RESUMO
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.
Assuntos
Antimetabólitos Antineoplásicos/isolamento & purificação , Antimetabólitos Antineoplásicos/toxicidade , Hemoperfusão , Metotrexato/isolamento & purificação , Metotrexato/toxicidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/sangue , Criança , Hemoperfusão/métodos , Humanos , Metotrexato/administração & dosagem , Metotrexato/sangueRESUMO
The evaluation of encapsulation efficiency is a regulatory requirement for the characterization of drug delivery systems. However, the difficulties in efficiently separating nanomedicines from the free drug may compromise the achievement of accurate determinations. Herein, ultrafiltration was exploited as a separative strategy towards the evaluation of methotrexate (MTX) encapsulation efficiency in nanostructured lipid carriers and polymeric nanoparticles. The effect of experimental conditions such as pH and the amount of surfactant present in the ultrafiltration media was addressed aiming at the selection of suitable conditions for the effective purification of nanocarriers. MTX-loaded nanoparticles were then submitted to ultrafiltration and the portions remaining in the upper compartment of the filtering device and in the ultrafiltrate were collected and analyzed by HPLC-UV using a reversed-phase (C18) monolithic column. A short centrifugation time (5 min) was suitable for establishing the amount of encapsulated MTX in nanostructured lipid carriers, based on the assumption that the free MTX concentration was the same in the upper compartment and in the ultrafiltrate. The defined conditions allowed the efficient separation of nanocarriers from the free drug, with recoveries of >85% even when nanoparticles were present in cell culture media and in pig skin surrogate from permeation assays.
Assuntos
Portadores de Fármacos , Metotrexato/química , Metotrexato/isolamento & purificação , Nanopartículas , Ultrafiltração , Portadores de Fármacos/química , Composição de Medicamentos , Sistemas de Liberação de Medicamentos , Metotrexato/farmacologia , Nanopartículas/química , Nanomedicina TeranósticaRESUMO
Methotrexate (MTX) is widely used for the treatment of many types of cancer. Folinic acid (FNA) and folic acid (FA) were usually simultaneously supplemented with MTX to reduce the side effects of a folate deficiency. This study, for the first time, included on-line sample preconcentration by stacking and sweeping techniques under reduced or enhanced electric conductivity in the sample region using short chain alkyl imidazolium ionic liquids (ILs) as micelle forming agents for analyte focusing. Both analyte focusing by micelle collapse (AFMC) and sweeping-MEKC had been investigated for the comparison of their effectiveness to examine simultaneously MTX, FNA and FA in plasma and urine under physiological conditions. In sweeping-MEKC, the sample solution without micelles was hydrodynamically injected as a long plug into a fused-silica capillary pre-filled with phosphate buffer containing 3.0 mol/L of 1-butyl-3-methylimidazolium bromide (BMIMBr). Using AFMC, the analytes were prepared in BMIMBr micellar matrix and hydrodynamically injected into the phosphate buffer without IL micelles. The conductivity ratio between BGE and sample (γ, BGE/sample) was optimized to be 3.0 in sweeping-MEKC and 0.33 in AFMC resulting the adequate separation of analytes within 4.0 min. To reduce the possibility of BMIMBr adsorption, an appropriate rinsing protocol was used. The limits of detection were calculated as 0.1 ng/mL MTX, 0.05 ng/mL FNA and 0.05 ng/mL FA by sweeping-MEKC and 0.5 ng/mL MTX, 0.3 ng/mL FNA and 0.3 ng/mL FA by AFMC. The accuracy was tested by recovery in plasma and urine matrices giving values ranging between 90 and 110%. Both stacking and sweeping by BMIMBr could be successfully used for the rapid, selective and sensitive determination of pharmaceuticals in complex matrices due to its fascinating properties, including high conductivity, good thermal stability and ability to form different types of interactions by electrostatic, hydrophobic, hydrogen bonding and π-π interactions. In sweeping-MEKC, the using of BMIMBr enhanced the γ factor, k retention factor and the injected amount of sample. Consequently, this technique offers particular potential for higher sensitivity by giving 22- and 5-fold sensitivity enhancement factors (SEFs) of MTX compared to CZE and AFMC, respectively.
