Serum Methotrexate Research Articles

Effects of hemoperfusion plus high-flux hemodialysis in a patient with methotrexate toxicity

Acute methotrexate toxicity resulting from methotrexate-induced renal failue is a medical emergency requiring extracorporeal removal of methotrexate. The optimum method of methotrexate removal has not yet been established. We report a case of osteosarcoma with lung metastasis that was treated with high-dose methotrexate as adjuvant chemotherapy. Although no problems occurred after the first 5 courses of methotrexate, methotrexate-induced renal failure and methotrexate toxicity appeared after the sixth course. The patient was treated, either with hemoperfusion plus high-flux hemodialysis, or hemoperfusion alone, and pre- and post-treatment serum methotrexate concentrations were monitored. The reduction in methotrexate by hemoperfusion alone for 2 hours was 54%, compared to a mean reduction of 59% by hemoperfusion combined with high-flux hemodialysis, for 3 hours. Rebound increases in methotrexate levels were small (less than 1 μmol/L) with either method. The combination of hemoperfusion and hemodialysis resulted in good control of volume status, as well as improvement in serum chemistry values.

Successful Rescue in a Patient with High Dose Methotrexate-Induced Nephrotoxicity and Acute Renal Failure

We describe the case of a 35-year old male who developed acute renal failure following high dose methotrexate therapy for Burkitt's non Hodgkin lymphoma. Serum methotrexate levels reached 37 μmol/l, and remained higher than 1 μmol/l for more than a week. Folinic acid rescue was intensified to 200–400 mg intravenously every 4 hours. As methotrexate binds markedly to proteins, plasma exchange was initially chosen, 4 sessions being performed from day 2 to day 4. The methotrexate pharmacokinetic profile was not significantly modified during plasma exchange, and serum drug level was 3 μmol/l. Continuous veno-venous hemodiafiltration was therefore performed from day 5 to day 10. This procedure also seemed ineffective, with evidence of low ultrafiltrate clearance. No extrarenal toxicity was observed in our patient. Thus, conventional extrarenal procedures appear to have a limited role in the setting of overexposure to methotrexate. The use of very high doses of folinic acid in our case probably played a major role in the eventual favorable outcome.

Determination of methotrexate in serum by high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method with ultraviolet detection was developed to quantitate methotrexate in serum-based calibrators, controls and patient samples. Sample clean-up was achieved with C 18 Sep-Pak Classic cartridges. The chromatographic separation was accomplished on a 5-μm Ultrasphere ODS Beckman column. 8-Chlorotheophylline was used as an internal standard. The method was validated by recovery, linearity, accuracy and precision studies. Two standard curves were constructed to cover the high and the low ends of the calibrator range (0.05–1.0 μmol/l). Response was found linear over the whole range of the calibrator set with a correlation coefficient of 0.999 and 1.00 for the low-level and the high-level curves, respectively. Accuracy varied from 12% at the lowest level to 1.2% at the highest level. The precision study showed a C.V. of 14.4% at the lowest level and 3.3% at the highest level.

Methotrexate serum concentration and histological response to multiagent primary chemotherapy for osteosarcoma of the limbs.

The authors investigated the influence of methotrexate (MTX) serum concentration on (histologically evaluated) tumor necrosis, induced by a primary multiagent chemotherapy, including MTX, for osteosarcoma. MTX serum peaks in 151 patients, preoperatively treated with MTX (8-12g/m2), cisplatin (120mg/m2) and Adriamycin (60mg/m2), were analyzed. Significantly (p < 0.01) higher serum MTX mean peaks were observed in patients with complete tumor necrosis (MTX 773.8 mumol/l) compared to patients with 90-99% tumor necrosis (639.8 mumol/l), 50-89% tumor necrosis (649.1 mumol/l) or less than 50% tumor necrosis (610 mumol/l). Complete tumor necrosis was observed in 9% of patients with MTX peaks of less than 600 mumol/l, in 27% of patients with serum MTX peaks between 600 and 699 mumol/l and in 37% of those with MTX peaks ranging from 700 to 799 mumol/l. Higher MTX peaks (800-899, 900-999, > 1000 mumol/l) were not associated with a further increase of cases with complete tumor necrosis. 40% of patients with an MTX peak greater than 700 mumol/l had complete tumor necrosis, compared to 15.5% of patients who did not reach this value (p < 0.002). At a multivariant analysis including age, sex, tumor site and volume, pretreatment serum alkaline phosphatase and lactic dehydrogenase levels, MTX peaks of 700 mumol/l and, less significantly, the histologic type (telangiectatic osteosarcoma), were independent factors influencing tumor necrosis. The authors conclude that MTX serum peaks significantly influence chemotherapy-induced tumor necrosis in osteosarcoma. In a primary treatment consisting of cisplatin, Adriamycin and MTX, complete tumor necrosis can be obtained in 40% of patients with MTX peak concentrations > or = 700 mumol/l.

