coherent and uncoherent visible and near ultraviolet (UV) light. Xe and Hg lamp and also a nitrogen pulsed laser radiation were used. The absorption spectra exhibited spectral bands between 225 nm and 450 nm. The 200 nm - 500 nm excitation spectra were measured with emission centred on 470 nm; fluorescence excitation was observed at 340 nm and 370 nm; emission fluorescence was detected between 400 nm and 600 nm, showing a maximum at 470 nm. Spectra modifications, non-linearly depending on exposure time (varying from 3 min to 180 min), indicated the photodissociation of MTX to the fluorescent compound 2,4 diamino-formylpteridine. In a preliminary in vivo experiment, rapid destruction of neovascularisation was observed in the MTX-impregnated conjunctive of the rabbit eye exposed to uncoherent UV and visible light.

Light activates reduction of methotrexate by NADPH in the ternary complex with Escherichia coli dihydrofolate reductase.

Methotrexate (MTX), a strong inhibitor of dihydrofolate reductase (DHFR), has been widely used for chemotherapy for many types of cancer as well as for juvenile rheumatoid arthritis. It mimics folate substrates and binds tightly to the active site of DHFR, perhaps in a conformation close to the transition state of the folate catalyzed reaction. Absorption, fluorescence and ultrasensitive Raman difference spectroscopies show that light-activated MTX reacts with NADPH in the enzyme active site, producing 5,8-dihydromethotrexate (5,8-dihydro-MTX) and NADP+. The reaction, which proceeds with a hydride transfer between C4 (pro-R side) of the nicotinamide ring and N5 of the pteridine ring, is similar to that between folate and NADPH except that the hydride is transferred to C6 in this case. Hence, MTX is catalytically competent in its excited state. Most experiments were performed on the Escherichia coli enzyme, but preliminary studies show that the reaction also occurs with human DHFR.

Time dependent inhibition of xanthine oxidase in irradiated solutions of folic acid, aminopterin and methotrexate.

The xanthine oxidase catalyzed oxidation of hypoxanthine was followed by monitoring the formation of uric acid at 290 nm. Inhibition of xanthine oxidase occurs in aqueous solutions of folic acid methotrexate and aminopterin. These compounds are known to dissociate upon exposure to ultraviolet light resulting in the formation of their respective 6-formylpteridine derivatives. The relative rates of dissociation were monitored spectrophotometrically by determining the absorbance of their 2,4-dinitrophenylhydrazine derivatives at 500 nm. When aqueous solutions of folic acid, aminopterin and methotrexate were exposed to uv light, a direct correlation was observed between the concentrations of the 6-formylpteridine derivatives existing in solution and the ability of these solutions to inhibit xanthine oxidase. The relative potency of the respective photolysis products were estimated.

Column liquid chromatography of methotrexate and its metabolites using a post-column photochemical reactor and fluorescence detection.

On irradiation with short-wavelength UV light in the presence of hydrogen peroxide, methotrexate and its metabolites 7-hydroxymethotrexate and 2,4-diamino-N10-methylpteroic acid are cleaved into highly fluorescent products. This reaction can be used for the sensitive and selective detection of the compounds in biological fluids, following reversed-phase high-performance liquid chromatographic separation. Study of the effect of the mobile phase composition and irradiation time on fluorescence signal intensity showed that a residence time of ca. 3 s in the on-line photochemical reactor was best. The detection limit for methotrexate was 0.4 ng, for 7-hydroxymethotrexate 1.0 ng and for 2,4-diamino-N10-methylpteroic acid 0.6 ng. The addition of dimethylformamide to the mobile phase enhanced the selectivity of separation.

Thermal and photolytic decomposition of methotrexate in aqueous solutions.

The chemical kinetics of thermal and photolytic degradation of methotrexate in aqueous solutions were studied. At above pH 7 and 85 degrees, methotrexate hydrolyzed to yield mainly N10-Methylpteroylglutamic acid. The hydrolysis rate followed first-order kinetics with respect to methotrexate concentration and increases rapidly above pH 9. When methotrexate solutions were kept under laboratory fluorescent light, the major degradation products were 2,4-diamino-6-pteridinecarbaldehyde, 2,4-diamino-6-pteridinecarboxylic acid, and p-aminobenzoylglutamic acid. The photolytic reaction followed zero-order kinetics with respect to methotrexate concentration and was catalyzed by bicarbonate ion. Evidence is presented for a proposed sequential cleavage of methotrexate by a free radical mechanism for the photolysis. Commercial parenteral methotrexate was found to be quite stable as marked when stored in the original vial at room temperature.

High-pressure liquid chromatographic analysis of methotrexate in presence of its degradation products.

Two high-pressure liquid chromatographic methods are described for the quantitative determination of methotrexate in the presence of its contaminants and degradation products. Method 1 takes less than 15 min and is recommended for routine assays of methotrexate in commercial products. Method 2 takes about 35 min and is the method of choice to detect and quantitate large amounts of degradation products. Quantitation to a level of 1 microgram of methotrexate/ml is feasible by these methods and thus provides potential applicability for the analysis of methotrexate in biological fluids.