Assuntos
Cromatografia Capilar Eletrocinética Micelar/métodos , Ácido Fólico/isolamento & purificação , Imidazóis/química , Líquidos Iônicos/química , Leucovorina/isolamento & purificação , Metotrexato/isolamento & purificação , Ácido Fólico/sangue , Ácido Fólico/química , Ácido Fólico/urina , Humanos , Leucovorina/sangue , Leucovorina/química , Leucovorina/urina , Limite de Detecção , Modelos Lineares , Metotrexato/sangue , Metotrexato/química , Metotrexato/urina , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
Antimetabólitos Antineoplásicos/sangue , Metotrexato/sangue , Ressonância de Plasmônio de Superfície , Antimetabólitos Antineoplásicos/isolamento & purificação , Antimetabólitos Antineoplásicos/uso terapêutico , Cromatografia Líquida de Alta Pressão , Monitoramento de Medicamentos , Imunoensaio de Fluorescência por Polarização , Ácido Fólico/química , Ouro/química , Humanos , Nanopartículas Metálicas/química , Metotrexato/isolamento & purificação , Metotrexato/uso terapêutico , Neoplasias/tratamento farmacológico , Extração em Fase Sólida , Espectrometria de Massas em Tandem , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
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.
Assuntos
Antimetabólitos Antineoplásicos/isolamento & purificação , Eletroforese Capilar/métodos , Antagonistas do Ácido Fólico/isolamento & purificação , Medições Luminescentes/métodos , Metotrexato/isolamento & purificação , Linhagem Celular Tumoral , Humanos , Preparações Farmacêuticas/química , EstereoisomerismoRESUMO
Methotrexate, as a folate antagonist, is one of the first anti-neoplasm drugs offered and is still used as an effective drug in the treatment of various malignancies. Methotrexate has a narrow treatment index and is associated with numerous side effects.In thisresearch, for the first time a double-solvent supramolecular system (DSS) was developed as an extractant without disperser solvent for dispersive liquid-liquid microextraction (DLLME). DSS - DLLME was applied to the extraction of methotrexate in plasma of children with acute leukemiaprior to itsdetermination by high-performance liquid chromatography-ultraviolet detection (HPLC - UV). In the present method, two long normal chain alcohols are mixed in a particular ratio, and then it is injected into the sample solution, which is on the magnetic stirrer. In this case, the mixture of the two alcohol changes to new supramolecular aggregate. This new supermolecule is used as an extractant, which has a higher extraction power than any of its components alone. Under the optimum conditions, the calibration graph was linear in the rage of 0.1-150 µg L-1 with detection limit of 0.03 µg L-1. Relative standard deviations (RSDs) including intra-day and inter-day of method based on7 replicate determinations of 100.0 µg L-1of methotrexate were 2.6% and 4.8%,respectively. The results proved that DSS - DLLME is a sensitive, very simple, inexpensive, environmental friendly, rapid and efficient method for the preconcentration of trace amount of drugs in biological samples.
Assuntos
Leucemia/tratamento farmacológico , Microextração em Fase Líquida/métodos , Metotrexato/sangue , Doença Aguda , Criança , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Limite de Detecção , Modelos Lineares , Metotrexato/isolamento & purificação , Metotrexato/uso terapêutico , Reprodutibilidade dos Testes , Solventes/químicaRESUMO
For biological assessing of hospital personnel occupationally exposed to antineoplastic drugs, highly sensitive and accurate methods are required. Methotrexate (MTX) is an anticancer agent that is widely used in a variety of human cancers. For the first time, dispersive-micro solid phase extraction (D-µ-SPE) has been applied for determination of low levels of MTX in saliva samples. The method is based on rapid extraction of MTX using graphene oxide adsorbent. The sample preparation time is decreased by the fact that the adsorbent dispersed in the sample solution and extraction equilibrium can be reached very fast. This significant feature which obtained with this method is of key interest for routine trace laboratory analysis. The influence of different variables on D-µ-SPE was investigated. Under optimum conditions, the calibration graph was linear over the range of 10-1000 ng/ml. The relative standard deviations are better than 9.0%. The proposed method was successfully applied for the determination of MTX in patient samples.