Activation and cytotoxicity of 2-α-aminoacyl prodrugs of methotrexate

In an effort to improve the selectivity of the anticancer drug methotrexate (MTX), a series of potential prodrugs in which the 2-amino group was acylated with various α-amino acids (as well as l-pyroglutamic acid) was synthesized. Such derivatives are anticipated to be hydrolysed to MTX by appropriate aminopeptidases localized (over-expressed naturally or targeted as anti-tumor antibody conjugates) in the vicinity of the tumor. The l-leucyl, l-valyl, l-isoleucyl, d-alanyl and l-pyroglutamyl derivatives were assessed as to their suitability as prodrugs. Except for the l-pyroglutamyl compound, all derivatives decomposed slowly when incubated in phosphate buffer, pH 7.3; the formation of MTX was minimal. No major differences were observed when serum was included in the incubation medium, except for the l-leucyl compound, which was hydrolysed to MTX. The l-leucyl, l-valyl and l-isoleucyl derivatives were hydrolysed readily to MTX by aminopeptidase M (EC 3.4.11.2), while the l-pyroglutamyl and d-alanyl compounds were activated by pyroglutamate aminopeptidase (EC 3.4.19.3) (from Bacillus amyloliquefaciens) and d-aminopeptidase (from Ochrobactrum anthropi), respectively. When tested for inhibition of the target enzyme dihydrofolate reductase (DHFR; EC 1.5.1.3), 2- l-valyl-MTX showed inhibition two orders of magnitude poorer than that given by MTX, in agreement with the expectation that acylation of the 2-amino group reduces binding to DHFR. After treatment of this derivative with aminopeptidase M, the extent of inhibition correlated with the amount of MTX formed. MTX derivatives alone or in combination with the complementary peptidase were tested for cytotoxicity on murine L1210 cells in culture. The above-listed derivatives were considerably less cytotoxic than MTX, except for the l-leucyl derivative which showed considerable cytotoxicity. When the appropriate exogenous peptidase was included, the cytotoxicity of the activated prodrugs approached that of MTX. These results indicate that 2- l-leucyl-MTX is unsuitable as a prodrug since it is activated prematurely by serum enzymes. Although the l-valyl and l-isoleucyl derivatives do not hydrolyse to MTX in serum and are readily activated, they are not ideal prodrugs since they decompose under physiological conditions; the properties of the decomposition product will have a bearing on the ultimate suitability of these compounds. 2- l-Pyroglutamyl-MTX is the best candidate prodrug, showing stability and ready activation by the appropriate aminopeptidase.

Microanalysis of methotrexate by high-performance liquid chromatography using a fluorescence detector

Methotrexate (N10-methyl-4-aminofolic acid) has been determined by HPLC after oxidizing it with permanganate to a fluorogenic derivative. The detection limit has been 0.01 μg/ml. The method has been applied for the determination of methotrexate in blood serum of rats.

