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.

Sensitive determination of methotrexate in plasma of children with acute leukemia using double-solvent supramolecular systemas a novel extractant for dispersive liquid-liquid microextraction.

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.

Dispersive Micro-Solid Phase Extraction for Sensitive Determination of Methotrexate from Human Saliva Followed by Spectrophotometric Method.

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.

Insights on Ultrafiltration-Based Separation for the Purification and Quantification of Methotrexate in Nanocarriers.

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.

A novel mixed hemimicelles dispersive micro-solid phase extraction using ionic liquid functionalized magnetic graphene oxide/polypyrrole for extraction and pre-concentration of methotrexate from urine samples followed by the spectrophotometric method.

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 -8.35 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 1000 ng/mL with a good correlation (R2 = 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 1 mg/kg/dose of MTX with acute lymphoblastic leukemia. The accuracy of the method was confirmed by HPLC measurements.

Use of short chain alkyl imidazolium ionic liquids for on-line stacking and sweeping of methotrexate, flinic acid and folic acid: their application to biological fluids.

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.

A novel dried blood spot-LCMS method for the quantification of methotrexate polyglutamates as a potential marker for methotrexate use in children.

OBJECTIVE: Development and validation of a selective and sensitive LCMS method for the determination of methotrexate polyglutamates in dried blood spots (DBS). METHODS: DBS samples [spiked or patient samples] were prepared by applying blood to Guthrie cards which was then dried at room temperature. The method utilised 6-mm disks punched from the DBS samples (equivalent to approximately 12 µl of whole blood). The simple treatment procedure was based on protein precipitation using perchloric acid followed by solid phase extraction using MAX cartridges. The extracted sample was chromatographed using a reversed phase system involving an Atlantis T3-C18 column (3 µm, 2.1 × 150 mm) preceded by Atlantis guard column of matching chemistry. Analytes were subjected to LCMS analysis using positive electrospray ionization. KEY RESULTS: The method was linear over the range 5-400 nmol/L. The limits of detection and quantification were 1.6 and 5 nmol/L for individual polyglutamates and 1.5 and 4.5 nmol/L for total polyglutamates, respectively. The method has been applied successfully to the determination of DBS finger-prick samples from 47 paediatric patients and results confirmed with concentrations measured in matched RBC samples using conventional HPLC-UV technique. CONCLUSIONS AND CLINICAL RELEVANCE: The methodology has a potential for application in a range of clinical studies (e.g. pharmacokinetic evaluations or medication adherence assessment) since it is minimally invasive and easy to perform, potentially allowing parents to take blood samples at home. The feasibility of using DBS sampling can be of major value for future clinical trials or clinical care in paediatric rheumatology.

Determination of methotrexate and folic acid by ion chromatography with electrochemical detection on a functionalized multi-wall carbon nanotube modified electrode.

A simple and sensitive method was developed for simultaneous determination of methotrexate (MTX) and folic acid (FA) by ion chromatography with electrochemical detection (IC-ECD). Quaternary amine functionalized multi-wall carbon nanotubes (q-MWNTs) modified glassy carbon electrode (GCE) was used as an amperometric sensor to determine MTX and FA. The electrochemical behaviors of MTX and FA at the q-MWNTs/GCE were studied by cyclic voltammetry (CV). Results indicated that this modified electrode exhibited electrocatalytic oxidation toward MTX and FA with high sensitivity, stability and long life. Good separation of MTX and FA was demonstrated by IC on an anion-exchange column with phosphate buffer solution (PBS) as eluent. Under the optimal conditions, the linear ranges were from 0.01 to 20mg/L for both MTX and FA with correlation coefficients r ≥ 0.9994. The obtained detection limits (LODs) for MTX and FA were 0.2 and 0.4 µg/L (S/N=3), respectively. The method has been successfully applied to the determination of MTX and FA in plasma and urine of patients of rheumatoid arthritis.

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.

Fabrication of electrolytic cell for online post-column electrochemical derivatization in ion chromatography.