Assuntos
Metotrexato/isolamento & purificação , Metotrexato/metabolismo , Saliva/metabolismo , Extração em Fase Sólida/métodos , Espectrofotometria/métodos , Grafite/química , Humanos , Concentração de Íons de Hidrogênio , Metotrexato/análise , Saliva/químicaRESUMO
Methotrexate (MTX) is an anticancer drug that is widely used in a variety of cancers including primary central nervous system lymphoma. It is also administrated in the treatment of some autoimmune diseases. A simple, accurate, sensitive, and precise mixed hemimicelles dispersive micro-solid phase extraction was proposed for MTX quantification in human urine samples. MTX was quantified by spectrophotometer after dispersive micro-solid phase extraction using ionic liquid functionalized magnetic graphene oxide/polypyrrole. Interactions of adsorbent and MTX were modeled by molecular docking and the interaction energy was predicted to be -8.35â¯kcal/mol. A larger absolute value of binding energy represents larger adsorption strength, indicating that graphene oxide nanosheets could perform higher adsorption strength toward MTX. The concentrations of MTX were proportional to analytical response in amounts ranging from 10 to 1000â¯ng/mL with a good correlation (R2â¯=â¯0.99). Inter- and intra-day precisions and accuracies were within the acceptable limit according to FDA guideline (15% for biological determination). The recoveries were ranging from 89 to 93% and the method was specific for routine analysis of MTX. This protocol was applied to the urine of two patients under MTX therapy received an intravenous administration of 1â¯mg/kg/dose of MTX with acute lymphoblastic leukemia. The accuracy of the method was confirmed by HPLC measurements.
Assuntos
Grafite/química , Líquidos Iônicos/química , Metotrexato/isolamento & purificação , Polímeros/química , Pirróis/química , Microextração em Fase Sólida , Humanos , Concentração de Íons de Hidrogênio , Fenômenos Magnéticos , Metotrexato/química , Metotrexato/urina , Modelos Moleculares , Estrutura Molecular , Nanocompostos/química , Espectrofotometria , Difração de Raios XRESUMO
Methotrexate from various commercial sources has been found to contain 0.5 to 48% (w/w) of the enantiomer D-methotrexate. The two methotrexate enantiomers were separated by using chiral high-performance liquid chromatography with an octadecyl silica column and a mobile phase containing L-proline and cupric nitrate. For the assay of D-methotrexate impurity in commercial methotrexate, L-methotrexate was hydrolyzed with carboxypeptidase G1, and the remaining D-methotrexate was quantitated by high-performance liquid chromatography. The biological effects of D-methotrexate were investigated and compared to that of L-methotrexate. D-Methotrexate was found to be a good inhibitor of dihydrofolate reductase from both murine and human tumor cells, but was a poor inhibitor of L1210 and CCRF-CEM cell growth. In animal experiments with dogs and mice, D-methotrexate was rapidly absorbed from the intestine and excreted by the kidneys.
Assuntos
Metotrexato/isolamento & purificação , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cães , Antagonistas do Ácido Fólico , Cinética , Leucemia L1210/fisiopatologia , Metotrexato/metabolismo , Metotrexato/toxicidade , Camundongos , Camundongos Endogâmicos , EstereoisomerismoRESUMO
Previous investigations have shown that untargeted liposomes, in which methotrexate is anchored to the lipid bilayers as methotrexate-gamma-dimyristoylphosphatidylethanolamine (methotrexate-gamma-DMPE), can inhibit in vitro cell proliferation. To test the possibility that this inhibition may involve extracellular metabolism of methotrexate-gamma-DMPE, we have degraded it chemically (dilute alkali) or enzymatically (phospholipase A2, phospholipase C, phospholipase C plus phosphatase), and assayed the products using human lymphoblastoid T cells or a subline that has a defective methotrexate transport system. Neither methotrexate-gamma-(1-myristoyl)-glycerophosphorylethanolamine, methotrexate-gamma-glycerophosphorylethanolamine, methotrexate-gamma-phosphorylethanolamine, nor methotrexate-gamma-ethanolamine resemble methotrexate-gamma-DMPE sensitized liposomes or the free derivative in their ability to block tritiated deoxyuridine incorporation into DNA. When added extracellularly, these putative metabolites manifest a higher ID50 concentration and/or, unlike the liposomes or unincorporated methotrexate-gamma-DMPE, utilize the methotrexate transport system to enter cells. Additionally, we have synthesized methotrexate-gamma-dihexadecylphosphatidylethanolamine and methotrexate-gamma-hexadecylphosphorylethanolamine, analogs of methotrexate-gamma-DMPE that cannot be hydrolyzed by phospholipases A2, C and D; liposomes prepared with these derivatives are markedly less potent cytotoxic agents than methotrexate-gamma-DMPE sensitized liposomes. All together, these results are consistent with the conclusion that methotrexate-gamma-DMPE must undergo intracellular metabolism to exert optimal inhibition; they also bear on possible mechanisms by which methotrexate-gamma-DMPE may enter cells.