High-performance liquid chromatographic determination of methotrexate, 7-hydroxymethotrexate, 5-methyltetrahydrofolic acid and folinic acid in serum and cerebrospinal fluid

A method for the simultaneous determination of the antifolates methotrexate and 7-hydroxymethotrexate as well as the folates 5-methyltetrahydrofolic acid and folinic acid (5-formyltetrahydrofolic acid) in serum and cerebrospinal fluid (CSF) is described. High-performance liquid chromatography with gradient elution and dual detection (ultraviolet absorption and fluorescence) was used to separate and quantitate the analytes. Serum samples containing high levels of the substances of interest and CSF samples were injected directly onto the HPLC column. For determination of low concentrations, serum samples were subjected to a solid-phase extraction method for clean-up and concentration purposes. The determination limits were 10 ng/ml for both antifolates, 100 ng/ml for folinic acid, and 0.1 ng/ml for the physiologically occurring methylated folate which is about 1 100 the serum concentration in healthy children. The suitability of the method for pharmacokinetic monitoring of high-dose methotrexate therapy combined with leucovorin rescue administered to children with acute lymphoblastic leukemia was demonstrated. Minimum values of the serum folate during treatment ranged from 0.2 to 3.1 ng/ml. Even those very low concentrations could be reliably measured.

Systemic treatment of ectopic pregnancies with single-dose methotrexate

Study Objective To evaluate the sucess rate, side effects, reproductive outcome, and possible pitfalls of systemic low-dose methotrexate (MTX) for treatment of ectopic pregnancy (EP). Design Longitudinal, nonrandomized trial. Setting Department of Obstetrics and Gynaecology at a teaching hospital in Germany. Patients and Interventions Forty women with EP were treated with a single dose of MTX 20 to 40 mg, intravenously. The highest pretreatment human chorionic gonadotropin (hCG) titer was 21,100 mlU/ml. Measurements and Main Results Thirty-seven patients (92.5%) were treated successfully. Thirty-four (85%) received a single dose of MTX. Six (15%) required a second dose about a week later. Three patients (7.5%) required surgical intervention. Serum MTX levels decreased below 0.1 μmol/L (45.4 μg/L) within 24 hours. In 53.8% of women hCG levels decreased directly after treatment; in 46.2% levels increased within the first 5 days after treatment. Conclusions In selected patients, systemic low-dose MTX is an effective way to treat EP, and has no demonstrable side effects.

The Effect of Low‐Dose Methotrexate on Bone Metabolism and Histomorphometry in Rats

To ascertain the effects of low-dose methotrexate (MTX) on bone metabolism and histomorphometry in rats. Female Sprague-Dawley rats (6 months old, n = 42) were divided into the following 4 groups: intraperitoneal (IP) injections of MTX, with and without ovariectomy, and IP saline (controls), with and without ovariectomy. Injections were given for 16 weeks. The MTX dose was equivalent to a standard dose for rheumatoid arthritis in humans that would yield similar serum MTX levels (0.6 +/- 0.1 mumoles). Bone formation (assessed by serum alkaline phosphatase and osteocalcin levels and histomorphometry) was significantly lower in the MTX groups, and bone resorption (assessed by urinary hydroxyproline levels and histomorphometry) was increased in the MTX groups. Bone mass was significantly diminished in the MTX groups. Prolonged administration of low-dose MTX in rats causes significant osteopenia via suppression of osteoblast activity and stimulation of osteoclast recruitment, which results in increased bone resorption.

Effect of protein-calorie malnutrition on methotrexate pharmacokinetics.

Methotrexate toxicity is increased in protein-calorie malnutrition. The influence of protein-calorie malnutrition on the pharmacokinetics and binding of methotrexate (MTX) and the formation of its major hepatic metabolite, 7-hydroxy-methotrexate was examined in 30 adult, female Lewis rats. Animals were randomized to receive either a standard diet (22.0% protein; 4.20 kcal/g) or a protein-depleted diet (PD) (0.03% protein; 4.27 kcal/g) ad libitum for 35 days. Animals were then separated into two groups for either methotrexate pharmacokinetics (n = 20) or serum protein binding (n = 10) studies. The mean weight loss in the PD group was 26% of their initial body weight, as compared with a 29% weight gain in the control group. In the protein binding study, a significant decrease in serum albumin (19%), uncorrected creatinine clearance (38%), and free fraction of MTX (15%) was found in the PD group. All animals in the pharmacokinetic study received a single intraperitoneal injection of MTX (10 mg/kg), and serum MTX and 7-hydroxy-methotrexate concentrations were determined using a specific, reversed phase, high-performance liquid chromatography assay. The mean AUC0-3 in the PD group was 43.6 +/- 3.9 micrograms/mL per hour compared with 15.8 +/- 1.1 micrograms/mL per hour in the control group (p < .001). The time to peak and the peak serum concentrations were significantly greater in the PD animals, which indicated delayed absorption and clearance. These results suggest that the increase in MTX toxicity observed in protein-calorie malnutrition is associated with a decrease in MTX clearance, and is not related to changes in protein binding or formation of 7-hydroxy-methotrexate.