An electrolytic cell (EC), composed of two ruthenium-plated titanium electrodes separated by cation-exchange membranes, was fabricated and evaluated for online postcolumn derivatization in ion chromatography (IC). Folic acid (FA) and methotrexate (MTX) were preliminarily used as prototype analytes to test the performance of EC. After separation by an anion exchange column, FA and MTX, which emit very weak fluorescence when excited, were electrochemically oxidized online in the anode chamber of the EC. The compounds with strong fluorescence, which are oxidation products, were detected by the fluorescence detector. The phosphate buffer solution (100 mM KH(2)PO(4)) served as an optimal eluent for anion exchange chromatographic separation and a suitable supporting electrolyte for electro-oxidation, leading to ideal compatibility between IC separation and the postcolumn electrochemical derivatization. For the presently proposed method, the linear ranges were from 0.01 mg L(-1) to 5 mg L(-1) for both FA and MTX. The detection limits of FA and MTX were 1.8 and 2.1 µg L(-1), and the relative standard deviations (RSD, n=7) were 2.9% and 3.6%, respectively. The method was applied for the simultaneous determination of FA and MTX in the plasma of patients being treated for rheumatoid arthritis. The determination of MTX in the urine of the patients of diffuse large B cell lymphoma was also demonstrated.

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.

Capillary electrophoretic determination of methotrexate, leucovorin and folic acid in human urine.

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.

Determination of the chiral and achiral related substances of methotrexate by cyclodextrin-modified micellar electrokinetic chromatography.

A cyclodextrin-modified micellar electrokinetic chromatographic (CD-MEKC) method for the determination of the most important potential impurities of methotrexate (MTX): 2,4-diamino-6-(hydroxymethyl)pteridine, aminopterine hydrate, 4-[N-(2-amino-4-hydroxy-6-pteridinylmethyl)-N-methylamino] benzoic acid, 4-[N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino] benzoic acid, and the distomer D-MTX is presented. The MEKC separation of these compounds was optimized by applying a step-by-step approach. The addition of beta-CD to a conventional MEKC system, based on sodium dodecyl sulfate (SDS) as surfactant, showed to be essential for the enantioresolution of racemic MTX as well as for the separation of the achiral impurities. To achieve high-resolution factor between the peaks adjacent to the main component (L-MTX), as required in the analysis of related impurities, the separation conditions were stressed; in particular, the addition of methanol to the CD-MEKC system resulted in a very effective choice. Under the optimized final conditions (100 mM SDS and 45 mM beta-CD in a mixture of 50 mM borate buffer, pH 9.30-methanol (75:25 v/v)), the method was validated showing a general adequate accuracy (93-106% recovery) in the determination of L-MTX related substances at the impurity level of 0.12% w/w with a relative standard deviation (RSD)% lower than 8% (n = 4). The method was successfully applied to the analysis of pharmaceuticals (tablets and injections) which showed to contain the distomer D-MTX as major impurity and aminopterine hydrate as a further related substance in the commercial tablets.

Separation of selected anticancer drugs using superheated water as the mobile phase.

Six anticancer drugs have been eluted on a polystyrene-divinylbenzene (PS-DVB) column with buffered superheated water as the mobile phase. The temperature range studied was from ambient temperature up to 160 degrees C, and the pH of the water was adjusted to 11.5 and 3.5 with phosphate buffer. It was possible to separate the substances 5-fluorouracil (5-FU), methotrexate (MTX), 7-hydroxymethotrexate (7-OH-MTX), and etoposide (VP-16) in one chromatographic run. The separation of these substances was optimized when adjusting the pH from 11.5 to 3.5, resulting in a total elution time of less than 13 min. Furthermore, retention factors of all of the investigated substances were measured at different temperatures and pH values.

Separation methods for methotrexate, its structural analogues and metabolites.