Assuntos
Divisão Celular/efeitos dos fármacos , Metotrexato/análogos & derivados , Fosfatidiletanolaminas/farmacologia , Linhagem Celular , Desoxiuridina/metabolismo , Humanos , Lipossomos/metabolismo , Metotrexato/isolamento & purificação , Metotrexato/metabolismo , Metotrexato/farmacologia , Fosfatidiletanolaminas/isolamento & purificação , Fosfatidiletanolaminas/metabolismo , Linfócitos T/metabolismoRESUMO
A simple, rapid and sensitive procedure using capillary zone electrophoresis (CZE) to measure methotrexate, folinic acid and folic acid in human urine has been developed and validated. Optimum separation of methotrexate, folinic acid and folic acid was obtained on a 60 cm x 75 microm capillary using a 15 mM phosphate buffer solution (pH 12.0), temperature and voltage 20 degrees C and 25 kV, respectively and hydrodynamic injection. Under these conditions the analysis takes approximately 9.0 min. Good results were obtained for different aspects including stability of the solutions, linearity, accuracy and precision. Before CZE determination, the urine samples were purified and enriched by means of a solid phase extraction step with a preconditioned C(18) cartridge and eluting the compound with a mixture 1:1 of methanol:water. A linear response over the urine concentration range 1.0-6.0 mgL(-1) for MTX and 0.5-6.0 mgL(-1) for folinic acid and folic acid was observed. Detection limits for the three compound in urine were 0.35 mgL(-1). CZE was shown to be a good method with regard to simplicity, satisfactory precision, and sensitivity.
Assuntos
Eletroforese Capilar/métodos , Ácido Fólico/urina , Leucovorina/urina , Metotrexato/urina , Biomarcadores/urina , Estabilidade de Medicamentos , Ácido Fólico/isolamento & purificação , Humanos , Concentração de Íons de Hidrogênio , Leucovorina/isolamento & purificação , Metotrexato/isolamento & purificação , Concentração Osmolar , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , TemperaturaRESUMO
Disposable microliter immunoabsorbent columns were constructed from pasteur pipets. The bed support was a cube of gelatin surgical sponge, which was tamped into the pipet tip. Column dead space, represented by the compressed volume of the sponge, was 5 microliters. The columns were used with protein A-Sepharose; settled bed volumes were 50 microliters. It was possible to pour columns that functioned as immunoabsorbents with bed volumes as small as 10 microliters. There was no gravity flow through these columns. Flow was achieved by touching column tips to absorbent paper if liquid was to be discarded or to 50 microliters capillary tubes for fluid collection. A simple capillary collection tube assembly was designed for operation of a row of 10 columns at a time. In a test system of [3H]methotrexate and IgG anti-methotrexate, 90% of applied antigen was bound to antibody columns, whereas 90% was recovered in the eluates from control columns. The columns were used in the initial step of screening uncloned hybridoma culture fluids for anti-MSP.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade/instrumentação , Imunoglobulina G/isolamento & purificação , Técnicas de Imunoadsorção/instrumentação , Microquímica/instrumentação , Animais , Anticorpos Monoclonais/imunologia , Quimiotaxia de Leucócito , Hibridomas/imunologia , Linfocinas/imunologia , Linfocinas/isolamento & purificação , Ativação de Macrófagos , Fatores Ativadores de Macrófagos , Macrófagos/imunologia , Metotrexato/imunologia , Metotrexato/isolamento & purificação , Camundongos , Sefarose , Proteína Estafilocócica ARESUMO
Affinity chromatography on methotrexate-Sepharose has been used to evaluate dissociation constants for interactions of methotrexate with dihydrofolate reductase from Lactobacillus casei. Equilibrium constants of 0.25 microM and 0.6 nM were obtained for dissociation of the inhibitor from the enzyme-methotrexate and enzyme-NADPH-methotrexate complexes, respectively, these estimates being in good agreement with the corresponding published values for dihydrofolate reductase from Streptococcus faecium. By employing a different method for evaluating the thermodynamic dissociation constant for the enzyme-NADPH-methotrexate interaction, this investigation provides independent support for the inference drawn from published fluorescence quenching studies that the interaction of methotrexate with dihydrofolate reductase-NADPH complex is governed by a dissociation constant in the vicinity of 600 pM, a value slightly higher than, but approaching, the inhibition constant of 50-60 pM obtained by enzyme kinetic techniques.