Evaluation of serious adverse events in patients treated with protocols including methotrexate infusions.

During the period 1979 to 1992 we treated 141 children for various malignant diseases with protocols including methotrexate (MTX) infusions in doses ranging from 0.5 to 33.6 g/m2. During a total of 922 courses, there were no fatal complications associated with MTX treatment. Serum MTX concentration and pharmacokinetic data were monitored continuously during the infusions. In this study, we evaluated the occurrence of serious untoward reactions to MTX infusions. Impaired renal function with delayed drug elimination was seen in seven patients, all boys, especially after short infusion times. All recovered completely without any serious clinical symptoms. In three leukemia patients who later died from resistant disease, we observed late neurological disturbances and computer tomography (CT) brain scan abnormalities. Pharmacokinetic data from the patients with complications are described and confirm that serial MTX concentration monitoring is the most important early indicator of renal toxicity.

Effect of neutropenia on gastric mucosal integrity and mucosal nitric oxide synthesis in the rat

The present study examined the interaction between neutrophils and gastric mucosal nitric oxide (NO) synthesis in the regulation of gastric mucosal integrity in the rat. Male Sprague-Dawley rats were made neutropenic by intraperitoneal injections of methotrexate (MT; 2.5 mg/kg) or antineutrophil serum (ANS; 100 microliters). Control rats were treated with saline. Neutropenia was confirmed by circulating neutrophil counts and tissue myeloperoxidase activity. Neutropenic or control rats were given 2 ml of ethanol (EtOH; 40% w/v intragastrically). Neutropenic rats were less susceptible to EtOH-induced mucosal damage when compared to control rats. Mucosal NO synthesis was increased in neutropenic rats. EtOH instillation reduced NO synthase in control rats. However, in MT-treated rats the reduced NO synthase activity was not different from that observed in untreated control rats. (1) EtOH-mediated gastric mucosal injury appears to be a neutrophil-mediated process. (2) Neutropenia results in an increase in mucosal NO synthesis. (3) MT treatment augments the degree of mucosal integrity. The increase in integrity is associated with a reduction in mucosal neutrophil infiltration as well as maintenance of NO synthase activity. ANS appears to influence mucosal integrity primarily by a reduction in circulating neutrophils.

Randomized comparison of 36-hour intermediate-dose versus 4-hour high-dose methotrexate infusions for remission induction in relapsed childhood acute lymphoblastic leukemia.

Pharmacokinetics, toxicity, and therapeutic efficacy of two different methotrexate (MTX) infusions for remission induction of relapsed childhood acute lymphoblastic leukemia (ALL) were investigated in a randomized multicenter trial. Sixty patients with early bone marrow relapse received a polychemotherapy induction protocol starting with either 12 g/m2 MTX as a 4-hour infusion (high-dose [HDM]) or 1 g/m2 as a 36-hour infusion (intermediate-dose [IDM]). In HDM, leucovorin (LCV) was administered orally (12 times, 15 mg/m2 every 6 hours), beginning at hour 24. In IDM, only two doses were administered at hours 48 and 54. Median serum MTX concentrations during infusion were 716 mumol/L in HDM and 7.2 mumol/L in IDM. In HDM, MTX serum levels at hour 24 (median, 2.8 mumol/L) were significantly less than steady-state levels of IDM. Concentrations greater than 1 mumol/L were maintained for 36 hours with HDM and 45 hours with IDM. General tolerance to treatment was better in the HDM group. Mucosal lesions occurred significantly more often and were more severe after IDM treatment. A median treatment delay of 3 days was required in the IDM group but not in the HDM group. At day 15, complete remission (CR) was documented in 45% of IDM- and 48% of HDM-treated patients. Persistent blasts (> 5%) appeared more frequently in HDM than in IDM (35% v 19% of patients; P = NS). After completion of induction therapy, 28 of 30 patients in each group achieved CR. Both regimens produced the same remission rates. The tendency to better antileukemic activity of IDM was accompanied by more severe side effects as a consequence of long-lasting cytotoxic MTX levels. Hence, long-term infusion of IDM followed by low-dose LCV is an effective treatment for recurrent ALL.