Methotrexate (MTX) is the prototype folate antagonist cytotoxic drug, employed in the therapy of solid tumors and leukaemias, and recently also as an immunosuppressive agent in organ transplantation, in the treatment of some autoimmune diseases and in the therapy of severe asthma. MTX is one of the very few antineoplastic drugs the therapeutic concentration monitoring of which is currently employed in clinical practice and can be routinely measured in biological samples by a number of different analytical techniques, among which are immunoenzymatic and chromatographic methods. Each technique has of course its own advantages in terms of sensitivity, specificity, speed, cost and level of expertise required. Along with therapeutic drug concentration monitoring and clinical pharmacology, fundamental research into the mechanism of action of antifolate drugs is still a field which requires the measurement of MTX, of its new analogues and of their metabolites in biological samples. This review summarizes the instrumental conditions and the performance of several published chromatographic methods employed to measure MTX, its metabolites and some analogues in clinical and biological research. More than 70 papers describing chromatographic assays for MTX and its metabolites have been published in the literature between 1975 and 2000. A wide array of experimental conditions for sample preparation, analyte separation and detection have been employed. According to their chemical properties, MTX, its metabolites and analogue drugs present in several biological samples (plasma, serum, saliva, urine, cerebrospinal fluid, tissue specimens) can be extracted, separated and detected under a variety of chromatographic conditions, i.e. on different stationary phases, under a wide choice of mobile phase conditions (acidic or neutral, employing ion-pair or micellar chromatography), followed by several detection techniques (UV-Vis spectrophotometry, pre- or post-column oxidation and fluorimetry, electrochemistry, mass spectrometry). Optimized methods allow simultaneous measurement within a few minutes of the plasma levels of MTX and its main metabolites at concentrations in the low-nM range. One special field which needs sensitive, fast and inexpensive methods for the detection and measurement of MTX is the monitoring of contamination in workplace environments, such as pharmaceutical industries and oncological hospital pharmacies, and in sewage waters. The measurement of the intracellular gamma-oligo-glutamate metabolites of biological folates, of MTX and of some analogue drugs is of great importance in basic pharmacological research. The existence of empirical quantitative relationships between the retention of individual oligomers under different chromatographic conditions and the number of added glutamic acid units allows identification of the metabolites even when authentic standards are not available.

Separation of etoposide phosphate and methotrexate by capillary zone electrophoresis using UV detection with a high sensitivity cell.

Etoposide phosphate and methotrexate are important anti-tumor chemotherapeutic agents. Our previously presented capillary zone electrophoresis (CZE) method using a high sensitivity cell (Z-cell) for quantitative analysis in biological media (urine, plasma) showed good precision and accuracy. The present results show that the investigation using a capillary with high sensitivity cell led to an approximately 10-fold improvement of the detection limit compared to standard capillaries. Plasma and urine samples were analyzed by using a calibration curve for drug concentrations between 0.1 and 100 microg/mL. Good detection limits and good relative standard deviations of the migration times and of the peak areas were observed in these experiments.

[Separation of methotrexate-polyglutamates by capillary electrophoresis and its application to the measurement of gamma-glutamyl hydrolase activity in human leukemia cells in culture].

We have applied capillary electrophoresis to the separation of methotrexate (MTX)-polyglutamates, and gamma-glutamyl hydrolase (GGH) activities in tumor cells were measured by using this new analytical method. MTX-polyglutamates were sufficiently separated in 15min by capillary electrophoresis with silica fused capillary (phi 50 microns x 75cm), being electrophoresed at 25kV and 30 degrees C in a buffer which contained 20mM sodium tetraborate, 20mM SDS and adjusted pH to 9.5. MTX-polyglutamates eluted were detected at 300nm UV. Cellular extracts obtained from the sensitive and antifolate-resistant human leukemia cell lines, MOLT-3 and K562, were incubated with MTX-glu5 at 37 degrees C for 1, 2 and 4 hr, and the amounts of the degradation products (glu1-glu4) were measured for GGH activity by capillary electrophoresis. There was no significant difference in the production of the metabolites between MOLT-3 and K562 cells (867 +/- 109 vs 799 +/- 56 pmol products/min/1 x 10(7) cells), however, the MTX-resistant MOLT-3 cells with a diminished polyglutamation of folates (MOLT-3/MTX.P-17) and the ZD1694-resistant K562 cells with the impaired membrane transport for reduced folates/MTX/ZD1694 (K562/ZD1694.C) showed decreased activities of GGH (519 +/- 52 and 680 +/- 99 pmol products/min/1 x 10(7) cells, respectively), suggesting the down-regulation of the enzyme in these antifolate-resistant cells concomitant with the intracellular substrate depletion. This study indicates that capillary electrophoresis is a rapid, cost-efficacious method with a sufficient reproducibility in the measurement of GGH activity and must be more suitable for the analysis of clinical samples than HPLC method which requires a large volume of the material.

Renal and hepatic toxicity after high-dose 7-hydroxymethotrexate in the rat.

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.

Concentration- and time-dependent cytostatic effects of methotrexate and etoposide on L1210 leukemic cells in vitro demonstrated by a new microperfusion method.

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.