Assuntos
Metotrexato/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Cromatografia de Afinidade/métodos , Cinética , Lacticaseibacillus casei/enzimologia , Metotrexato/isolamento & purificação , Ligação Proteica , Tetra-Hidrofolato Desidrogenase/isolamento & purificação , TermodinâmicaRESUMO
To study the influence of the level of dihydrofolate reductase (DHFR) on methotrexate (MTX) metabolism, the formation of methotrexate polyglutamates (MTXPGs) and the retention of the drug were examined in Chinese hamster ovary cells (DUKXB11) lacking DHFR and in control cells (CHO-UTC). Both cells accumulated MTXPGs poorly. After a 24-hr incubation with 1.0 microM [3H]MTX, the level of total MTX in DUKXB11 cells was 40% of that in CHO-UTC cells, reflecting the lack of DHFR-bound MTX and MTXPGs in the mutant cells. MTXPGs accounted for a higher proportion of the intracellular MTX in DUKXB11 than in CHO-UTC cells (25 vs 18%). Following exposure to 3.0 microM MTX for 24 hr, total drug levels were similar in both cell lines, and MTXPGs constituted even more of the intracellular drug in DUKXB11 cells compared to CHO-UTC cells (34 vs 23%). DUKXB11 cells accumulated longer MTXPGs (MTXG1u3,4) compared to CHO-UTC cells (MTXG1u2,3), following exposure to both 1.0 and 3.0 microM MTX. The longer MTXPGs in the mutant cells may have resulted from the lack of DHFR in them. Binding of MTXPGs to DHFR in CHO-UTC may interfere with their further polyglutamylation. When cells were resuspended in drug-free buffer for 1 hr following a 24-hr incubation with MTX, the retention of drug was less in DUKXB11 cells (46%) than in CHO-UTC cells (78%), due mainly to a greater loss of unmetabolized MTX in the mutant cells (89%) than in control cells (26%). Nevertheless, the amount of non-exchangeable unmetabolized MTX retained in DUKXB11 cells following exposure to 3.0 microM MTX exceeded the MTX-binding capacity. These studies demonstrate that DHFR-deficient cells accumulated more and longer MTXPGs than control cells. In addition, they suggest that some unmetabolized MTX was retained in cells not bound to DHFR.
Assuntos
Metotrexato/metabolismo , Mutação , Tetra-Hidrofolato Desidrogenase/deficiência , Animais , Linhagem Celular , Cricetinae , Cricetulus , Feminino , Cinética , Metotrexato/análogos & derivados , Metotrexato/isolamento & purificação , Ovário , Ácido Poliglutâmico/análogos & derivados , Ácido Poliglutâmico/isolamento & purificação , Ácido Poliglutâmico/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
Previous studies have demonstrated that insulin augments methotrexate transport and enhances its cytotoxicity to human breast cancer cells. We therefore investigated the effects of insulin on methotrexate polyglutamate synthesis and binding to dihydrofolate reductase (DHFR) in two human breast cancer cell lines, MCF-7 and MDA-MB-231. Cells were exposed to 2 microM [3H]MTX and varying insulin concentrations for the desired time before determination of the polyglutamate content by high-performance liquid chromatography (HPLC). DHFR-bound drug was separated from free intracellular drug by chromatography on DEAE-Sephacel minicolumns prior to HPLC analysis. Incubation of MCF-7 cells with 2.5 nM insulin for 48 h before exposure to 2 microM [3H]MTX for a further 24 h resulted in a significant increase in both total drug and total polyglutamates compared with control cells. Increasing the insulin concentration in the medium yielded further increases in polyglutamylation so that at 250 nM insulin and above total polyglutamates were increased by 64% compared with control cells. Further evaluation of the effects of physiologic insulin levels on polyglutamate synthesis revealed that 2.5 nM insulin caused an increase in the net glutamylation rate for each polyglutamate derivative during the final 12 h of a 24 h exposure to MTX. Analysis of the effects of insulin on polyglutamate binding to DHFR revealed that exposure to 2.5 nM insulin resulted in the preferential binding of higher polyglutamates to DHFR. In MDA-231 cells, a breast cancer cell line with a poor capacity for polyglutamate synthesis, insulin exposure resulted in an increase in the cellular accumulation of each polyglutamate derivative, with the greatest proportionate increases occurring in the cellular levels of higher polyglutamates. These data suggest that insulin augmentation of MTX polyglutamate synthesis may account for its previously observed ability to enhance MTX cytotoxicity.