Serum Methotrexate (MTX) Concentrations and Prognosis in Patients with Osteosarcoma of the Extremities Treated with a Multidrug Neoadjuvant Regimen

The relationship between the serum concentration of methotrexate and the prognosis has been studied in 108 patients with osteosarcoma of the extremities treated from September 1986 to December 1989 at the Chemotherapy Department of Rizzoli Hospital. The protocol of neoadjuvant chemotherapy included high doses of methotrexate (HDMTX) adriamycin, cisplatinum, ifosfamide and VP-16. After a median follow-up of 40.4 months (range 24-62), 84 (77.7%) of the patients studied remained continuously disease-free (CDF) and 24 relapsed. Significantly higher mean serum MTX concentrations were observed in the patients who remained CDF (669.5 mumol/l) than in the patients who relapsed (571.9 mumol/l) (p < .004). The breaking point of prognostic significance for the serum MTX levels seems to be 700 mumol/l. In fact, according to the mean MTX concentrations, the patients with less than 700 mumol/l showed a significantly lower disease-free survival than the patients with higher mean MTX concentrations (68.12% vs 94.87% p < .0013). The distribution of prognostic variables between the two groups was the same in terms of site and histological type of tumor and alkaline phosphatase serum levels at diagnosis. In the group which had more than 700 mumol/l MTX, a higher percentage of good histological response after primary chemotherapy was observed. This is probably independent from the MTX because no significant preoperative MTX serum levels between good and partially responding patients were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Dry Reagent Technology. Kodak Ektachem 700 XR in Clinical Enzymology

The comparability of results for enzyme activity concentrations estimated with the methods recommended by the Scandinavian Committee on Enzymes(SCE) or the subcommittee on Enzymes of the European Committee for Clinical Laboratory Standards(ECCLS), and with the Kodak Ektachem methods, are discussed. As the Ektachem system in principle uses the IFCC methods at 37 degrees C as a reference, the results compare reasonably well for the alanine amino-transferase(ALAT), aspartate aminotransferase(ASAT), creatine kinase(CK) and gamma-glutamyltransferase(GT). The Ektachem reference method for lactate dehydrogenase(LD) is in principle the SCE method. The reasons for lower values with the SCE method are discussed. The interference on the Ektachem alkaline phosphatase (ALP) method from high concentrations of methotrexate(MTX) in serum, is shown. Results from our investigations on the interference by heparin in enzyme determinations on Ektachem are given. The problems with CK determinations in cancer patients regarding myocardial diseases are well known. When CK-BB is present in serum, the CK-B concentration is apparently lower using the Ektachem CK method than with use of the CK-MB slide, probably relating to the fact that chelator is omitted from the CK slide, while EGTA is supplied in the CK-MB slide. The user's dependence on the manufacturer for the choice of methods and the quality of the reagents, makes it important for laboratories and the Kodak Company to collaborate with the intention to continuously improve the Ektachem methodologies. The future possibilities of this technology seem to be nearly unlimited.

Fluorometric Determination of Methotrexate in Serum by High-Performance Liquid Chromatography Using In-Line Oxidation with Hydrogen Peroxide

A fluorometric method has been developed for the determination of methotrexate in serum by high-performance liquid chromatography. Methotrexate after deproteinization with 2 M perchloric acid was separated by reversed-phase chromatography with a neutral mobile phase (pH 7.0, 8% acetonitrile in 25 mM phosphate buffer) containing 25 mM hydrogen peroxide as a fluorogenic reagent, and was fluorometrically detected (Ex 379 nm and Em 457 nm) via in-line oxidation at high temperature (160°C), The calibration curve could be made linear over the range 1 × 10-6 - 1 × 10-5 M by injecting a volume of 20 μl of deproteinized serum. The detection limit (signal-to-noise ratio=3) for methotrexate in serum was 2 × 10-8 M using a 50-μl aliquot of deproteinized serum. Comparison with the fluorescence polarization immunoassay gave a good correlation coefficient of 0.949.

The effect of food on the absorption of methotrexate sodium tablets in healthy volunteers

To examine the effect of food on the absorption and bioavailability of low-dose orally administered methotrexate sodium tablets. In this randomized, 2-way crossover study, a 7.5-mg dose of methotrexate (three 2.5-mg tablets) was administered to 12 healthy male volunteers after an overnight fast or within 10 minutes of consuming a high fat-content breakfast. Serum methotrexate concentrations over the next 24 hours were used to determine the area under the concentration-time curve (AUC), the maximum concentration, the time to maximum concentration (tmax), and the serum half-life for each phase. Food delayed the tmax by approximately 30 minutes, but the extent of absorption, as measured by the AUC, for both phases was similar. These results demonstrate that the bioavailability of low-dose orally administered methotrexate sodium tablets is not influenced by food.

The influence of serum methotrexate concentrations and drug dosage on outcome in childhood acute lymphoblastic leukaemia

Sequential methotrexate (Mtx) absorption studies were undertaken in 127 children undergoing treatment for childhood non-T acute lymphoblastic leukaemia (ALL) to determine whether serum drug concentration, clearance and dosage affect event free survival (EFS). Higher serum concentration and area under the plasma concentration curve (AUC) were not associated with an improved EFS. Methotrexate clearance was not found to be of prognostic significance. Patients who tolerated only low 6-mercaptopurine (6-MP) doses because of neutropaenia and those who randomly were prescribed higher doses of Mtx had a lower rate of leukaemia relapse after the completion of therapy. This suggests that the use of maintenance therapy in maximally tolerated doses may be associated with an increased survival in childhood ALL.

High-performance liquid chromatography method for serum methotrexate levels in children with severe steroid-dependent asthma

Monitoring of low-dose methotrexate (MTX), as used in severe steroid-dependent asthma, requires a sensitive and reproducible technique which has hitherto not been available. A high-performance liquid chromatographic method for the determination of MTX in serum is reported. The method involves deproteinization with acetone followed by addition of butanol and diethyl ether. The percentage recovery with this method compared with others was high (90 versus 70%). The samples were chromatographed on a reversed-phase ODS column and monitored at 313 nm. The retention time for MTX was 14.7 min. Pharmacokinetics of MTX was studied in five patients (age 3–15 years) with severe asthma who received a weekly oral dose of 10 mg/m 2 body surface area. Following administration, the serum disappearance was monophasic with a half-life of 5 h. A metabolite, 7-hydroxymethotrexate was detected in serum after 2 h and reached a maximum concentration after 6 h. This new method will facilitate monitoring of asthmatic patients on methotrexate and allow for dose response and toxicity studies to be conducted.

Methotrexate Disposition Following Disruption of the Blood-Brain Barrier

Intracarotid drug administration after osmotic blood-brain barrier disruption (BBBD) enhances drug delivery to brain tumors. Despite clinical use, the pharmacokinetics of drugs following BBBD has not been described to date. Since methotrexate exhibits a concentration-toxicity response relationship, methotrexate disposition was determined following BBBD and intracarotid administration in seven patients with nonoperable brain tumors. Following a 1.5-5 g intracarotid methotrexate injection, 12 blood samples and 9 urine collections were obtained. Methotrexate concentrations in serum and urine were determined by fluorescence polarization immunoassay. The serum concentration-time data were best described by a three-compartment model. Systemic and renal clearances were consistent with previous studies. However, a prolonged mean terminal half-life of 51.5 h was observed. Serum methotrexate concentrations at 72 h exceeded 0.1 mumol/L in five of seven patients. Stomatitis occurred in one patient. Relative to previous reports, prolonged methotrexate half-life and cytotoxic methotrexate concentrations were observed in the serum of patients receiving intracarotid methotrexate following BBBD. Due to the prolonged cytotoxic methotrexate concentrations observed, extended leucovorin therapy may be indicated following BBBD and intracarotid methotrexate.