Assuntos
Neoplasias da Mama/metabolismo , Insulina/farmacologia , Metotrexato/análogos & derivados , Peptídeos/metabolismo , Ácido Poliglutâmico/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Linhagem Celular , Feminino , Humanos , Cinética , Metotrexato/isolamento & purificação , Metotrexato/metabolismo , Ácido Poliglutâmico/análogos & derivados , Ácido Poliglutâmico/isolamento & purificação , Ligação ProteicaRESUMO
To examine directly the hepatic and renal toxicity of 7-hydroxymethotrexate (7-OH-MTX) without interference of the parent compound methotrexate (MTX), we purified and gave 100 mg/kg 7-OH-MTX to rats, a dose resulting in serum levels of 7-OH-MTX comparable with those achieved in the clinic after the administration of high-dose MTX (HD-MTX). After only 5 h, the 7-OH-MTX-treated rats demonstrated 2.6-fold increases in serum creatinine values and 2-fold elevations in serum aspartate aminotransferase (ASAT) levels as compared with the controls. Morphologic evidence of toxicity, however, was apparent only in the kidneys. Intraluminal cellular debris containing membranous material and deteriorated organelles was seen, but no precipitate of the delivered drug. The peak serum concentration of 7-OH was up to 939 microM, and concentrations of 7-OH-MTX declined triphasically, showing a t1/2 alpha value of 2.45 min, a t1/2 beta value of 30.5 min, and a terminal half-life (t1/2 gamma) of 240 min. The total clearance value was 14.5 ml min-1 kg, and the postdistributional volume of distribution (V beta) was 5070 ml/kg. Our results may indicate a direct toxic effect of 7-OH-MTX on kidney and liver cells.
Assuntos
Antagonistas do Ácido Fólico/toxicidade , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Metotrexato/análogos & derivados , Animais , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Antagonistas do Ácido Fólico/administração & dosagem , Antagonistas do Ácido Fólico/análise , Antagonistas do Ácido Fólico/isolamento & purificação , Antagonistas do Ácido Fólico/farmacocinética , Humanos , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Metotrexato/administração & dosagem , Metotrexato/análise , Metotrexato/isolamento & purificação , Metotrexato/farmacocinética , Metotrexato/toxicidade , Ratos , Ratos Wistar , Fatores de Tempo , Distribuição TecidualRESUMO
The conversion of methotrexate to poly-gamma-glutamyl derivatives in Ehrlich ascites carcinoma cells which are characterized by different pools of endogenous folates is described. The cells in which folate pool was high (the 5-fluorodeoxy-uridine-resistant cell line) the ability to convert methotrexate to its polyglutamate derivatives was much lower than in the cells in which folate pool was smaller (the parental cell line). When the cellular folate pool was reduced by treatment of the cells with lysolecithin, a similar methotrexate polyglutamate concentration in both cell lines was observed. These data suggest that cellular folate pool has a regulatory effect on methotrexate polyglutamate synthesis.
Assuntos
Carcinoma de Ehrlich/metabolismo , Ácido Fólico/metabolismo , Metotrexato/análogos & derivados , Biossíntese Peptídica , Ácido Poliglutâmico/biossíntese , Animais , Cinética , Metotrexato/biossíntese , Metotrexato/isolamento & purificação , Metotrexato/metabolismo , Camundongos , Ácido Poliglutâmico/análogos & derivados , Ácido Poliglutâmico/isolamento & purificaçãoRESUMO
For validation of a new microperfusion system to follow the influence of pharmacokinetic parameters of cytostatic drugs on cultured tumor cells, we investigated the effects of methotrexate (MTX) and etoposide (VP16-213) on L1210 colony growth. Inhibition kinetics of cells were compared to those obtained by suspension culture exposure. We found good correlations between the IC50 values measured with either method. Evaluation of the kinetics under constant drug concentrations (steady-state) showed that the microperfusion method is comparable to other methods. A simple HPLC column-switching method was developed to determine drug concentrations in serum-free medium. Under these conditions solutions of etoposide were found to be unstable. Its quantity decreased biphasically with a concentration-dependent first-order kinetic in the initial phase. The relevance of exposure dose (cxt) for the mode of drug action has been shown, especially for instable solutions of drugs (VP16-213). The microperfusion method might substantially improve in vitro screening assays of anticancer drugs.
Assuntos
Etoposídeo/farmacologia , Etoposídeo/farmacocinética , Leucemia L1210/tratamento farmacológico , Leucemia L1210/metabolismo , Metotrexato/farmacologia , Metotrexato/farmacocinética , Animais , Cromatografia , Estabilidade de Medicamentos , Etoposídeo/isolamento & purificação , Cinética , Metotrexato/isolamento & purificação , Camundongos , Microquímica/métodos , Perfusão , Células Tumorais CultivadasRESUMO
Chiral high performance liquid chromatography was used for the enantiometric separation of leucovorin. The optimal separation conditions recommended after optimizing the mobile phase composition, flow rate, and temperature are described. Achiral reversed-phase chromatographic method was applied for the simultaneous separation of methotrexate and leucovorin in clinical samples. Column-switching system including achiral short pre-column and chiral analytical column based on immobilized bovine serum albumin were used for simultaneous determination of leucovorin and methotrexate patients treated at the National Cancer Institute.
Assuntos
Leucovorina/química , Metotrexato/química , Cromatografia Líquida de Alta Pressão , Humanos , Indicadores e Reagentes , Leucovorina/sangue , Leucovorina/isolamento & purificação , Metotrexato/sangue , Metotrexato/isolamento & purificação , EstereoisomerismoRESUMO
Synthesis of poly-gamma-glutamyl metabolites of methotrexate was demonstrated in mouse small intestine, liver and bone marrow, and in L1210 leukemia, Sarcoma 180 and Ehrlich tumor cells after sc injections of [3H]methotrexate to tumor bearing mice. Ion exchange chromatography of tissue extracts resolved six peaks of radioactivity believed to represent methotrexate and metabolites with up to 4 additional glutamyl residues. Polyglutamate formation in L1210 cells and small intestine was shown to be independent of dose at least to 400 mg/kg as long as intracellular levels of drug in excess of the dihydrofolate reductase binding capacity (exchangeable) were maintained. Both the total amount of polyglutamates and the average length of the polyglutamyl chain increased with time as long as exchangeable level of drug were present intracellularily. The results also showed differences in the extent of metabolism of methotrexate polyglutamates among the tissues examined. Although, these differences were at times very large, there was no consistent correlation between these differences and other pharmacologic parameters or cytoxicity. Tumor cells appeared to synthesize more polyglutamates than the normal tissues examined. However, differences in total drug persistence and sensitivity to drug among tumor cells and among normal tissues did not reflect the relative extent of polyglutamate synthesis in each group. We observed no selective retention of polyglutamates as compared to methotrexate by L1210 cells in vitro as indicated by the extracellular accumulation during efflux of methotrexate and the polyglutamates. This could only be demonstrated by allowing efflux of intracellular drug in the presence of extracellular dihydrofolate reductase, which averted hydrolysis of the polyglutamates. It is concluded that the extent of polyglutamate synthesis per se may not be a determinant of drug sensitivity in murine tissues. However, the accumulation of these metabolites may contribute in some way to overall therapeutic response or relative cytotoxicity.